Identification of RUNX1 as a Mediator of AberrantRetinal AngiogenesisJonathan D Lam1 Daniel J Oh1 Lindsay L Wong1 Dhanesh Amarnani1 Cindy Park-Windhol1

Angie V Sanchez1 Jonathan Cardona-Velez12 Declan McGuone3 Anat O Stemmer-Rachamimov3

Dean Eliott4 Diane R Bielenberg5 Tave van Zyl4 Lishuang Shen6 Xiaowu Gai6 Patricia A DrsquoAmore17

Leo A Kim14 and Joseph F Arboleda-Velasquez1

Diabetes 2017661950ndash1956 | httpsdoiorg102337db16-1035

Proliferative diabetic retinopathy (PDR) is a commoncause of blindness in the developed worldrsquos working adultpopulation and affects those with type 1 and type 2 diabe-tes We identified Runt-related transcription factor1 (RUNX1) as a gene upregulated in CD311 vascular en-dothelial cells obtained from human PDR fibrovascularmembranes (FVMs) via transcriptomic analysis In vitrostudies using human retinal microvascular endothelialcells (HRMECs) showed increased RUNX1 RNA andprotein expression in response to high glucose whereasRUNX1 inhibition reduced HRMEC migration prolifera-tion and tube formation Immunohistochemical stainingfor RUNX1 showed reactivity in vessels of patient-derivedFVMs and angiogenic tufts in the retina of mice with ox-ygen-induced retinopathy suggesting that RUNX1 upre-gulation is a hallmark of aberrant retinal angiogenesisInhibition of RUNX1 activity with the Ro5ndash3335 small mol-ecule resulted in a significant reduction of neovasculartufts in oxygen-induced retinopathy supporting the feasi-bility of targeting RUNX1 in aberrant retinal angiogenesis

Neovascularization is a pathological feature of prolifera-tive diabetic retinopathy (PDR) wet age-related maculardegeneration retinopathy of prematurity cancer andother conditions (1) Antiangiogenic therapies typically

target vascular endothelial growth factor (VEGF) and areeffective treatments for a number of neovascular oculardiseases and some solid tumors (2) Despite the successof anti-VEGF therapies considerable interest remains inidentifying new therapeutic targets for aberrant angiogen-esis as anti-VEGF therapies may acutely trigger hemor-rhages and tractional retinal detachments in patients withPDR whereas extended therapy may lead to tissue atrophyischemia and reperfusion injury (3ndash6)

A major challenge to understanding neovascularization inPDR is that animal models of diabetes do not develop theproliferative phase of diabetic retinopathy consistently (7) Fi-brovascular membranes (FVMs) in patients are surgicallyremoved to relieve retinal traction with associated retinaldetachment and remain largely understudied as they areoften discarded after ocular surgery To establish platformsfor discovering the mechanisms underlying aberrant angio-genesis we developed methods for the isolation andcharacterization of vascular endothelial cells (ECs) from pa-tient-derived PDR FVMs (8)

RESEARCH DESIGN AND METHODS

The Internal Review Board of Massachusetts Eye and Ear(MEE) approved this study Research protocols adhered to theARVO Statement on Human Subjects and the tenets of the

1Department of Ophthalmology Schepens Eye Research InstituteMassachusettsEye and Ear Harvard Medical School Boston MA2Universidad Pontificia Bolivariana Medellin Colombia3CS Kubik Laboratory for Neuropathology Massachusetts General Hospital Boston MA4Retina Service Department of Ophthalmology Massachusetts Eye and EarHarvard Medical School Boston MA5Vascular Biology Program Department of Surgery Boston Childrenrsquos HospitalHarvard Medical School Boston MA6Center for Personalized Medicine Childrenrsquos Hospital Los Angeles Los Angeles CA7Department of Pathology Harvard Medical School Boston MA

Corresponding authors Joseph F Arboleda-Velasquez joseph_arboledameeiharvardedu Leo A Kim leo_kimmeeiharvardedu and Patricia A DrsquoAmorepatricia_damoremeeiharvardedu

Received 26 August 2016 and accepted 16 March 2017

This article contains Supplementary Data online at httpdiabetesdiabetesjournalsorglookupsuppldoi102337db16-1035-DC1

JDL and DJO contributed equally to this work

JFA-V LAK and PAD contributed equally to this work

copy 2017 by the American Diabetes Association Readers may use this article aslong as the work is properly cited the use is educational and not for profit and thework is not altered More information is available at httpwwwdiabetesjournalsorgcontentlicense

1950 Diabetes Volume 66 July 2017

PATHOPHYSIO

LOGY

Declaration of Helsinki All participants gave informed consentprior to surgery and inclusion in the study Surgical sampleswere collected at MEE Control retinal samples were obtainedfrom cadaver eyes of subjects without a diagnosis of diabetesthrough an approved internal review board protocol fromMassachusetts General Hospital (Supplementary Table 1)

Whole-Transcriptome SequencingCD311 cells were isolated from FVMs as previously de-scribed (8) RNA-sequencing was performed using a HiSEq2000 (Illumina) aligned to reference genome UCSChg19GRCh37 with TopHat and analyzed using PartekFlow (Partek) CuffLinks EdgeR and DESeq2 (9) A mixed-model ANOVA was used with a threshold false discoveryrate of 005 and fold change $ 62 for significance Geneontology was determined using the Database for Annota-tion Visualization and Integrated Discovery (DAVID) (10)

Oxygen-Induced Retinopathy ModelMouse care and experimental procedures were in accordancewith MEE Institutional Animal Care and Use Committeeregulations Oxygen-induced retinopathy (OIR) was inducedin wild-type C57BL6J mice as previously described (11)Intravitreal injections with 1 mL of 75 mmolL Ro5ndash3335Runt-related transcription factor 1 (RUNX1) inhibitor orDMSO were performed on left eyes only at postnatal day(P)13 and P15 under ketaminexylazine anesthesia Pupswere euthanized at P17 and eyes were collected fixed in4 paraformaldehyde and used for retinal flat mounts(nvehicle 5 7 nRo5ndash3335 5 9) Avascular and neovascularareas were quantified using Photoshop (Adobe Systems)as previously described (12)

