Genetic/Epigenetic Modulation, Ocular Diseases, and ...downloads.hindawi.com/journals/specialissues/945685.pdf · Journal of Ophthalmology Genetic/Epigenetic Modulation, Ocular Diseases,

Journal of Ophthalmology

GeneticEpigenetic Modulation Ocular Diseases and Therapeutic Prospective

Guest Editors Jingsheng Tuo Lai Wei and Nan Hu

GeneticEpigenetic ModulationOcular Diseases and Therapeutic Prospective




Copyright copy 2013 Hindawi Publishing Corporation All rights reserved

This is a special issue published in ldquoJournal of Ophthalmologyrdquo All articles are open access articles distributed under the Creative Com-mons Attribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Editorial Board

Monica L Acosta New ZealandHee Bae Ahn KoreaLuis Amselem SpainUsha P Andley USASiamak Ansari Shahrezaei AustriaTaras Ardan Czech RepublicFrancisco Arnalich-Montiel SpainTakayuki Baba JapanAntonio Benito SpainSusanne Binder AustriaMehmet Borazan TurkeyGary C Brown USADavid J Calkins USAFrancis Carbonaro MaltaChi-Chao Chan USAHaoyu Chen ChinaLingyun Cheng USAChung-Jung Chiu USADaniel C Chung USAC I Clement AustraliaDavid K Coats USAMiguel Cordero-Coma SpainLucian Del Priore USAVasilios F Diakonis USAPriyanka P Doctor IndiaEdgar M Espana USAMichel Eid Farah BrazilPaolo Fogagnolo ItalyFarzin Forooghian CanadaBrian A Francis USAJoel Gambrelle FranceM-A Gamulescu GermanyIan Grierson UKKoray Gumus Turkey

Vishali Gupta IndiaAlon B Harris USATakaaki Hayashi JapanTakeshi Ide JapanVishal Jhanji Hong KongThomas Klink GermanyNaoshi Kondo JapanBobby S Korn USAOzlem Gurbuz Koz TurkeyRachel W Kuchtey USAHiroshi Kunikata JapanToshihide Kurihara JapanGeorgios Kymionis GreecePierre Lachapelle CanadaTimothy Y Lai Hong KongVan Charles Lansingh USATheodore Leng USAChristopher Leung Hong KongKin Sheng Lim UKPaloma B Liton USAMarco Lombardo ItalyTamer A Macky EgyptEdward Manche USAFlavio Mantelli ItalyEnrique Mencia-Gutierrez SpainMarcel N Menke SwitzerlandLawrence S Morse USADarius M Moshfeghi USAMajid M Moshirfar USAHermann Mucke AustriaRamon Naranjo-Tackman MexicoKristina Narfstrm USAMagella M Neveu UKNeville Osborne UK

Mahesh Palanivelu IndiaSuresh Kumar Pandey IndiaJijing Pang USAEnrico Peiretti ItalyPai-Huei Peng TaiwanDavid P Pinero SpainPawan Prasher IndiaYi Qu ChinaAntonio Queiros PortugalEduardo Buchele Rodrigues BrazilDirk Sandner GermanyAna R Santiago PortugalPatrik Schatz SwedenKyoung Yul Seo Republic of KoreaWisam A Shihadeh USAIngeborg Stalmans BelgiumKatsuyoshi Suzuki JapanS K Swamynathan USASuphi Taneri GermanyChristoph Tappeiner SwitzerlandStephen C Teoh SingaporeP G Theodossiadis GreeceBiju B Thomas USALisa Toto ItalyDavid A Wilkie USAWai T Wong USAVictoria WYWong Hong KongS C Wong UKHuseyin Yetik TurkeyTerri L Young USAHyeong-Gon Yu Republic of KoreaHunter Yuen Hong KongVicente Zanon-Moreno Spain

Contents

GeneticEpigenetic Modulation Ocular Diseases andTherapeutic Prospective Jingsheng Tuo Lai Weiand Nan HuVolume 2013 Article ID 980608 2 pages

Systems Biology Profiling of AMD on the Basis of Gene Expression Mones S Abu-Asab Jose SalazarJingsheng Tuo and Chi-Chao ChanVolume 2013 Article ID 453934 7 pages

RNA Interference Targeting Connective Tissue Growth Factor Inhibits the Transforming GrowthFactor-120573

2Induced Proliferation in Human Tenon Capsule Fibroblasts Jiaona Jing Ping Li Tiejun Li

Yuncheng Sun and Huaijin GuanVolume 2013 Article ID 354798 9 pages

An Extensive Replication Study onThree New Susceptibility Loci of Primary Angle Closure Glaucomain Han Chinese Jiangsu Eye Study Haihong Shi Rongrong Zhu Nan Hu Jian Shi Junfang ZhangLinjuan Jiang Hong Jiang and Huaijin GuanVolume 2013 Article ID 641596 5 pages

RNA Interference Targeting Snail Inhibits the Transforming Growth Factor 1205732-InducedEpithelial-Mesenchymal Transition in Human Lens Epithelial Cells Ping Li Jiaona Jing Jianyan HuTiejun Li Yuncheng Sun and Huaijin GuanVolume 2013 Article ID 869101 8 pages

Vascular Adhesion Protein 1 in the Eye Wenting Luo Fang Xie Zhongyu Zhang and Dawei SunVolume 2013 Article ID 925267 8 pages

Hindawi Publishing CorporationJournal of OphthalmologyVolume 2013 Article ID 980608 2 pageshttpdxdoiorg1011552013980608

EditorialGeneticEpigenetic Modulation Ocular Diseasesand Therapeutic Prospective

Jingsheng Tuo1 Lai Wei2 and Nan Hu3

1 Laboratory of Immunology National Eye Institute National Institutes of Health Bethesda MD 20892-1857 USA2 State Key Laboratory of Ophthalmology Zhongshan Ophthalmic Center Sun Yat-sen University Guangdong China3 Eye Institute Affiliated Hospital of Nantong University Nantong China

Correspondence should be addressed to Jingsheng Tuo tuojneinihgov

Received 27 November 2013 Accepted 27 November 2013

Copyright copy 2013 Jingsheng Tuo et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Complex eye diseases often have significant genetic compo-nents Previous work exploring the genetic contributions ofocular diseases has implicated numerous genomic regionsand a variety of candidate genes as modulators of thedisease susceptibility including cataract age-related maculardegeneration (AMD) diabetic retinopathy (DR) glaucomahigh myopia and others With the advance of techniquesboth on genotyping and phenotyping additional genes witha role in complex eye disease are waiting to be discoveredIn contrast it is apparent that a significant portion of theheritability of ocular disease cannot be explained through thealteration of DNA sequencesThe field of epigenetics pursuesthe changes in gene expression or cellular phenotypes causedby mechanisms other than changes in the underlying DNAsequence In general epigenetic changes pertain to DNAmethylation and histone modification Aberrant epigeneticchanges are associatedwith genomic instability andhave beenimplicated in various human diseases Recent advances inhigh-throughput platforms can generate voluminous datawhich requires desperately the tools of system biologyto effectively elucidate the true pictures underlying themKnowledge and understanding of these genetic componentsand pathways have led to the development of promisingtherapies including small inference RNA (siRNA)

This special issue contains 5 articles the contents of whichare summarized as follows

In the original paper ldquoAn extensive replication study onthree new susceptibility loci of primary angle closure glaucomain Han Chinese Jiangsu Eye Studyrdquo by A Shi et al the authorstried to replicate recent findings of three new susceptibility

loci for primary angle closure glaucoma (PACG) reportedby a genome-wide association study For a long time thegenetic study on glaucomahas been focused onprimary angleopen glaucoma Instead of using clinical diagnosis of PACGas the phenotype to study the authors chose a preclinicalcondition primary angle closure (PAC) and same anatomicalfeatures of eyes to investigate This community-based studydid not find any significant association between the definedphenotypes and the single nucleotide polymorphisms inPLEKHA7 COL11A1 and PCMTD1-ST18

In the reviewpaper ldquoVascular adhesion protein 1 in the eyerdquoby W Luo et al the authors gave an overview on the newresearch progresses of VAP-1 in the ocular diseases includinguveitis AMD DR and ocular tumor Based on the propertiesand results obtained so far from preclinical and clinicalstudies VAP-1 may provide a novel research direction or apotent therapeutic strategy for ophthalmological diseases

In the original paper ldquoRNA interference targeting con-nective tissue growth factor inhibits the transforming growthfactor-1205732 induced proliferation in humanTenon capsule fibrob-lastsrdquo by J Jing et al the authors showed that siRNA couldefficiently prevent TGF-1205732 induced proliferation of humanTenon capsule fibroblast through targeting CTGF geneexpression Therefore a siRNA based therapeutic approachwas proposed for eliminating filtration bleb scarring afterglaucoma filtration surgery

In the original paper ldquoRNA interference targeting snailinhibits the transforming growth factor 1205732-induced epithelial-mesenchymal transition in human lens epithelial cellsrdquo by PLi et al the authors tested the concept to use Snail targeting

2 Journal of Ophthalmology

siRNA to block TGF 1205732-induced proliferation in human lensepithelial cells The results show that epithelial-mesenchymaltransition was inhibited by Snail targeting siRNA in themodel system that the article described accompanied by thesuppression on snail expression The finding is informativefor the design of the preventive strategy on posterior capsuleopacification after cataract surgery

In the original paper ldquoSystems biology profiling of AMDon the basis of gene expressionrdquo by M S Abu-Asab et ala systems biology analytical paradigm called parsimonyphylogenetics was used to reveal the various transcriptomicprofiles of AMDrsquos subtypes Genetic pathways underlying theinitiation and progression of AMD and the correlations ofAMDrsquos genotypes phenotypes and disease spectrum wereinvestigated

On the whole the papers contained in this special issuecovered the most active fields of genetic studies on complexeye diseases

Jingsheng TuoLai WeiNan Hu


Research ArticleSystems Biology Profiling of AMD on the Basisof Gene Expression

Mones S Abu-Asab Jose Salazar Jingsheng Tuo and Chi-Chao Chan

Laboratory of Immunology National Eye Institute National Institutes of Health Bethesda MD 20892 USA

Correspondence should be addressed to Mones S Abu-Asab monesmailnihgov

Received 15 July 2013 Revised 18 August 2013 Accepted 22 August 2013

Academic Editor Nan Hu

Copyright copy 2013 Mones S Abu-Asab et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Genetic pathways underlying the initiation and progression of age-related macular degeneration (AMD) have not been yetsufficiently revealed and the correlations of AMDrsquos genotypes phenotypes and disease spectrum are still awaiting resolution Weare tackling both problems with systems biology phylogenetic parsimony analysis Gene expression data (GSE29801 NCBI Geo)of macular and extramacular specimens of the retinas and retinal pigment epithelium (RPE) choroid complexes representing dryAMD without geographic atrophy (GA) choroidal neovascularization (CNV) GA as well as pre-AMD and subclinical pre-AMDwere polarized against their respective normal specimens and then processed through the parsimony program MIX to producephylogenetic cladograms Gene lists from cladogramsrsquo nodes were processed in Genomatix GePS to reveal the affected signalingpathway networks Cladograms exposed a highly heterogeneous transcriptomic profiles within all the conventional phenotypesMoreover clades and nodal synapomorphies did not support the classical AMD phenotypes as valid transcriptomal genotypesGene lists defined by cladogram nodes showed that the AMD-related deregulations occurring in the neural retina were differentfrom those in RPE-choroidal tissue Our analysis suggests a more complex transcriptional profile of the phenotypes than expectedEvaluation of the disease in much earlier stages is needed to elucidate the initial events of AMD

1 Introduction

Age-related macular degeneration (AMD) is the main causeof permanent central blindness in the developed countries [1]It manifests in drusen formation and degenerationatrophyof the retinal pigmented epithelium (RPE) and neural retinaas well as the formation of abnormal choroidal capillaries [23] In addition to aging as the principal risk factor there areothers such as smoking diet and genetic predisposition [34] However it is not yet sufficiently resolved the exact geneticpathways underlying the initiation and progression of AMDand the relationship between its genotypes and phenotypes[1]

Although amore recent clinical classification of AMDhasbeen published recently [5] we are using that of Newmanet al [1] since the study specimens were categorized inthe public data according to their phenotypes (see Table 1for details) these encompass (1) dry AMD (2) choroidalneovascularization (CNV) or Wet AMD (3) geographic

atrophy (GA) in macular region of RPE (4) GACNV (5)pre-AMD and (6) subclinical pre-AMD These phenotypesare typically the progressing manifestations of the diseaseand their gene expressions may not harbor the early eventsresponsible for the initiation and progression of the diseaseA transcriptomic profiling of these phenotypes will elucidatethe affected signaling pathways reveal their similarities anddifferences and clarify whether AMDrsquos phenotypes representa single disease or entities of an assemblage of diseases Inthis studywe used systems biology analytical paradigmcalledparsimony phylogenetics to reveal the various transcriptomicprofiles of AMDrsquos subtypes

Further specific objectives of this analysis are to find outif gene expression profiling supports the current classifica-tion of phenotypes to identify the shared gene expressionaberrations among AMDrsquos phenotypes to find out if thetransformations in the neural retina are similar to those inRPE-choroidal region and to carry out class discovery inorder to subtypeAMDon the basis of gene expression profiles


Table 1 Description of AMD phenotypic subtypes according to Newman et al [1] Abbreviated names in the first column are used in labelingthe cladogramsrsquo legends in Figures 1 and 2

AMD phenotype Alternative name DescriptionMD1 Pre-AMD Hard macular drusen (lt63120583m) only

MD2 Subclinicalpre-AMD

Soft distinct macular drusen (gt63120583m)Macular pigmentary irregularities without soft drusen

Dry AMD Dry AMD(non-GA)

Soft indistinct (gt125120583m) or reticular macular drusenSoft distinct macular drusen (gt63 120583m) with pigmentary changesSoft indistinct macular drusen with pigmentary changes

GA Geographicatrophy

Sharply demarcated area of apparent absence of the RPE (gt175120583m)involving central macular region

CNV Wet AMD Subretinal choroidal neovascularizationGACNV Geographic atrophy with choroidal neovascularization

and answer whether it is a single disease or different diseaseentities

To reach the above stated objectives we have selectedparsimony phylogenetics as the best systems biology tool toanalyze microarray gene expression data of AMD obtainedfrompublic domains Parsimony is an evolutionary analyticalmethod that has been applied to mass spectrometry dataof cancer [6] gene-expression of various diseases [7 8]vaccine analysis [9] and systematics biology of taxa [10]Parsimony algorithms are capable of utilizing shared derivedgene expression aberrations to subtype specimens they arevery suitable for high dimensional heterogeneous data (iewith 10000s of variables) [11]

2 Materials and Methods

Our analytical strategy can be summarized in the followingsteps classify the patient specimens into clades (a clusterof specimens located on the cladogram) onto cladogramthrough parsimony analysis of their gene-expression dataidentify shared genes with abnormal expression (termedsynapomorphies in phylogenetic vocabulary) for each cladeand identify genetic pathways affected by abnormal geneexpression for all AMD specimens andor for each clade

Dataset GSE29801 was downloaded fromGeoDatasets ofNCBI (httpwwwncbinlmnihgovgeoqueryacccgiacc=GSE29801) The gene expression dataset of macular andextramacular encompassed specimens of retinas (55 normal13 pre-AMD and 47 AMD) and retinal pigment epithelium(RPE-) choroid complexes (96 normal 21 pre-AMD and60 AMD) [1] The AMD specimens encompassed dry AMDwithout geographic atrophy (GA) choroidal neovasculariza-tion (CNV) and GA (Table 2)

Pre-AMD and AMD gene expression values of reti-nal and RPE-choroidal specimens were polarized sepa-rately against their respective normal specimens (eg RPE-choroid data was polarized using normal RPE-choroidspecimens data) and the new polarized data matriceswere processed separately through MIX [12] a parsimonyprogram of the PHYLIP package (httpevolutiongenet-icswashingtoneduphyliphtml) to produce phylogeneticcladograms for both datasets (for details of this process see [7

Table 2 The study collectionrsquos clinical phenotypes and the numberof their specimens Data source GSE29801 at Geo Datasets of NCBI(httpwwwncbinlmnihgovgeoqueryacccgiacc=GSE29801)

Dx RetinaMacular Extramacular

Normal (119899 = 55) 28 27

Pre-AMD (119899 = 13) MD1 = 4 MD1 = 4MD2 = 3 MD2 = 2

AMD (119899 = 47)

Dry = 15 Dry = 16CNV = 5 CNV = 4GA = 1 GA = 1

GACNV = 3 GACNV = 2RPE-choroid

Normal (119899 = 96) 48 48

Pre-AMD (119899 = 21) MD1 = 6 MD1 = 5MD2 = 4 MD2 = 4

AMD (119899 = 60)


GACNV = 2 GACNV = 2Undetermined = 6 Undetermined = 6

13]) The resulting cladograms were studied for meaningfulinterpretations and to fulfill the objectives stated in the intro-ductionGene lists extracted from the cladograms nodeswereprocessed in Genomatix GePS (httpwwwgenomatixde)to reveal the affected gene signaling pathway networks

3 Results

For amoremeaningful interpretation of the affected signalingpathways our analysis focused on sampling different regionsof the cladograms to reveal the diversity of the affectedsignaling pathways within AMD lesions After the extractionof the synapomorphies at several locations of cladograms 1and 2 we extrapolated from the synapomorphies the affectedsignaling pathways (Tables 3 and 4) by modeling the list of

Journal of Ophthalmology 3

Table 3 Affected retinal signaling pathways at different locations of cladogram in Figure 1 Sample identification follows httpwwwncbinlmnihgovgeoqueryacccgiacc=GSE29801

First nodeShared by all retinalspecimens

RetMD1-106(Sample GSM738713)Lower part of thecladogram

Specimen RetDRY98(Sample GSM738705)Middle part of the cladogram

Specimen RetDRY70(Sample GSM738677)Upper part of thecladogram

(1) Apoptosis(2) Cell cycle(3) Cytoskeleton(4) Differentiation(5) Growth(6) Insulin metabolism

(1) Apoptosis(2) Cell cycle(3) Development(4) Growth(5) Neurotransmission(6) Transcription activation(7) Tumor suppression

(1) Cytokine receptor degradationsignaling(2) Cytosolic calcium ionconcentration elevation (through IP3receptor) (GPCR signaling (G alphaq))(3) EGFR1(4) ERK cascade GPCR signaling (Galpha s PKA and ERK)(5) Protein binding(6) Proteolysis

(1) Amyloid metabolism(2) Apoptosis(3) Cell cycle(4) Cytoskeleton(5) Immunoregulation(6) Inflammation(7) Lipid metabolism(8) Retinoid metabolism(9) Ribosomal proteins(10) Telomere metabolism

Table 4 Affected RPE-choroidal signaling pathways at different locations of cladogram in Figure 2 Sample identification followshttpwwwncbinlmnihgovgeoqueryacccgiacc=GSE29801 Updates on genesrsquo functions can be obtained from httpwwwncbinlmnihgovgene

Dry 135(Sample GSM738566)Lower part of the cladogram

Dry 145(Sample GSM738575)Middle part of the cladogram

Dry 136(Sample GSM738567)Upper part of the cladogram

(1) CXCL12 activates lymphocytes(2) GDNF promotes the survivaland differentiation ofdopaminergic neurons(3) MAPK1 proliferationdifferentiation transcriptionregulation and development(4) PIK3CA oncogenic(5) SFRP1 soluble modulator ofWnt signaling(6) SOD1 superoxide dismutase 1

(1) ABL1 protooncogene implicated in celldifferentiation division adhesion and stressresponse(2) CAV1 cell cycle(3) CCL20 inflammation(4) CREB1 a transcription factor cAMP pathway(5) CRY2 insulin metabolism(6) ERCC1 DNA repair(7) ESR1 hormone binding DNA binding andactivation of transcription(8) IL8 inflammatory response(9) INS insulin(10) MSN cytoskeleton(11) MT1A cytoskeleton and so forth(12) PML tumor suppressor(13) SERPINE1 inhibitor of fibrinolysis(14) TBP assembly of transcription complex andacts as a channel for regulatory signals(15) TMSB4X cytoskeleton proliferationmigration and differentiation

(1) CAV1 cell cycle(2) CCL5 inflammation(3) CXCL12 activates lymphocytes(4) EGF growth proliferation anddifferentiation(5) PPARA peroxisomeproliferator-activated receptor alpha

synapomorphies into Genomatix GePS The sampled loca-tions represented the basal the middle and upper sectionsof both cladograms

Each dataset analysis with MIX produced over 100cladograms and only one cladogram was selected (usuallythe first since the differences between the cladograms werein the upper minor branches) to represent each analysis(Figures 1 and 2) Interestingly the analysis revealed the highheterogeneity of the specimensrsquo gene expression irrespectiveof their phenotype in both retina and RPE-choroid complexThiswas evident by the large number of cladograms produced(over 100) by the two datasets Usually the fewer the numberof cladograms produced the lower the heterogeneity and thehigher the confidence in the results Also supporting this

conclusion were several aspects of the cladograms such asthe terminal distribution of gene expression aberrations (seebelow)

The specimens of each AMD phenotype did not clustertogether to form a clade (a clade is a group of specimenssharing one or more abnormal gene expressions) but ratherformed mixed clades that encompassed several phenotypes(Figures 1 and 2)Therefore AMD phenotypes seemed not tobe distinct entities according to their transcriptomic profilesof the retina or RPE-choroid complex suggesting that theclinically recognized phenotypes may not be supported by aclassification based on gene expression abnormalities

Macular and temporal extra-macular tissues of the samepatient separated in most of the retinal and RPE-choroid


MD1

MD2

Dry AMD

CNVGAGACNV

407 synp rarrlarr 10 synp

larr 1 synp

larr 1 synplarr 1 synp

larr 1 synp

larr 2 synp

larr 118 synplarr 786 synplarr 239 synp

larr 0 synp

larr 2 synp

larr 2 synp

larr 0 synp

larr 0 synp

larr 14 synp

larr 0 synp

larr 0 synp


Figure 1 Cladogram of retinal specimens The number of synapomorphies for major nodes is indicated to the right of the nodes as well asfor some specimens used as examples in the pathways analysis (numbers in red) Colors indicate AMD phenotypic subtypes

complex sets but some clustered together (12ndash15) indicatingsimilar changes in both locations (macular and extramacu-lar) This could be attributed to the diversity of the diseaseitself where it is similar in both locations in some patients anddifferent in others or could be due to sampling from similarlocations

The two cladograms (Figures 1 and 2) demonstrate thatthe AMD retina and RPE-choroid complex had slightlymore transcriptomic subtypes than the currently recognizedclinical phenotypes for example the number of clades withineach cladogram is larger than the number of currentlyrecognized phenotypes

Except for the majority of the retina AMD specimens(both macular and extramacular) that shared 113 synapo-morphies (shared gene expression aberrations) most of thegenetic aberrations were specimen-specific however therewere a few synapomorphies defining a number of cladesSince AMD phenotypes did not form their respective cladesthere were not any synapomorphies that defined any ofthe phenotype While the retina clade was defined by 113synapomorphies the RPE-choroid complex clade had onlytwo synapomorphies these are located at the basal section ofthe cladograms (Figures 1 and 2)

Tables 3 and 4 summarized the affected signaling path-ways of the retina and RPE-choroid complex datasets respec-tivelyDifferent signaling pathwayswere affected in the neuraland nonneural tissues Furthermore the sampled sections ofeach cladogram had differently affected signaling pathwaysdespite some minor overlap While the changes in the retinawere highlighted in apoptosis cell cycle cytoskeleton andgrowth signaling pathway those of the RPE-choroid com-plex showed affected signaling pathways of oxidative stressinflammation cell differentiation and oncogenecity

The samples of Table 4 were selected to represent thevarious locations of the cladogram of Figure 2 in order toexplore the affected pathways among various clades Someof the affected genes included C-X-C motif chemokine12 (CXCL12) that is a chemokine strongly chemotacticfor lymphocytes [14] glial cell-derived neurotrophic factor(GDNF) that strongly promotes the survival of neurons [15]and prevents apoptosis of motor neurons secreted frizzled-related protein 1 (SFRP1) that acts as a biphasic modulatorof Wnt signaling counteracting Wnt-induced effects at highconcentrations and promoting them at lower concentrations[16] which may also affect the differentiation of photoreceptors [17] and superoxide dismutase 1 (SOD1) that is


948 synp rarr 397 synp rarr

71 synp rarr14 synp rarrlarr 15 synp larr 190 synp


1 synp rarr

3 synp rarr

larr 3 synp

larr 0 synp


larr 0 synp

larr 0 synp


MD1

MD2

Dry AMD

CNVGAAMD

larr 354 synp

Figure 2 Cladogram of RPE-choroidal specimens The number of synapomorphies for major nodes is indicated to the right of the nodes aswell as for some specimens used as examples in the pathways analysis (numbers in red) Colors indicate AMD phenotypic subtypes

associated with macular degeneration when its levels dropsbelow normal [18] More updates on other genesrsquo functionscan be obtained from httpwwwncbinlmnihgovgeneUnfortunately since the cladograms of Figures 1 and 2 showthat their clades do not have commonly shared aberrationsalong the axis of the cladograms nothing can be said aboutdirectionality of gene change inAMD from these cladogramsThe amount of heterogeneity in AMD advanced phenotypesseems to be vast and random

4 Discussion

This study is the first transcriptomal analysis of the retinaand RPE-choroid complex tissues from AMD patients andnormal subjects by means of phylogenetic parsimony Themethod is a data-based (not specimen-based) analyticalparadigm that produces a hierarchical modeling of thespecimens into clades (phylogenetic clusters) defined bytheir shared aberrations which when identified reveal theaffected signaling pathways The parsimony cladogram ismultidimensional tool that exposes the characteristics of itsdata In this study the large number of equally parsimonious

cladograms that were produced from the two datasets dis-played the massive heterogeneity of the expression patternwithin or across the clinical classification of AMD Eachdataset produced over 100 cladograms an unusually highnumber of cladograms for a dataset of anatomically-relatedspecimens However such diversity in advanced degenerativedisease could be expected since these diseases are a downhillpath toward undifferentiation due to the deregulation of dif-ferentiation pathways and their phenotypes can be reachedthrough several ontogenic pathways AMD follows the samepattern and it should not be unexpected that its specimenshave shown this considerable heterogeneity

However it may be surprising to find that the transcrip-tional profiles of both datasets did not support the currentclassification of the AMDs phenotypes and that the neuralretina is different from the RPE-choroid complex in theirderegulated pathwaysThe clades produced by the parsimonyalgorithm did not even come close to the classification ofNewman et al [1] as evident in the cladograms of Fig-ures 1 and 2 Further analyses of other data sets such asmetabolomic and proteomic data are needed to confirm thefindings


Pathological aberrations in general are usually dividedinto driver (clonal) and passenger (nonexpanded) [19] On acladogram the driver aberrations are usually modeled at thebasal nodes of the cladogram while the passenger ones areat the terminal level of the clades or randomly distributed onthe cladogram In this study the vast majority of aberrationsare at the terminal level that is specimen-specific Thisrevelation that most of the gene expression aberrations arespecimen-specific points out to two conclusions the first isthat the change is mostly patient-specific and the second isthat there are probably multiple etiologies for AMD

Our analysis is fundamentally different from that ofNewman et al who mainly used fold change (ge15) as theircriteria to identify significantly expressed genes in AMDphenotypes Ours differs in that we used the normal range ofgene expression (minimum and maximum values of healthyspecimens) as the cutoff for determining the under-andoverexpressed genes per specimen This was followed by aphylogenetic stratification of AMD retinal and RPE-choroidspecimens to find the natural clusters (clades) and theiraffected pathways for each of the two groups of specimensSince these two methods belong to two different schools ofthought (specimen-based versus data-based) the congruenceof their results was very weak Therefore gene lists andpathways of Newman et al differed from ours Furthermorewhile Newman et al claimed that their results supported thecurrent phenotypic classification of AMD we think that ourunsupervised analysis did not support AMDrsquos phenotypes[1] Newman et al maps of significant genes are the bestindicators of gene expression heterogeneity within AMDrsquosphenotypes and the difficulty in declaring any as globalbiomarkers the vastmajority of their claimed globally signifi-cant genes (Newman et al Figure 2) are actually insignificantexcept for LOC100294179 in retina that is significant in dryAMD GA and CNV and C10orf18 in RPE-choroid that issignificant in CNV and MD Our analysis indicated that thetranscriptomal changes within the neural retina as a groupof specimens were different from those in the RPE-choroidspecimens and these two sets of tissues differ from each otherin their aberrations therefore it is most likely that there areno global biomarkers for AMDrsquos phenotypes as defined inTable 1This conclusion highlights the necessity of stratifying(subtyping) the disease as a priori to declare any aberrationsas the global biomarkers of the disease subtypes [19] As ouranalysis has shown here there were different transcriptomalsubtypes than the clinical ones