Cell CultureHuman retinal microvascular ECs (HRMECs) (Cell Systems)and human umbilical vein ECs (HUVECs) (Lonza) weregrown at 37degC with 5 CO2 using endothelial growth mediaplus antibiotics (Lonza) and 2 FBS (Atlanta Biologicals)For the quantitative RT-PCR (qRT-PCR) gene candidatescreen in high glucose cells were treated for 48 h with en-dothelial basal media-2 with 2 platelet-poor plasma-derivedserum (Alfa Aesar) and D-glucose (Sigma-Aldrich) or osmoticcontrol (mannitol and L-glucose Sigma-Aldrich)

Small Interfering RNA Gene KnockdownSmall interfering RNA (siRNA) (75 nmolL) (IntegratedDNA Technologies sequences in Supplementary Table 2)were transfected for 6ndash8 h using DharmaFECT 1 (GE LifeSciencesDharmacon) in Opti-MEM (Life Technologies)

ImmunostainingFVMs and cells were incubated with mouse anti-RUNX1(Santa Cruz Biotechnology) followed by biotinylated sec-ondary antibody (Vector Laboratories) horseradishperoxidasendashlabeled avidin (PerkinElmer) tyramide signalamplification (PerkinElmer) Vector Red chromogenic sub-strate (Vector Laboratories) and Gill no 3 hematoxylincounterstain (Sigma-Aldrich) Immunofluorescent stainingfor CD31 (mouse anti-CD31 DakoCytomation) and Ki67(rabbit anti-Ki67 Novus Biologicals) was performed as

previously described (8) The number of Ki67-positive nu-clei was averaged for three fields of view per sample at320original magnification

Mouse retinal whole mounts were blocked with 1 BSA01 Triton X-100 and 3 serum in PBlec buffer in-cubated with primary (isolectin IB4 Alexa Fluor 488 Conju-gate Life Technologies rat anti-CD31 BD Pharmingen andrabbit anti-RUNX1 LifeSpan BioSciences) and secondaryantibodies (donkey anti-rat Alexa Fluor 594 and donkeyanti-rabbit Alexa Fluor 647 or goat anti-rabbit Alexa Fluor594 Life Technologies) Samples were imaged with an Axi-oskop 2 MOT Plus microscope (Carl Zeiss) or TCS-SP5 con-focal microscope (Leica Microsystems)

qRT-PCR AnalysisRNA was extracted using RNeasy Mini Kits (Qiagen)Primers were purchased from Integrated DNA Technologiesfor 200 candidate genes (Supplementary Table 2) Geneswere excluded from further analysis if there was noamplification or amplification was outside the linear rangeThis resulted in a final list of 101 genes analyzed by qRT-PCR cDNA was prepared using the iScript cDNA synthesiskit (Bio-Rad Laboratories) and probed using FastStartUniversal SYBR Green Master (Hoffmann-La Roche) Fluo-rescent intensities were normalized to spike-in controls(ERCC RNA Spike-In Mix Life Technologies) hypoxanthinephosphoribosyltransferase 1 and B2-microglobulin

Western Blot AnalysisCells were lysed using RIPA buffer (Cell Signaling Technol-ogy) Samples were separated on a 4ndash15 SDS-PAGE trans-ferred to polyvinylidene difluoride membranes blockedwith Odyssey Blocking Buffer (LI-COR Biosciences LincolnNE) and probed with mouse anti-RUNX1 (Santa Cruz Bio-technology Inc) rabbit antindashb-actin (Cell Signaling Tech-nology) IRDye 680RD donkey anti-rabbit and IRDye800CW donkey anti-mouse (LI-COR Biosciences) antibodiesImmunoreactive bands were visualized using the OdysseyInfrared Imaging System and band intensities normalizedto b-actin were quantified using Image Studio version 21(LI-COR Biosciences)

Scratch-Wound Migration AssayMigration was assessed with the scratch-wound assay (13)One scratch was generated per well and imaged on an EVOSimaging system every 3 h for 12 h Images were analyzedwith the TScratch Matlab module (14)

Tube-Forming AssayHRMECs were treated overnight with 75 mmolL Ro5ndash3335 or DMSO before plating 20000 cells onto wells pre-coated with basement membrane extract (Trevigen) Cellswere imaged 6 h after plating and tube formation wasquantified using the Angiogenesis Analyzer plugin forImageJ (National Institutes of Health)

Statistical AnalysisResults are presented as mean 6 SEM The Student t testwas performed for comparisons between two groups and

diabetesdiabetesjournalsorg Lam and Associates 1951

one-way ANOVA (Kruskal-Wallis test) was used for compar-isons between multiple groups A P value 005 was con-sidered significant

RESULTS

RNA Sequencing of CD311 Cells From FVMsWhole transcriptomic profiles were constructed for CD311

cells from FVMs and compared with transcriptomes ofCD311 cells from postmortem retinas isolated from indi-viduals without diabetes (Supplementary Fig 1 and Supple-mentary Table 1) Postmortem retinas were used to defineexpression baseline because there is no normal correlate ofFVMs CD311 cells were identified as vascular ECs based ontheir expression of five vascular endothelial markers CD93CD31 KLF4 ESAM and VEGFR1 (Supplementary Table 3)However because a single marker namely CD31 was usedfor cell isolation the presence of other cell types cannot beruled out Principal component analysis of the tran-scriptomes demonstrated congruent expression profilesfor control samples whereas profiles of cells from theFVM samples had more variable gene expression pat-terns (Fig 1A) The data sets are accessible through the

National Center for Biotechnology Informationrsquos GeneExpression Omnibus (GSE94019) (15)

Two hundred genes were identified as differentiallyexpressed in CD311 cells from FVMs involving gene ontol-ogy categories related to wound response vessel develop-ment angiogenesis and other categories (Fig 1B andSupplementary Table 4)

RUNX1 Is Upregulated in Cultured ECs Exposedto High GlucoseHyperglycemia is a major risk factor for progression toPDR in patients with diabetes (16) High glucose-dependentgenes from the candidate gene list were identified in pri-mary HRMEC culture using a qRT-PCR screen Four out of101 genes were glucose responsive consistent with tran-scriptomic data (fold change 6 SEM) RUNX1 (29 6 02)peptidyl-prolyl cis-trans isomerase F (PPIF) (36 6 05)serinethreonine-protein kinase PIM3 (PIM3) (39 6 10)and CD44 (35 6 04) (Fig 2A and B) We focused onRUNX1 for further studies because of its documented rolesin EC biology (17ndash19) Exposure of HRMEC and HUVECcultures to high glucose induced a 30 increase inRUNX1 protein whereas osmotic controls had no significant

Figure 1mdashPrinciple component analysis of transcriptomes and gene ontology of candidate genes A Two-dimensional representations of thethree most significant principle components (PCs) PC1 PC2 and PC3 account for 4537 of the observed sample variance The widedistribution of transcriptomes from FVM-derived CD311 cells contrasts the congruity of transcriptomes of cells from normal retinal samplesindicating significant variability in FVM gene expression levels B The top 20 pathways are presented in order of descending significance withthe number of genes from the candidate gene list enriching the pathway

1952 Role of RUNX1 in Aberrant Retinal Angiogenesis Diabetes Volume 66 July 2017

effect in HRMEC and HUVECs (Fig 2C and D) Lastly weconfirmed increased RUNX1 protein expression in vesselsfrom FVMs compared with retina from control subjectswithout diabetes using immunohistochemical staining(Fig 2E)

RUNX1 Regulates Migration and Proliferation ofEndothelial Cells In VitrosiRNA knockdown of RUNX1 resulted in a 60 decrease inwound closure rate indicating a significant role of RUNX1

in endothelial cell migration (Fig 3A) The coefficient ofdetermination (r2 value) for the simple linear regressionwas calculated comparing wound closure (dependent vari-able) to time (independent variable) (control r2 5 099scramble siRNA r2 5 097 and RUNX1 siRNA r2 5 097)This experiment was validated with a second siRNA target-ing RUNX1 to rule out possible off-target effects (Supple-mentary Fig 2B) ECs transfected with RUNX1 siRNA had40 fewer Ki67-positive cells compared with controls 24 h

Figure 2mdashEffect of elevated glucose on HRMEC gene expression A Results of FVM RNA-sequencing (reads per kilobase of transcript permillion mapped reads) show increased expression of RUNX1 PPIF PIM3 and CD44 in ECs from patients with PDR compared with patientswithout diabetes CXCL10 did not exhibit increased expression in FVMs (ncontrols 5 4 nPDR-FVM 5 8) B Corresponding gene expression ofRUNX1 PPIF PIM3 CD44 and CXCL10 measured by qRT-PCR (HG high D-glucose [30 mmolL] NG normal D-glucose [5 mmolL]) Eachcandidate gene had a marked increase in response to D-glucose but no statistically significant changes in response to osmotic controls(L-glucose or mannitol) CXCL10 is not glucose responsive consistent with RNA-sequencing (n5 3 the experiment was performed in triplicate)C Increasing D-glucose led to a dose-dependent increase of RUNX1 protein expression in HRMECs (top) and HUVECs (bottom) as determinedby Western blot (n5 3 experiment performed in triplicate) D The increase in RUNX1 protein in HRMECs induced by 30 mmolL D-glucose wasindependent of osmotic forces (n 5 3 experiment performed in triplicate) E Normal retinal vessels showed no staining of RUNX1 (L vessellumen RPE retinal pigment epithelium) (left panel) Scale bars 5 100 mm A subset of vessels in FVM stained positively for RUNX1 (arrows)Asterisks denote nonstaining vessels (left) Scale bar 5 50 mm P lt 005 P lt 001 P lt 0001

diabetesdiabetesjournalsorg Lam and Associates 1953

after transfection indicating RUNX1 also contributes toretinal EC proliferation (Fig 3B)

Ro5ndash3335 RUNX1-Core Binding Factor b InhibitorBlocks EC Tube Formation In Vitro and AberrantAngiogenesis In VivoTo investigate the potential therapeutic relevance ofRUNX1 the small molecule Ro5ndash3335 RUNX1ndashcore bindingfactor b inhibitor II was tested in vitro and in vivo (20)Treatment with RUNX1 inhibitor reduced total tube length(18 reduction) nodes (35 reduction) and meshes (46reduction) supporting RUNX1rsquos role in vascular morpho-genesis (Fig 3C)

Neovascular tufts of P17 C57BL6J mice with OIRidentified by isolectin B4 and CD31 staining showedincreased expression of RUNX1 whereas RUNX1 stainingwas absent from the underlying normal retinal vasculatureindicating a role for RUNX1 in active pathological angio-genesis (Fig 4A) In vivo testing of the effects on aberrantangiogenesis of RUNX1 inhibition was conducted by inject-ing Ro5ndash3335 intravitreally in mice with OIR There was nosignificant change in the extent of vaso-obliteration and noeffect was observed in the contralateral eye (data not shown)

Eyes treated with inhibitor exhibited a 50 reductionin neovascularization compared with vehicle-treated eyes(Fig 4B)

DISCUSSION

Our analyses of CD311 cells from FVMs of patients withPDR in conjunction with in vitro and in vivo validationassays suggest a role for RUNX1 in the proliferation mi-gration and morphogenesis of ECs in vitro as well as inaberrant angiogenesis in vivo RUNX proteins have pleio-tropic functions in vascular development hematopoiesisand cancer through direct transcriptional regulation oftarget genes and complex interactions with fundamentalsignaling mechanisms including Notch and transforminggrowth factor-b pathways (2122) RUNX1 is a major con-tributor to cancer progression and metastasis most notablyin acute myeloid leukemia and also plays a role in angio-genesis (1823ndash26)

Previous studies in model organisms have shown thatglucose levels trigger RUNX1 expression via reactive oxygenspeciesndashmediated upregulation of hypoxia-inducible factor1 and regulate hematopoietic stem cell production by