AMD like all degenerative diseases can be bioinformat-ically modeled on a cladogram as a spectrum that rangesfrom early stages with initial events to advanced stageswith later events When specimens representing all stages ofAMD are used to construct a cladogram the ones harboringearly stages of the disease will occupy the basal location ofthe cladogram while later stages follow Therefore revealingearly events of AMD (ie gene expression deregulations thatprobably are not associated with morphological changes)requires the study of specimens that are less advanced intheir pathology [19] In this study the identification ofearly events was not possible this may be attributed to thelack of specimens with asymptomatic stages or relatively

normal pathology of the disease The presence of drusen inpre-AMD and subclinical specimens (see Table 1) may alsorepresent part of an advanced stage of the disease ratherthan a pre-AMD or sub-clinical diagnosis since drusen maysignify an advanced dysfunction of the mitochondria [20]Although ophthalmologists rely on morphological criteriathat appear to represent advanced events for AMD diagnosisearly detection of AMD transformations should be carriedout on the basis of gene-expression profiling according toour analysis Such early gene-expression profiles of AMDtransformations have not yet been determined Additionallythe subtyping of AMDmay have to be delayed till early gene-expression profiles become available

In spite of some slight overlap the affected signalingpathways in AMD are different in the retina and RPE-choroid complex (Tables 3 and 4) In general the retinaspecimens shared aberrations within apoptosis cell cyclecytoskeleton and growth signaling pathways and the RPE-choroid complexes showed aberrations related to inflamma-tion differentiation hypoxia and oncogenecity It appearsfrom the list of affected signaling pathways that the two tissuetypes are exposed to different stressors and therefore areresponding in a different manner Tables 3 and 4 detail theaffected signaling pathways in the retina and RPE-choroidcomplex of AMD lesions

In conclusion AMD appears to be a diverse disease thatinvolves two major independent but parallel pathologicalprocesses one within the neural retina and the other withinthe RPE-choroid complex In both areas the transcriptomalchanges are very heterogeneous and seem to be mostlypatient-specific and involve various signaling pathways Fur-thermore the transcriptomal profiles seem to be incongruentwith the clinical phenotypes and the early gene expressionevents of AMD cannot be deciphered from the advancedphenotypes of the disease

Conflict of Interests

There is no conflict of interests for any of the authors

References

[1] A M Newman N B Gallo L S Hancox et al ldquoSystems-level analysis of age-related macular degeneration reveals glob-al biomarkers and phenotype-specific functional networksrdquoGenome Medicine vol 4 article 16 2012

[2] C A Curcio N EMedeiros andC LMillican ldquoPhotoreceptorloss in age-relatedmacular degenerationrdquo InvestigativeOphthal-mology and Visual Science vol 37 no 7 pp 1236ndash1249 1996

[3] X Ding M Patel and C-C Chan ldquoMolecular pathology ofage-related macular degenerationrdquo Progress in Retinal and EyeResearch vol 28 no 1 pp 1ndash18 2009

[4] Age-Related Eye Disease Study Research Group ldquoRisk fac-tors associated with age-related macular degeneration a case-control study in the age-related eye disease study age-relatedeye disease study report number 3rdquoOphthalmology vol 107 no12 pp 2224ndash2232 2000


[5] F L Ferris III C PWilkinson A Bird et al ldquoClinical classifica-tion of age-related macular degenerationrdquo Ophthalmology vol120 no 4 pp 844ndash851 2013

[6] M Abu-Asab M Chaouchi and H Amri ldquoPhyloproteomicswhat phylogenetic analysis reveals about serum proteomicsrdquoJournal of Proteome Research vol 5 no 9 pp 2236ndash2240 2006

[7] M S Abu-Asab M Chaouchi and H Amri ldquoPhylogeneticmodeling of heterogeneous gene-expression microarray datafrom cancerous specimensrdquo OMICS vol 12 no 3 pp 183ndash1992008

[8] M Abu-Asab M Zhang D Amini N Abu-Asab and H AmrildquoEndometriosis gene expression heterogeneity and biosigna-ture a phylogenetic analysisrdquo Obstetrics and Gynecology Inter-national vol 2011 Article ID 719059 12 pages 2011

[9] M S Abu-Asab M Laassri and H Amri ldquoAlgorithmic assess-ment of vaccine-induced selective pressure and its implicationson future vaccine candidatesrdquo Advances in Bioinformatics vol2010 Article ID 178069 6 pages 2010

[10] EOWiley andB S LiebermanPhylogeneticsTheory and Prac-tice of Phylogenetics Systematics Wiley-Blackwell Hoboken NJUSA 2011

[11] M Abu-Asab M Chaouchi and H Amri ldquoEvolutionarymedicine a meaningful connection between omics diseaseand treatmentrdquo Proteomics vol 2 no 2 pp 122ndash134 2008

[12] J Felsenstein ldquoPHYLIP phylogeny inference package (version3 2)rdquo Cladistics vol 5 pp 164ndash166 1989

[13] M S Abu-Asab M Chaouchi S Alesci et al ldquoBiomarkers inthe age of omics time for a systems biology approachrdquo OMICSvol 15 no 3 pp 105ndash112 2011

[14] Q Ma D Jones P R Borghesani et al ldquoImpaired B-iymphopoiesis myelopoiesis and derailed cerebellar neuronmigration in CXCR4- and SDF-1-deficient micerdquo Proceedings ofthe National Academy of Sciences of the United States of Americavol 95 no 16 pp 9448ndash9453 1998

[15] SWang P Ren YGuan C Zou L Fu andY Zhang ldquoInducibleregulation of GDNF expression in human neural stem cellsrdquoScience China Life Sciences vol 56 no 1 pp 32ndash39 2013

[16] X Zhong T Desilva L Lin et al ldquoRegulation of secretedFrizzled-related protein-1 by heparinrdquo Journal of BiologicalChemistry vol 282 no 28 pp 20523ndash20533 2007

[17] P Esteve A Sandonıs M Cardozo et al ldquoSFRPs act as nega-tive modulators of ADAM10 to regulate retinal neurogenesisrdquoNature Neuroscience vol 14 no 5 pp 562ndash569 2011

[18] F L Muller M S Lustgarten Y Jang A Richardson and Hvan Remmen ldquoTrends in oxidative aging theoriesrdquo Free RadicalBiology and Medicine vol 43 no 4 pp 477ndash503 2007

[19] M S Abu-Asab N Abu-Asab C A Loffredo R Clarke andH Amri ldquoIdentifying early events of gene expression in breastcancer with systems biology phylogeneticsrdquo Cytogenetic andGenome Research vol 139 no 3 pp 206ndash214 2013

[20] J Bereiter-Hahn ldquoDo we age because we have mitochondriardquoProtoplasma 2013


Research ArticleRNA Interference Targeting Connective Tissue GrowthFactor Inhibits the Transforming Growth Factor-1205732 InducedProliferation in Human Tenon Capsule Fibroblasts

Jiaona Jing12 Ping Li1 Tiejun Li34 Yuncheng Sun34 and Huaijin Guan1

1 Eye Institute Affiliated Hospital of Nantong University 20 Xisi Road Nantong Jiangsu Province 226001 China2Nanjing Governmental Hospital 116 Chengxian Street Nanjing Jiangsu Province 210018 China3Department of Life Science Center Biomics Biotechnologies Co Ltd 76 Changxing Road EampT Development AreaNantong Jiangsu Province 226016 China

4 Small RNA Technology and Application Institute Nantong University 76 Changxing Road EampT Development AreaNantong Jiangsu Province 226016 China

Correspondence should be addressed to Huaijin Guan gtnantongeyegmailcom

Received 26 June 2013 Accepted 8 September 2013

Academic Editor Lai Wei

Copyright copy 2013 Jiaona Jing et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

PurposeThis studywas to determine the effect of CTGF-small interferingRNA (siRNA) onTGF-1205732-induced proliferation in human

Tenon capsule fibroblasts (HTFs) Methods HTFs were transfected with four of CTGF-siRNAs separately for screening of genesilencing efficacy that was determined by transcript level measured by quantitative real-time PCR (qRT-PCR) Recombinant TGF-1205732was added into the culture to stimulate the proliferation of HTFs The gene silencing efficacy of the siRNAs was evaluated by

qRT-PCR and immunofluorescence of CTGF transcript and protein levels The viability of HTFs was determined by cell countingkit-8 (CCK-8) FCMwas used to assess cell cycle after CTGF-siRNA transfectionResultsThe expression of CTGF and proliferationof HTFs were increased significantly by TGF-120573

2stimulationThe transfection of CTGF-siRNA abolished the upregulation of CTGF

and cell proliferation induced by TGF-1205732 The analysis of cell cycle indicated that CTGF-siRNA treatment stimulated cells from S

phase to G0G1 phase in comparison with the inverse physiologic function of TGF-1205732 Conclusion CTGF targeting siRNA could

effectively suppress the expression of CTGF and attenuate the proliferation ofHTFsThe siRNA approachmay provide a therapeuticoption for eliminating filtration bleb scarring after glaucoma filtration surgery (GFS)

1 Introduction

Glaucoma filtration surgery (GFS) is often required whenmedication fails to control intraocular pressure (IOP) ade-quately Though this method has an immediate effect onreducing IOP the long-term success is often impaired by thepostoperative wound-healing process [1ndash3] Previous studieshave shown that human Tenon capsule fibroblasts (HTFs)located in the incision area play amajor role in scar formationvia the proliferation migration and synthesis of extracellularmatrix (ECM) [4 5] Although antiscarring agents such asmitomycin C and 5-fluorouracil can prevent postoperativescarring and improve the success rate of trabeculectomy theirapplication is associated with relatively less specificity and anincreased incidence of severe complications [6 7]

Cytokines play crucial roles in scar formation of the bleb[8] Among the cytokines transforming growth factor-120573(TGF-120573) is an important profibrotic factor and is found inaqueous humor and other eye tissue [9ndash11] TGF-120573

2plays an

important role in bleb scarring which is one of the majorreasons for the failure of GFS [12] However the completedsuppression of TGF-120573 may result in significant adverse sideeffects because it plays broad physiological functions such asintercellular signaling and immune regulation [13]Moreoverthe existence of certain levels of antiproliferativemechanismsis required for homeostasis of epithelial cells and tumor sup-pressionTherefore it is necessary to search for an alternativetarget for antifibrotic therapy after trabeculectomy

Connective tissue growth factor (CTGF) is a secretedpeptide which acts as a downstream mediator of TGF-120573 and


Table 1 Targets and duplex sequences for human CTGF specific siRNAs and control siRNA

siRNA duplex CTGF target sequence(51015840-31015840) siRNA duplex sequences

CTGF-siRNA1 (1024)GCACCAGCATGAAGACATACC 51015840-GCACCAGCAUGAAGACAUACCdTdT-31015840

51015840-GGUAUGUCUUCAUGCUGGUGCdTdT-31015840

CTGF-siRNA2 (862)CCCGGGTTACCAATGACAACG 51015840-CCCGGGUUACCAAUGACAACGdTdT-31015840

51015840-CGUUGUCAUUGGUAACCCGGGdTdT-31015840

CTGF-siRNA3 (883)CCTCCTGCAGGCTAGAGAAGC 51015840-CCUCCUGCAGGCUAGAGAAGCdTdT-31015840

51015840-CCAAGCCUAUCAAGUUUGAGCdTdT-31015840

CTGF-siRNA4 (994)CCAAGCCTATCAAGTTTGAGC 51015840-CCAAGCCUAUCAAGUUUGAGCdTdT-31015840

51015840-GCUCAAACUUGAUAGGCUUGGdTdT-31015840

control siRNA 51015840-UUCUCCGAACGUGUCACGUdTdT-31015840

51015840-ACUCCUCGCAGCAUUUCCCGGdTdT-31015840

Four siRNAs were designed from the coding sequence of human CTGF gene The target sequences (51015840-31015840) and the siRNA duplex sequences are listed with theposition of the first nucleotide in CTGF sequence shown in parentheses A nonspecific scrambled siRNA duplex as control siRNA was used as a control

thus also as a profibrotic factor [13] Without blocking otherphysiological effects onTGF-120573 such as suppression on epithe-lial cellsrsquo growth andmodulation of immune or inflammatorycells inhibition of CTGF might specifically suppress thetissue scarring In fibroblasts CTGF is crucial in pathologicalfibrosis by promoting fibroblast proliferation inducing ECMremodeling and initiating myofibroblast differentiation [1415] CTGF also stimulates chemotaxis and the expression ofintegrin promotes endothelial cell growth migration adhe-sion and survival and is thus implicated in endothelial cellfunction and angiogenesis [13]The increase of CTGF expres-sion has been proved to have contributed to many ocularfibrosis diseases including pterygium cataract and prolifer-ative vitreoretinopathy [16ndash18]

RNA interference (RNAi) is an evolutionally conservedmechanism for regulating targeted gene expression [19]RNAi is initiated by the conversion of double strain RNA into21ndash23 nucleotide fragments termed small interfering RNAs(siRNAs) [20] In this process siRNAs subsequently degradetheir target mRNA in a sequence-dependence manner Syn-thesized siRNA has been extensively used for manipulatinggene expression in vitro and in vivo [20 21] The therapeuticapplication of siRNA has opened a new avenue for drugdevelopment for various diseases including ocular disorders[22 23]

In this study we tested the effect of synthesized CTGF-siRNA on the inhibition of CTGF expression and prolifera-tion of HTFs stimulated by TGF-120573

2

2 Material and Methods

21 Cell Culture and Identification Human subconjuncti-val Tenon capsule samples were obtained from individualsundergoing strabismus surgery The human tissue was usedin strict accordance with the tenets of the Declaration ofHelsinki and institutional human experimentation com-mittee approval was granted Each donor signed informedwritten consent The patients had no history of systemic orconjunctival diseases and did not take any topical ocularmedications HTFs were obtained as an expansion culture ofthe Tenon capsule explants of 1 times 1 cm3 and were propagated

in Dulbeccorsquos modified Eagle medium (DMEM InvitrogenCarlsbad CA USA) supplemented with 15 heat-inactivatedfetal bovine serum (FBS Hyclone Logan UT USA)100UmL penicillin and 100120583gmL streptomycin (Sigma-Aldrich Saint-Louis Missouri USA) in 5 CO

2humidified

atmosphere at 37∘C HTFs of passage 3 to 6 were used in theexperiments Cells were identified by immunocytochemistryof fibroblast marker vimentin (monoclonal antivimentinfrom Santa Cruz CA USA) and epithelial cells markerkeratin (monoclonal antikeratin fromCell Signaling BeverlyMA USA)

22 CTGF-siRNA Sequences siRNAs were derived from thecoding region of the human CTGF gene (NM 001901) Thedesign was based on the software (siRNA Target Finder)from Ambion (Austin TX USA) and the sequences wereBLASTed against the Genbank for excluding potential homo-logs The target sequences (51015840 to 31015840) and the duplexes of 4relevant siRNAs are listed in Table 1 These siRNAs weresynthesized and purified by Invitrogen (Carlsbad CA USA)In addition a FAM-labeled nonspecific siRNA (BiomicsNantong China) was used for evaluating efficacy of transfec-tion and as control siRNA as well

23 siRNA Transfection and TGF-1205732Treatment The cells

were seeded in plates with a density of 4 times 105 cellsmL in thecomplete culture medium without antibiotics After 24 h theculture media were then replaced with DMEM without bothantibiotics and serum for 2 hours before transfection TheHTFswere transfected with CTGF-siRNA (50 nM) or controlsiRNA (50 nM) using Lipofectamine 2000 (Invitrogen Carls-bad CA USA) following the manufacturerrsquos protocol After24 h the medium was replaced with the antibiotic- serum-free DMEM with or without human TGF-120573

2(5 ngmL)

(PeproTech Rocky Hill NJ USA) The cells were harvestedafter 24 or 48 h of the treatment The controls HTFs wereeither untreated or treated with Lipofectamine 2000 only

24 Transfection Efficiency of siRNA A FAM-labeled controlsiRNA (green fluorescence) was used for verifying transfec-tion efficiencyThe siRNAwas transfected as described above


The transfection efficacy was evaluated by observation of thegreen fluorescence cells versus total cells using fluorescencemicroscope and flow cytometry (Becton Dickinson andCompany Franklin Lakes NJ USA) The untreated HTFswere used as control For flow cytometry at least 1 times 104 cellsin each samplewere analyzedThe experiments were repeatedfor at least 3 times

25 Quantitative Real-Time PCR Quantitative real-timePCRwas used to determine the level ofCTGFmRNAofHTFsafter various treatments Total RNA was isolated from HTFsusing RISO reagent (Biomics Nantong China) and treatedwith DNase I cDNAwas synthesized by reverse transcriptasefrom total RNA with oligo-d (T) primers Quantitative real-time PCR analysis was performed with the Bio-Rad IQ5 real-time PCR detection system (Bio-Rad Hercules CA USA)using the SYBR Master mixture (Biomics Nantong China)The PCR reactions were performed in triplicate on eachcDNA template along with triplicate reactions of a house-keeping gene GAPDH We used the following primers forCTGF forward (51015840-ACTATGATTAGAGCCAACTG-31015840) andreverse (51015840-TGTTCTCTTCCAGGTCAG-31015840) for GAPDHforward (51015840-GAAGGTGAAGGTCGGAGTC-31015840) and reverse(51015840-GAAGATGGTGATGGGATTTC-31015840)The specific ampli-fication was verified by melting curve analysis The datawere normalized against GAPDHThe expression levels weredetermined using the ΔΔCT method (IQ5 software version20 Bio-Rad) and presented as fold changes Experimentswere performed in triplicate with 3 biological samples fromeach treatment

26 Immunocytochemistry HTFs were seeded in coverslipsbefore transfection of siRNA After being stimulated by TGF-1205732for 48 h the cells on coverslips were washed three times

with PBS and fixed with freshly prepared 4 paraformalde-hyde solution in 001M PBS for 30min at room temperatureThe fixed samples were incubated with primary antibodiesmouse monoclonal antivimentin (1 50 dilution) mousemonoclonal antikeratin (1 400 dilution) or mouse mono-clonal anti-CTGF (1 100 dilution Santa Cruz CA USA)overnight at 4∘C in a humidified chamber After beingwashedthree times with PBS the samples were further reacted withsecond antibodies Alexa Fluor 488 goat anti-mouse (1 200dilution Invitrogen Carlsbad CA USA) for 2 h at 37∘Cand counterstained with 5 120583gmL of Hoechst 33342 (Sigma-Aldrich Saint-Louis Missouri USA) The cells were viewedand photographed under a fluorescence microscope

27 CCK-8Assay Theeffect of CTGF-siRNAonHTFs viabil-ity after TGF-120573

2treatment was determined by cell counting

kit-8 (CCK-8 Biomics Nantong China) assay This assay isbased on the cleavage of the tetrazolium salt WST-8 by mito-chondrial dehydrogenase in viable cells After various treat-ments HTFs in an exponential phase of growth were har-vested and seeded in five 96-well plates at a density of 1 times105 cellsmL in a total volume of 100 120583L per well After 0 2448 72 and 96 h of incubation the viability of HTFs was ana-lyzed by CCK-8 assay The media were replaced by 100 120583L of

DMEM containing CCK-8 (10 120583L) to each well After 35 h ofincubation at 37∘C the absorbance at 450 nm was measuredwith a Thermomax microplate reader The experiment wasrepeated three times

28 Flow Cytometry After being transfected with siRNAand treated with TGF-120573

2for 48 h cell cycle was checked by

flow cytometry The HTFs were collected by trypsinizationand washed twice with PBS before being resuspended at1 times 106 cellsmL in PBS and fixed in 70 ice-cold ethanol(vv) overnight at 4∘C Fixed cells were stained with 05mLof propidium iodide (Sigma-Aldrich Saint-Louis MissouriUSA)RNase staining buffer (BD Pharmingen San DiegoCA USA) in the dark at 4∘C for 30minThe numbers of cellsat G0G1 S and G2M fractions were analyzed using a flowcytometer (BD FACSCalibur BD Bioscience USA) Prolif-eration index was calculated according to PI = (G2M +S)(G0G1 + S + G2M)

29 Statistical Analysis Statistical analysis was performedusing SPSS software (SPSS V 140 SPSS Inc) All results arepresented as the meanplusmn SD One way ANOVA was per-formed for comparing the differences among groups Differ-ences with 119875 lt 005 were considered statistically significant

3 Results

31 Identification of Human Tenon Capsule FibroblastsVimentin and keratin are cell surface markers for fibroblastand epithelium respectively The cultured cells were stainedpositive for vimentin and negative for keratin (Figure 1) Theresults excluded the possible contamination of conjunctivalepithelia during the cell culture

32 Transfection Efficiency of siRNA The results indicatedthat most HTFs displayed green fluorescence after the trans-fection of FAM-labeled control siRNA (Figure 2(a)) HTFsshowed the highest transfection efficiency of siRNA by beingobserved under fluorescence microscopy The transfectionwas efficient in that 837 of the cells displayed green fluores-cence detected by FCM (data not shown) (Figure 2(b)) Thetransfection efficiency implied that Lipofectamine 2000 couldeffectively introduce siRNA into HTFs

33 Suppression of CTGF mRNA Expression After TGF-1205732

induction the HTFs transfected with CTGF-siRNA1 CTGF-siRNA3 or CTGF-siRNA4 but not CTGF-siRNA2 demon-strated the reducedCTGFgene expressionA 579 reductionin CTGF transcript level was observed after being transfectedwith CTGF-siRNA1 (119875 lt 001) while CTGF-siRNA3 andCTGF-siRNA4 caused 273 (119875 lt 005) and 284 (119875 lt 001)reductions of the CTGF transcript levels respectively (Fig-ure 3(a)) in comparison with that from HTFs withouttransfectionTherefore CTGF-siRNA1 was used in follow-upexperiments named CTGF-siRNA The CTGF mRNA levelincreased significantly after TGF-120573

2treatment for 24 h com-

pared with that of TGF-1205732(minus) group (119875 lt 001 Figure 3(b))

There was no significant difference among the control siRNA


Hoechst

Hoechst

Vimentin

Cytokeratin Merge

Merge

Figure 1 Identification of human Tenon capsule fibroblasts A vimentin and cytokeratin immunostaining technique was used to detectfibroblast feature of the cultured cells Fibroblast produced vimentin constitutively with the cytoplasm staining positively (in green) Butcytokeratin staining in the fibroblast is negative Nuclei stained with Hoechst were seen in blue

Bright Fluorescent Merge

(a)

Control siRNAControl

200

0

Cou

nts

Data001

M1M2

FL1-H10

010

110

210

310

4

200

0

Cou

nts

Data002

M1M2

FL1-H10

010

110

210

310

4

(b)

Figure 2 Transfection efficiency of siRNA (a) Transfection efficiency of HTFs transfected with FAM-labeled control siRNA was observedby a fluorescence microscope Green staining in cells stands for effective transfection (b) FCMwas used to analyze the transfection efficiencyof siRNA HTFs transfected withwithout control siRNA were counted by FCM Untransfected cells were marked with M1 and FAM-labeledcells were marked with M2 (here we just show one of the results)


Table 2 Effect of CTGF-siRNA on cell cycle of HTFs

Group G0G1 () S () G2M ()Control 94917 plusmn 1063 1613 plusmn 0372 3470 plusmn 1131

TGF-1205732(+) 88290 plusmn 0335lowast 9037 plusmn 0258lowast 2673 plusmn 0153

CTGF-siRNA + TGF-1205732(+) 91177 plusmn 1064 5410 plusmn 0589 3413 plusmn 0533

Control siRNA + TGF-1205732(+) 88390 plusmn 1074 9047 plusmn 0284 2563 plusmn 0825

Serum starved HTFs were transfected with CTGF-siRNA or control siRNA before being stimulated with TGF-1205732 for 48 h Flow cytometry was used to analyzethe effect of CTGF-siRNA on cell cycle (G0G1 S G2M phase) after various treatments Data were from three experiments lowast119875 lt 001 versus control group119875 lt 005 versus TGF-1205732(+) group

0

5

10

15

20

25

30

35

Relat

ive C

TGF

mRN

A ex

pres

sion

lowast lowastlowast

lowastlowast

lowastlowast

Con

trol

TGF-1205732(+)

CTG

F-siR

NA

1+

TGF-1205732(+)

CTG

F-siR

NA

2+

TGF-1205732(+)

CTG

F-siR

NA

3+

TGF-1205732(+)

CTG

F-siR

NA

4+

TGF-1205732(+)

(a)

0

5

10

15

20

25

30

35

40

Control CTGF-siRNA Control siRNA Lipofectamine2000

Relat

ive C

TGF

mRN

A ex

pres

sion

TGF-1205732(minus)TGF-1205732(+)

lowast

(b)

Figure 3 siRNA inhibition of CTGF mRNA expression Serumstarved HTFs were transfected with CTGF-siRNAs (siRNA1ndashsiRNA5) or control siRNA respectively before being stimulatedwith TGF-120573

2for 24 h (a) Comparison of relative expression of

CTGF mRNA in cultured HTFs transfected with different siRNAsData were from three experiments lowast119875 lt 005 lowastlowast119875 lt 001 versusTGF-120573

2(+) (b) Comparison of transcription levels of CTGF in

HTFs under different conditionsDatawere from three experiments119875 lt 001 versus HTFs stimulated without TGF-120573

2in control group

lowast119875 lt 001 versus HTFs treated with TGF-1205732only

group Lipofectamine 2000 group and the control groupstimulated with TGF-120573

2(Figure 3(b))

34 Suppression of CTGF Protein Expression The effect ofthe CTGF-siRNA on expression of CTGF protein was deter-mined by immunocytochemical staining As shown in Fig-ure 4 control HTFs exhibited a weak green punctiform stain-ing in the cytoplasm After treatment with TGF-120573

2 a distin-

guished strong pattern of punctuate patches of staining wasdisplayed in cells indicating enhancedCTGF expressionThetreatment of CTGF-siRNA with the TGF-120573

2stimulated cells

led to a considerable reduction of fluorescence staining inten-sity compared with that of TGF-120573

2(+) group HTFs treated

with control siRNA exhibited a similar staining intensity andpattern as that of the TGF-120573

2treated cells

35 CTGF-siRNA Inhibits Viability of HTFs The viability ofHTFs was detected by CCK-8 As shown in Figure 5 the cellgrowth showed that exogenous TGF-120573

2might offer a growth

advantage for HTFs In contrast to only TGF-1205732stimulation

group the CTGF-siRNA treatment reduced the viability ofTGF-120573

2stimulated cells by 788 (119875 lt 001) and 1011 (119875 lt

001) at the time points of 48 h and 72 h respectively AfterTGF-120573

2treatment the cell viability ofHTFs treatedwith con-

trol siRNA or Lipofectamine 2000 was similar to that of TGF-1205732-treated cells indicating a low cytotoxicity by Lipofec-

tamine 2000 There was no significant difference in HTFsviability between the TGF-120573

2(+) group and the CTGF-siRNA

group (119875 gt 005) at the time points of 24 h and 96 h Thisindicated that CTGF-siRNA could effectively inhibit the pro-liferation of HTFs at the time points of 48 h and 72 h

36 Effect of CTGF-siRNA on Cell Cycle The effect of CTGF-siRNA on the cell cycle was evaluated by flow cytometry(Table 2)Thepercentage ofHTFs inG0G1 phase in theTGF-1205732(+) group (88290 plusmn 0335) was significantly reduced

compared with the control group (94917 plusmn 1063) (119875 lt001) and was higher in the CTGF-siRNA group (91177 plusmn1064) than the TGF-120573