Figure 3mdashRole of RUNX1 in EC function A Scratch-wound assay using HRMECs at 0 (left column) 6 (middle column) and 12 h (rightcolumn) with scramble siRNA (top) and RUNX1 siRNA (bottom) treatment Dark gray regions denote wound areas Scale bar 5400 mm Quantification of wound closure rates shows that knockdown of RUNX1 effectively inhibits wound closure (n 5 10 experimentperformed in duplicate) B Ki67 staining 48 h posttransfection demonstrates significant reduction in cell number and proliferative capacity ofRUNX1 siRNA-treated cells compared with cells treated with scramble siRNA Scale bar 5 200 mm Quantification of percentage of DAPI-positive nuclei colocalized with Ki67 stain (n 5 6 experiment performed in duplicate) C HRMECs treated with Ro5ndash3335 RUNX1 inhibitorovernight exhibited reduced tube-forming capacity compared with vehicle-treated cells at 6 h after plating There was statistically significantreduction in tube length meshes and nodes Scale bar 5 500 mm n 5 4 experiment performed in duplicate P lt 005 P lt 001 P lt0001

1954 Role of RUNX1 in Aberrant Retinal Angiogenesis Diabetes Volume 66 July 2017

endothelial to hematopoietic transition in a RUNX1-dependentmanner (2728) Consistent with these observations ourresults demonstrate that high glucose regulates RUNX1 andimplicates RUNX1 in aberrant retinal angiogenesis

Previous studies of PDR have mainly focused on geneexpression in FVMs or biomarkers in the vitreous (2930)Although FVMs from PDR may be highly informative path-ological tissues they are largely unstudied The window forprocessing samples postsurgery is narrow and intraopera-tive confounders such as FVM size location and involve-ment of sensory retina further complicate successful sampleacquisition CD311 cells from FVMs do not grow readily inculture making additional experimentation difficult Lim-ited sample size may also skew our gene ontology analysis

We report elevated RUNX1 expression in ECs of patient-derived FVMs from patients with PDR Also we demon-strate a role for RUNX1 in EC migration proliferation andvascular morphogenesis Furthermore we report selectivelyenhanced expression of RUNX1 in neovascular tufts in anexperimental model of OIR and that inhibition of RUNX1function reduced retinal neovascularization These findings

including the high glucosendashdependent expression of RUNX1identify a novel pathway of potential therapeutic interestand implicate RUNX1 in aberrant angiogenesis in multipleconditions

Acknowledgments The authors thank Dr Magali Saint-Geniez for insightfuldiscussions and providing mouse retinal samples Dr Eric Ng Mark Graham andDr Arturo Machuca-Parra for helpful discussions and Dr Christina Marko fortechnical contributions (all from Schepens Eye Research Institute Boston MA) Theauthors also thank Kristin Waraska and Andrew Gagne of the Harvard BiopolymersFacility Harvard Medical School and the Ocular Genetics Institute core facilityMassachusetts Eye and Ear (Boston MA) for their insights and workFunding The work performed in the manuscript was supported by NationalInstitutes of Health grants R01-EY-005318 (to PAD) R00-EY-021624 (to JFA-V)UH2NS100121-01 (to JFA-V) R21-EY-027061 (to LAK) K12-EY-16335 (toLAK) and Center Core grant P30-EY-003790 American Diabetes AssociationInnovation Award 7ndash12-IN-11 (to PAD) Massachusetts Lions Eye Research Fund(to JFA-V) E Matilda Ziegler Foundation for the Blind (to LAK) Karl KirchgessnerFoundation (to LAK) Department of Ophthalmology Harvard Medical School(to JFA-V and LAK) the Howard Hughes Medical Institute Medical ResearchFellows Program (to DJO) and the Vascular Biology Program at Boston ChildrenrsquosHospital (to DRB)

Figure 4mdashRUNX1 inhibition reduces neovascularization in the OIR model A Retina of P17 C57BL6J mice with OIR costained for RUNX1 andvessels (IB4 or CD31) showing positive RUNX1 staining conforming to neovascular tufts (arrows) and not to normal underlying vasculature Scalebars5 50 mm B Retinal flat mounts of P17 C57BL6J mice following OIR induction and intravitreal injection of 75 mmolL Ro5ndash3335 or vehicleat P13 and P15 (red overlay identifies the avascular area and neovascularization respectively) Scale bars 5 1 mm There was a nonsignificantdownward trend in avascular area but a significant reduction in neovascularization in the treated group compared with vehicle-treated group(nvehicle 5 7 nRo5ndash3335 5 9) Experiment performed in triplicate P lt 001

diabetesdiabetesjournalsorg Lam and Associates 1955

Duality of Interest PAD LAK and JFA-V have filed a provisional patentapplication on RUNX1 regulation for the treatment of aberrant angiogenesis and PDRNo other potential conflicts of interest relevant to this article were reportedAuthor Contributions JDL and DJO performed the experimentsanalyzed the data and wrote the manuscript LLW DA CP-W AVS andJC-V performed experiments DM AOS-R DE and DRB provided tissuesamples and reviewed and edited the manuscript TvZ LS and XG obtaineddata and reviewed the manuscript PAD LAK and JFA-V conceived theproject analyzed the data and wrote the manuscript PAD LAK and JFA-V arethe guarantors of this work and as such had full access to all the data in the studyand take responsibility for the integrity of the data and the accuracy of the dataanalysis

References1 Chung AS Ferrara N Developmental and pathological angiogenesis Annu RevCell Dev Biol 201127563ndash5842 Kim LA DrsquoAmore PA A brief history of anti-VEGF for the treatment of ocularangiogenesis Am J Pathol 2012181376ndash3793 Hsu YJ Hsieh YT Yeh PT Huang JY Yang CM Combined tractional andrhegmatogenous retinal detachment in proliferative diabetic retinopathy in the anti-VEGF era J Ophthalmol 201420149173754 Kuiper EJ Van Nieuwenhoven FA de Smet MD et al The angio-fibrotic switchof VEGF and CTGF in proliferative diabetic retinopathy PLoS One 20083e26755 Saint-Geniez M Maharaj AS Walshe TE et al Endogenous VEGF is required forvisual function evidence for a survival role on muumlller cells and photoreceptors PLoSOne 20083e35546 Sang DN DrsquoAmore PA Is blockade of vascular endothelial growth factorbeneficial for all types of diabetic retinopathy Diabetologia 2008511570ndash15737 Jo DH Cho CS Kim JH Jun HO Kim JH Animal models of diabetic reti-nopathy doors to investigate pathogenesis and potential therapeutics J Biomed Sci201320388 Kim LA Wong LL Amarnani DS et al Characterization of cells from patient-derived fibrovascular membranes in proliferative diabetic retinopathy Mol Vis 201521673ndash6879 Trapnell C Pachter L Salzberg SL TopHat discovering splice junctions withRNA-Seq Bioinformatics 2009251105ndash111110 Huang da W Sherman BT Lempicki RA Bioinformatics enrichment tools pathstoward the comprehensive functional analysis of large gene lists Nucleic Acids Res2009371ndash1311 Smith LE Wesolowski E McLellan A et al Oxygen-induced retinopathy in themouse Invest Ophthalmol Vis Sci 199435101ndash11112 Connor KM Krah NM Dennison RJ et al Quantification of oxygen-inducedretinopathy in the mouse a model of vessel loss vessel regrowth and pathologicalangiogenesis Nat Protoc 200941565ndash157313 Liang CC Park AY Guan JL In vitro scratch assay a convenient and in-expensive method for analysis of cell migration in vitro Nat Protoc 20072329ndash33314 Gebaumlck T Schulz MM Koumoutsakos P Detmar M TScratch a novel andsimple software tool for automated analysis of monolayer wound healing assaysBiotechniques 200946265ndash274

15 Edgar R Domrachev M Lash AE Gene Expression Omnibus NCBI gene ex-

pression and hybridization array data repository Nucleic Acids Res 200230207ndash

21016 The Diabetes Control and Complications Trial Research Group The relationship

of glycemic exposure (HbA1c) to the risk of development and progression of reti-

nopathy in the diabetes control and complications trial Diabetes 199544968ndash98317 McLeod DS Hasegawa T Baba T et al From blood islands to blood vessels

morphologic observations and expression of key molecules during hyaloid vascular

system development Invest Ophthalmol Vis Sci 2012537912ndash792718 Iwatsuki K Tanaka K Kaneko T et al Runx1 promotes angiogenesis by

downregulation of insulin-like growth factor-binding protein-3 Oncogene 200524

1129ndash113719 Namba K Abe M Saito S et al Indispensable role of the transcription factor

PEBP2CBF in angiogenic activity of a murine endothelial cell MSS31 Oncogene

200019106ndash11420 Cunningham L Finckbeiner S Hyde RK et al Identification of benzodiazepine

Ro5-3335 as an inhibitor of CBF leukemia through quantitative high throughput

screen against RUNX1-CBFb interaction Proc Natl Acad Sci U S A 201210914592ndash

1459721 Burns CE Traver D Mayhall E Shepard JL Zon LI Hematopoietic stem cell fate

is established by the Notch-Runx pathway Genes Dev 2005192331ndash234222 Ito Y Miyazono K RUNX transcription factors as key targets of TGF-beta su-

perfamily signaling Curr Opin Genet Dev 20031343ndash4723 Doll A Gonzalez M Abal M et al An orthotopic endometrial cancer mouse

model demonstrates a role for RUNX1 in distant metastasis Int J Cancer 2009125

257ndash26324 Scheitz CJ Lee TS McDermitt DJ Tumbar T Defining a tissue stem cell-driven

Runx1Stat3 signalling axis in epithelial cancer EMBO J 2012314124ndash413925 Planagumagrave J Diacuteaz-Fuertes M Gil-Moreno A et al A differential gene ex-

pression profile reveals overexpression of RUNX1AML1 in invasive endometrioid

carcinoma Cancer Res 2004648846ndash885326 Nagamachi A Htun PW Ma F et al A 59 untranslated region containing the

IRES element in the Runx1 gene is required for angiogenesis hematopoiesis and

leukemogenesis in a knock-in mouse model Dev Biol 2010345226ndash23627 Harris JM Esain V Frechette GM et al Glucose metabolism impacts the

spatiotemporal onset and magnitude of HSC induction in vivo Blood 2013121

2483ndash249328 Imanirad P Solaimani Kartalaei P Crisan M et al HIF1a is a regulator of

hematopoietic progenitor and stem cell development in hypoxic sites of the mouse

embryo Stem Cell Res (Amst) 20141224ndash3529 Ishikawa K Yoshida S Kobayashi Y et al Microarray analysis of gene ex-

pression in fibrovascular membranes excised from patients with proliferative diabetic

retinopathy Invest Ophthalmol Vis Sci 201556932ndash94630 McAuley AK Sanfilippo PG Hewitt AW et al Vitreous biomarkers in diabetic

retinopathy a systematic review and meta-analysis J Diabetes Complications 2014

28419ndash425

1956 Role of RUNX1 in Aberrant Retinal Angiogenesis Diabetes Volume 66 July 2017

one-way ANOVA (Kruskal-Wallis test) was used for compar-isons between multiple groups A P value 005 was con-sidered significant

RESULTS

RNA Sequencing of CD311 Cells From FVMsWhole transcriptomic profiles were constructed for CD311

cells from FVMs and compared with transcriptomes ofCD311 cells from postmortem retinas isolated from indi-viduals without diabetes (Supplementary Fig 1 and Supple-mentary Table 1) Postmortem retinas were used to defineexpression baseline because there is no normal correlate ofFVMs CD311 cells were identified as vascular ECs based ontheir expression of five vascular endothelial markers CD93CD31 KLF4 ESAM and VEGFR1 (Supplementary Table 3)However because a single marker namely CD31 was usedfor cell isolation the presence of other cell types cannot beruled out Principal component analysis of the tran-scriptomes demonstrated congruent expression profilesfor control samples whereas profiles of cells from theFVM samples had more variable gene expression pat-terns (Fig 1A) The data sets are accessible through the

National Center for Biotechnology Informationrsquos GeneExpression Omnibus (GSE94019) (15)

Two hundred genes were identified as differentiallyexpressed in CD311 cells from FVMs involving gene ontol-ogy categories related to wound response vessel develop-ment angiogenesis and other categories (Fig 1B andSupplementary Table 4)

RUNX1 Is Upregulated in Cultured ECs Exposedto High GlucoseHyperglycemia is a major risk factor for progression toPDR in patients with diabetes (16) High glucose-dependentgenes from the candidate gene list were identified in pri-mary HRMEC culture using a qRT-PCR screen Four out of101 genes were glucose responsive consistent with tran-scriptomic data (fold change 6 SEM) RUNX1 (29 6 02)peptidyl-prolyl cis-trans isomerase F (PPIF) (36 6 05)serinethreonine-protein kinase PIM3 (PIM3) (39 6 10)and CD44 (35 6 04) (Fig 2A and B) We focused onRUNX1 for further studies because of its documented rolesin EC biology (17ndash19) Exposure of HRMEC and HUVECcultures to high glucose induced a 30 increase inRUNX1 protein whereas osmotic controls had no significant