2(+) group (119875 lt 005) On the con-

trary the percentage of HTFs in S phase in the TGF-1205732(+)

group (9037 plusmn 0258) was increased compared with thecontrol group (1613 plusmn 0372) (119875 lt 001) and was lower intheCTGF-siRNAgroup (5410plusmn 0589) than the TGF-120573

2(+)

group (119875 lt 005)Therewas no significant difference betweenthe TGF-120573

2(+) group and the control siRNA group in G0G1

phase or S phase (119875 gt 005)Flow cytometry showed that the cells treatedwithTGF-120573

2

had a higher value in proliferation index (PI) than the controlgroup (119875 lt 001) (Figure 6) However the pretreatment with


Hoechst CTGF Merge

Control

CTGF-siRNA

Control-siRNA

+TGF-1205732(+)

+ TGF-1205732(+)

+ TGF-1205732(+)

Figure 4 Suppression of CTGF protein expression inHTFs by siRNAHTFs were stimulated with TGF-1205732for 48 h after cells were transfected

with CTGF-siRNA or control siRNA Immunofluorescence analysis of HTFs was performed to visualize the CTGF protein in cell matrix (ingreen) after various treatments Nuclei stained with Hoechst were seen in blue

CTGF-siRNA decreased the PI of TGF-1205732treated cells (119875 lt

005)

4 Discussion

The scar formation after GFS is consistent with the produc-tion of connective tissue during wound repairing TGF-120573 isknown to be themost potent growth factor involved inwoundhealing and also a key modulator in the process of bleb fibro-sis [24ndash26]There are three isoforms of TGF-120573 in human andthe level of TGF-120573

2is the highest in aqueous humor and other

eye tissues After filtering operations aqueous humor comesinto direct contact with the connective tissue of the subcon-junctiva and stimulates fibroblasts proliferation This mightbe responsible for the failure of trabeculectomy Our studyshows that HTFs treated with TGF-120573

2had increased viability

These cells also had an increased portion in S phase adecreased portion in G0G1 phase and higher value in PIthan the control group These results indicated that TGF-120573

2

could promote the proliferation of HTFs significantly Recentstudies have proved that treating TGF-120573

2with monoclonal

antibodies or antisense nucleotides could inhibit fibroblastproliferation and prolong the survival of experimental filter-ing blebs in animal models [27 28]

Researches have suggested that CTGF may mediate thekey actions of TGF-120573 in scar formation such as stimulation ofcell proliferation extracellular matrix protein synthesis andmyofibroblast differentiation in fibroblasts [29ndash32] Blockadeof CTGF expression or its functionmay effectively inhibit theeffects of TGF-120573 Treating CTGF with antisense oligonu-cleotides or neutralizing antibodies could decrease TGF-120573-mediated collagen synthesis in human corneal fibroblast


Control

00

02

04

06

08

10

12

14

16

18

0 24 48 72 96

OD

val

ue

lowastlowast

lowastlowastlowast

lowastlowast

+TGF-1205732(+)

Lipofectamine 2000 + TGF-1205732(+)

CTGF-siRNA + TGF-1205732(+)Control siRNA + TGF-1205732(+)

TGF-1205732 stimulated time (hours)

Figure 5 CTGF-siRNA reduces the viability of HTFs Serumstarved HTFs were transfected withCTGF-siRNA control siRNAor Lipofectamine 2000 before being stimulated with TGF-120573

2for 0

24 48 72 and 96 h The viability of HTFs was analyzed by CCK-8assay CTGF-siRNA suppressed the viability of TGF-120573

2stimulated

cells at the time points of 48 h and 72 h respectively Data were fromthree experiments lowast119875 lt 005 lowastlowast119875 lt 001

CTGF antisense oligodeoxynucleotide could inhibit TGF-1205731-mediated myofibroblast differentiation and corneal-

fibroblast-seeded collagen lattices (FSCL) contraction [3334] In our study we further illustrated that siRNA targetingCTGF could attenuate the proliferation of HTFs

Double-stranded siRNA is an effective approach toinduce gene silencing in cells [35] Inhibition of geneexpression through siRNA is superior to conventional gene-blocking approaches due to the following reasons (1) inhib-itory effect is more potent and stable [36 37] (2) targeting ofgene expression ismore specific [38] (3) blocking efficacy canbe passed on for multiple generations [37] Therefore thereare more potential clinical applications for siRNA [35] Pre-vious reports have shown that TGF-120573

2coupled with CTGF

mediated the bleb-scarring process [8 27 39] In the presentstudy we treated the normal HTFs with exogenous TGF-120573

2

to simulate cell proliferation that mimic bleb formation afterfiltration surgeryWe came to a conclusion that TGF-120573

2could

increase the expression ofCTGF inHTFs and this effect couldbe abolished by pretreatment with CTGF-siRNA

The induction of proliferation byCTGFhas been found insome mesenchymal cells [13] Ishibuchi et al demonstratedthat the proliferation was constantly suppressed by CTGF-silencing in normal and systemic sclerosis fibroblast [40]

000

200

400

600

800

1000

1200

1400

Con

trol

PI (

)

CTG

F-siR

NA+

Con

trol-s

iRN

A+

TGF-1205732(+)

TGF-1205732(+)

TGF-1205732(+)

lowastlowast

lowast

Figure 6 CTGF-siRNA decreases proliferation index of HTFsHTFs were stimulated with TGF-120573

2for 48 h after cells were

transfected with CTGF-siRNA or control siRNA PI of HTFs wascalculated according to cell cycle analyzed by flow cytometry CTGF-siRNA decreased the PI of TGF-120573

2treated cells Data were from

three independent experiments lowast119875 lt 005 lowastlowast119875 lt 001 versus TGF-1205732(+) group

Another study also showed that CTGF induced corneastroma fibroblasts proliferation [41] In our study the analysisof cell cycle revealed that CTGF-siRNA treatment resulted inan increased proportion inG0G1 phase and an inverse one inS phase The reduction of the viability of HTFs was alsodetected by CCK-8 assay These results suggested that down-regulation of CTGF expression could induce the cell cycle ofHTFs to arrest in G0G1 phase and might prevent its DNAsynthesis which might be the mechanism of inhibition ofcell proliferation after transfection of siRNA-CTGF in HTFsSome studies have also suggested that reduction of ECMaccumulationmay attenuate cell proliferation To validate thishypothesis the effect of CTGF-siRNA on ECM in HTFs andthe relationship between ECM and proliferation are neededto be conducted

5 Conclusions

In summary we showed that siRNA targeting CTGF could besuccessfully transfected into HTFs in vitro and could sub-sequently inhibit the proliferation of HTFs These resultssuggested that specific inhibitors of CTGF could have ben-eficial effects on preventing pathogenic fibrosis in bleb afterglaucoma filtration surgery


The authors declare that there is no conflict of interestsregarding the publication of this paper

Acknowledgment

This work was supported by Research Fund of Nantong Uni-versity China


References

[1] E M Addicks H A Quigley W R Green and A L RobinldquoHistologic characteristics of filtering blebs in glaucomatouseyesrdquo Archives of Ophthalmology vol 101 no 5 pp 795ndash7981983

[2] R A Hitchings and I Grierson ldquoClinico pathological correla-tion in eyes with failed fistulizing surgeryrdquo Transactions of theOphthalmological Societies of the United Kingdom vol 103 part1 pp 84ndash88 1983

[3] H D Jampel L J B McGuigan G R Dunkelberger N LLrsquoHernault and H A Quigley ldquoCellular proliferation afterexperimental glaucoma filtration surgeryrdquo Archives of Ophthal-mology vol 106 no 1 pp 89ndash94 1988

[4] P T Khaw N L Occleston G Schultz I Grierson M BSherwood and G Larkin ldquoActivation and suppression of fibro-blast functionrdquo Eye vol 8 part 2 pp 188ndash195 1994

[5] N L Occleston J T Daniels R W Tarnuzzer et al ldquoSingleexposures to antiproliferatives long-term effects on ocularfibroblast wound-healing behaviorrdquo Investigative Ophthalmol-ogy amp Visual Science vol 38 no 10 pp 1998ndash2007 1997

[6] J G Crowston A N Akbar P H Constable N L Occleston JT Daniels and P T Khaw ldquoAntimetabolite-induced apoptosisin Tenonrsquos capsule fibroblastsrdquo Investigative Ophthalmology ampVisual Science vol 39 no 2 pp 449ndash454 1998

[7] R L StamperM GMcMenemy andM F Lieberman ldquoHypot-onous maculopathy after trabeculectomy with subconjunctival5-fluorouracilrdquo The American Journal of Ophthalmology vol114 no 5 pp 544ndash553 1992

[8] D W Esson A Neelakantan S A Iyer et al ldquoExpression ofconnective tissue growth factor after glaucomafiltration surgeryin a rabbitmodelrdquo InvestigativeOphthalmologyampVisual Sciencevol 45 no 2 pp 485ndash491 2004

[9] S Saika ldquoTGF120573 pathobiology in the eyerdquo Laboratory Investiga-tion vol 86 no 2 pp 106ndash115 2006

[10] F Verrecchia and A Mauviel ldquoTransforming growth factor-120573and fibrosisrdquo World Journal of Gastroenterology vol 13 no 22pp 3056ndash3062 2007

[11] G A Lutty C Merges A B Threlkeld S Crone and D SMcLeod ldquoHeterogeneity in localization of isoforms of TGF-120573 inhuman retina vitreous and choroidrdquo Investigative Ophthalmol-ogy amp Visual Science vol 34 no 3 pp 477ndash487 1993

[12] D W Esson M P Popp L Liu G S Schultz and M B Sher-wood ldquoMicroarray analysis of the failure of filtering blebs in arat model of glaucoma filtering surgeryrdquo Investigative Ophthal-mology amp Visual Science vol 45 no 12 pp 4450ndash4462 2004

[13] I E Blom R Goldschmeding and A Leask ldquoGene regulationof connective tissue growth factor new targets for antifibrotictherapyrdquoMatrix Biology vol 21 no 6 pp 473ndash482 2002

[14] G R Grotendorst ldquoConnective tissue growth factor amediatorof TGf-120573 action on fibroblastsrdquo Cytokine amp Growth FactorReviews vol 8 no 3 pp 171ndash179 1997

[15] G R Grotendorst and M R Duncan ldquoIndividual domains ofconnective tissue growth factor regulate fibroblast proliferationand myofibroblast differentiationrdquo FASEB Journal vol 19 no 7pp 729ndash738 2005

[16] G van SettenM Aspiotis T D Blalock G Grotendorst andGSchultz ldquoConnective tissue growth factor in pterygium simul-taneous presence with vascular endothelial growth factormdashpossible contributing factor to conjunctival scarringrdquo GraefersquosArchive for Clinical and Experimental Ophthalmology vol 241no 2 pp 135ndash139 2003

[17] KWunderlichM Pech A N Eberle MMihatsch J Flammerand P Meyer ldquoExpression of connective tissue growth factor(CTGF) mRNA in plaques of human anterior subcapsularcataracts and membranes of posterior capsule opacificationrdquoCurrent Eye Research vol 21 no 2 pp 627ndash636 2000

[18] D R Hinton S He M L Jin E Barron and S J Ryan ldquoNovelgrowth factors involved in the pathogenesis of proliferativevitreoretinopathyrdquo Eye vol 16 no 4 pp 422ndash428 2002

[19] A Fire S Xu M K Montgomery S A Kostas S E Driver andC CMello ldquoPotent and specific genetic interference by double-stranded RNA in Caenorhabditis elegansrdquo Nature vol 391 no6669 pp 806ndash811 1998

[20] S M Elbashir J Harborth W Lendeckel A Yalcin K Weberand T Tuschl ldquoDuplexes of 21-nucleotide RNAs mediate RNAinterference in cultured mammalian cellsrdquo Nature vol 411 no6836 pp 494ndash498 2001

[21] D H Chitwood and M C Timmermans ldquoSmall RNAs are onthe moverdquo Nature vol 467 no 7314 pp 415ndash419 2010

[22] DH Kim and J J Rossi ldquoStrategies for silencing human diseaseusing RNA interferencerdquo Nature Reviews Genetics vol 8 no 3pp 173ndash184 2007

[23] P A Campochiaro ldquoPotential applications for RNAi to probepathogenesis and develop new treatments for ocular disordersrdquoGene Therapy vol 13 no 6 pp 559ndash562 2006

[24] G S Ashcroft J Dodsworth E van Boxtel et al ldquoEstro-gen accelerates cutaneous wound healing associated with anincrease in TGF-1205731 levelsrdquo Nature Medicine vol 3 no 11 pp1209ndash1215 1997

[25] M Shah D M Foreman and M W Ferguson ldquoNeutralisationof TGF-1205731 and TGF-1205732 or exogenous addition of TGF-1205733 tocutaneous rat wounds reduces scarringrdquo Journal of Cell Sciencevol 108 part 3 pp 985ndash1002 1995

[26] A Leask and D J Abraham ldquoTGF-120573 signaling and the fibroticresponserdquo FASEB Journal vol 18 no 7 pp 816ndash827 2004

[27] M F Cordeiro A Mead R R Ali et al ldquoNovel antisenseoligonucleotides targeting TGF-120573 inhibit in vivo scarring andimprove surgical outcomerdquo GeneTherapy vol 10 no 1 pp 59ndash71 2003

[28] A L Mead T T Wong M F Cordeiro I K Anderson andP T Khaw ldquoEvaluation of anti-TGF-1205732 antibody as a new post-operative anti-scarring agent in glaucoma surgeryrdquo InvestigativeOphthalmology amp Visual Science vol 44 no 8 pp 3394ndash34012003

[29] D Kothapalli K S Frazier A Welply P R Segarini andG R Grotendorst ldquoTransforming growth factor 120573 inducesanchorage-independent growth of NRK fibroblasts via a con-nective tissue growth factor-dependent signaling pathwayrdquo CellGrowth amp Differentiation vol 8 no 1 pp 61ndash68 1997

[30] M R Duncan K S Frazier S Abramson et al ldquoConnectivetissue growth factor mediates transforming growth factor 120573-induced collagen synthesis down-regulation by cAMPrdquo FASEBJournal vol 13 no 13 pp 1774ndash1786 1999

[31] G RGrotendorstH Rahmanie andMRDuncan ldquoCombina-torial signaling pathways determine fibroblast proliferation andmyofibroblast differentiationrdquo FASEB Journal vol 18 no 3 pp469ndash479 2004

[32] O Yamanaka S Saika K Ikeda K Miyazaki A Kitano and YOhnishi ldquoConnective tissue growth factor modulates extracel-lular matrix production in human subconjunctival fibroblastsand their proliferation and migration in vitrordquo Japanese Journalof Ophthalmology vol 52 no 1 pp 8ndash15 2008


[33] T D Blalock M R Duncan J C Varela et al ldquoConnectivetissue growth factor expression and action in human cornealfibroblast cultures and rat corneas after photorefractive kerate-ctomyrdquo Investigative Ophthalmology and Visual Science vol 44no 5 pp 1879ndash1887 2003

[34] Q Garrett P T Khaw T D Blalock G S Schultz G R Gro-tendorst and J T Daniels ldquoInvolvement of CTGF in TGF-1205731-stimulation ofmyofibroblast differentiation and collagenmatrixcontraction in the presence of mechanical stressrdquo InvestigativeOphthalmology amp Visual Science vol 45 no 4 pp 1109ndash11162004

[35] D M Dykxhoorn C D Novina and P A Sharp ldquoKilling themessenger short RNAs that silence gene expressionrdquo NatureReviews Molecular Cell Biology vol 4 no 6 pp 457ndash467 2003

[36] J R BertrandM Pottier A Vekris P Opolon AMaksimenkoand C Malvy ldquoComparison of antisense oligonucleotides andsiRNAs in cell culture and in vivordquo Biochemical and BiophysicalResearch Communications vol 296 no 4 pp 1000ndash1004 2002

[37] T R Brummelkamp R Bernards and R Agami ldquoA systemfor stable expression of short interfering RNAs in mammaliancellsrdquo Science vol 296 no 5567 pp 550ndash553 2002

[38] AMCelotto andB RGraveley ldquoExon-specificRNAi a tool fordissecting the functional relevance of alternative splicingrdquoRNAvol 8 no 6 pp 718ndash724 2002

[39] M F Cordeiro J A Gay and P T Khaw ldquoHuman anti-transforming growth factor-1205732 antibody a new glaucoma anti-scarring agentrdquo Investigative Ophthalmology amp Visual Sciencevol 40 no 10 pp 2225ndash2234 1999

[40] H IshibuchiMAbe Y Yokoyama andO Ishikawa ldquoInductionof matrix metalloproteinase-1 by small interfering RNA target-ing connective tissue growth factor in dermal fibroblasts frompatients with systemic sclerosisrdquo Experimental Dermatologyvol 19 no 8 pp e111ndashe116 2010

[41] Y Chang and X Y Wu ldquoJNK12 siRNA inhibits transforming-growth factor-1205731-induced connective tissue growth factorexpression and fibrotic function in THSFsrdquo Molecular andCellular Biochemistry vol 335 no 1-2 pp 83ndash89 2010


Research ArticleAn Extensive Replication Study on ThreeNew Susceptibility Loci of Primary Angle ClosureGlaucoma in Han Chinese Jiangsu Eye Study

Haihong Shi Rongrong Zhu Nan Hu Jian Shi Junfang ZhangLinjuan Jiang Hong Jiang and Huaijin Guan

Eye Institute Affiliated Hospital of Nantong University 20 Xisi Road Nantong 226001 Jiangsu China


Received 12 July 2013 Revised 15 September 2013 Accepted 15 September 2013

Academic Editor Jingsheng Tuo

Copyright copy 2013 Haihong Shi et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Genome-wide association study (GWAS) analysis identified three new susceptibility loci for PACG In this study we aimed toinvestigate whether these three loci in PLEKHA7 COL11A1 and PCMTD1-ST18 are associated with PAC and ocular biometriccharacteristics such as axial length (AL) anterior chamber depth (ACD) and diopter of spherical power (DS)The study was a partof the Jiangsu Eye Study The samples were collected from 232 PAC subjects and 306 controls from a population-based prevalencesurvey conducted in Funing County of Jiangsu China The single nucleotide polymorphisms (SNPs) of rs11024102 in PLEKHA7rs3753841 in COL11A1 and rs1015213 in PCMTD1-ST18 were genotyped by TaqMan-MGB probe using the RT-PCR system Noneof the three polymorphisms showed differences in the distribution of genotypes and allele frequencies between the PAC groupand the control group No significant association was determined between the 3 SNPs and AL ACD or DS of PAC subjects Weconcluded that even though PLEKHA7 rs11024102 COL11A1 rs3753841 and PCMTD1-ST18 rs1015213 are associated with PACGthose sequence variations are not associated with PAC in a Han Chinese population Our results also did not support a significantrole for these three SNPs in ocular biometry such as AL ACD and DS

1 Introduction

Glaucoma is the second leading cause of irreversible blind-ness worldwide Clinically primary glaucoma presents twomajor subtypes primary open-angle glaucoma (POAG) andprimary angle closure glaucoma (PACG) The classificationrelies mainly on the anterior segment anatomy particularlythat of the anterior chamber angle PACG is characterizedby obstruction of aqueous fluid drainage through the trabec-ular meshwork from the anterior chamber of the eye Theanterior chamber depth (ACD) is a main factor affecting thedrainage of aqueous humor PACG affects as many as 45million people in China and it has been reported that Asianpopulations are at higher risk of developing PACG than otherethnic groups [1]

Eyes with PACG usually display characteristic anatomicalfeatures such as a shorter corneal diameter a steeper corneal

curvature a shallower anterior chamber a thicker and moreanteriorly positioned lens and a shortened eyeball oftenaccompanied by hyperopic refraction error [2] The riskfactors for developing PACG include age family history andbeing female [3] First-degree relatives were found to have a6- to 9-fold increased risk of developing PACG [4] Siblingsof Chinese patients with PAC or PACG have almost a 50probability of having narrow angles and aremore than 7 timesmore likely to have narrow angles than the general population[5] Ethnic differences are also associatedwith PACGThere isalso a higher prevalence among Inuits and Asians comparedto Caucasians suggesting a genetic predisposition for thedisorder [6]

Because the ocular anatomic features are predisposingfactors for PACG genes involved in regulation of axiallength and structural remodeling of connective tissues maycontribute to development of PACG Some tissue remodeling


Table 1 Demographics of study participants

Demographic features Control 119899 () PAC 119899 () 119875

Female 248 (8105) 191 (8233) 070Male 58 (1895) 41 (1767)Mean age (year) plusmn SD 6508 plusmn 753 6484 plusmn 859 074Age range 50ndash85 50ndash83Hypertension 66 (1964) 46 (1983) 069Diabetes 24 (736) 20 (86) 076Cardiovascular 10 (327) 4 (172) 041

genes including membrane frizzled-related protein (MFRP)[7 8] extracellular matrix metalloprotease-9 (MMP-9) [9ndash11] and methylenetetrahydrofolate reductase (MTHFR) [12]have been reported to be associated with PACG Even thoughheat shock protein 70 (HSP70) does not regulate tissueremolding directly it regulates the expression of matrix met-alloproteases (MMPs) and is thought to be associated withPACG [13] However the above findings remain controversialand have not been replicated by independent studies

Recently a genome-wide association study (GWAS) iden-tified three new susceptibility loci for PACG includingrs11024102 in PLEKHA7 rs3753841 in COL11A1 and rs1015213in PCMTD1-ST18 [14] However the mechanism of thesegenes in PACG pathogenesis is unclear and the biologi-cal plausibility is absent We hypothesized that PLEKHA7COL11A1 and PCMTD1-ST18 might contribute to PACG byinfluencing ocular biometryThus in this study we attemptedto replicate the association between these three loci withprimary angle closure (PAC) instead of PACG to investigatewhether the SNPs of these three genes are associated withocular biometry PAC is the earlier stage of PACG andshares the same anatomical features however PAC doesnot present glaucomatous optic neuropathy Our definitionof PAC includes asymptomatic individuals with occludableangles who have not had an acute attack as well as thosewho have had an attack but received prompt treatment andsuffered no detectable nerve damage

2 Methods

21 Study Subjects The study was a part of the JiangsuEye Study and was conducted according to the Declarationof Helsinki and approved by the Ethics Committee of theAffiliated Hospital of Nantong University Each participantwas fully informed of the purpose and procedures involvedin the study and signed the Informed Consent Form Thegeneral demographic information of the participants is listedin Table 1 All participants were recruited from a population-based prevalence survey on eye diseases using a clusterrandom sampling strategy in Funing County of JiangsuChina Of the 6032 people screened 232 people with PACand 306 controls were enrolled in the study PAC subjects andcontrols were matched in groups for sex and age and wereethnically homogenous The participants were unrelated andself-identifiedHan ChineseThere was no difference between

the control group and the PAC group in gender age orsystemic disease distribution

All study participants were residents of Funing Countyof Jiangsu China aged 50 years and above Each participantreceived a thorough ophthalmic examination included best-corrected visual acuity anterior segment photography Gold-mann applanation tonometry fundus examination optic discphotography visual field objective refraction and subjectiverefraction The depth of the peripheral anterior chamberwas determined using Van Herick technique [15] The sub-jects with a peripheral chamber depth less than one-thirdof corneal thickness were invited for gonioscopy A-scanultrasonography and ultrasound biomicroscopy (UBM SW-3200S SUOER China) examinations UBM examinationswere conducted in light and dark conditions in eight posi-tions The detailed protocol for gonioscopy and UBM wasreported previously by Barkana et al [16] Three measure-ments of ACD and AL were made by A-scan to get meanvalues and mean values of binoculus were used for statisticalanalyses

PACwas defined according to the International Society ofGeographical and Epidemiologic Ophthalmology (ISGEO)classification by Foster et al [17] (1) either eye has thepresence of an occluded angle (at least 180 degrees of closedangle in which the trabecular meshwork is not visible ongonioscopy or iris apposition to the trabecular meshworkmore than 180 degrees on UBM) (2) at least one of the fol-lowing features was detected peripheral anterior synechiaeintraocular pressure gt21mmHg excessive pigment deposi-tion on the superior trabecular meshwork iris whirling his-tory of symptoms or intraocular pressure elevated ge8mmHgafter UBM examination in dark conditions (3) no signs ofsecondary angle closure (4) no signs of glaucomatous opticneuropathy and peripheral visual loss (5) no previous ocularsurgery or laser therapy The clinical features of the PACsubjects are listed in Table 2

The criteria for enrollment of the control group were (1)peripheral chamber depth more than one-third of cornealthickness (2) intraocular pressure less than 21mmHg (3)normal optic nerve heads with cup-to-cup ratio less than 05(4) normal visual field (5) no family history of glaucoma(6) no ophthalmic diseases except slight cataract and (7)refractive error less than three diopters

22 SNP Genotyping Genomic DNA was extracted from theperipheral blood of each individual using the Qiagen BloodDNA Mini Kit (Qiagen Valencia CA) according to themanufacturerrsquos instructions and stored at minus20∘C

The samples were genotyped by TaqMan AenotypingAssay (Applied Biosystems Foster City CA USA) using theReal-time PCR 7500 system (Applied Biosystems Foster CityCA USA) The assay IDs are C 2981015 10 for rs11024102(an SNP in intron region) C 2947954 10 for rs3753841 (amissense SNP) and C 7479939 10 for rs1015213 (a SNP inintergenic region) PCR reactions were performed in a totalvolume of 10 120583L containing 1120583L (10 ng) DNA 5 120583L TaqManUniversal Master Mix 020120583L TaqMan SNP GenotypingAssay Mix (40x) and 38 120583L Dnase-free sterile filtered water


Table 2 Clinical features of PAC subjects

Right eye (mean plusmn SD) Left eye (mean plusmn SD) Mean of both eyes (mean plusmn SD)Axial length (mm) 2217 plusmn 083 2217 plusmn 082 2217 plusmn 083

ACD (mm) 249 plusmn 029 245 plusmn 030 247 plusmn 029

Refractive (diopter) 053 plusmn 185 068 plusmn 187 058 plusmn 184

Tonometry (mmHg) 1518 plusmn 431 1578 plusmn 446 1552 plusmn 439

Table 3 Allele frequency of SNPs in control and PAC subjects

SNP Allele distributionminormajor (minor )119875 OR (95 CI)

Control PACPLEKHA7 rs11024102 (TC) 245367 (400) 199265 (429) 0346 113 (088ndash144)COL11A1 rs3753841 (AG) 195417 (319) 136328 (293) 0369 088 (068ndash115)PCMTD1-ST18 rs1015213 (CT) 13599 (21) 11453 (24) 0786 112 (050ndash251)All HWE 119875 values gt 005 except for PCMTD1-ST18 in controls

Amplification was carried out with an initial denaturation at95∘C for 5min followed by 40 cycles of denaturation at 95∘Cfor 30 s and annealing at 60∘C for 30 s

23 Statistical Analysis Statistical analysis was performedwith SPSS version 150 softwareDifferences in age and genderbetween PAC subjects and controls were assessed usingt-test and Chi-Square test respectively Hardy-Weinbergequilibrium was tested using Chi-Square test To analyzethe association of these three SNPs with PAC and controlsthe frequency of genotypes and alleles were evaluated usingChi-Square test 119875 valueslt 005 were considered statisticallysignificant Logistic regression analysis was performed tocalculate the odds ratio (OR) value the 95 confidenceinterval (95 CI) and to adjust the confounding effects ofage and gender If any positive association was found inthe initial analysis Bonferroni correction was performedThree genetic models were analyzed the additive modeldefined as minor allele homozygotes versus heterozygotesversus common allele homozygotes the dominant model asheterozygotes plus minor allele homozygotes versus com-mon allele homozygotes and the recessive model as minorallele homozygotes versus common allele homozygotes plusheterozygotes The association of these three SNPs withAL ACD and DS was also assessed under the additivegenetic model dominant model and recessive model using119905-test

3 Results

Thecall rates of all SNP genotypingwere 100 and the call ac-curacies were 100 in a randomly selected 10 sample All 3SNPs conformed to Hardy-Weinberg equilibrium (119875 gt 005)except for PCMTD1-ST18 rs1015213 in controls

None of the three polymorphisms showed differences inthe distribution of allele frequencies (Table 3) and genotypes(Table 4) between the cases and controls

All three SNPs were not significantly associated withbiometric parameters including ACD AL and DS (Table 5)

4 Discussion

This study to the best of our knowledge is the firstpopulation-based study to investigate the association ofrs11024102 rs3753841 and rs1015213 with PAC and PACrelevant biometric parameters such as ACD AL and DS ina Han Chinese population The design of a population-basedstudy can minimize sample selection bias often present inhospital-based case-control study We attempted to replicatethe association between these three loci with PAC insteadof PACG to verify the relationship between these SNPs andanatomic features The results show that the variations ofPLEKHA7 rs11024102 COL11A1 rs3753841 and PCMTD1-ST18 rs1015213 were not associated with either PAC orbiometric factors in Han Chinese population

PLEKHA7 encodes pleckstrin homology domain-containing protein 7 which is involved in the maintenanceand stability of epithelial and endothelial adherens junctions[18] PLEKHA7 is expressed in the cornea iris and trabecularmeshwork (TM) Increased resistance to drainage of aqueoushumor through the pressure-dependent TM is believed tobe responsible for POAG [19] However the pathogenesisof PACG is distinct from that of POAG Eyes with PACGtend to share certain anatomic biometric characteristics andhave nothing to do with aqueous humor outflow facility Inour present study we did not find any association betweenrs11024102 and PAC nor did we find any association betweenrs11024102 and biometric parameters

COL11A1 gene codes for one of the two120572-chains of typeXIcollagens TypeXI collagen is aminor fibril-forming collagencontrolling fibril growth diameter and assembly of majorcollagens It is expressed primarily in the articular cartilageand the ocular vitreous [20] Mutations in COL11A1 causeMarshall syndrome Stickler syndrome and Stickler-likesyndrome these disorders are all characterized by midfacialhypoplasia sensorineural hearing deficit and nonprogressiveaxial myopia [21] Hyperopic and shorter axial length but notaxial myopia is well-known predisposing factor for PACG Inour present study the distribution of genotypes of rs3753841was similar in the PAC and in the control group as were thebiometric parameters


Table 4 Genotype frequency of SNPs in control and PAC subjects

SNP Genotype distribution 119899 () General 119875 value Dominant pOR (95 CI) Recessive pOR (95 CI)Control PAC

PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)

Table 5 The relationship of biometric parameters with genotypes of rs1015213 rs375384 and rs11024102 in PAC group

Genotype AL (mm) (mean plusmn SD) ACD (mm) (mean plusmn SD) Refrative power (D) (mean plusmn SD)

PLEKHA7 rs11024102 TT 2216 plusmn 070 244 plusmn 023 064 plusmn 129

TC + CC 2215 plusmn 076 247 plusmn 022 074 plusmn 163

119875 0958 0448 0663

COL11A1 rs3753841 AA 2211 plusmn 072 246 plusmn 023 071 plusmn 146

AG + GG 2220 plusmn 076 246 plusmn 022 070 plusmn 158

119875 0366 0924 0945

PCMTD1-ST18 rs1015213 CC 2215 plusmn 072 246 plusmn 022 070 plusmn 155

CT + TT 2229 plusmn 099 242 plusmn 027 080 plusmn 068

119875 0528 0617 0835

Rs1015213 is located upstream of PCMTD1 and down-stream of ST18 PCMTD1 encodes protein-l-isoaspartateO-methyltransferase domain-containing protein 1 that isexpressed in the cornea iris and TM ST18 encodes the sup-pression of tumorigenicity 18 protein expressed in the corneaand lens but not in the TM [14] In our study the minorallele frequency of rs1015213 was low which is consistent withprevious reports [14 22] Little is known about the functionof PCMTD1 or ST-18 There was no significant differencebetween the two groups in the genotype frequency or allelesfor rs1015213 nor any significant difference between rs1015213and biometric parameters

Our results were not in line with Vithana et alrsquos report[14] that reported the three loci susceptible for PACG bya GWAS study with a two-stage strategy Sample size andethnic distribution are two main factors that can influencethe results of genotype association studies Vithnanrsquos studyincluded 1854PACGcases fromanAsian population in stage 1and 1917 PACGcases from6 sample collections (two inChinaand one each in UK Singapore India and Saudi Arabia)The power analysis based on their data indicated that ourstudy is underpowered (lt50) to detect any association ofthe 3 tested SNPs However all subjects included in this studyare Han Chinese and subjects in both groups were age andgender matched Moreover the study was community basedthus decreasing the confounding of possible populationstratification We believe that our sample size is reasonableto detect a biologically meaningful association if it exists

Another possible reason that we did not replicate theVithanarsquos reportmight be due to the different definition of the

phenotypes PAC in our study and PACG in Vithanarsquos studyBecause the number of PACG patients in this communitycohort did not meet the basic requirements to conduct anindependent association study we excluded this phenotypeDay et al [22] conducted a genotype-phenotype analysis ofthese three SNPs with the ocular biometry of 988 Europeanpeople They found that the A allele of rs1015213 was nom-inally associated with ACD (119875 = 0046) but not associatedwith AL or corneal keratometry Rs11024102 and rs1015213were not associated with ocular biometry which is consistentwith our results

Another limitation in our study is that AL and ACDparameters are only available for the PAC group It is timeconsuming and technically demanding to invite all 6032participants for UBM gonioscopy and A-scan examinationsAdditionally the development of PACG is complex andlikely depends on polygenic inheritance It appears that eachanatomic characteristic is not determined by a series of inde-pendent genes acting with no relation to other componentsbut is instead an additive outcome of the action of a largenumber of genes The effect of each gene would be small anddifficult to distinguish individually

5 Conclusion

The sequence variants of PLEKHA7 rs11024102 COL11A1rs3753841 and PCMTD1-ST18 rs1015213 do not appear tobe associated with PAC and ocular biometry in our studyBecause the PLEKHA7 rs11024102 COL11A1 rs3753841 and


PCMTD1-ST18 rs1015213 were reported to be associated withPACG the lack of association of these SNPs may be due to adifferent phenotype being assessed


The authors declare no conflict of interestsThe authors aloneare responsible for the content and writing of the paper

Acknowledgments

The authors thank all the patients and family members fortheir participation They appreciate the great contributionof the Funing Health Bureau Funing CDC Shizhuang EyeHospital of Funing and the Peoplersquos Hospital of Funing tostudy coordination and participant recruitment The studywas supported by the National Natural Science Founda-tion of China (no 81070718) the 333 Project of JiangsuProvince (no BRA2010173) and the NantongMunicipal Spe-cial Project of Major Scientific and Technologic Innovation(no XA2009001-8)

References

[1] P J Foster and G J Johnson ldquoGlaucoma in china how big isthe problemrdquo British Journal of Ophthalmology vol 85 no 11pp 1277ndash1282 2001

[2] R SihotaNC LakshmaiahHCAgarwal RM Pandey and JS Titiyal ldquoOcular parameters in the subgroups of angle closureglaucomardquo Clinical and Experimental Ophthalmology vol 28no 4 pp 253ndash258 2000

[3] N Amerasinghe and T Aung ldquoAngle-closure risk factorsdiagnosis and treatmentrdquo Progress in Brain Research vol 173pp 31ndash45 2008

[4] N Wang H Wu and Z Fan ldquoPrimary angle closure glaucomain Chinese and western populationsrdquo Chinese Medical Journalvol 115 no 11 pp 1706ndash1715 2002

[5] N Amerasinghe J Zhang AThalamuthu et al ldquoThe heritabil-ity and sibling risk of angle closure in Asiansrdquo Ophthalmologyvol 118 no 3 pp 480ndash485 2011

[6] P H Alsbirk ldquoPrimary angle-closure glaucoma Oculometryepidemiology and genetics in a high risk populationrdquo ActaOphthalmologica no 127 pp 5ndash31 1976

[7] T Aung M C C Lim T T L Wong et al ldquoMolecular analysisof CHX10 and MFRP in Chinese subjects with primary angleclosure glaucoma and short axial length eyesrdquoMolecular Visionvol 14 pp 1313ndash1318 2008

[8] I-J Wang S Lin T-H Chiang et al ldquoThe association ofmembrane frizzled-related protein (MFRP) gene with acuteangle-closure glaucomamdasha pilot studyrdquo Molecular Vision vol14 pp 1673ndash1679 2008

[9] T Aung V H K Yong M C C Lim et al ldquoLack of associationbetween the rs2664538 polymorphism in the MMP-9 geneand primary angle closure glaucoma in singaporean subjectsrdquoJournal of Glaucoma vol 17 no 4 pp 257ndash258 2008

[10] Y Cong X Guo X Liu et al ldquoAssociation of the singlenucleotide polymorphisms in the extracellular matrix met-alloprotease-9 gene with PACG in southern Chinardquo MolecularVision vol 15 pp 1412ndash1417 2009

[11] I-J Wang T-H Chiang Y-F Shih et al ldquoThe association ofsingle nucleotide polymorphisms in the MMP-9 genes withsusceptibility to acute primary angle closure glaucoma inTaiwanese patientsrdquo Molecular Vision vol 12 pp 1223ndash12322006

[12] S Michael R Qamar F Akhtar W A Khan and AAhmed ldquoC677T polymorphism in the methylenetetrahydro-folate reductase gene is associated with primary closed angleglaucomardquoMolecular Vision vol 14 pp 661ndash665 2008

[13] H Ayub M I Khan S Micheal et al ldquoAssociation of eNOSand HSP70 gene polymorphisms with glaucoma in PakistanicohortsrdquoMolecular Vision vol 16 pp 18ndash25 2010

[14] E N Vithana C C Khor C Qiao M E Nongpiur R Georgeet al ldquoGenome-wide association analyses identify three newsusceptibility loci for primary angle closure glaucomardquo NatureGenetics vol 44 pp 1142ndash1146 2012

[15] P J Foster J G Devereux P H Alsbirk et al ldquoDetection ofgonioscopically occludable angles and primary angle closureglaucoma by estimation of limbal chamber depth in Asiansmodified grading schemerdquo British Journal of Ophthalmologyvol 84 no 2 pp 186ndash192 2000

[16] Y Barkana S K Dorairaj Y Gerber J M Liebmann and RRitch ldquoAgreement between gonioscopy and ultrasound biomi-croscopy in detecting iridotrabecular appositionrdquo Archives ofOphthalmology vol 125 no 10 pp 1331ndash1335 2007

[17] P J Foster R Buhrmann H A Quigley andG J Johnson ldquoThedefinition and classification of glaucoma in prevalence surveysrdquoBritish Journal of Ophthalmology vol 86 no 2 pp 238ndash2422002

[18] P Pulimeno S Paschoud and S Citi ldquoA role for ZO-1 andPLEKHA7 in recruiting paracingulin to tight and adherensjunctions of epithelial cellsrdquo Journal of Biological Chemistry vol286 no 19 pp 16743ndash16750 2011

[19] P V Rao Y K Peterson T Inoue and P J Casey ldquoEffects ofpharmacologic inhibition of protein geranylgeranyltransferasetype I on aqueous humor outflow through the trabecularmeshworkrdquo Investigative Ophthalmology andVisual Science vol49 no 6 pp 2464ndash2471 2008

[20] S Annunen J Korkko M Czarny et al ldquoSplicing mutationsof 54-bp exons in the COL11A1 gene cause Marshall syndromebut other mutations cause overlapping MarshallStickler phe-notypesrdquo American Journal of Human Genetics vol 65 no 4pp 974ndash983 1999

[21] R A Kahler S M C Yingst L H Hoeppner et al ldquoCollagen11a1 is indirectly activated by lymphocyte enhancer-bindingfactor 1 (Lef1) and negatively regulates osteoblast maturationrdquoMatrix Biology vol 27 no 4 pp 330ndash338 2008

[22] A C Day R Luben A P Khawaja S Low S Hayat et alldquoGenotype-phenotype analysis of SNPs associatedwith primaryangle closure glaucoma (rs1015213 rs3753841 and rs11024102)and ocular biometry in the EPIC-Norfolk Eye Studyrdquo BritishJournal of Ophthalmology vol 97 pp 704ndash707 2013


Research ArticleRNA Interference Targeting Snail Inhibits the TransformingGrowth Factor 1205732-Induced Epithelial-Mesenchymal Transitionin Human Lens Epithelial Cells

Ping Li12 Jiaona Jing1 Jianyan Hu1 Tiejun Li34 Yuncheng Sun34 and Huaijin Guan1

1 Department of Ophthalmology Affiliated Hospital of Nantong University 20 Xisi Road Nantong Jiangsu 226001 China2Department of Ophthalmology Yixing Hospital of Traditional Chinese Medicine 128 Yangquan East Road YixingWuxi Jiangsu 214200 China

3Department of Life Science Center Biomics Biotechnologies Co Ltd 76 Changxing Road EampT Development AreaNantong Jiangsu 226016 China

4 Small RNA Technology and Application Institute Nantong University 76 Changxing Road EampT Development AreaNantong Jiangsu 226016 China


Received 27 June 2013 Revised 5 August 2013 Accepted 14 August 2013


Copyright copy 2013 Ping Li et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Epithelial-msenchymal transition (EMT) contributes to posterior capsule opacification (PCO) type of cataract Transcriptionfactors Snail is a key trigger of EMT activated by transforming growth factor 120573 (TGF120573)This study was done to investigate the effectof Snail targeting siRNA on TGF1205732-induced EMT in human lens epithelial cells TGF1205732 treatment of cultured human epithelial cellline (HLEB3) upregulated the expression of Snail and the EMT relevant molecules such as vimentin and 120572-SMA but downregulatedthe expression of keratin and E-cadherin After the stimulation of TGF1205732 the HLEB3 cells became fibroblast-like in morphologyand the junctions of cell-cell disappeared TGF1205732 treatment also enhanced migration ability of HLEB3 cells TGF1205732-induced Snailexpression and EMT were significantly inhibited by Snail siRNA By analyzing the response characteristics of HLEB3 in TGF1205732-induced EMTmodel withwithout Snail-specific siRNA we concluded that Snail is an element in the EMT of HLEB3 cells inducedby TGF1205732 Snail siRNA targeting can block the induced EMT and therefore has the potential to suppress the development of PCO

1 Introduction

Epithelial-mesenchymal transition (EMT) is a programmeddevelopment of biological cells characterized by loss of celladhesion repression of E-cadherin expression increasedcell mobility and change of morphology EMT is a highlyconserved and fundamental process not only in developmentbut also in fibrosis metastasis of tumor cells and woundhealing [1ndash4] In cataract surgery where entire lens contentis removed lens epithelial cells (LECs) can undergo EMTmigrate to the posterior capsular surface and result in fibrosisof the posterior capsule as well as the residual anteriorcapsule [4ndash6] Clinically the EMT of LECs after cataractlens removal usually results in secondary cataract that can

present as anterior polar cataracts andor posterior capsularopacification [7 8]

During EMT epithelium cells undergo transdifferentia-tion toward a myofibroblastic phenotype The two cell typeshave different skeletal proteins keratin for epithelium andvimentin for myofibroblastic The cells derived from surfaceectoderm always express E-cadherin to form adherence toeach other The EMT process involves transcriptional repro-gramming of a series of genes that include 120572-SMAknown as amaker ofmyofibroblast cellsTherefore except for the distinctexpression of keratin and vimentin the 120572-SMA expression isconsidered as the feature of LECs transdifferentiation as wellas the loss of E-cadherin production [9ndash11]


TGF120573 is composed of homodimeric polypeptides thatregulate many aspects of cellular function including cellgrowth differentiation inflammation and wound healing[12ndash14] Numerous in vitro and in vivo studies have indicatedthe role of active TGF120573 in promoting an aberrant differenti-ation pathway and EMT of various epithelial tissues [15 16]Although five members of the TGF120573 family have currentlybeen identified only TGF120573 isoforms 1 2 and 3 have beendetected in mammals [17] TGF1205731 and TGF1205732 are expressedin human lens and release abundantly in the ocular media[18] The predominant form of TGF1205731 and TGF1205732 is in thelatent [19] but can be activated under pathological conditionssuch as inflammation fibrosis trauma and surgery after a 25-kDa dimer cleaved from its latent precursor [20]The amountof TGF120573 in aqueous humor after cataract surgery withintraocular lens implantation ranged from 23 to 81 ngmLwith 61 of it present in the active form [21] Normally theactivity of TGF120573 in the eye appears to be highly regulatedby vitreous containing molecules [8] TGF1205732 is expressed atmuch higher levels than the other isoforms in the aqueoushumor and vitreous and thus is likely to be a major mediatorof EMT in LECs in vivo [22 23]

The Snail family members are a group of transcriptionfactors that are involved in regulation of EMT induced byTGF120573 during embryonic development and tumor prog-ression [24ndash28] They are involved in many embryonic pro-cesses such as the ingression of the early mesodermal cellsat gastrulation and the delamination of the neural crest fromthe neural tube [29] In adult Snail was mainly expressedin heart lung brain and skeletal muscle but there is noexpression in most normal organstissues including eyes[30] However Snail can be expressed in ocular tissueunder pathological conditions especially fibrotic diseasessuch as corneal scarring [31] subcapsular cataract [32] andproliferative vitreoretinopathy (PVR) [33] Indeed Snail isactivated to induce EMT inmammalian cells and suppress theexpression of E-cadherin [8 34 35] Cho et al have reportedthe role of Snail in ETM of mouse lens epithelial cells [36]

In the present study we sought to confirm the involve-ment of Snail gene in TGF1205732-induced EMT of human LECsand to test a novel hypothesis that the inhibition of Snailexpression by siRNA can block TGF1205732-induced EMT


21 Cells and Cell Culture Human lens epithelial cell lineHLEB3 was purchased from ATCC Cells were cultured inDulbeccorsquos modified Eaglersquos medium (DMEM InvitrogenCA USA) supplemented with 15 fetal bovine serum (FBSInvitrogen CA USA) All culture medium contained noantibiotics The TGF1205732 treatment was carried out after thecells were incubated in serum-free medium for 24 hours and10 ngmL of TGF1205732 was added to the culture medium for theindicated times

22 Reagents and Antibodies Recombinant human TGF1205732was purchased from Peprotech (Rocky Hill NJ USA) Anti-E-cadherin and keratin antibodies were purchased from Cell

Table 1 siRNA sequences for snail targeting and negative control

siRNAduplex siRNA duplex sequences (51015840-31015840)

P1 Sense GAAUGUCCCUGCUCCACAAGCdTdTAntisense GCUUGUGGAGCAGGGACAUUCdTdT

P2 Sense GCGAGCUGCAGGACUCUAAUCdTdTAntisense GAUUAGAGUCCUGCAGCUCGCdTdT

P3 Sense CCUUCGUCCUUCUCCUCUACUdTdTAntisense AGUAGAGGAGAAGGACGAAGGdTdT

P4 Sense CAGAUGUCAAGAAGUACCAGUdTdTAntisense ACUGGUACUUCUUGACAUCUGdTdT

P5 Sense UUCUCCGAACGUGUCACGUdTdTAntisense ACGUGACACGUUCGGAGAAdTdT

Four siRNAs (P1ndashP4) were designed from the coding sequence of the humanSnail geneThe siRNA duplex sequences are listed A nonspecific scrambledsiRNA duplex as negative control (P5) was used as a control

Signaling (Beverly MA USA) Anti-Snail and vimentin anti-bodies were obtained from Santa Cruz Biotechnology (SantaCruz CA USA) Anti-120572-SMA antibody was purchased fromAbcam (CambridgeMAUSA) CY3FITC tagged secondaryantibodies were from BOSTER (Wuhan China)

23 siRNA and Transfection According to Elbashirrsquos prin-ciple [37] four siRNAs (P1ndashP4) targeting human Snailand one negative control siRNA (P5) were designed usingweb-based software (httpwwwambioncomtechlibmiscsiRNA finderhtml) and synthesized chemically (BiomicsNantong China) (Table 1) The siRNAs were transfectedinto HLEB3 cells by liposome Lipofectamine 2000 accordingto the manufacturerrsquos protocol (Invitrogen CA USA) ThesiRNA treatment was performed before the TGF1205732 stimula-tion

24 Quantification of Snail mRNA Total RNA of HLEB3cells was extracted for cDNA synthesis using RISO reagent(RISO Biomics Nantong China) cDNA was synthesized byMLV reverse transcriptase using 2 120583g total RNA in a totalvolume of 20 120583L (QuantiTect Qiagen Germany) The Snailtranscript was detected by quantitative RT-PCR using iCy-cler iQ System (Bio-Rad Laboratories Hercules CA USA)and SYBR Green QPCR Master Mix (Biomics NantongChina) The primers for snail are forward 51015840-TCGTCC-TTCTCCTCTACTTCAG-31015840 and reverse 51015840-CGTGTGGCT-TCGGATGTG-31015840 which amplify a 201 bp target For theinternal control GAPDH was amplified using primers for-ward 51015840-GAAGGTGAAGGTCGGAGTC-31015840 and reverse 51015840-GAAGATGGTGATGGGATTTC-31015840 which amplify a 226 bptarget Following PCR a thermal melt profile was per-formed for amplicon identification The specificity of theamplification reactions was also confirmed by agarose gelelectrophoresis The relative expression was presented as foldchanges after normalizing to the GAPDH control

25 Immunofluorescent Staining HLEB3 cells were grownon glass coverslips before siRNAs were transfected and then


GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)

Figure 1 TGF1205732-induced expression of Snail mRNA in a dose-dependent manner (a) Representative agarose gel electrophoresis images ofSnail and house gene expression after TGF1205732 treatment (b) The summary of triplicated experiments lowast119875 lt 005 compared with TGF1205732 (minus)(0 ngmL) 119875 lt 005 compared with the group treated with 10 ngmL TGF1205732

0 1 4 8 12

GAPDH

Time (h) 05TGF1205732 (10ngmL)

Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)

Figure 2 The time course of TGF1205732-induced expression of Snail mRNA (a) Representative agarose gel electrophoresis images revealedTGF1205732-induced early expression of Snail (b)The summary of triplicated experiments lowast119875 lt 005 compared with TGF1205732 (minus) (0 h) 119875 lt 005compared with TGF1205732 (+) (1 h)

exposed to 10 ngmL of TGF1205732 for 1 hour Cells were fixedwith 4 paraformaldehyde for 30min at 4∘C followed byincubation with 01 Triton X-100 and 3 BSA for 2 hin room temperature for permeabilization and blockingThe primary antibodies (1 100) against Snail vimentin E-cadherin keratin or 120572-SMA diluted in PBS were placedon cells for overnight at 4∘C respectively followed byincubation with CY3-conjugated goat anti-rabbit or FITC-conjugated goat anti-mouse immunoglobulin (1 200) for 2hours at 37∘C in the dark The nuclei were counterstainedwith Hoechst 33258 (Invitrogen CA USA) Images wereacquired with a fluorescence microscope (DM4000B LeicaGermany)

26 Transwell Assay Transwell apparatuswith 8120583mpore sizemembrane (Costar CambridgeMAUSA)was used to detectthe migration ability of HLEB3 cells The siRNAs-treatedHLEB3 cells were exposed to 10 ngmL of TGF1205732 for 48 hSerum-free DMEM containing 1 times 105 cells in 100 120583L wasadded into the upper chamber the lower chamber contained500120583L of 15 FBS-containing medium After incubation at37∘C for 24 h membranes were swabbed with a cotton swabsoaked in 01 crystal violet for 10min and thenwashedwithPBS The number of cells attached to the lower surface of the

polycarbonate filter was counted at 100x magnification undera light microscope

27 Statistical Analysis All results are expressed as themean plusmn SDThe data were analyzed with ANOVA and SNK-qtest using SPSS170119875 lt 005was considered to be statisticallysignificant

3 Results

31 Expression of Snail Induced by TGF1205732 To determinewhether the expression of Snail is regulated by TGF1205732 weexamined the expression and intracellular localization ofSnail in HLEB3 cells RT-PCR results indicated that in theabsence of TGF1205732 there was no Snail expression in HLEB3cells whereas the level of Snail mRNA was significantlyelevated in cells stimulated with TGF1205732 TGF1205732-inducedSnail expression was does dependent and the expression wasdetected as early as 05 h after the treatment (Figures 1 and2)

Consistent with the mRNA expression Snail proteinsynthesis was induced after stimulation by TGF1205732 In theabsence of TGF1205732 the cells showed no immunoreactivity forthe protein However Snail protein production was greatly


Hoechst Merge

Control

TGF1205732

Snail

Figure 3 TGF1205732-induced expression of Snail protein HLEB3 cellswere incubated in the absence or presence of 10 ngmL TGF1205732After 8 hours of culture cells were immunofluorescence stainedwithanti-Snail antibody (red) and counterstained with Hoechst (blue)Snail were expressed after TGF1205732 treatment and located in nuclear(400x)

0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)

Figure 4 Efficiency of four siRNAs (P1ndashP4) on Snail expressionSerum starved HLEB3 cells were transfected with human SnailsiRNAs (P1ndashP4) and negative control (P5) before being stimulat-ed with TGF1205732 for 1 hour Snail expressions were significantlydecreased with the siRNA treatmentThe data were collected from 3experiments lowast119875 lt 005 compared with siRNA (minus)TGF1205732 (+)(10 ngmL)

increased in the presence of TGF1205732 and immunostainingwas detected mainly in the nucleus and nearby cytosol(Figure 3)

32 Efficiency of siRNAs Inhibition of Snail Expression FourSnail siRNAs (P1ndashP4) inhibited the expression of SnailmRNAexpression after TGF1205732 treatment by 5500 (P1) 7485(P2) 4985 (P3) and 4398 (P4) respectively (119875 lt 005)while the negative control siRNA (P5) showed no effects(Figure 4) Because P2was themost efficient in the inhibitionit was used in the following experiments

33 Role of Snail in TGF1205732-Induced EMT of HLEB3 TheSnail siRNA (P2) reduced the Snail protein expression as wellas the mRNA level induced by TGF1205732 (Figure 5) AlthoughLECs are derived from surface ectoderm they expressvimentin [38] as well as the epithelial surface marker keratin

siRNA P2 P5 +++

minusminus

minusTGF1205732

Figure 5 siRNA inhibition of Snail protein expression Serumstarved HLEB3 cells were transfected with human Snail siRNA(P2) and negative control (P5) before being stimulated with TGF1205732for 8 hours Cells were stained with anti-Snail antibody (red)and counterstained with Hoechst (blue) Images were taken byfluorescence microscope (400x)

siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732

Figure 6 siRNA inhibition of EMT relevant molecules Serumstarved HLEB3 cells were transfected with human Snail siRNA (P2)negative control siRNA (P5)Then cells were stimulatedwith TGF1205732for 24 hours Various cellular proteins were detected by immunoflu-orescence staining Images were taken by fluorescence microscope(400x)

and E-cadherin The vimentin is expressed physiologically inan appropriate amount while overexpression is an evidenceof EMT Immunofluorescence analysis for EMT relevantproteins revealed that keratin E-cadherin and vimentinwere expressed in normal HLEB3 cells but not 120572-SMAThe TGF1205732-induced repression of keratin and E-cadherinproduction was significantly abolished by the Snail targetingsiRNA The increase of vimentin and 120572-SMA by TGF1205732 wasinhibited by the siRNA treatment (Figure 6)

The observation of the morphology of HLEB3 cellsshowed that untreated HLEB3 cells were polygonal with tightjunction After the stimulation of TGF1205732 the cells becamelonger and slimmer spindly shaped as fibroblast and thejunctions of cell-cell were lost Snail targeting siRNA reversedthose morphological changes (Figure 7)


Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)

Figure 7 Morphological changes of HLEB3 cells Serum starved HLEB3 cells were transfected with human Snail siRNA (P2) and negativecontrol siRNA (P5) before the cells were stimulated with TGF1205732 The morphology of the cells was observed under inverted microscope (a)TGF1205732-induced cells became spindly shaped (b) Snail targeting siRNA prevented the cells from the TGF1205732-induced morphological change(200x)

siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe

rsiRNA P2 P5minusminus

lowast

lowast

TGF1205732 (10ngmL)(b)