Figure 1mdashPrinciple component analysis of transcriptomes and gene ontology of candidate genes A Two-dimensional representations of thethree most significant principle components (PCs) PC1 PC2 and PC3 account for 4537 of the observed sample variance The widedistribution of transcriptomes from FVM-derived CD311 cells contrasts the congruity of transcriptomes of cells from normal retinal samplesindicating significant variability in FVM gene expression levels B The top 20 pathways are presented in order of descending significance withthe number of genes from the candidate gene list enriching the pathway

1952 Role of RUNX1 in Aberrant Retinal Angiogenesis Diabetes Volume 66 July 2017

effect in HRMEC and HUVECs (Fig 2C and D) Lastly weconfirmed increased RUNX1 protein expression in vesselsfrom FVMs compared with retina from control subjectswithout diabetes using immunohistochemical staining(Fig 2E)

RUNX1 Regulates Migration and Proliferation ofEndothelial Cells In VitrosiRNA knockdown of RUNX1 resulted in a 60 decrease inwound closure rate indicating a significant role of RUNX1

in endothelial cell migration (Fig 3A) The coefficient ofdetermination (r2 value) for the simple linear regressionwas calculated comparing wound closure (dependent vari-able) to time (independent variable) (control r2 5 099scramble siRNA r2 5 097 and RUNX1 siRNA r2 5 097)This experiment was validated with a second siRNA target-ing RUNX1 to rule out possible off-target effects (Supple-mentary Fig 2B) ECs transfected with RUNX1 siRNA had40 fewer Ki67-positive cells compared with controls 24 h

Figure 2mdashEffect of elevated glucose on HRMEC gene expression A Results of FVM RNA-sequencing (reads per kilobase of transcript permillion mapped reads) show increased expression of RUNX1 PPIF PIM3 and CD44 in ECs from patients with PDR compared with patientswithout diabetes CXCL10 did not exhibit increased expression in FVMs (ncontrols 5 4 nPDR-FVM 5 8) B Corresponding gene expression ofRUNX1 PPIF PIM3 CD44 and CXCL10 measured by qRT-PCR (HG high D-glucose [30 mmolL] NG normal D-glucose [5 mmolL]) Eachcandidate gene had a marked increase in response to D-glucose but no statistically significant changes in response to osmotic controls(L-glucose or mannitol) CXCL10 is not glucose responsive consistent with RNA-sequencing (n5 3 the experiment was performed in triplicate)C Increasing D-glucose led to a dose-dependent increase of RUNX1 protein expression in HRMECs (top) and HUVECs (bottom) as determinedby Western blot (n5 3 experiment performed in triplicate) D The increase in RUNX1 protein in HRMECs induced by 30 mmolL D-glucose wasindependent of osmotic forces (n 5 3 experiment performed in triplicate) E Normal retinal vessels showed no staining of RUNX1 (L vessellumen RPE retinal pigment epithelium) (left panel) Scale bars 5 100 mm A subset of vessels in FVM stained positively for RUNX1 (arrows)Asterisks denote nonstaining vessels (left) Scale bar 5 50 mm P lt 005 P lt 001 P lt 0001

diabetesdiabetesjournalsorg Lam and Associates 1953

after transfection indicating RUNX1 also contributes toretinal EC proliferation (Fig 3B)

Ro5ndash3335 RUNX1-Core Binding Factor b InhibitorBlocks EC Tube Formation In Vitro and AberrantAngiogenesis In VivoTo investigate the potential therapeutic relevance ofRUNX1 the small molecule Ro5ndash3335 RUNX1ndashcore bindingfactor b inhibitor II was tested in vitro and in vivo (20)Treatment with RUNX1 inhibitor reduced total tube length(18 reduction) nodes (35 reduction) and meshes (46reduction) supporting RUNX1rsquos role in vascular morpho-genesis (Fig 3C)

Neovascular tufts of P17 C57BL6J mice with OIRidentified by isolectin B4 and CD31 staining showedincreased expression of RUNX1 whereas RUNX1 stainingwas absent from the underlying normal retinal vasculatureindicating a role for RUNX1 in active pathological angio-genesis (Fig 4A) In vivo testing of the effects on aberrantangiogenesis of RUNX1 inhibition was conducted by inject-ing Ro5ndash3335 intravitreally in mice with OIR There was nosignificant change in the extent of vaso-obliteration and noeffect was observed in the contralateral eye (data not shown)

Eyes treated with inhibitor exhibited a 50 reductionin neovascularization compared with vehicle-treated eyes(Fig 4B)

DISCUSSION

Our analyses of CD311 cells from FVMs of patients withPDR in conjunction with in vitro and in vivo validationassays suggest a role for RUNX1 in the proliferation mi-gration and morphogenesis of ECs in vitro as well as inaberrant angiogenesis in vivo RUNX proteins have pleio-tropic functions in vascular development hematopoiesisand cancer through direct transcriptional regulation oftarget genes and complex interactions with fundamentalsignaling mechanisms including Notch and transforminggrowth factor-b pathways (2122) RUNX1 is a major con-tributor to cancer progression and metastasis most notablyin acute myeloid leukemia and also plays a role in angio-genesis (1823ndash26)

Previous studies in model organisms have shown thatglucose levels trigger RUNX1 expression via reactive oxygenspeciesndashmediated upregulation of hypoxia-inducible factor1 and regulate hematopoietic stem cell production by