Figure 8 Inhibition of migration ability by Snail siRNA Serum starved HLEB3 cells were transfected with human Snail siRNA (P2)negative control siRNA (P5) before the cells were stimulated with TGF1205732 for 48 h Transwell assay was used to detect the migration ability ofcells (a) Crystal violet stained transmembrane cells under light microscope (100x) (b) The count of migrated HLEB3 cells from triplicatedexperimentslowast119875 lt 005 compared with siRNA (minus)TGF1205732 (+) (10 ngmL)

There were few untreated HLEB3 cells that passedthrough the polycarbonate The migration of TGF1205732-treatedcells was markedly higher than the untreated cells (119875 lt005)The treatment of Snail siRNA (P2) significantly blockedthe increased migration stimulated by TGF1205732 (119875 lt 005)(Figure 8)

4 Discussion

In this study we successfully established a human LEC EMTmodel and found that Snail targeting siRNA can efficientlyinhibit TGF1205732-induced EMT of human LECs which has notbeen reported previously The data indicated the potential touse siRNA approach to suppress development of PCO aftercataract surgery

At present surgery is the only effective treatment of cata-ract to restore impaired vision Unfortunately many patientssuffer a secondary loss of vision over time because of PCOPCO is themost common long-term complication of cataractsurgeryThe incidence of PCO is approximately 50 in adultsand 100 in children [39ndash42] It usually causes a decreasein visual acuity by blocking the visual axis and striae orfolds in the posterior capsule In addition traction-inducedintraocular lens (IOL) malposition which needed furthercorrective surgery can occur during PCO

PCO is usually caused by the proliferation migra-tion EMT collagen deposition and lens fiber regenerationof residual LECs [43ndash46] because the surgery induces a

wound-healing response in the lens Usually proliferationof the remaining LECs starts within a few hours aftercataract surgery [47] Proliferation and migration of LECsmay precede EMT and the two events are thought to beindependently regulated [48 49] Therefore postsurgicalmedical inhibition of LECsrsquo proliferation migration andEMT would be an option for preventing PCO

Myofibroblasts play a central role in the process of tissuefibrosis and scarring This cell type is derived from both acti-vated fibroblasts and epithelial cells including LECs Expres-sion of 120572-SMA a marker for fibroblast-myofibroblast con-version is mediated by Smads [50] The transdifferentiationin which an epithelial cell changes its phenotype to amyofibroblast involves many transcription factors includingZEB (Sip1dEF1) bHLH (E47Twist) and Snail12 [51ndash54]These transcription factors are upregulated by TGF120573 anddirectly suppress E-cadherin promoter which is essential inthe maintenance of epithelial phenotype Expression of Snailthe master transcription factor involved in an early step ofthe EMT is considered as an important factor in the tissuefibrosis in the eye [7]

We focus on Snail because of its relation in cellular pro-liferation and differentiation Snail is a member of a family ofzinc finger-containing transcriptional repressors Snail familyis implicated in the transcriptional repression of E-cadherinby interacting with the E-box sequence in the proximal E-cadherin promoter So the function of the gene is associatedwith suppression of the epithelial phenotype [55] The gene


had been shown to be a master gene for early stage of EMT[51 56 57]

Cho et al had reported that TGF120573 induced Snail expres-sion in mouse lens epithelial cells [36] It is also reportedthat Slug (Snail2 another member of Snail superfamily)was expressed in anterior polar cataracts and human lensepithelial cell lines [58]

RNA interference has become a standard method forin vitro knockdown of any target gene of interest siRNAcan incorporate into a protein complex that recognizes andcleaves target mRNA [59] Compared to small chemicals forthe purpose of inhibition siRNA mimics RNAi that is acommon phenomenon in living creature and is believed tobe safe and efficient in the inhibition of a specific gene expres-sion Four siRNAs against Snail were used to avoid off-targeteffects Our data suggested that all the designed siRNAsinhibited the expression of Snail notably

In this study we have demonstrated that Snail is anearly responder of TGF120573 in EMT of human LECs TGF1205732-treated HLEB3 cells lose their epithelium character and gainmesenchymal feature Snails are implicated in the repressionby interacting with the E-box sequence in the proximal E-cadherin promoter which is associated with morphologicchanges in cells that occur during EMT in embryonic devel-opment and in tumor cell invasion [27 34 35]We confirmedthe similar mechanism in HLEB3 cells TGF1205732 changed thepolygonal LECs to elongated shape and lost contact with theirneighbors These cells gained notable migration ability Wepresumed that the loss of cellsrsquo junction is caused by Snail-induced E-cadherinrsquos reduction and the contractive propertyof 120572-SMA contributes to the migration We found that allthese EMT relevant changes were blocked by targeting Snail

In conclusion our data indicated that TGF1205732 inducesSnail expression and EMT of human LECs and Snail is anessential factor in this process Snail targeting siRNA inhibitsSnail expression and EMT in human LECs and might be acandidate strategy to prevent subcapsular cataract includingPCO


The authors declare that there is no conflict of interestsThe authors have no commercial interest in any materialsdiscussed in this paper

Acknowledgments

This research was supported by the Research Program ofNantong University The authors greatly thank Dr Yuan-yuan Zhu and his team of Biomics for scientific advice andtechnical assistance

References

[1] E D Hay ldquoAn overview of epithelio-mesenchymal transforma-tionrdquo Acta Anatomica vol 154 no 1 pp 8ndash20 1995

[2] P Savagner ldquoLeaving the neighborhood molecular mech-anisms involved during epithelial-mesenchymal transitionrdquoBioEssays vol 23 no 10 pp 912ndash923 2001

[3] J PThiery ldquoEpithelial-mesenchymal transitions in cancer onsetand progressionrdquo Bulletin de lrsquoAcademie Nationale de Medecinevol 193 no 9 pp 1969ndash1979 2009

[4] D Sun S Baur and E D Hay ldquoEpithelial-mesenchymal trans-formation is the mechanism for fusion of the craniofacialprimordia involved in morphogenesis of the chicken liprdquoDevelopmental Biology vol 228 no 2 pp 337ndash349 2000

[5] S Saika Y Okada T Miyamoto Y Ohnishi A Ooshima andJ W McAvoy ldquoSmad translocation and growth suppressionin lens epithelial cells by endogenous TGF1205732 during woundrepairrdquo Experimental Eye Research vol 72 no 6 pp 679ndash6862001

[6] S Saika TMiyamoto S Tanaka et al ldquoResponse of lens epithe-lial cells to injury role of lumican in epithelial-mesenchymaltransitionrdquo Investigative Ophthalmology and Visual Science vol44 no 5 pp 2094ndash2102 2003

[7] S Saika S Kono-Saika Y Ohnishi et al ldquoSmad3 signaling isrequired for epithelial-mesenchymal transition of lens epithe-lium after injuryrdquoAmerican Journal of Pathology vol 164 no 2pp 651ndash663 2004

[8] R U de Iongh E Wederell F J Lovicu and J W McAvoyldquoTransforming growth factor-120573-induced epithelial-mesenchy-mal transition in the lens a model for cataract formationrdquo CellsTissues Organs vol 179 no 1-2 pp 43ndash55 2005

[9] M B Vaughan E W Howard and J J Tomasek ldquoTransform-ing growth factor-1205731 promotes the morphological and func-tional differentiation of the myofibroblastrdquo Experimental CellResearch vol 257 no 1 pp 180ndash189 2000

[10] G Serini M Bochaton-Piallat P Ropraz et al ldquoThe fibronectindomain ED-A is crucial for myofibroblastic phenotype induc-tion by transforming growth factor-1205731rdquo Journal of Cell Biologyvol 142 no 3 pp 873ndash881 1998

[11] A Vernon and C LaBonne ldquoTumor metastasis a new twiston epithelial-mesenchymal transitionsrdquoCurrent Biology vol 14no 17 pp R719ndashR721 2004

[12] J Massague ldquoTGF-beta signal transductionrdquo Annual Review ofBiochemistry vol 67 pp 753ndash791 1998

[13] C M Zimmerman and R W Padgett ldquoTransforming growthfactor 120573 signaling mediators and modulatorsrdquo Gene vol 249no 1-2 pp 17ndash30 2000

[14] K Miyazono P Ten Dijke and C Heldin ldquoTGF-120573 signaling bySmad proteinsrdquo Advances in Immunology vol 75 pp 115ndash1572000

[15] A Moustakas K Pardali A Gaal and C Heldin ldquoMechanismsof TGF-120573 signaling in regulation of cell growth and differentia-tionrdquo Immunology Letters vol 82 no 1-2 pp 85ndash91 2002

[16] P Ten Dijke M J Goumans F Itoh and S Itoh ldquoRegulationof cell proliferation by Smad proteinsrdquo Journal of CellularPhysiology vol 191 no 1 pp 1ndash16 2002

[17] A B Roberts and M B Sporn ldquoDifferential expression of theTGF-120573 isoforms in embryogenesis suggests specific roles indeveloping and adult tissuesrdquo Molecular Reproduction andDevelopment vol 32 no 2 pp 91ndash98 1992

[18] C Gordon-Thomson R U de Iongh A M Hales C G Cham-berlain and J W McAvoy ldquoDifferential cataractogenic potencyof tgf-1205731 1205732 and -1205733 and their expression in the postnatal rateyerdquo Investigative Ophthalmology and Visual Science vol 39 no8 pp 1399ndash1409 1998

[19] T Ashish C K T Jonathan S Ajay G Rangan and RM RajivldquoRole of transforming growth factor beta in corneal functionbiology and pathologyrdquo Current Molecular Medicine vol 10 no6 pp 565ndash578 2010


[20] K Ohta S Yamagami A W Taylor and J W Streilein ldquoIL-6 antagonizes TGF-120573 and abolishes immune privilege in eyeswith endotoxin-induced uveitisrdquo Investigative Ophthalmologyand Visual Science vol 41 no 9 pp 2591ndash2599 2000

[21] H D Jampel N Roche W J Stark and A B Roberts ldquoTrans-forming growth factor-120573 in human aqueous humorrdquo CurrentEye Research vol 9 no 10 pp 963ndash969 1990

[22] SWCousinsMMMcCabeDDanielpour and JW StreileinldquoIdentification of transforming growth factor-beta as an imm-unosuppressive factor in aqueous humorrdquo Investigative Oph-thalmology and Visual Science vol 32 no 8 pp 2201ndash2211 1991

[23] T Kita Y Hata R Arita et al ldquoRole of TGF-120573 in proliferativevitreoretinal diseases and ROCK as a therapeutic targetrdquo Pro-ceedings of the National Academy of Sciences of the United Statesof America vol 105 no 45 pp 17504ndash17509 2008

[24] T Kokudo Y Suzuki Y Yoshimatsu T Yamazaki T Watabeand K Miyazono ldquoSnail is required for TGF120573-inducedendothelial-mesenchymal transition of embryonic stem cell-derived endothelial cellsrdquo Journal of Cell Science vol 121 no20 pp 3317ndash3324 2008

[25] C Come V Arnoux F Bibeau and P Savagner ldquoRoles of thetranscription factors Snail and slug during mammary morpho-genesis and breast carcinomaprogressionrdquo Journal ofMammaryGland Biology and Neoplasia vol 9 no 2 pp 183ndash193 2004

[26] E Rosivatz I Becker K Specht et al ldquoDifferential expressionof the epithelial-mesenchymal transition regulators Snail SIP1and twist in gastric cancerrdquo American Journal of Pathology vol161 no 5 pp 1881ndash1891 2002

[27] E Batlle E Sancho C Franci et al ldquoThe transcription factorSnail is a repressor of E-cadherin gene expression in epithelialtumour cellsrdquoNature Cell Biology vol 2 no 2 pp 84ndash89 2000

[28] H G Palmer M J Larriba J M Garcıa et al ldquoThe transcrip-tion factor Snail represses vitamin D receptor expression andresponsiveness in human colon cancerrdquo Nature Medicine vol10 no 9 pp 917ndash919 2004

[29] S A Murray and T Gridley ldquoSnail1 gene function during earlyembryo patterning in micerdquo Cell Cycle vol 5 no 22 pp 2566ndash2570 2006

[30] W A Paznekas K Okajima M Schertzer S Wood and E WJabs ldquoGenomic organization expression and chromosomelocation of the human Snail gene (SNAI1) and a related pro-cessed pseudogene (SNAI1P)rdquo Genomics vol 62 no 1 pp 42ndash49 1999

[31] K Aomatsu T Arao K Sugioka et al ldquoTGF-120573 inducessustained upregulation of SNAI1 and SNAI2 through smad andnon-smad pathways in a human corneal epithelial cell linerdquoInvestigative Ophthalmology and Visual Science vol 52 no 5pp 2437ndash2443 2011

[32] K Shirai S Saika T Tanaka et al ldquoA new model of anteriorsubcapsular cataract involvement of TGF120573Smad signalingrdquoMolecular Vision vol 12 pp 681ndash691 2006

[33] A M Abu El-Asrar L Missotten and K Geboes ldquoExpressionof myofibroblast activation molecules in proliferative vitreo-retinopathy epiretinal membranesrdquo Acta Ophthalmologica vol89 no 2 pp e115ndashe121 2011

[34] A Cano M A Perez-Moreno I Rodrigo et al ldquoThe transcrip-tion factor Snail controls epithelial-mesenchymal transitions byrepressing E-cadherin expressionrdquo Nature Cell Biology vol 2no 2 pp 76ndash83 2000


[36] H J Cho K E Baek S Saika M Jeong and J Yoo ldquoSnail isrequired for transforming growth factor-120573-induced epithelial-mesenchymal transition by activating PI3 kinaseAkt signalpathwayrdquo Biochemical and Biophysical Research Communica-tions vol 353 no 2 pp 337ndash343 2007

[37] S M Elbashir W Lendeckel and T Tuschl ldquoRNA interferenceis mediated by 21- and 22-nucleotide RNAsrdquo Genes and Devel-opment vol 15 no 2 pp 188ndash200 2001

[38] C M Sax F X Farrell Z E Zehner and J Piatigorsky ldquoRegu-lation of vimentin gene expression in the ocular lensrdquo Develop-mental Biology vol 139 no 1 pp 56ndash64 1990

[39] D S Clark ldquoPosterior capsule opacificationrdquo Current Opinionin Ophthalmology vol 11 no 1 pp 56ndash64 2000

[40] D A Schaumberg M R DanaW G Christen and R J GlynnldquoA systematic overview of the incidence of posterior capsuleopacificationrdquo Ophthalmology vol 105 no 7 pp 1213ndash12211998

[41] P J McDonnell M A Zarbin and W R Green ldquoPosteriorcapsule opacification in pseudophakic eyesrdquo Ophthalmologyvol 90 no 12 pp 1548ndash1553 1983

[42] S Dewey ldquoPosterior capsule opacificationrdquo Current Opinion inOphthalmology vol 17 no 1 pp 45ndash53 2006

[43] L M Cobo E Ohsawa and D Chandler ldquoPathogenesis ofcapsular opacification after extracapsular cataract extractionAn animal modelrdquo Ophthalmology vol 91 no 7 pp 857ndash8631984

[44] I M Wormstone ldquoPosterior capsule opacification a cell bio-logical perspectiverdquo Experimental Eye Research vol 74 no 3pp 337ndash347 2002

[45] R Frezzotti A Caporossi D Mastrangelo et al ldquoPathogenesisof posterior capsular opacification Part II histopathologicaland in vitro culture findingsrdquo Journal of Cataract and RefractiveSurgery vol 16 no 3 pp 353ndash360 1990

[46] J P Kappelhof and G F Vrensen ldquoThe pathology of after-cataract A minireviewrdquoActa ophthalmologica supplement 205pp 13ndash24 1992

[47] N Awasthi and B J Wagner ldquoSuppression of human lensepithelial cell proliferation by proteasome inhibition a potentialdefense against posterior capsular opacificationrdquo InvestigativeOphthalmology and Visual Science vol 47 no 10 pp 4482ndash4489 2006

[48] IMWormstone C S C Liu J Rakic JMMarcantonio G F JM Vrensen and G Duncan ldquoHuman lens epithelial cell prolif-eration in a protein-free mediumrdquo Investigative Ophthalmologyand Visual Science vol 38 no 2 pp 396ndash404 1997

[49] J L Walker I MWolff L Zhang and A S Menko ldquoActivationof Src kinases signals induction of posterior capsule opacifica-tionrdquo Investigative Ophthalmology and Visual Science vol 48no 5 pp 2214ndash2223 2007

[50] D Javelaud and A Mauviel ldquoCrosstalk mechanisms betweenthe mitogen-activated protein kinase pathways and Smad sig-naling downstream of TGF-120573 implications for carcinogenesisrdquoOncogene vol 24 no 37 pp 5742ndash5750 2005

[51] M A Nieto ldquoThe Snail superfamily of zinc-finger transcriptionfactorsrdquoNature Reviews Molecular Cell Biology vol 3 no 3 pp155ndash166 2002

[52] R Kalluri and E G Neilson ldquoEpithelial-mesenchymal tran-sition and its implications for fibrosisrdquo Journal of ClinicalInvestigation vol 112 no 12 pp 1776ndash1784 2003

[53] M A Huber N Azoitei B Baumann et al ldquoNF-120581B is essen-tial for epithelial-mesenchymal transition and metastasis in a


model of breast cancer progressionrdquo Journal of Clinical Investi-gation vol 114 no 4 pp 569ndash581 2004

[54] J M Lee S Dedhar R Kalluri and E W Thompson ldquoTheepithelial-mesenchymal transition new insights in signalingdevelopment and diseaserdquo Journal of Cell Biology vol 172 no7 pp 973ndash981 2006

[55] M A Nieto ldquoEpithelial-Mesenchymal Transitions in develop-ment and disease old views and new perspectivesrdquo Interna-tional Journal of Developmental Biology vol 53 no 8ndash10 pp1541ndash1547 2009

[56] G A Barrallo and M A Nieto ldquoThe Snail genes as inducers ofcell movement and survival implications in development andcancerrdquo Development vol 132 no 14 pp 3151ndash3161 2005

[57] B De Craene F Van Roy and G Berx ldquoUnraveling signallingcascades for the Snail family of transcription factorsrdquo CellularSignalling vol 17 no 5 pp 535ndash547 2005

[58] J Choi Y P Sun and C Joo ldquoTransforming growth factor-1205731 represses E-cadherin production via Slug expression in lensepithelial cellsrdquo Investigative Ophthalmology and Visual Sciencevol 48 no 6 pp 2708ndash2718 2007

[59] M T McManus and P A Sharp ldquoGene silencing in mammalsby small interfering RNAsrdquo Nature Reviews Genetics vol 3 no10 pp 737ndash747 2002


Review ArticleVascular Adhesion Protein 1 in the Eye

Wenting Luo12 Fang Xie23 Zhongyu Zhang12 and Dawei Sun12

1 Department of Ophthalmology 2nd Affiliated Hospital of Harbin Medical University 246 Xuefu Road Harbin 150001 China2Harbin Medical University-The Key Laboratory of Myocardial Ischemia Chinese Ministry of Education Harbin 150001 China3Department of Ophthalmology 1st Affiliated Hospital of Harbin Medical University Harbin 150001 China

Correspondence should be addressed to Dawei Sun drsundwgmailcom

Received 17 January 2013 Revised 17 April 2013 Accepted 14 May 2013


Copyright copy 2013 Wenting Luo et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Semicarbazide-sensitive amine oxidasevascular adhesion protein-1 (SSAOVAP-1) a dual-function molecule with adhesive andenzymatic properties is expressed on the surface of vascular endothelial cells of mammals It also exists as a soluble form(sVAP-1) which is implicated in oxidative stress via its enzymatic activity and can be a prognostic biomarker Recent evidencesuggests that VAP-1 is an important therapeutic target for several inflammation-related ocular diseases such as uveitis age-related macular degeneration (AMD) and diabetic retinopathy (DR) by involving in the recruitment of leukocytes at sites ofinflammation Furthermore VAP-1 plays an important role in the pathogenesis of conjunctival inflammatory diseases such aspyogenic granulomas and the progression of conjunctival lymphoma VAP-1 may be an alternative therapeutic target in oculardiseases The in vivo imaging of inflammation using VAP-1 as a target molecule is a novel approach with a potential for earlydetection and characterization of inflammatory diseasesThis paper reviews the critical roles of VAP-1 in ophthalmological diseaseswhich may provide a novel research direction or a potent therapeutic strategy

1 Introduction

Vascular adhesion protein-1 (VAP-1) is a homodimeric sia-lylated glycoprotein originally discovered in inflamed syn-ovial vessels by Salmi and Jalkanen in 1992 [1] VAP-1 is amultifunctional molecule that possesses enzymatic activityknown as semicarbazide-sensitive amine oxidase (SSAO) andis involved in the leukocyte recruitment cascade The VAP-1molecule consists of an extracellular part which harbors thecatalytic site a transmembrane segment and a short intracel-lular N-terminal tail [2 3] On the plasma membrane VAP-1normally forms a homodimer of two 90 kDa glycoproteinsThe extracellular part of each monomer consists of threedomains (D2ndashD4) VAP-1 has a relatively narrow substratechannel formed by domains D4 and D3 and a key leucine(469 in human) guards the entry of substrates The large D4domains from each subunit form the dimer interface andeach also contains a catalytic site buried at the base of a deepcleft

VAP-1 exists as membrane-bound and soluble forms inthe plasma Its major sources are endothelial cells smooth

muscle cells and the adipocytes [4] VAP-1 is expressed on theendothelium of human tissues such as skin brain lung liverand heart under both normal and inflamed conditions [4ndash8]In the ocular tissues of humans and rats VAP-1 is localizedon the endothelial cells of retinal and choroidal vessels [9ndash12]VAP-1 labeling showed the highest intensity in both arteriesand veins of neuronal tissues retina and optic nerve themoderate intensity in scleral and choroidal vessels and thelowest intensity in the iris vasculature [10] Moreover VAP-1intensity was significantly higher in the arteries compared toveins [10]

Under normal conditions VAP-1 is mainly absent fromthe endothelial cell surface and is stored within intracellulargranules while on inflammation it is rapidly translocatedto the endothelial cell surface and facilitates the recruit-ment of leukocytes into the inflamed tissues together withother leukocyte adhesion molecules [13] (Figure 1) In factprevious studies have elucidated that VAP-1 is involved inthe molecular mechanisms of acute ocular inflammation[11] inflammation-associated ocular angiogenesis [12] andleukostasis under diabetic conditions [10] Indeed VAP-1


Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow

Figure 1Under normal conditions VAP-1 ismainly absent from theendothelial cell surface and is stored within intracellular granuleswhile on inflammation it is rapidly translocated to the endothelialcell surface and facilitates the recruitment of leukocytes into the in-flamed tissues together with other leukocyte adhesion molecules

inhibition may be a novel and potent therapeutic strategyin the treatment of ocular inflammatory diseases NotablySSAOVAP-1 contributes to inflammation not only throughits role as an adhesion molecule but also through its functionas an enzyme by causing the formation of cytotoxicmoleculessuch as hydrogen peroxide aldehyde and ammonia [14]These molecules are involved in the pathophysiology of ocu-lar inflammation [15 16] and their inhibition for instancethrough antioxidants recovers the integrity of the blood-aqueous barrier in endotoxin-induced uveitis (EIU) animals[17]

Here we give an overview on the new research progressesof VAP-1 in the ocular diseases including uveitis age-relatedmacular degeneration (AMD) diabetic retinopathy (DR)and ocular tumorThe connection between VAP-1 and oculardiseases will be elucidated and may provide a new researchdirection for the diagnosis and treatment of these ocular dis-eases

2 VAP-1 in Acute Inflammation ofEndotoxin-Induced Uveitis

Uveitis is regarded as a sight-threatening disease Compli-cations such as cystoid macular edema glaucoma vascularocclusion and proliferative vitreoretinopathy are commoncauses of permanent vision loss [18ndash21] EIU is one of animalmodels to establish new therapeutic targets for treatinghuman uveitis which is marked by a vasodilatation of theiris and vascular changes in the ciliary body accompaniedby an increased vascular permeability and breakdown of theblood-aqueous barrier [22ndash24] The leukocytes infiltrate intothe anterior chamber vitreous cavity and retina from ciliarybody and iris in conjunction with protein extravasation into

the aqueous humor As part of this inflammatory responseendothelial adhesion molecules are upregulated For exam-ple endothelial P-selectin which mediates the first stepof the leukocyte recruitment the tethering and rollingis upregulated in retinal vessels of EIU animals [25 26]Furthermore intercellular adhesion molecule-1 (ICAM-1)which mediates the subsequent step of firm leukocyte adhe-sion to the vascular endothelium is increased in the retina ofEIU animals [27 28] Functional inhibition of P-selectin [29]or ICAM-1 [28] prevents the infiltration of leukocytes into theinflamed ocular tissues during EIU and thus attenuates theinflammatory response at the early stages of rolling and firmadhesion

In 2008 Noda et al investigated the role of VAP-1 in anestablished model of EIU VAP-1 is constitutively expressedin the normal retina and its expression is elevated togetherwith SSAO activity during EIU [11] Their data also indicatethat VAP-1 inhibition substantially suppresses retinal inflam-mation during EIU on a molecular cellular and organ levelFor example VAP-1 inhibition in EIU animals significantlysuppressed leukocytes recruitment to the anterior chambervitreous and retina as well as retinal endothelial P-selectinexpression The diameter of the retinal veins and arteries ofEIU animals 24 h after LPS injection was significantly largerthan the corresponding retinal vessels in normal animalsHowever VAP-1 inhibition reduced the diameter of corre-sponding retinal veins and arteries 24 h after LPS injectioncompared with vehicle-treated rats even though the differ-ence did not reach statistical significance To sum up VAP-1 is crucially involved in leukocyte infiltration into oculartissues during acute inflammation of EIU VAP-1 inhibitionmay even prevent leukocyte recruitment at the early stage ofrolling and become a novel strategy in the treatment of uveitis(Table 1)

3 VAP-1 in the Choroidal Neovascularization

Choroidal neovascularization (CNV) is the main cause ofsevere vision loss in patients with age-related macular degen-eration (AMD) [30] Inflammation plays a critical role inthe formation of CNV lesions and may contribute to thepathogenesis of both the nonexudative and exudative formsof AMD [31 32] For example inflammatory cells are foundin surgically excised CNV lesions from AMD patients [33ndash36] and in autopsied eyes with CNV [37ndash39] In particularmacrophages have been implicated in the pathogenesis ofAMDdue to their spatiotemporal distribution in the proxim-ity of the CNV lesions in experimental models and humans[40ndash42] Macrophages are a source of proangiogenic andinflammatory cytokines such as vascular endothelial growthfactor (VEGF) [43] and tumor necrosis factor (TNF)-120572 [44]both of which significantly contribute to the pathogenesis ofCNV [45 46] Furthermore druse which has proven to beone of the earliest signs of AMDcontainsmany inflammatorymolecules [47 48] Some inflammatorymolecules such as thecomplement components C3a and C5a are proinflammatoryand can induce VEGF [49]

As an endothelial adhesion molecule involved in leuko-cyte recruitment under inflammatory conditions VAP-1


Table 1 The function of vascular adhesion protein-1 in ocular diseases

Eye diseases Possible role of VAP-1 ReferenceUveitis VAP-1 is involved in leukocyte infiltration into ocular tissues [11]

NVC During acute inflammation VAP-1 regulates both firm adhesion and transmigration VAP-1 contributes to therecruitment of macrophages to CNV lesions and has a novel link with angiogenesis

[12 50]

DR In chronic low-grade inflammation VAP-1 may only regulate transmigration sVAP-1 is increased andcorrelated with oxidative stress in the vitreous fluid [10 64]

Tumor VAP-1 is correlated with the angiogenesis and tumor growth [71 72]VAP-1 vascular adhesion protein-1

was recently showed to contribute to the recruitment ofmacrophages to CNV lesions in a rat laser-induced AMDmodel and had a novel link with angiogenesis [12] In theirstudy VAP-1 was found to be expressed in the choroid andretina exclusively in the vessels and localized in the vessels ofthe CNV lesions Inhibition of VAP-1 significantly decreasedCNV size fluorescein angiography leakage and the accu-mulation of macrophages in CNV lesions [12] Further-more VAP-1 blockade significantly reduced the expression ofinflammation-associated molecules such as tumor necrosisfactor (TNF)-120572 monocyte chemoattractant protein (MCP)-1 and intercellular adhesion molecule (ICAM)-1 [12]

Most recently in a mouse laser-induced CNV modelVAP-1 inhibition significantly attenuated CNV formation ina dose-dependent manner and reduced macrophage infiltra-tion into CNV lesions [50] Furthermore VAP-1 blockadedecreased the expression of ICAM-1 and MCP-1 both ofwhich played a pivotal role in macrophage recruitment [50]Thus VAP-1 blockade reduced macrophage recruitment intoCNV lesion indirectly via suppression of other adhesionmolecules Previous studies have demonstrated that markedsuppression of VEGF is crucial for the suppression of CNVformation in the laser-inducedCNVmodel [51 52] Howeverin this study VAP-1 blockade showed weak inhibitory effectson VEGF a key molecule for angiogenesis whereas CNVformation was significantly suppressed It may indicate thatVAP-1 inhibition ameliorates ocular angiogenesis throughmechanism(s) other than VEGF expression Further evalu-ation is needed to elucidate the detailed mechanism(s) Inconclusion the current data suggest that VAP-1 may be anattractive molecular target in the treatment of CNV forma-tion of AMD (Table 1)

4 VAP-1 in Chronic Low-Grade Inflammationof Diabetic Retinopathy

Diabetic retinopathy (DR) is one of the main microvascularcomplications of diabetes and a leading cause of adult visionloss [53 54] Recent studies have elucidated that chroniclow-grade inflammation underlies much of the vascularcomplications of DR [55 56]Manymolecular and functionalchanges that are characteristics of inflammation have beendetected inDRThe recruitment of leukocytes has been foundto be significantly increased in retinas of diabetic animals[57ndash59] andmight contribute to the capillary nonperfusion ofdiabetic retinopathy Leukocytes firmly adhering to capillary

endothelial cells via adhesion molecules induce apoptoticchanges in retinal endothelial cells

As demonstrated through several lines of evidence VAP-1 seems to be a key player in the inflammation associatedwith DR In 2009 Noda et al investigated the role of VAP-1in DR Contrastively retinal VAP-1 expression was higher indiabetic animals compared to the normal controls howeverthe difference did not reach statistical significance [10] Theirresults also suggested that VAP-1 principally regulated thestep of leukocyte transmigration with little influence on thepreceding step of firm adhesion [10] This provides a cleardistinction between the role of VAP-1 in acute and chronicinflammation During acute inflammation VAP-1 regulatesboth firm adhesion and transmigration [11] while in chroniclow-grade inflammation such as found during diabetesVAP-1may only regulate transmigration In conclusion VAP-1 contributes to the inflammatory outcome of DR VAP-1inhibition may be beneficial in the treatment and preventionof DR Further investigation may provide a better under-standing of the role of VAP-1 in DR

VAP-1 also exists as a soluble form in serumwhich retainsits enzymatic function [60] Like other soluble adhesionmolecules sVAP-1 modulates lymphocyte adherence In factsVAP-1 appears to augment lymphocyte binding to endothe-lial cells [61] Much attention has recently been paid to theelevated serum concentration of sVAP-1 in patients with type1 and type 2 diabetes [61 62] In type 2 diabetes sVAP-1 evenserves as an independent prognostic marker for the diabeticcomplications and predicts the risk for cardiovascular andcancer mortality in these patients [63] Moreover patientswith DR display significantly higher plasma SSAO activitiescompared to patients without DR [61] (Table 1)

In a recent clinical study Murata et al [64] demonstratedthat sVAP-1 is increased and correlated with oxidative stressin the vitreous fluid of patients with PDR Furthermore reti-nal capillary endothelial cells produce the membrane-boundform of VAP-1 and release sVAP-1 when stimulated with highglucose or inflammatory cytokines such as TNF-120572 and IL-1120573 MMP-2 (matrix metalloproteinases-2) and MMP-9 candegrade type IV collagen laminin and fibronectin the mainconstituents of the basement membrane thereby MMPs playa crucial role in the degradation of basement membraneduring angiogenesis [65 66] MMP-2 and MMP-9 are theproteinases predominantly responsible for VAP-1 sheddingfrom retinal capillary endothelial cells [64] The present dataprovide evidence on the link between sVAP-1 and type IV


collagenases in the pathogenesis of PDR Therefore furtherstudies are needed to clarify the relationship between sVAP-1and other ocular diseases

5 VAP-1 in Ocular Tumor

The relationships between VAP-1 and tumors have beenreported In human skin melanoma VAP-1 protein expres-sion was significantly decreased in intratumoral vessels [67]It has been demonstrated that the 5-year survival of melano-ma patients with low VAP-1 protein expression in intratu-moral blood vessels was lower than that of those patientswith high VAP-1 expression [67] Strong expression of VAP-1 on tumor endothelium could distinguish human hepato-cellular carcinoma from colorectal hepatic metastases [68]Furthermore some studies indicate that patients with lowsVAP-1 levels have significantly worse prognosis of colorectalcancer and that sVAP-1 is an independent marker of hepaticand lymph node metastasis in these patients [69] A similarcorrelationwith low sVAP-1 and poor prognosis was reportedin gastric cancer [70]

Lately Fukuhara et al examined the immunolocalizationof VAP-1 in pyogenic granuloma and extranodal marginalzone B-cell lymphoma (EMZL) as common human con-junctival tumors They showed strong expression of VAP-1protein in intratumoral blood vessels of pyogenic granulomaa benign inflammatory conjunctival tumor and relativelylower expression in EMZL a malignant inflammatory tumor[71] Moreover the microvessel density was high in pyogenicgranuloma compared to that in EMZL [71]Their data suggestthat VAP-1 plays an important role in the pathogenesis anddevelopment of conjunctival inflammatory diseases such aspyogenic granulomas whereas the relatively lower expressionof VAP-1 in intratumoral microvessels might be correlatedwith the progression of conjunctival lymphoma

Furthermore VAP-1 is involved in angiogenesis andtumor growth via controlling the migration of Gr-1+CD11b+myeloid cells which comprise immature macrophages anddendritic cells playing a pivotal role in tumor angiogenesis[72] VAP-1 may support tumor progression VAP-1 deficientmice melanoma and lymphoma tumors grew more slowlythan in wild-type animals [72] The tumors in VAP-1minusminushost had defective angiogenesis and impaired recruitment ofmyeloid-derived suppressor cells (MDSCs) Notably if theMDSCs were ablated from the mice VAP-1 deficiency nolonger protected the animals Moreover genetic experimentswith transgenic mice expressing an enzymatically inactivemutant of VAP-1 showed that the effects onMDSC accumula-tion were dependent on the oxidase activity of VAP-1 There-fore VAP-1 enhances local malignant lymphoma growth byincreasing the recruitment of myeloid leukocytes into thetumors These data suggest that VAP-1 contributes to thedevelopment of conjunctival EMZL Since tumor cells utilizethe catalytic activity of VAP-1 to recruit myeloid cells intotumors and to support tumor progression small-moleculeVAP-1 inhibitors could be an effective immunotherapy forthe inhibition of tumor progression [73] Currently Salmiand Jalkanen [74] hypothesize that the VAP-1 express-ing in neoangiogenic vessels of the tumor bind MDSC As

a consequence the intratumoral numbers of this particularprotumorigenic leukocyte subtype are selectively increasedwith a concomitant stimulation of the neoangiogenesis andenhancement of the immunosuppressing gene signature ofthe tumor microenvironment In conclusions VAP-1 may bean alternative therapeutic target in ocular tumors (Table 1)

6 The Role of VAP-1 in Molecular Imaging

The special structure of the eye provides a unique oppor-tunity for noninvasive light-based imaging of fundus vas-culature Using adhesion-molecule-conjugated fluorescentmicrospheres (MSs) in live animals researchers showed earlyendothelial changes in ocular microvessels at an early stage[75] which were previously detectable only by the most sen-sitive in vitro techniques such as immunohistochemistry orPCR This novel method also allows evaluation of leukocyte-endothelial interaction in the retinal and choroidal capillariesflow or identification of specific molecular changes duringdisease Molecular imaging is defined as the ability to visual-ize and quantitatively measure the function of biological andcellular processes in vivo [76 77] In vivo molecular imaginghas a great potential to impact medicine by detecting diseasesor screening diseases in early stages identifying extent ofdisease selecting disease- and patient-specific therapeutictreatment applying a directed or targeted therapy and mea-suring molecular-specific effects of treatment Inflammationand tracing of inflammatory cells have been a key topic inmolecular imaging in recent years An ideal target for invivo imaging of inflammation would be a molecule that isnormally absent from the endothelium of healthy tissues butis induced at the onset of inflammation

According to our previous summarization VAP-1 may besuitable as an imaging target in the diagnosis and treatmentof ocular inflammatory diseases A recent paper using thetechnique of in vivo molecular imaging showed that VAP-1was expressed in the resting and angiogenic corneal bloodvessel endothelial cells but not in lymphatic vessels [78]Moreover the study demonstrated a higher VAP-1 expressionin angiogenic than normal blood vessels which revealed thekey role of VAP-1 in angiogenesis-related diseases [78] Inthe study IL-1ndashinducedM2macrophage infiltration as well aslymph-and angiogenesis were blocked by VAP-1 inhibitionwhereas VEGF-A-induced lymph- and angiogenesis wereunaffected by VAP-1 inhibition [78] These results indicatea critical role for VAP-1 in lymph- and angiogenesis-relatedmacrophage recruitment To sum up VAP-1 might becomea new target for the treatment of inflammatory lymph- andangiogenic diseases including cancer

The proof of concept regarding the use of VAP-1 as animaging target was also obtained with iodinated monoclonalantibodies against VAP-1 They were used to detect skinand joint inflammation in the pig [79] Currently VAP-1was investigated as a potential target for in vivo imagingof inflammation by means of PET [80] Panning of phagedisplay libraries with recombinant VAP-1 has led to theidentification of the first cellular counter-receptors of VAP-1 These experiments showed that VAP-1 binds to Siglec-9 and Siglec-10 proteins both in cell free protein-protein


interaction assays and in different cell-based models [80ndash83]Siglecs belong to a family of lectin molecules which bindto sialic acids and mediate various adhesive and signalingevents both within the immune system and elsewhere inthe body [84] The cellular distributions of Siglec-9 and -10are very different Siglec-9 is expressed on all granulocyteswhereas Siglec-10 is present mainly on B-cells Based onmolecular modeling it is plausible that both Siglecs canpresent specific arginine residues into the enzymatic cavityof VAP-1 Although the side chain of arginine terminates ina complex guanidinium structure rather than in normal pri-mary amine the arginine 293 of Siglec-10 has been experi-mentally demonstrated to function as a substrate of VAP-1[81] Thus these molecules can apparently serve as surface-bound substrates of VAP-1 Siglec-VAP-1 interaction can beutilized for the imaging of inflammation and cancer in vivo[82] Short synthetic Siglec-9 peptides (containing the VAP-1 interacting core sequence) localize selectively to sites ofinflammation in vivo in VAP-1 expressing transgenic micebut not in VAP-1 deficient mice From the clinical point ofview a VAP-1-specific imaging agent could be valuable for thedetection of infectioninflammation during its early stagesAs a diagnostic tool the method could differentiate betweeninflammation and cancerous growth or bacterial infectionfrom sterile inflammation [85]

7 Conclusions and Future Perspectives

Aberrant leukocyte trafficking to sites of inflammation isoften harmful leading to tissue damageTherefore moleculesresponsible for the harmful traffic are theoretically excellenttargets to prevent inflammations VAP-1 acts via direct inter-actions with its counter-receptors and more importantlyexerts its effects via the end-products of its enzymaticactivity The inhibitors of VAP-1 may be anti-inflammatoryand antiangiogenic agents to decrease the inflammation inophthalmological diseases The end-products of VAP-1 areproinflammatory so they would be beneficial to suppressVAP-1 and alleviate inflammatory reactions In comparisonto other trafficking-associated molecules VAP-1 providespharmaceutical industry with unique targets for the design ofnovel molecule-targeted therapies of inflammatory diseasesMoreover VAP-1 may be an alternative therapeutic target intumors The in vivo imaging of inflammation using VAP-1 asa targetmolecule is a novel approachwith a potential for earlydetection and characterization of inflammatory diseases andhas obvious clinical significance Based on the properties andresults obtained so far from preclinical and clinical studiesVAP-1 may provide a novel research direction or a potenttherapeutic strategy for ophthalmological diseases includinginflammatory lymph- and angiogenic diseases includingcancer

Acknowledgments

Support by National Natural Science Foundation of ChinaGrant 81171381 Heilongjiang Science Grant LC2011C27 andMinistry of Education fund 20112307120019 was granted toD Sun

References

[1] M Salmi and S Jalkanen ldquoA 90-kilodalton endothelial cellmolecule mediating lymphocyte binding in humansrdquo Sciencevol 257 no 5075 pp 1407ndash1409 1992

[2] T T Airenne Y Nymalm H Kidron et al ldquoCrystal structureof the human vascular adhesion protein-1 unique structuralfeatures with functional implicationsrdquo Protein Science vol 14no 8 pp 1964ndash1974 2005

[3] K Ernberg A P McGrath T S Peat et al ldquoA new crystal formof human vascular adhesion protein 1rdquo Acta CrystallographicaF vol 66 part 12 pp 1572ndash1578 2010

[4] M Salmi K Kalimo and S Jalkanen ldquoInduction and functionof vascular adhesion protein-1 at sites of inflammationrdquo Journalof Experimental Medicine vol 178 no 6 pp 2255ndash2260 1993

[5] K Koskinen P J Vainio D J Smith et al ldquoGranulocytetransmigration through the endothelium is regulated by theoxidase activity of vascular adhesion protein-1 (VAP-1)rdquo Bloodvol 103 no 9 pp 3388ndash3395 2004

[6] E Akin J Aversa and A C Steere ldquoExpression of adhesionmolecules in synovia of patients with treatment-resistant lymearthritisrdquo Infection and Immunity vol 69 no 3 pp 1774ndash17802001

[7] K Jaakkola S Jalkanen K Kaunismaki et al ldquoVascular adhe-sion protein-1 intercellular adhesion molecule-1 and P-selectinmediate leukocyte binding to ischemic heart in humansrdquoJournal of the American College of Cardiology vol 36 no 1 pp122ndash129 2000

[8] B Singh T Tschernig M van Griensven A Fieguth and RPabst ldquoExpression of vascular adhesion protein-1 in normaland inflamed mice lungs and normal human lungsrdquo VirchowsArchiv vol 442 no 5 pp 491ndash495 2003

[9] L Almulki K Noda S Nakao T Hisatomi K L Thomasand A Hafezi-Moghadam ldquoLocalization of vascular adhesionprotein-1 (VAP-1) in the human eyerdquoExperimental Eye Researchvol 90 no 1 pp 26ndash32 2010

[10] K Noda S Nakao S Zandi V Engelstadter Y Mashima andA Hafezi-Moghadam ldquoVascular adhesion protein-1 regulatesleukocyte transmigration rate in the retina during diabetesrdquoExperimental Eye Research vol 89 no 5 pp 774ndash781 2009

[11] K Noda SMiyahara T Nakazawa et al ldquoInhibition of vascularadhesion protein-1 suppresses endotoxin-induced uveitisrdquo TheFASEB Journal vol 22 no 4 pp 1094ndash1103 2008

[12] KNodaH She T Nakazawa et al ldquoVascular adhesion protein-1 blockade suppresses choroidal neovascularizationrdquoTheFASEBJournal vol 22 no 8 pp 2928ndash2935 2008

[13] M Salmi P Rajala and S Jalkanen ldquoHoming of mucosalleukocytes to joints distinct endothelial ligands in synoviummediate leukocyte-subtype specific adhesionrdquo Journal of Clin-ical Investigation vol 99 no 9 pp 2165ndash2172 1997

[14] P H Yu SWright E H Fan Z Lun and D Gubisne-HarberleldquoPhysiological and pathological implications of semicarbazide-sensitive amine oxidaserdquo Biochimica et Biophysica Acta vol1647 no 1-2 pp 193ndash199 2003

[15] H Izuta N Matsunaga M Shimazawa T Sugiyama T Ikedaand H Hara ldquoProliferative diabetic retinopathy and relationsamong antioxidant activity oxidative stress and VEGF in thevitreous bodyrdquoMolecular Vision vol 16 pp 130ndash136 2010

[16] A Belkhiri C Richards M Whaley S A McQueen andF W Orr ldquoIncreased expression of activated matrix metallo-proteinase-2 by human endothelial cells after sublethal H

2O2


exposurerdquo Laboratory Investigation vol 77 no 5 pp 533ndash5391997

[17] M C A Duyndam T M Hulscher D Fontijn H M Pinedoand E Boven ldquoInduction of vascular endothelial growth factorexpression and hypoxia-inducible factor 1120572 protein by theoxidative stressor arseniterdquo Journal of Biological Chemistry vol276 no 51 pp 48066ndash48076 2001

[18] A Rothova T T J M Berendschot K Probst B van Kooijand G S Baarsma ldquoBirdshot chorioretinopathy long-termmanifestations and visual prognosisrdquo Ophthalmology vol 111no 5 pp 954ndash959 2004

[19] O M Durrani N N Tehrani J E Marr P Moradi P Stavrouand P I Murray ldquoDegree duration and causes of visual lossin uveitisrdquo British Journal of Ophthalmology vol 88 no 9 pp1159ndash1162 2004

[20] N Vidovic-Valentincic A Kraut M Hawlina S Stunf andA Rothova ldquoIntermediate uveitis long-term course and visualoutcomerdquo British Journal of Ophthalmology vol 93 no 4 pp477ndash480 2009

[21] T I Tugal S Onal Y R Altan H H Altunbas and MUrgancioglu ldquoUveitis in Behcet disease an analysis of 880patientsrdquoAmerican Journal of Ophthalmology vol 138 no 3 pp373ndash380 2004

[22] P Bhattacherjee ldquoProstaglandins and inflammatory reactionsin the eyerdquo Methods and Findings in Experimental and ClinicalPharmacology vol 2 no 1 pp 17ndash31 1980

[23] A F de Vos M A C van Haren C Verhagen R Hoekzemaand A Kijlstra ldquoKinetics of intraocular tumor necrosis factorand interleukin-6 in endotoxin-induced uveitis in the ratrdquo In-vestigative Ophthalmology and Visual Science vol 35 no 3 pp1100ndash1106 1994

[24] A Okumura M Mochizuki M Nishi and C P HerbortldquoEndotoxin-induced uveitis (EIU) in the rat a study of inflam-matory and immunological mechanismsrdquo International Oph-thalmology vol 14 no 1 pp 31ndash36 1990

[25] C C Chan R R Caspi M Ni et al ldquoPathology of experimentalautoimmune uveoretinitis in micerdquo Journal of Autoimmunityvol 3 no 3 pp 247ndash255 1990

[26] H R Jiang L Lumsden and J V Forrester ldquoMacrophages anddendritic cells in IRBP-induced experimental autoimmune uve-oretinitis in B10RIII micerdquo Investigative Ophthalmology andVisual Science vol 40 no 13 pp 3177ndash3185 1999

[27] L AtallaM Linker-Israeli L Steinman andN A Rao ldquoInhibi-tion of autoimmune uveitis by anti-CD4 antibodyrdquo InvestigativeOphthalmology and Visual Science vol 31 no 7 pp 1264ndash12701990

[28] R R Caspi C CChan Y Fujino et al ldquoRecruitment of antigen-nonspecific cells plays a pivotal role in the pathogenesis of a Tcell-mediated organ-specific autoimmune disease experimen-tal autoimmune uveoretinitisrdquo Journal of Neuroimmunologyvol 47 no 2 pp 177ndash188 1993

[29] AKAbbas J Lohr andBKnoechel ldquoBalancing autoaggressiveand protective T cell responsesrdquo Journal of Autoimmunity vol28 no 2-3 pp 59ndash61 2007

[30] E S Gragoudas A P Adamis E T Cunningham et al ldquoPegap-tanib for neovascular age-related macular degenerationrdquo TheNewEngland Journal ofMedicine vol 351 no 27 pp 2805ndash28162004

[31] D H Anderson R F Mullins G S Hageman and L VJohnson ldquoA role for local inflammation in the formation ofdrusen in the aging eyerdquo American Journal of Ophthalmologyvol 134 no 3 pp 411ndash431 2002

[32] L A Donoso D Kim A Frost A Callahan and G HagemanldquoThe role of inflammation in the pathogenesis of age-relatedmacular degenerationrdquo Survey of Ophthalmology vol 51 no 2pp 137ndash152 2006

[33] K Dastgheib and W R Green ldquoGranulomatous reactionto Bruchrsquos membrane in age-related macular degenerationrdquoArchives of Ophthalmology vol 112 no 6 pp 813ndash818 1994

[34] M C Killingsworth J P Sarks and S H Sarks ldquoMacrophagesrelated to Bruchrsquos membrane in age-related macular degenera-tionrdquo Eye vol 4 part 4 pp 613ndash621 1990

[35] P L Penfold M C Killingsworth and S H Sarks ldquoSenile mac-ular degeneration the involvement of immunocompetent cellsrdquoGraefersquos Archive for Clinical and Experimental Ophthalmologyvol 223 no 2 pp 69ndash76 1985

[36] M A Zarbin ldquoCurrent concepts in the pathogenesis of age-related macular degenerationrdquo Archives of Ophthalmology vol122 no 4 pp 598ndash614 2004

[37] H E Grossniklaus P H Miskala W R Green et al ldquoHis-topathologic and ultrastructural features of surgically excisedsubfoveal choroidal neovascular lesions submacular surgerytrials report no 7rdquo Archives of Ophthalmology vol 123 no 7pp 914ndash921 2005

[38] A K Hutchinson H E Grossniklaus and A Z CaponeldquoGiant-cell reaction in surgically excised subretinal neovascularmembranerdquo Archives of Ophthalmology vol 111 no 6 pp 734ndash735 1993

[39] S Seregard P V Algvere and L Berglin ldquoImmunohistochemi-cal characterization of surgically removed subfoveal fibrovascu-lar membranesrdquo Graefersquos Archive for Clinical and ExperimentalOphthalmology vol 232 no 6 pp 325ndash329 1994

[40] D G Espinosa-Heidmann I J Suner E P Hernandez DMon-roy K G Csaky and S W Cousins ldquoMacrophage depletiondiminishes lesion size and severity in experimental choroidalneovascularizationrdquo Investigative Ophthalmology and VisualScience vol 44 no 8 pp 3586ndash3592 2003

[41] E Sakurai A Anand B K Ambati N van Rooijen and JAmbati ldquoMacrophage depletion inhibits experimental choroid-al neovascularizationrdquo Investigative Ophthalmology and VisualScience vol 44 no 8 pp 3578ndash3585 2003

[42] C Tsutsumi K H Sonoda K Egashira et al ldquoThe criticalrole of ocular-infiltrating macrophages in the development ofchoroidal neovascularizationrdquo Journal of Leukocyte Biology vol74 no 1 pp 25ndash32 2003

[43] H E Grossniklaus J X Ling T MWallace et al ldquoMacrophageand retinal pigment epithelium expression of angiogeniccytokines in choroidal neovascularizationrdquo Molecular Visionvol 8 pp 119ndash126 2002

[44] H Oh H Takagi C Takagi et al ldquoThe potential angiogenicrole of macrophages in the formation of choroidal neovascularmembranesrdquo Investigative Ophthalmology and Visual Sciencevol 40 no 9 pp 1891ndash1898 1999

[45] N N Markomichelakis P G Theodossiadis and P P SfikakisldquoRegression of neovascular age-related macular degenerationfollowing infliximab therapyrdquo American Journal of Ophthalmol-ogy vol 139 no 3 pp 537ndash540 2005

[46] X Shi I Semkova P S Muther S Della N Kocioka and AM Joussena ldquoInhibition of TNF-alpha reduces laser-inducedchoroidal neovascularizationrdquo Experimental Eye Research vol83 no 6 pp 1325ndash1334 2006

[47] R F Mullins S R Russell D H Anderson et al ldquoDrusenassociated with aging and age-related macular degeneration


contain proteins common to extracellular deposits associatedwith atherosclerosis elastosis amyloidosis and dense depositdiseaserdquo Journal of the Federation of American Societies forExperimental Biology vol 14 no 7 pp 835ndash846 2000

[48] L V Johnson W P Leitner M K Staples and D H AndersonldquoComplement activation and inflammatory processes in drusenformation and age related macular degenerationrdquo ExperimentalEye Research vol 73 no 6 pp 887ndash896 2001

[49] M Nozaki B J Raisler E Sakurai et al ldquoDrusen complementcomponents C3a and C5a promote choroidal neovasculariza-tionrdquo Proceedings of the National Academy of Sciences of theUnited States of America vol 103 no 7 pp 2328ndash2333 2006

[50] N Yoshikawa K Noda Y Ozawa K Tsubota Y Mashima andS Ishida ldquoBlockade of vascular adhesion protein-1 attenuateschoroidal neovascularizationrdquo Molecular Vision vol 18 pp593ndash600 2012

[51] N Nagai Y Oike K Izumi-Nagai et al ldquoAngiotensin II type 1receptor-mediated inflammation is required for choroidal neo-vascularizationrdquoArteriosclerosisThrombosis andVascular Biol-ogy vol 26 no 10 pp 2252ndash2259 2006

[52] T Koto N Nagai H Mochimaru et al ldquoEicosapentaenoic acidis anti-inflammatory in preventing choroidal neovasculariza-tion in micerdquo Investigative Ophthalmology and Visual Sciencevol 48 no 9 pp 4328ndash4334 2007

[53] T C Moore J E Moore Y Kaji et al ldquoThe role of advancedglycation end products in retinal microvascular leukostasisrdquoInvestigative Ophthalmology and Visual Science vol 44 no 10pp 4457ndash4464 2003

[54] M J Sheetz and G L King ldquoMolecular understanding of hy-perglycemiarsquos adverse effects for diabetic complicationsrdquo Jama-Journal of the AmericanMedical Association vol 288 no 20 pp2579ndash2588 2002

[55] D A Antonetti A J Barber and S K Bronsonetal ldquoDiabeticretinopathy seeing beyond glucose-inducedmicrovascular dis-easerdquo Diabetes vol 55 no 9 pp 2401ndash2411 2006

[56] T W Gardner D A Antonetti A J Barber K F LaNoue andS W Levison ldquoDiabetic retinopathy more than meets the eyerdquoSurvey of Ophthalmology vol 47 supplement 2 pp s253ndashs2622002

[57] R TadayoniM Paques AGaudric andEVicaut ldquoErythrocyteand leukocyte dynamics in the retinal capillaries of diabeticmicerdquo Experimental Eye Research vol 77 no 4 pp 497ndash5042003

[58] A M Joussen V Poulaki M L Le et al ldquoA central role forinflammation in the pathogenesis of diabetic retinopathyrdquo TheFASEB Journal vol 18 no 12 pp 1450ndash1452 2004

[59] H Tamura K Miyamoto J Kiryu et al ldquoIntravitreal injectionof corticosteroid attenuates leukostasis and vascular leakage inexperimental diabetic retinardquo Investigative Ophthalmology andVisual Science vol 46 no 4 pp 1440ndash1444 2005

[60] R Kurkijarvi D H Adams R Leino T Mottonen S Jalkanenand M Salmi ldquoCirculating form of human vascular adhesionprotein-1 (VAP-1) increased serum levels in inflammatory liverdiseasesrdquo Journal of Immunology vol 161 no 3 pp 1549ndash15571998

[61] F Boomsma A H van den Meiracker S Winkel et al ldquoCircu-lating semicarbazide-sensitive amine oxidase is raised both intype I (insulin-dependent) in type II (non-insulin-dependent)diabetes mellitus and even in childhood type I diabetes at firstclinical diagnosisrdquoDiabetologia vol 42 no 2 pp 233ndash237 1999