Figure 3mdashRole of RUNX1 in EC function A Scratch-wound assay using HRMECs at 0 (left column) 6 (middle column) and 12 h (rightcolumn) with scramble siRNA (top) and RUNX1 siRNA (bottom) treatment Dark gray regions denote wound areas Scale bar 5400 mm Quantification of wound closure rates shows that knockdown of RUNX1 effectively inhibits wound closure (n 5 10 experimentperformed in duplicate) B Ki67 staining 48 h posttransfection demonstrates significant reduction in cell number and proliferative capacity ofRUNX1 siRNA-treated cells compared with cells treated with scramble siRNA Scale bar 5 200 mm Quantification of percentage of DAPI-positive nuclei colocalized with Ki67 stain (n 5 6 experiment performed in duplicate) C HRMECs treated with Ro5ndash3335 RUNX1 inhibitorovernight exhibited reduced tube-forming capacity compared with vehicle-treated cells at 6 h after plating There was statistically significantreduction in tube length meshes and nodes Scale bar 5 500 mm n 5 4 experiment performed in duplicate P lt 005 P lt 001 P lt0001

1954 Role of RUNX1 in Aberrant Retinal Angiogenesis Diabetes Volume 66 July 2017

endothelial to hematopoietic transition in a RUNX1-dependentmanner (2728) Consistent with these observations ourresults demonstrate that high glucose regulates RUNX1 andimplicates RUNX1 in aberrant retinal angiogenesis

Previous studies of PDR have mainly focused on geneexpression in FVMs or biomarkers in the vitreous (2930)Although FVMs from PDR may be highly informative path-ological tissues they are largely unstudied The window forprocessing samples postsurgery is narrow and intraopera-tive confounders such as FVM size location and involve-ment of sensory retina further complicate successful sampleacquisition CD311 cells from FVMs do not grow readily inculture making additional experimentation difficult Lim-ited sample size may also skew our gene ontology analysis

We report elevated RUNX1 expression in ECs of patient-derived FVMs from patients with PDR Also we demon-strate a role for RUNX1 in EC migration proliferation andvascular morphogenesis Furthermore we report selectivelyenhanced expression of RUNX1 in neovascular tufts in anexperimental model of OIR and that inhibition of RUNX1function reduced retinal neovascularization These findings

including the high glucosendashdependent expression of RUNX1identify a novel pathway of potential therapeutic interestand implicate RUNX1 in aberrant angiogenesis in multipleconditions

Acknowledgments The authors thank Dr Magali Saint-Geniez for insightfuldiscussions and providing mouse retinal samples Dr Eric Ng Mark Graham andDr Arturo Machuca-Parra for helpful discussions and Dr Christina Marko fortechnical contributions (all from Schepens Eye Research Institute Boston MA) Theauthors also thank Kristin Waraska and Andrew Gagne of the Harvard BiopolymersFacility Harvard Medical School and the Ocular Genetics Institute core facilityMassachusetts Eye and Ear (Boston MA) for their insights and workFunding The work performed in the manuscript was supported by NationalInstitutes of Health grants R01-EY-005318 (to PAD) R00-EY-021624 (to JFA-V)UH2NS100121-01 (to JFA-V) R21-EY-027061 (to LAK) K12-EY-16335 (toLAK) and Center Core grant P30-EY-003790 American Diabetes AssociationInnovation Award 7ndash12-IN-11 (to PAD) Massachusetts Lions Eye Research Fund(to JFA-V) E Matilda Ziegler Foundation for the Blind (to LAK) Karl KirchgessnerFoundation (to LAK) Department of Ophthalmology Harvard Medical School(to JFA-V and LAK) the Howard Hughes Medical Institute Medical ResearchFellows Program (to DJO) and the Vascular Biology Program at Boston ChildrenrsquosHospital (to DRB)

Figure 4mdashRUNX1 inhibition reduces neovascularization in the OIR model A Retina of P17 C57BL6J mice with OIR costained for RUNX1 andvessels (IB4 or CD31) showing positive RUNX1 staining conforming to neovascular tufts (arrows) and not to normal underlying vasculature Scalebars5 50 mm B Retinal flat mounts of P17 C57BL6J mice following OIR induction and intravitreal injection of 75 mmolL Ro5ndash3335 or vehicleat P13 and P15 (red overlay identifies the avascular area and neovascularization respectively) Scale bars 5 1 mm There was a nonsignificantdownward trend in avascular area but a significant reduction in neovascularization in the treated group compared with vehicle-treated group(nvehicle 5 7 nRo5ndash3335 5 9) Experiment performed in triplicate P lt 001

diabetesdiabetesjournalsorg Lam and Associates 1955

Duality of Interest PAD LAK and JFA-V have filed a provisional patentapplication on RUNX1 regulation for the treatment of aberrant angiogenesis and PDRNo other potential conflicts of interest relevant to this article were reportedAuthor Contributions JDL and DJO performed the experimentsanalyzed the data and wrote the manuscript LLW DA CP-W AVS andJC-V performed experiments DM AOS-R DE and DRB provided tissuesamples and reviewed and edited the manuscript TvZ LS and XG obtaineddata and reviewed the manuscript PAD LAK and JFA-V conceived theproject analyzed the data and wrote the manuscript PAD LAK and JFA-V arethe guarantors of this work and as such had full access to all the data in the studyand take responsibility for the integrity of the data and the accuracy of the dataanalysis

References1 Chung AS Ferrara N Developmental and pathological angiogenesis Annu RevCell Dev Biol 201127563ndash5842 Kim LA DrsquoAmore PA A brief history of anti-VEGF for the treatment of ocularangiogenesis Am J Pathol 2012181376ndash3793 Hsu YJ Hsieh YT Yeh PT Huang JY Yang CM Combined tractional andrhegmatogenous retinal detachment in proliferative diabetic retinopathy in the anti-VEGF era J Ophthalmol 201420149173754 Kuiper EJ Van Nieuwenhoven FA de Smet MD et al The angio-fibrotic switchof VEGF and CTGF in proliferative diabetic retinopathy PLoS One 20083e26755 Saint-Geniez M Maharaj AS Walshe TE et al Endogenous VEGF is required forvisual function evidence for a survival role on muumlller cells and photoreceptors PLoSOne 20083e35546 Sang DN DrsquoAmore PA Is blockade of vascular endothelial growth factorbeneficial for all types of diabetic retinopathy Diabetologia 2008511570ndash15737 Jo DH Cho CS Kim JH Jun HO Kim JH Animal models of diabetic reti-nopathy doors to investigate pathogenesis and potential therapeutics J Biomed Sci201320388 Kim LA Wong LL Amarnani DS et al Characterization of cells from patient-derived fibrovascular membranes in proliferative diabetic retinopathy Mol Vis 201521673ndash6879 Trapnell C Pachter L Salzberg SL TopHat discovering splice junctions withRNA-Seq Bioinformatics 2009251105ndash111110 Huang da W Sherman BT Lempicki RA Bioinformatics enrichment tools pathstoward the comprehensive functional analysis of large gene lists Nucleic Acids Res2009371ndash1311 Smith LE Wesolowski E McLellan A et al Oxygen-induced retinopathy in themouse Invest Ophthalmol Vis Sci 199435101ndash11112 Connor KM Krah NM Dennison RJ et al Quantification of oxygen-inducedretinopathy in the mouse a model of vessel loss vessel regrowth and pathologicalangiogenesis Nat Protoc 200941565ndash157313 Liang CC Park AY Guan JL In vitro scratch assay a convenient and in-expensive method for analysis of cell migration in vitro Nat Protoc 20072329ndash33314 Gebaumlck T Schulz MM Koumoutsakos P Detmar M TScratch a novel andsimple software tool for automated analysis of monolayer wound healing assaysBiotechniques 200946265ndash274