[62] H Garpenstrand J Ekblom L B Backlund L Oreland andU Rosenqvist ldquoElevated plasma semicarbazide-sensitive amine

oxidase (SSAO) activity in type 2 diabetes mellitus complicatedby retinopathyrdquo Diabetic Medicine vol 16 no 6 pp 514ndash5211999

[63] S TohkaM L Laukkanen S Jalkanen andM Salmi ldquoVascularadhesion protein 1 (VAP-1) functions as a molecular brakeduring granulocyte rolling and mediates recruitment in vivordquoThe FASEB Journal vol 15 no 2 pp 373ndash382 2001

[64] M Murata K Noda J Fukuhara et al ldquoSoluble vascular adhe-sion protein-1 accumulates in proliferative diabetic retinopa-thyrdquo Investigative Ophthalmology and Visual Science vol 53 no7 pp 4055ndash4062 2012

[65] T Itoh M Tanioka H Yoshida et al ldquoReduced angiogenesisand tumor progression in gelatinase A-deficient micerdquo CancerResearch vol 58 no 5 pp 1048ndash1051 1998

[66] T H Vu J M Shipley G Bergers et al ldquoMMP-9gelatinase Bis a key regulator of growth plate angiogenesis and apoptosis ofhypertrophic chondrocytesrdquo Cell vol 93 pp 411ndash422 1998

[67] C Forster-Horvath B Dome S Paku et al ldquoLoss of vascularadhesion protein-1 expression in intratumoral microvessels ofhuman skin melanomardquo Melanoma Research vol 14 no 2 pp135ndash140 2004

[68] K F Yoong G McNab S G Hubscher and D H AdamsldquoVascular adhesion protein-1 and ICAM-1 support the adhesionof tumor- infiltrating lymphocytes to tumor endothelium inhuman hepatocellular carcinomardquo Journal of Immunology vol160 no 8 pp 3978ndash3988 1998

[69] O Kemik A Sumer A S Kemik et al ldquoHuman vascular adhe-sion proteidotlessn-1 (VAP-1) serum levels for hepatocellularcarcinoma in non-alcoholic and alcoholic fatty liver diseaserdquoWorld Journal of Surgical Oncology vol 8 article 83 2010

[70] H Yasuda Y Toiyama M Ohi Y Mohri C Miki and MKusunoki ldquoSerum soluble vascular adhesion protein-1 is avaluable prognosticmarker in gastric cancerrdquo Journal of SurgicalOncology vol 103 no 7 pp 695ndash699 2011

[71] J Fukuhara S Kase K Noda et al ldquoImmunolocalization ofvascular adhesion protein-1 in human conjunctival tumorsrdquoOphthalmic Research vol 48 no 1 pp 33ndash37 2012

[72] F Marttila-Ichihara K Auvinen K Elima S Jalkanen and MSalmi ldquoVascular adhesion protein-1 enhances tumor growthby supporting recruitment of Gr-1+CD11b+ myeloid cells intotumorsrdquo Cancer Research vol 69 no 19 pp 7875ndash7883 2009

[73] F Marttila-Ichihara K Castermans K Auvinen et al ldquoSmall-molecule inhibitors of vascular adhesion protein-1 reduce theaccumulation of myeloid cells into tumors and attenuate tumorgrowth in micerdquo Journal of Immunology vol 184 no 6 pp3164ndash3173 2010

[74] M Salmi and S Jalkanen ldquoHoming-associatedmolecules CD73and VAP-1 as targets to prevent harmful inflammations andcancer spreadrdquo FEBS Letters vol 585 no 11 pp 1543ndash1550 2011

[75] D Sun S Nakao F Xie S Zandi A Schering and A Hafezi-Moghadam ldquoSuperior sensitivity of novel molecular imagingprobe simultaneously targeting two types of endothelial injurymarkersrdquoThe FASEB Journal vol 24 no 5 pp 1532ndash1540 2010

[76] F Xie D Sun A Schering et al ldquoNovel molecular imagingapproach for subclinical detection of iritis and evaluation oftherapeutic successrdquoAmerican Journal of Pathology vol 177 no1 pp 39ndash48 2010

[77] R C Garland D Sun S Zandi et al ldquoNoninvasive molecularimaging reveals role of PAF in leukocyte-endothelial interactionin LPS-induced ocular vascular injuryrdquoThe FASEB Journal vol25 no 4 pp 1284ndash1294 2011


[78] S Nakao K Noda S Zandi et al ldquoVAP-1-mediated M2macro-phage infiltration underlies IL-1120573- but not VEGF-A-inducedlymph- and angiogenesisrdquo American Journal of Pathology vol178 no 4 pp 1913ndash1921 2011

[79] K Jaakkola T Nikula R Holopainen et al ldquoIn vivo detectionof vascular adhesion protein-1 in experimental inflammationrdquoAmerican Journal of Pathology vol 157 no 2 pp 463ndash471 2000

[80] T Ujula S Salomaki P Virsu et al ldquoSynthesis 68Ga labelingand preliminary evaluation of DOTA peptide binding vascularadhesion protein-1 a potential PET imaging agent for diagnos-ing osteomyelitisrdquo Nuclear Medicine and Biology vol 36 no 6pp 631ndash641 2009

[81] E Kivi K Elima K Aalto et al ldquoHuman Siglec-10 can bind tovascular adhesion protein-1 and serves as its substraterdquo Bloodvol 114 no 26 pp 5385ndash5392 2009

[82] K Aalto A Autio E A Kiss et al ldquoSiglec-9 is a novel leukocyteligand for vascular adhesion protein-1 and can be used in PETimaging of inflammation and cancerrdquo Blood vol 118 no 13 pp3725ndash3733 2011

[83] A Autio T Henttinen H J Sipila S Jalkanen and ARoivainen ldquoMini-PEG spacering of VAP-1-targeting 68Ga-DOTAVAP-P1 peptide improves PET imaging of inflamma-tionrdquo EJNMMI Research vol 1 no 1 p 10 2011

[84] P R Crocker J C Paulson andA Varki ldquoSiglecs and their rolesin the immune systemrdquo Nature Reviews Immunology vol 7 no4 pp 255ndash266 2007

[85] A Roivainen S Jalkanen andCNanni ldquoGallium-labelled pep-tides for imaging of inflammationrdquo European Journal of NuclearMedicine andMolecular Imaging vol 39 supplement 1 pp s68ndashs77 2012







Editorial Board




Contents





































1 Introduction























Normal (119899 = 55) 28 27

Pre-AMD (119899 = 13) MD1 = 4 MD1 = 4MD2 = 3 MD2 = 2

AMD (119899 = 47)



Normal (119899 = 96) 48 48

Pre-AMD (119899 = 21) MD1 = 6 MD1 = 5MD2 = 4 MD2 = 4

AMD (119899 = 60)




3 Results

























MD1

MD2

Dry AMD

CNVGAGACNV


larr 1 synp


larr 1 synp

larr 2 synp


larr 0 synp

larr 2 synp

larr 2 synp

larr 0 synp

larr 0 synp

larr 14 synp

larr 0 synp

larr 0 synp












1 synp rarr

3 synp rarr

larr 3 synp

larr 0 synp


larr 0 synp

larr 0 synp


MD1

MD2

Dry AMD

CNVGAAMD

larr 354 synp



4 Discussion













References






































1 Introduction



2plays an




















2




2humidified





2(5 ngmL)
















3 Results









Hoechst

Hoechst

Vimentin

Cytokeratin Merge

Merge



(a)


200

0

Cou

nts

Data001

M1M2

FL1-H10

010

110

210

310

4

200

0

Cou

nts

Data002

M1M2

FL1-H10

010

110

210

310

4

(b)









0

5

10

15

20

25

30

35

Relat

ive C

TGF

mRN

A ex

pres

sion

lowast lowastlowast

lowastlowast

lowastlowast

Con

trol

TGF-1205732(+)

CTG

F-siR

NA

1+

TGF-1205732(+)

CTG

F-siR

NA

2+

TGF-1205732(+)

CTG

F-siR

NA

3+

TGF-1205732(+)

CTG

F-siR

NA

4+

TGF-1205732(+)

(a)

0

5

10

15

20

25

30

35

40


Relat

ive C

TGF

mRN

A ex

pres

sion

TGF-1205732(minus)TGF-1205732(+)

lowast

(b)






2in control group



2(Figure 3(b))


2 a distin-


2stimulated cells




2treated cells

















2(+)




2



Hoechst CTGF Merge

Control

CTGF-siRNA

Control-siRNA

+TGF-1205732(+)

+ TGF-1205732(+)

+ TGF-1205732(+)




005)

4 Discussion






2


2with monoclonal




Control

00

02

04

06

08

10

12

14

16

18

0 24 48 72 96

OD

val

ue

lowastlowast

lowastlowastlowast

lowastlowast

+TGF-1205732(+)





2for 0


2stimulated





2coupled with CTGF


2


2could



000

200

400

600

800

1000

1200

1400

Con

trol

PI (

)

CTG

F-siR

NA+

Con

trol-s

iRN

A+

TGF-1205732(+)

TGF-1205732(+)

TGF-1205732(+)

lowastlowast

lowast







5 Conclusions




Acknowledgment



References




















































1 Introduction











2 Methods



















3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2

















































































Editorial Board




Contents





































1 Introduction























Normal (119899 = 55) 28 27

Pre-AMD (119899 = 13) MD1 = 4 MD1 = 4MD2 = 3 MD2 = 2

AMD (119899 = 47)



Normal (119899 = 96) 48 48

Pre-AMD (119899 = 21) MD1 = 6 MD1 = 5MD2 = 4 MD2 = 4

AMD (119899 = 60)




3 Results

























MD1

MD2

Dry AMD

CNVGAGACNV


larr 1 synp


larr 1 synp

larr 2 synp


larr 0 synp

larr 2 synp

larr 2 synp

larr 0 synp

larr 0 synp

larr 14 synp

larr 0 synp

larr 0 synp












1 synp rarr

3 synp rarr

larr 3 synp

larr 0 synp


larr 0 synp

larr 0 synp


MD1

MD2

Dry AMD

CNVGAAMD

larr 354 synp



4 Discussion













References






































1 Introduction



2plays an




















2




2humidified





2(5 ngmL)
















3 Results









Hoechst

Hoechst

Vimentin

Cytokeratin Merge

Merge



(a)


200

0

Cou

nts

Data001

M1M2

FL1-H10

010

110

210

310

4

200

0

Cou

nts

Data002

M1M2

FL1-H10

010

110

210

310

4

(b)









0

5

10

15

20

25

30

35

Relat

ive C

TGF

mRN

A ex

pres

sion

lowast lowastlowast

lowastlowast

lowastlowast

Con

trol

TGF-1205732(+)

CTG

F-siR

NA

1+

TGF-1205732(+)

CTG

F-siR

NA

2+

TGF-1205732(+)

CTG

F-siR

NA

3+

TGF-1205732(+)

CTG

F-siR

NA

4+

TGF-1205732(+)

(a)

0

5

10

15

20

25

30

35

40


Relat

ive C

TGF

mRN

A ex

pres

sion

TGF-1205732(minus)TGF-1205732(+)

lowast

(b)






2in control group



2(Figure 3(b))


2 a distin-


2stimulated cells




2treated cells

















2(+)




2



Hoechst CTGF Merge

Control

CTGF-siRNA

Control-siRNA

+TGF-1205732(+)

+ TGF-1205732(+)

+ TGF-1205732(+)




005)

4 Discussion






2


2with monoclonal




Control

00

02

04

06

08

10

12

14

16

18

0 24 48 72 96

OD

val

ue

lowastlowast

lowastlowastlowast

lowastlowast

+TGF-1205732(+)





2for 0


2stimulated





2coupled with CTGF


2


2could



000

200

400

600

800

1000

1200

1400

Con

trol

PI (

)

CTG

F-siR

NA+

Con

trol-s

iRN

A+

TGF-1205732(+)

TGF-1205732(+)

TGF-1205732(+)

lowastlowast

lowast







5 Conclusions




Acknowledgment



References




















































1 Introduction











2 Methods



















3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2














































































Editorial Board




Contents





































1 Introduction























Normal (119899 = 55) 28 27

Pre-AMD (119899 = 13) MD1 = 4 MD1 = 4MD2 = 3 MD2 = 2

AMD (119899 = 47)



Normal (119899 = 96) 48 48

Pre-AMD (119899 = 21) MD1 = 6 MD1 = 5MD2 = 4 MD2 = 4

AMD (119899 = 60)




3 Results

























MD1

MD2

Dry AMD

CNVGAGACNV


larr 1 synp


larr 1 synp

larr 2 synp


larr 0 synp

larr 2 synp

larr 2 synp

larr 0 synp

larr 0 synp

larr 14 synp

larr 0 synp

larr 0 synp












1 synp rarr

3 synp rarr

larr 3 synp

larr 0 synp


larr 0 synp

larr 0 synp


MD1

MD2

Dry AMD

CNVGAAMD

larr 354 synp



4 Discussion













References






































1 Introduction



2plays an




















2




2humidified





2(5 ngmL)
















3 Results









Hoechst

Hoechst

Vimentin

Cytokeratin Merge

Merge



(a)


200

0

Cou

nts

Data001

M1M2

FL1-H10

010

110

210

310

4

200

0

Cou

nts

Data002

M1M2

FL1-H10

010

110

210

310

4

(b)









0

5

10

15

20

25

30

35

Relat

ive C

TGF

mRN

A ex

pres

sion

lowast lowastlowast

lowastlowast

lowastlowast

Con

trol

TGF-1205732(+)

CTG

F-siR

NA

1+

TGF-1205732(+)

CTG

F-siR

NA

2+

TGF-1205732(+)

CTG

F-siR

NA

3+

TGF-1205732(+)

CTG

F-siR

NA

4+

TGF-1205732(+)

(a)

0

5

10

15

20

25

30

35

40


Relat

ive C

TGF

mRN

A ex

pres

sion

TGF-1205732(minus)TGF-1205732(+)

lowast

(b)






2in control group



2(Figure 3(b))


2 a distin-


2stimulated cells




2treated cells

















2(+)




2



Hoechst CTGF Merge

Control

CTGF-siRNA

Control-siRNA

+TGF-1205732(+)

+ TGF-1205732(+)

+ TGF-1205732(+)




005)

4 Discussion






2


2with monoclonal




Control

00

02

04

06

08

10

12

14

16

18

0 24 48 72 96

OD

val

ue

lowastlowast

lowastlowastlowast

lowastlowast

+TGF-1205732(+)





2for 0


2stimulated





2coupled with CTGF


2


2could



000

200

400

600

800

1000

1200

1400

Con

trol

PI (

)

CTG

F-siR

NA+

Con

trol-s

iRN

A+

TGF-1205732(+)

TGF-1205732(+)

TGF-1205732(+)

lowastlowast

lowast







5 Conclusions




Acknowledgment



References




















































1 Introduction











2 Methods



















3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2












































































Editorial Board




Contents





































1 Introduction























Normal (119899 = 55) 28 27

Pre-AMD (119899 = 13) MD1 = 4 MD1 = 4MD2 = 3 MD2 = 2

AMD (119899 = 47)



Normal (119899 = 96) 48 48

Pre-AMD (119899 = 21) MD1 = 6 MD1 = 5MD2 = 4 MD2 = 4

AMD (119899 = 60)




3 Results

























MD1

MD2

Dry AMD

CNVGAGACNV


larr 1 synp


larr 1 synp

larr 2 synp


larr 0 synp

larr 2 synp

larr 2 synp

larr 0 synp

larr 0 synp

larr 14 synp

larr 0 synp

larr 0 synp












1 synp rarr

3 synp rarr

larr 3 synp

larr 0 synp


larr 0 synp

larr 0 synp


MD1

MD2

Dry AMD

CNVGAAMD

larr 354 synp



4 Discussion













References






































1 Introduction



2plays an




















2




2humidified





2(5 ngmL)
















3 Results









Hoechst

Hoechst

Vimentin

Cytokeratin Merge

Merge



(a)


200

0

Cou

nts

Data001

M1M2

FL1-H10

010

110

210

310

4

200

0

Cou

nts

Data002

M1M2

FL1-H10

010

110

210

310

4

(b)









0

5

10

15

20

25

30

35

Relat

ive C

TGF

mRN

A ex

pres

sion

lowast lowastlowast

lowastlowast

lowastlowast

Con

trol

TGF-1205732(+)

CTG

F-siR

NA

1+

TGF-1205732(+)

CTG

F-siR

NA

2+

TGF-1205732(+)

CTG

F-siR

NA

3+

TGF-1205732(+)

CTG

F-siR

NA

4+

TGF-1205732(+)

(a)

0

5

10

15

20

25

30

35

40


Relat

ive C

TGF

mRN

A ex

pres

sion

TGF-1205732(minus)TGF-1205732(+)

lowast

(b)






2in control group



2(Figure 3(b))


2 a distin-


2stimulated cells




2treated cells

















2(+)




2



Hoechst CTGF Merge

Control

CTGF-siRNA

Control-siRNA

+TGF-1205732(+)

+ TGF-1205732(+)

+ TGF-1205732(+)




005)

4 Discussion






2


2with monoclonal




Control

00

02

04

06

08

10

12

14

16

18

0 24 48 72 96

OD

val

ue

lowastlowast

lowastlowastlowast

lowastlowast

+TGF-1205732(+)





2for 0


2stimulated





2coupled with CTGF


2


2could



000

200

400

600

800

1000

1200

1400

Con

trol

PI (

)

CTG

F-siR

NA+

Con

trol-s

iRN

A+

TGF-1205732(+)

TGF-1205732(+)

TGF-1205732(+)

lowastlowast

lowast







5 Conclusions




Acknowledgment



References




















































1 Introduction











2 Methods



















3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2












































































Contents





































1 Introduction























Normal (119899 = 55) 28 27

Pre-AMD (119899 = 13) MD1 = 4 MD1 = 4MD2 = 3 MD2 = 2

AMD (119899 = 47)



Normal (119899 = 96) 48 48

Pre-AMD (119899 = 21) MD1 = 6 MD1 = 5MD2 = 4 MD2 = 4

AMD (119899 = 60)




3 Results

























MD1

MD2

Dry AMD

CNVGAGACNV


larr 1 synp


larr 1 synp

larr 2 synp


larr 0 synp

larr 2 synp

larr 2 synp

larr 0 synp

larr 0 synp

larr 14 synp

larr 0 synp

larr 0 synp












1 synp rarr

3 synp rarr

larr 3 synp

larr 0 synp


larr 0 synp

larr 0 synp


MD1

MD2

Dry AMD

CNVGAAMD

larr 354 synp



4 Discussion













References






































1 Introduction



2plays an




















2




2humidified





2(5 ngmL)
















3 Results









Hoechst

Hoechst

Vimentin

Cytokeratin Merge

Merge



(a)


200

0

Cou

nts

Data001

M1M2

FL1-H10

010

110

210

310

4

200

0

Cou

nts

Data002

M1M2

FL1-H10

010

110

210

310

4

(b)









0

5

10

15

20

25

30

35

Relat

ive C

TGF

mRN

A ex

pres

sion

lowast lowastlowast

lowastlowast

lowastlowast

Con

trol

TGF-1205732(+)

CTG

F-siR

NA

1+

TGF-1205732(+)

CTG

F-siR

NA

2+

TGF-1205732(+)

CTG

F-siR

NA

3+

TGF-1205732(+)

CTG

F-siR

NA

4+

TGF-1205732(+)

(a)

0

5

10

15

20

25

30

35

40


Relat

ive C

TGF

mRN

A ex

pres

sion

TGF-1205732(minus)TGF-1205732(+)

lowast

(b)






2in control group



2(Figure 3(b))


2 a distin-


2stimulated cells




2treated cells

















2(+)




2



Hoechst CTGF Merge

Control

CTGF-siRNA

Control-siRNA

+TGF-1205732(+)

+ TGF-1205732(+)

+ TGF-1205732(+)




005)

4 Discussion






2


2with monoclonal




Control

00

02

04

06

08

10

12

14

16

18

0 24 48 72 96

OD

val

ue

lowastlowast

lowastlowastlowast

lowastlowast

+TGF-1205732(+)





2for 0


2stimulated





2coupled with CTGF


2


2could



000

200

400

600

800

1000

1200

1400

Con

trol

PI (

)

CTG

F-siR

NA+

Con

trol-s

iRN

A+

TGF-1205732(+)

TGF-1205732(+)

TGF-1205732(+)

lowastlowast

lowast







5 Conclusions




Acknowledgment



References




















































1 Introduction











2 Methods



















3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2








































































































1 Introduction























Normal (119899 = 55) 28 27

Pre-AMD (119899 = 13) MD1 = 4 MD1 = 4MD2 = 3 MD2 = 2

AMD (119899 = 47)



Normal (119899 = 96) 48 48

Pre-AMD (119899 = 21) MD1 = 6 MD1 = 5MD2 = 4 MD2 = 4

AMD (119899 = 60)




3 Results

























MD1

MD2

Dry AMD

CNVGAGACNV


larr 1 synp


larr 1 synp

larr 2 synp


larr 0 synp

larr 2 synp

larr 2 synp

larr 0 synp

larr 0 synp

larr 14 synp

larr 0 synp

larr 0 synp












1 synp rarr

3 synp rarr

larr 3 synp

larr 0 synp


larr 0 synp

larr 0 synp


MD1

MD2

Dry AMD

CNVGAAMD

larr 354 synp



4 Discussion













References






































1 Introduction



2plays an




















2




2humidified





2(5 ngmL)
















3 Results









Hoechst

Hoechst

Vimentin

Cytokeratin Merge

Merge



(a)


200

0

Cou

nts

Data001

M1M2

FL1-H10

010

110

210

310

4

200

0

Cou

nts

Data002

M1M2

FL1-H10

010

110

210

310

4

(b)









0

5

10

15

20

25

30

35

Relat

ive C

TGF

mRN

A ex

pres

sion

lowast lowastlowast

lowastlowast

lowastlowast

Con

trol

TGF-1205732(+)

CTG

F-siR

NA

1+

TGF-1205732(+)

CTG

F-siR

NA

2+

TGF-1205732(+)

CTG

F-siR

NA

3+

TGF-1205732(+)

CTG

F-siR

NA

4+

TGF-1205732(+)

(a)

0

5

10

15

20

25

30

35

40


Relat

ive C

TGF

mRN

A ex

pres

sion

TGF-1205732(minus)TGF-1205732(+)

lowast

(b)






2in control group



2(Figure 3(b))


2 a distin-


2stimulated cells




2treated cells

















2(+)




2



Hoechst CTGF Merge

Control

CTGF-siRNA

Control-siRNA

+TGF-1205732(+)

+ TGF-1205732(+)

+ TGF-1205732(+)




005)

4 Discussion






2


2with monoclonal




Control

00

02

04

06

08

10

12

14

16

18

0 24 48 72 96

OD

val

ue

lowastlowast

lowastlowastlowast

lowastlowast

+TGF-1205732(+)





2for 0


2stimulated





2coupled with CTGF


2


2could



000

200

400

600

800

1000

1200

1400

Con

trol

PI (

)

CTG

F-siR

NA+

Con

trol-s

iRN

A+

TGF-1205732(+)

TGF-1205732(+)

TGF-1205732(+)

lowastlowast

lowast







5 Conclusions




Acknowledgment



References




















































1 Introduction











2 Methods



















3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2


























































































1 Introduction























Normal (119899 = 55) 28 27

Pre-AMD (119899 = 13) MD1 = 4 MD1 = 4MD2 = 3 MD2 = 2

AMD (119899 = 47)



Normal (119899 = 96) 48 48

Pre-AMD (119899 = 21) MD1 = 6 MD1 = 5MD2 = 4 MD2 = 4

AMD (119899 = 60)




3 Results

























MD1

MD2

Dry AMD

CNVGAGACNV


larr 1 synp


larr 1 synp

larr 2 synp


larr 0 synp

larr 2 synp

larr 2 synp

larr 0 synp

larr 0 synp

larr 14 synp

larr 0 synp

larr 0 synp












1 synp rarr

3 synp rarr

larr 3 synp

larr 0 synp


larr 0 synp

larr 0 synp


MD1

MD2

Dry AMD

CNVGAAMD

larr 354 synp



4 Discussion













References






































1 Introduction



2plays an




















2




2humidified





2(5 ngmL)
















3 Results









Hoechst

Hoechst

Vimentin

Cytokeratin Merge

Merge



(a)


200

0

Cou

nts

Data001

M1M2

FL1-H10

010

110

210

310

4

200

0

Cou

nts

Data002

M1M2

FL1-H10

010

110

210

310

4

(b)









0

5

10

15

20

25

30

35

Relat

ive C

TGF

mRN

A ex

pres

sion

lowast lowastlowast

lowastlowast

lowastlowast

Con

trol

TGF-1205732(+)

CTG

F-siR

NA

1+

TGF-1205732(+)

CTG

F-siR

NA

2+

TGF-1205732(+)

CTG

F-siR

NA

3+

TGF-1205732(+)

CTG

F-siR

NA

4+

TGF-1205732(+)

(a)

0

5

10

15

20

25

30

35

40


Relat

ive C

TGF

mRN

A ex

pres

sion

TGF-1205732(minus)TGF-1205732(+)

lowast

(b)






2in control group



2(Figure 3(b))


2 a distin-


2stimulated cells




2treated cells

















2(+)




2



Hoechst CTGF Merge

Control

CTGF-siRNA

Control-siRNA

+TGF-1205732(+)

+ TGF-1205732(+)

+ TGF-1205732(+)




005)

4 Discussion






2


2with monoclonal




Control

00

02

04

06

08

10

12

14

16

18

0 24 48 72 96

OD

val

ue

lowastlowast

lowastlowastlowast

lowastlowast

+TGF-1205732(+)





2for 0


2stimulated





2coupled with CTGF


2


2could



000

200

400

600

800

1000

1200

1400

Con

trol

PI (

)

CTG

F-siR

NA+

Con

trol-s

iRN

A+

TGF-1205732(+)

TGF-1205732(+)

TGF-1205732(+)

lowastlowast

lowast







5 Conclusions




Acknowledgment



References




















































1 Introduction











2 Methods



















3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2





















































































1 Introduction























Normal (119899 = 55) 28 27

Pre-AMD (119899 = 13) MD1 = 4 MD1 = 4MD2 = 3 MD2 = 2

AMD (119899 = 47)



Normal (119899 = 96) 48 48

Pre-AMD (119899 = 21) MD1 = 6 MD1 = 5MD2 = 4 MD2 = 4

AMD (119899 = 60)




3 Results

























MD1

MD2

Dry AMD

CNVGAGACNV


larr 1 synp


larr 1 synp

larr 2 synp


larr 0 synp

larr 2 synp

larr 2 synp

larr 0 synp

larr 0 synp

larr 14 synp

larr 0 synp

larr 0 synp












1 synp rarr

3 synp rarr

larr 3 synp

larr 0 synp


larr 0 synp

larr 0 synp


MD1

MD2

Dry AMD

CNVGAAMD

larr 354 synp



4 Discussion













References






































1 Introduction



2plays an




















2




2humidified





2(5 ngmL)
















3 Results









Hoechst

Hoechst

Vimentin

Cytokeratin Merge

Merge



(a)


200

0

Cou

nts

Data001

M1M2

FL1-H10

010

110

210

310

4

200

0

Cou

nts

Data002

M1M2

FL1-H10

010

110

210

310

4

(b)









0

5

10

15

20

25

30

35

Relat

ive C

TGF

mRN

A ex

pres

sion

lowast lowastlowast

lowastlowast

lowastlowast

Con

trol

TGF-1205732(+)

CTG

F-siR

NA

1+

TGF-1205732(+)

CTG

F-siR

NA

2+

TGF-1205732(+)

CTG

F-siR

NA

3+

TGF-1205732(+)

CTG

F-siR

NA

4+

TGF-1205732(+)

(a)

0

5

10

15

20

25

30

35

40


Relat

ive C

TGF

mRN

A ex

pres

sion

TGF-1205732(minus)TGF-1205732(+)

lowast

(b)






2in control group



2(Figure 3(b))


2 a distin-


2stimulated cells




2treated cells

















2(+)




2



Hoechst CTGF Merge

Control

CTGF-siRNA

Control-siRNA

+TGF-1205732(+)

+ TGF-1205732(+)

+ TGF-1205732(+)




005)

4 Discussion






2


2with monoclonal




Control

00

02

04

06

08

10

12

14

16

18

0 24 48 72 96

OD

val

ue

lowastlowast

lowastlowastlowast

lowastlowast

+TGF-1205732(+)





2for 0


2stimulated





2coupled with CTGF


2


2could



000

200

400

600

800

1000

1200

1400

Con

trol

PI (

)

CTG

F-siR

NA+

Con

trol-s

iRN

A+

TGF-1205732(+)

TGF-1205732(+)

TGF-1205732(+)

lowastlowast

lowast







5 Conclusions




Acknowledgment



References




















































1 Introduction











2 Methods



















3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2






























































































Normal (119899 = 55) 28 27

Pre-AMD (119899 = 13) MD1 = 4 MD1 = 4MD2 = 3 MD2 = 2

AMD (119899 = 47)



Normal (119899 = 96) 48 48

Pre-AMD (119899 = 21) MD1 = 6 MD1 = 5MD2 = 4 MD2 = 4

AMD (119899 = 60)




3 Results

























MD1

MD2

Dry AMD

CNVGAGACNV


larr 1 synp


larr 1 synp

larr 2 synp


larr 0 synp

larr 2 synp

larr 2 synp

larr 0 synp

larr 0 synp

larr 14 synp

larr 0 synp

larr 0 synp












1 synp rarr

3 synp rarr

larr 3 synp

larr 0 synp


larr 0 synp

larr 0 synp


MD1

MD2

Dry AMD

CNVGAAMD

larr 354 synp



4 Discussion













References






































1 Introduction



2plays an




















2




2humidified





2(5 ngmL)
















3 Results









Hoechst

Hoechst

Vimentin

Cytokeratin Merge

Merge



(a)


200

0

Cou

nts

Data001

M1M2

FL1-H10

010

110

210

310

4

200

0

Cou

nts

Data002

M1M2

FL1-H10

010

110

210

310

4

(b)









0

5

10

15

20

25

30

35

Relat

ive C

TGF

mRN

A ex

pres

sion

lowast lowastlowast

lowastlowast

lowastlowast

Con

trol

TGF-1205732(+)

CTG

F-siR

NA

1+

TGF-1205732(+)

CTG

F-siR

NA

2+

TGF-1205732(+)

CTG

F-siR

NA

3+

TGF-1205732(+)

CTG

F-siR

NA

4+

TGF-1205732(+)

(a)

0

5

10

15

20

25

30

35

40


Relat

ive C

TGF

mRN

A ex

pres

sion

TGF-1205732(minus)TGF-1205732(+)

lowast

(b)






2in control group



2(Figure 3(b))


2 a distin-


2stimulated cells




2treated cells

















2(+)




2



Hoechst CTGF Merge

Control

CTGF-siRNA

Control-siRNA

+TGF-1205732(+)

+ TGF-1205732(+)

+ TGF-1205732(+)




005)

4 Discussion






2


2with monoclonal




Control

00

02

04

06

08

10

12

14

16

18

0 24 48 72 96

OD

val

ue

lowastlowast

lowastlowastlowast

lowastlowast

+TGF-1205732(+)





2for 0


2stimulated





2coupled with CTGF


2


2could



000

200

400

600

800

1000

1200

1400

Con

trol

PI (

)

CTG

F-siR

NA+

Con

trol-s

iRN

A+

TGF-1205732(+)

TGF-1205732(+)

TGF-1205732(+)

lowastlowast

lowast







5 Conclusions




Acknowledgment



References




















































1 Introduction











2 Methods



















3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2



































































































MD1

MD2

Dry AMD

CNVGAGACNV


larr 1 synp


larr 1 synp

larr 2 synp


larr 0 synp

larr 2 synp

larr 2 synp

larr 0 synp

larr 0 synp

larr 14 synp

larr 0 synp

larr 0 synp












1 synp rarr

3 synp rarr

larr 3 synp

larr 0 synp


larr 0 synp

larr 0 synp


MD1

MD2

Dry AMD

CNVGAAMD

larr 354 synp



4 Discussion













References






































1 Introduction



2plays an




















2




2humidified





2(5 ngmL)
















3 Results









Hoechst

Hoechst

Vimentin

Cytokeratin Merge

Merge



(a)


200

0

Cou

nts

Data001

M1M2

FL1-H10

010

110

210

310

4

200

0

Cou

nts

Data002

M1M2

FL1-H10

010

110

210

310

4

(b)









0

5

10

15

20

25

30

35

Relat

ive C

TGF

mRN

A ex

pres

sion

lowast lowastlowast

lowastlowast

lowastlowast

Con

trol

TGF-1205732(+)

CTG

F-siR

NA

1+

TGF-1205732(+)

CTG

F-siR

NA

2+

TGF-1205732(+)

CTG

F-siR

NA

3+

TGF-1205732(+)

CTG

F-siR

NA

4+

TGF-1205732(+)

(a)

0

5

10

15

20

25

30

35

40


Relat

ive C

TGF

mRN

A ex

pres

sion

TGF-1205732(minus)TGF-1205732(+)

lowast

(b)






2in control group



2(Figure 3(b))


2 a distin-


2stimulated cells




2treated cells

















2(+)




2



Hoechst CTGF Merge

Control

CTGF-siRNA

Control-siRNA

+TGF-1205732(+)

+ TGF-1205732(+)

+ TGF-1205732(+)




005)

4 Discussion






2


2with monoclonal




Control

00

02

04

06

08

10

12

14

16

18

0 24 48 72 96

OD

val

ue

lowastlowast

lowastlowastlowast

lowastlowast

+TGF-1205732(+)





2for 0


2stimulated





2coupled with CTGF


2


2could



000

200

400

600

800

1000

1200

1400

Con

trol

PI (

)

CTG

F-siR

NA+

Con

trol-s

iRN

A+

TGF-1205732(+)

TGF-1205732(+)

TGF-1205732(+)

lowastlowast

lowast







5 Conclusions




Acknowledgment



References




















































1 Introduction











2 Methods



















3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2













































































MD1

MD2

Dry AMD

CNVGAGACNV


larr 1 synp


larr 1 synp

larr 2 synp


larr 0 synp

larr 2 synp

larr 2 synp

larr 0 synp

larr 0 synp

larr 14 synp

larr 0 synp

larr 0 synp












1 synp rarr

3 synp rarr

larr 3 synp

larr 0 synp


larr 0 synp

larr 0 synp


MD1

MD2

Dry AMD

CNVGAAMD

larr 354 synp



4 Discussion













References






































1 Introduction



2plays an




















2




2humidified





2(5 ngmL)
















3 Results









Hoechst

Hoechst

Vimentin

Cytokeratin Merge

Merge



(a)


200

0

Cou

nts

Data001

M1M2

FL1-H10

010

110

210

310

4

200

0

Cou

nts

Data002

M1M2

FL1-H10

010

110

210

310

4

(b)









0

5

10

15

20

25

30

35

Relat

ive C

TGF

mRN

A ex

pres

sion

lowast lowastlowast

lowastlowast

lowastlowast

Con

trol

TGF-1205732(+)

CTG

F-siR

NA

1+

TGF-1205732(+)

CTG

F-siR

NA

2+

TGF-1205732(+)

CTG

F-siR

NA

3+

TGF-1205732(+)

CTG

F-siR

NA

4+

TGF-1205732(+)

(a)

0

5

10

15

20

25

30

35

40


Relat

ive C

TGF

mRN

A ex

pres

sion

TGF-1205732(minus)TGF-1205732(+)

lowast

(b)






2in control group



2(Figure 3(b))


2 a distin-


2stimulated cells




2treated cells

















2(+)




2



Hoechst CTGF Merge

Control

CTGF-siRNA

Control-siRNA

+TGF-1205732(+)

+ TGF-1205732(+)

+ TGF-1205732(+)




005)

4 Discussion






2


2with monoclonal




Control

00

02

04

06

08

10

12

14

16

18

0 24 48 72 96

OD

val

ue

lowastlowast

lowastlowastlowast

lowastlowast

+TGF-1205732(+)





2for 0


2stimulated





2coupled with CTGF


2


2could



000

200

400

600

800

1000

1200

1400

Con

trol

PI (

)

CTG

F-siR

NA+

Con

trol-s

iRN

A+

TGF-1205732(+)

TGF-1205732(+)

TGF-1205732(+)

lowastlowast

lowast







5 Conclusions




Acknowledgment



References




















































1 Introduction











2 Methods



















3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2
















































































1 synp rarr

3 synp rarr

larr 3 synp

larr 0 synp


larr 0 synp

larr 0 synp


MD1

MD2

Dry AMD

CNVGAAMD

larr 354 synp



4 Discussion













References






































1 Introduction



2plays an




















2




2humidified





2(5 ngmL)
















3 Results









Hoechst

Hoechst

Vimentin

Cytokeratin Merge

Merge



(a)


200

0

Cou

nts

Data001

M1M2

FL1-H10

010

110

210

310

4

200

0

Cou

nts

Data002

M1M2

FL1-H10

010

110

210

310

4

(b)









0

5

10

15

20

25

30

35

Relat

ive C

TGF

mRN

A ex

pres

sion

lowast lowastlowast

lowastlowast

lowastlowast

Con

trol

TGF-1205732(+)

CTG

F-siR

NA

1+

TGF-1205732(+)

CTG

F-siR

NA

2+

TGF-1205732(+)

CTG

F-siR

NA

3+

TGF-1205732(+)

CTG

F-siR

NA

4+

TGF-1205732(+)

(a)

0

5

10

15

20

25

30

35

40


Relat

ive C

TGF

mRN

A ex

pres

sion

TGF-1205732(minus)TGF-1205732(+)

lowast

(b)






2in control group



2(Figure 3(b))


2 a distin-


2stimulated cells




2treated cells

















2(+)




2



Hoechst CTGF Merge

Control

CTGF-siRNA

Control-siRNA

+TGF-1205732(+)

+ TGF-1205732(+)

+ TGF-1205732(+)




005)

4 Discussion






2


2with monoclonal




Control

00

02

04

06

08

10

12

14

16

18

0 24 48 72 96

OD

val

ue

lowastlowast

lowastlowastlowast

lowastlowast

+TGF-1205732(+)





2for 0


2stimulated





2coupled with CTGF


2


2could



000

200

400

600

800

1000

1200

1400

Con

trol

PI (

)

CTG

F-siR

NA+

Con

trol-s

iRN

A+

TGF-1205732(+)

TGF-1205732(+)

TGF-1205732(+)

lowastlowast

lowast







5 Conclusions




Acknowledgment



References




















































1 Introduction











2 Methods



















3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2





















































































References






































1 Introduction



2plays an




















2




2humidified





2(5 ngmL)
















3 Results









Hoechst

Hoechst

Vimentin

Cytokeratin Merge

Merge



(a)


200

0

Cou

nts

Data001

M1M2

FL1-H10

010

110

210

310

4

200

0

Cou

nts

Data002

M1M2

FL1-H10

010

110

210

310

4

(b)









0

5

10

15

20

25

30

35

Relat

ive C

TGF

mRN

A ex

pres

sion

lowast lowastlowast

lowastlowast

lowastlowast

Con

trol

TGF-1205732(+)

CTG

F-siR

NA

1+

TGF-1205732(+)

CTG

F-siR

NA

2+

TGF-1205732(+)

CTG

F-siR

NA

3+

TGF-1205732(+)

CTG

F-siR

NA

4+

TGF-1205732(+)

(a)

0

5

10

15

20

25

30

35

40


Relat

ive C

TGF

mRN

A ex

pres

sion

TGF-1205732(minus)TGF-1205732(+)

lowast

(b)






2in control group



2(Figure 3(b))


2 a distin-


2stimulated cells




2treated cells

















2(+)




2



Hoechst CTGF Merge

Control

CTGF-siRNA

Control-siRNA

+TGF-1205732(+)

+ TGF-1205732(+)

+ TGF-1205732(+)




005)

4 Discussion






2


2with monoclonal




Control

00

02

04

06

08

10

12

14

16

18

0 24 48 72 96

OD

val

ue

lowastlowast

lowastlowastlowast

lowastlowast

+TGF-1205732(+)





2for 0


2stimulated





2coupled with CTGF


2


2could



000

200

400

600

800

1000

1200

1400

Con

trol

PI (

)

CTG

F-siR

NA+

Con

trol-s

iRN

A+

TGF-1205732(+)

TGF-1205732(+)

TGF-1205732(+)

lowastlowast

lowast







5 Conclusions




Acknowledgment



References




















































1 Introduction











2 Methods



















3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2













































































































1 Introduction



2plays an




















2




2humidified





2(5 ngmL)
















3 Results









Hoechst

Hoechst

Vimentin

Cytokeratin Merge

Merge



(a)


200

0

Cou

nts

Data001

M1M2

FL1-H10

010

110

210

310

4

200

0

Cou

nts

Data002

M1M2

FL1-H10

010

110

210

310

4

(b)









0

5

10

15

20

25

30

35

Relat

ive C

TGF

mRN

A ex

pres

sion

lowast lowastlowast

lowastlowast

lowastlowast

Con

trol

TGF-1205732(+)

CTG

F-siR

NA

1+

TGF-1205732(+)

CTG

F-siR

NA

2+

TGF-1205732(+)

CTG

F-siR

NA

3+

TGF-1205732(+)

CTG

F-siR

NA

4+

TGF-1205732(+)

(a)

0

5

10

15

20

25

30

35

40


Relat

ive C

TGF

mRN

A ex

pres

sion

TGF-1205732(minus)TGF-1205732(+)

lowast

(b)






2in control group



2(Figure 3(b))


2 a distin-


2stimulated cells




2treated cells

















2(+)




2



Hoechst CTGF Merge

Control

CTGF-siRNA

Control-siRNA

+TGF-1205732(+)

+ TGF-1205732(+)

+ TGF-1205732(+)




005)

4 Discussion






2


2with monoclonal




Control

00

02

04

06

08

10

12

14

16

18

0 24 48 72 96

OD

val

ue

lowastlowast

lowastlowastlowast

lowastlowast

+TGF-1205732(+)





2for 0


2stimulated





2coupled with CTGF


2


2could



000

200

400

600

800

1000

1200

1400

Con

trol

PI (

)

CTG

F-siR

NA+

Con

trol-s

iRN

A+

TGF-1205732(+)

TGF-1205732(+)

TGF-1205732(+)

lowastlowast

lowast







5 Conclusions




Acknowledgment



References




















































1 Introduction











2 Methods



















3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2




























































































1 Introduction



2plays an




















2




2humidified





2(5 ngmL)
















3 Results









Hoechst

Hoechst

Vimentin

Cytokeratin Merge

Merge



(a)


200

0

Cou

nts

Data001

M1M2

FL1-H10

010

110

210

310

4

200

0

Cou

nts

Data002

M1M2

FL1-H10

010

110

210

310

4

(b)









0

5

10

15

20

25

30

35

Relat

ive C

TGF

mRN

A ex

pres

sion

lowast lowastlowast

lowastlowast

lowastlowast

Con

trol

TGF-1205732(+)

CTG

F-siR

NA

1+

TGF-1205732(+)

CTG

F-siR

NA

2+

TGF-1205732(+)

CTG

F-siR

NA

3+

TGF-1205732(+)

CTG

F-siR

NA

4+

TGF-1205732(+)

(a)

0

5

10

15

20

25

30

35

40


Relat

ive C

TGF

mRN

A ex

pres

sion

TGF-1205732(minus)TGF-1205732(+)

lowast

(b)






2in control group



2(Figure 3(b))


2 a distin-


2stimulated cells




2treated cells

















2(+)




2



Hoechst CTGF Merge

Control

CTGF-siRNA

Control-siRNA

+TGF-1205732(+)

+ TGF-1205732(+)

+ TGF-1205732(+)




005)

4 Discussion






2


2with monoclonal




Control

00

02

04

06

08

10

12

14

16

18

0 24 48 72 96

OD

val

ue

lowastlowast

lowastlowastlowast

lowastlowast

+TGF-1205732(+)





2for 0


2stimulated





2coupled with CTGF


2


2could



000

200

400

600

800

1000

1200

1400

Con

trol

PI (

)

CTG

F-siR

NA+

Con

trol-s

iRN

A+

TGF-1205732(+)

TGF-1205732(+)

TGF-1205732(+)

lowastlowast

lowast







5 Conclusions




Acknowledgment



References




















































1 Introduction











2 Methods



















3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2





























































































2




2humidified





2(5 ngmL)
















3 Results









Hoechst

Hoechst

Vimentin

Cytokeratin Merge

Merge



(a)


200

0

Cou

nts

Data001

M1M2

FL1-H10

010

110

210

310

4

200

0

Cou

nts

Data002

M1M2

FL1-H10

010

110

210

310

4

(b)









0

5

10

15

20

25

30

35

Relat

ive C

TGF

mRN

A ex

pres

sion

lowast lowastlowast

lowastlowast

lowastlowast

Con

trol

TGF-1205732(+)

CTG

F-siR

NA

1+

TGF-1205732(+)

CTG

F-siR

NA

2+

TGF-1205732(+)

CTG

F-siR

NA

3+

TGF-1205732(+)

CTG

F-siR

NA

4+

TGF-1205732(+)

(a)

0

5

10

15

20

25

30

35

40


Relat

ive C

TGF

mRN

A ex

pres

sion

TGF-1205732(minus)TGF-1205732(+)

lowast

(b)






2in control group



2(Figure 3(b))


2 a distin-


2stimulated cells




2treated cells

















2(+)




2



Hoechst CTGF Merge

Control

CTGF-siRNA

Control-siRNA

+TGF-1205732(+)

+ TGF-1205732(+)

+ TGF-1205732(+)




005)

4 Discussion






2


2with monoclonal




Control

00

02

04

06

08

10

12

14

16

18

0 24 48 72 96

OD

val

ue

lowastlowast

lowastlowastlowast

lowastlowast

+TGF-1205732(+)





2for 0


2stimulated





2coupled with CTGF


2


2could



000

200

400

600

800

1000

1200

1400

Con

trol

PI (

)

CTG

F-siR

NA+

Con

trol-s

iRN

A+

TGF-1205732(+)

TGF-1205732(+)

TGF-1205732(+)

lowastlowast

lowast







5 Conclusions




Acknowledgment



References




















































1 Introduction











2 Methods



















3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2

























































































3 Results









Hoechst

Hoechst

Vimentin

Cytokeratin Merge

Merge



(a)


200

0

Cou

nts

Data001

M1M2

FL1-H10

010

110

210

310

4

200

0

Cou

nts

Data002

M1M2

FL1-H10

010

110

210

310

4

(b)









0

5

10

15

20

25

30

35

Relat

ive C

TGF

mRN

A ex

pres

sion

lowast lowastlowast

lowastlowast

lowastlowast

Con

trol

TGF-1205732(+)

CTG

F-siR

NA

1+

TGF-1205732(+)

CTG

F-siR

NA

2+

TGF-1205732(+)

CTG

F-siR

NA

3+

TGF-1205732(+)

CTG

F-siR

NA

4+

TGF-1205732(+)

(a)

0

5

10

15

20

25

30

35

40


Relat

ive C

TGF

mRN

A ex

pres

sion

TGF-1205732(minus)TGF-1205732(+)

lowast

(b)






2in control group



2(Figure 3(b))


2 a distin-


2stimulated cells




2treated cells

















2(+)




2



Hoechst CTGF Merge

Control

CTGF-siRNA

Control-siRNA

+TGF-1205732(+)

+ TGF-1205732(+)

+ TGF-1205732(+)




005)

4 Discussion






2


2with monoclonal




Control

00

02

04

06

08

10

12

14

16

18

0 24 48 72 96

OD

val

ue

lowastlowast

lowastlowastlowast

lowastlowast

+TGF-1205732(+)





2for 0


2stimulated





2coupled with CTGF


2


2could



000

200

400

600

800

1000

1200

1400

Con

trol

PI (

)

CTG

F-siR

NA+

Con

trol-s

iRN

A+

TGF-1205732(+)

TGF-1205732(+)

TGF-1205732(+)

lowastlowast

lowast







5 Conclusions




Acknowledgment



References




















































1 Introduction











2 Methods



















3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2













































































Hoechst

Hoechst

Vimentin

Cytokeratin Merge

Merge



(a)


200

0

Cou

nts

Data001

M1M2

FL1-H10

010

110

210

310

4

200

0

Cou

nts

Data002

M1M2

FL1-H10

010

110

210

310

4

(b)









0

5

10

15

20

25

30

35

Relat

ive C

TGF

mRN

A ex

pres

sion

lowast lowastlowast

lowastlowast

lowastlowast

Con

trol

TGF-1205732(+)

CTG

F-siR

NA

1+

TGF-1205732(+)

CTG

F-siR

NA

2+

TGF-1205732(+)

CTG

F-siR

NA

3+

TGF-1205732(+)

CTG

F-siR

NA

4+

TGF-1205732(+)

(a)

0

5

10

15

20

25

30

35

40


Relat

ive C

TGF

mRN

A ex

pres

sion

TGF-1205732(minus)TGF-1205732(+)

lowast

(b)






2in control group



2(Figure 3(b))


2 a distin-


2stimulated cells




2treated cells

















2(+)




2



Hoechst CTGF Merge

Control

CTGF-siRNA

Control-siRNA

+TGF-1205732(+)

+ TGF-1205732(+)

+ TGF-1205732(+)




005)

4 Discussion






2


2with monoclonal




Control

00

02

04

06

08

10

12

14

16

18

0 24 48 72 96

OD

val

ue

lowastlowast

lowastlowastlowast

lowastlowast

+TGF-1205732(+)





2for 0


2stimulated





2coupled with CTGF


2


2could



000

200

400

600

800

1000

1200

1400

Con

trol

PI (

)

CTG

F-siR

NA+

Con

trol-s

iRN

A+

TGF-1205732(+)

TGF-1205732(+)

TGF-1205732(+)

lowastlowast

lowast







5 Conclusions




Acknowledgment



References




















































1 Introduction











2 Methods



















3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2



















































































0

5

10

15

20

25

30

35

Relat

ive C

TGF

mRN

A ex

pres

sion

lowast lowastlowast

lowastlowast

lowastlowast

Con

trol

TGF-1205732(+)

CTG

F-siR

NA

1+

TGF-1205732(+)

CTG

F-siR

NA

2+

TGF-1205732(+)

CTG

F-siR

NA

3+

TGF-1205732(+)

CTG

F-siR

NA

4+

TGF-1205732(+)

(a)

0

5

10

15

20

25

30

35

40


Relat

ive C

TGF

mRN

A ex

pres

sion

TGF-1205732(minus)TGF-1205732(+)

lowast

(b)






2in control group



2(Figure 3(b))


2 a distin-


2stimulated cells




2treated cells

















2(+)




2



Hoechst CTGF Merge

Control

CTGF-siRNA

Control-siRNA

+TGF-1205732(+)

+ TGF-1205732(+)

+ TGF-1205732(+)




005)

4 Discussion






2


2with monoclonal




Control

00

02

04

06

08

10

12

14

16

18

0 24 48 72 96

OD

val

ue

lowastlowast

lowastlowastlowast

lowastlowast

+TGF-1205732(+)





2for 0


2stimulated





2coupled with CTGF


2


2could



000

200

400

600

800

1000

1200

1400

Con

trol

PI (

)

CTG

F-siR

NA+

Con

trol-s

iRN

A+

TGF-1205732(+)

TGF-1205732(+)

TGF-1205732(+)

lowastlowast

lowast







5 Conclusions




Acknowledgment



References




















































1 Introduction











2 Methods



















3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2













































































Hoechst CTGF Merge

Control

CTGF-siRNA

Control-siRNA

+TGF-1205732(+)

+ TGF-1205732(+)

+ TGF-1205732(+)




005)

4 Discussion






2


2with monoclonal




Control

00

02

04

06

08

10

12

14

16

18

0 24 48 72 96

OD

val

ue

lowastlowast

lowastlowastlowast

lowastlowast

+TGF-1205732(+)





2for 0


2stimulated





2coupled with CTGF


2


2could



000

200

400

600

800

1000

1200

1400

Con

trol

PI (

)

CTG

F-siR

NA+

Con

trol-s

iRN

A+

TGF-1205732(+)

TGF-1205732(+)

TGF-1205732(+)

lowastlowast

lowast







5 Conclusions




Acknowledgment



References




















































1 Introduction











2 Methods



















3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2













































































Control

00

02

04

06

08

10

12

14

16

18

0 24 48 72 96

OD

val

ue

lowastlowast

lowastlowastlowast

lowastlowast

+TGF-1205732(+)





2for 0


2stimulated





2coupled with CTGF


2


2could



000

200

400

600

800

1000

1200

1400

Con

trol

PI (

)

CTG

F-siR

NA+

Con

trol-s

iRN

A+

TGF-1205732(+)

TGF-1205732(+)

TGF-1205732(+)

lowastlowast

lowast







5 Conclusions




Acknowledgment



References




















































1 Introduction











2 Methods



















3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2













































































References




















































1 Introduction











2 Methods



















3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2































































































1 Introduction











2 Methods



















3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2





















































































1 Introduction











2 Methods



















3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2


















































































2 Methods



















3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2























































































3 Results




4 Discussion







PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2















































































PLEKHA7 rs11024102 (TC)TT 105 (343) 78 (336)

0283 087103 (072ndash148) 012143 (091ndash226)TC 157 (513) 109 (470)CC 44 (144) 45 (144)

COL11A1 rs3753841 (AG)AA 145 (474) 116 (194)

0606 055090 (064ndash127) 034075 (042ndash140)AG 127 (415) 96 (414)GG 34 (111) 20 (86)

PCMTD1-ST18 rs1015213 (CT)CC 295 (964) 221 (953)

0261 051134 (056ndash314) 051026 (001ndash549)CT 9 (29) 11 (47)TT 2 (06) 0 (00)





119875 0958 0448 0663



119875 0366 0924 0945



119875 0528 0617 0835






5 Conclusion






Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2
















































































Acknowledgments


References


































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2























































































1 Introduction
























GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2
































































































GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2













































































GAPDH

0 05 1 5 10 20(ngmL) Time (1h)

TGF1205732

Snail

(a)

002040608

1

0 05 1 5 10 20

(ngmL)

Rala

tive S

nail

mRN

A le

vel

Time (1h)

lowast

lowast

lowast lowastlowast

TGF1205732

(b)


0 1 4 8 12

GAPDH


Snail

(a)

0010203040506070809

0 05 1 4 8 12Time (h)

Rala

tive S

nail

mRN

A le

vel

lowast

lowast

lowast

lowast

lowast

TGF1205732 (10ngmL)

(b)






3 Results




Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2













































































Hoechst Merge

Control

TGF1205732

Snail


0

02

04

06

08

1

12

siRNA

Rala

tive S

nail

siRN

A le

vel

P1 P2 P3 P4 P5

lowastlowast

lowast

lowast

lowast

minusminus

TGF1205732 (10ngmL)





siRNA P2 P5 +++

minusminus

minusTGF1205732


siRNA P2 P5 +++

minusminus

minus

Keratin

E-Cadherin

Vimentin

120572-SAM

TGF1205732





Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2













































































Time (h) 72+

48+

24+

0minusTGF1205732

(a)

siRNA P5 +

P2++

minusminus

minusTGF1205732

(b)


siRNA P2 P5 +++

minusminus

minusTGF1205732

(a)

050

100150200250300

Mig

rato

ry ce

ll nu

mbe


lowast

lowast

TGF1205732 (10ngmL)(b)



4 Discussion















Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2




















































































Acknowledgments


References








































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2
































































































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2





























































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2





















































































1 Introduction






Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2













































































Rolling

Endothelial cell

Adhesion

Migration

Leukocyte

Granules with VAP-1

VAP-1

Blood flow















[12 50]


























Acknowledgments


References

















2O2
















































































[12 50]


























Acknowledgments


References

















2O2



























































































Acknowledgments


References

















2O2
















































































Acknowledgments


References

















2O2










































































































































































































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