15 Edgar R Domrachev M Lash AE Gene Expression Omnibus NCBI gene ex-

pression and hybridization array data repository Nucleic Acids Res 200230207ndash

21016 The Diabetes Control and Complications Trial Research Group The relationship

of glycemic exposure (HbA1c) to the risk of development and progression of reti-

nopathy in the diabetes control and complications trial Diabetes 199544968ndash98317 McLeod DS Hasegawa T Baba T et al From blood islands to blood vessels

morphologic observations and expression of key molecules during hyaloid vascular

system development Invest Ophthalmol Vis Sci 2012537912ndash792718 Iwatsuki K Tanaka K Kaneko T et al Runx1 promotes angiogenesis by

downregulation of insulin-like growth factor-binding protein-3 Oncogene 200524

1129ndash113719 Namba K Abe M Saito S et al Indispensable role of the transcription factor

PEBP2CBF in angiogenic activity of a murine endothelial cell MSS31 Oncogene

200019106ndash11420 Cunningham L Finckbeiner S Hyde RK et al Identification of benzodiazepine

Ro5-3335 as an inhibitor of CBF leukemia through quantitative high throughput

screen against RUNX1-CBFb interaction Proc Natl Acad Sci U S A 201210914592ndash

1459721 Burns CE Traver D Mayhall E Shepard JL Zon LI Hematopoietic stem cell fate

is established by the Notch-Runx pathway Genes Dev 2005192331ndash234222 Ito Y Miyazono K RUNX transcription factors as key targets of TGF-beta su-

perfamily signaling Curr Opin Genet Dev 20031343ndash4723 Doll A Gonzalez M Abal M et al An orthotopic endometrial cancer mouse

model demonstrates a role for RUNX1 in distant metastasis Int J Cancer 2009125

257ndash26324 Scheitz CJ Lee TS McDermitt DJ Tumbar T Defining a tissue stem cell-driven

Runx1Stat3 signalling axis in epithelial cancer EMBO J 2012314124ndash413925 Planagumagrave J Diacuteaz-Fuertes M Gil-Moreno A et al A differential gene ex-

pression profile reveals overexpression of RUNX1AML1 in invasive endometrioid

carcinoma Cancer Res 2004648846ndash885326 Nagamachi A Htun PW Ma F et al A 59 untranslated region containing the

IRES element in the Runx1 gene is required for angiogenesis hematopoiesis and

leukemogenesis in a knock-in mouse model Dev Biol 2010345226ndash23627 Harris JM Esain V Frechette GM et al Glucose metabolism impacts the

spatiotemporal onset and magnitude of HSC induction in vivo Blood 2013121

2483ndash249328 Imanirad P Solaimani Kartalaei P Crisan M et al HIF1a is a regulator of

hematopoietic progenitor and stem cell development in hypoxic sites of the mouse

embryo Stem Cell Res (Amst) 20141224ndash3529 Ishikawa K Yoshida S Kobayashi Y et al Microarray analysis of gene ex-

pression in fibrovascular membranes excised from patients with proliferative diabetic

retinopathy Invest Ophthalmol Vis Sci 201556932ndash94630 McAuley AK Sanfilippo PG Hewitt AW et al Vitreous biomarkers in diabetic

retinopathy a systematic review and meta-analysis J Diabetes Complications 2014

28419ndash425

1956 Role of RUNX1 in Aberrant Retinal Angiogenesis Diabetes Volume 66 July 2017

effect in HRMEC and HUVECs (Fig 2C and D) Lastly weconfirmed increased RUNX1 protein expression in vesselsfrom FVMs compared with retina from control subjectswithout diabetes using immunohistochemical staining(Fig 2E)

RUNX1 Regulates Migration and Proliferation ofEndothelial Cells In VitrosiRNA knockdown of RUNX1 resulted in a 60 decrease inwound closure rate indicating a significant role of RUNX1

in endothelial cell migration (Fig 3A) The coefficient ofdetermination (r2 value) for the simple linear regressionwas calculated comparing wound closure (dependent vari-able) to time (independent variable) (control r2 5 099scramble siRNA r2 5 097 and RUNX1 siRNA r2 5 097)This experiment was validated with a second siRNA target-ing RUNX1 to rule out possible off-target effects (Supple-mentary Fig 2B) ECs transfected with RUNX1 siRNA had40 fewer Ki67-positive cells compared with controls 24 h

Figure 2mdashEffect of elevated glucose on HRMEC gene expression A Results of FVM RNA-sequencing (reads per kilobase of transcript permillion mapped reads) show increased expression of RUNX1 PPIF PIM3 and CD44 in ECs from patients with PDR compared with patientswithout diabetes CXCL10 did not exhibit increased expression in FVMs (ncontrols 5 4 nPDR-FVM 5 8) B Corresponding gene expression ofRUNX1 PPIF PIM3 CD44 and CXCL10 measured by qRT-PCR (HG high D-glucose [30 mmolL] NG normal D-glucose [5 mmolL]) Eachcandidate gene had a marked increase in response to D-glucose but no statistically significant changes in response to osmotic controls(L-glucose or mannitol) CXCL10 is not glucose responsive consistent with RNA-sequencing (n5 3 the experiment was performed in triplicate)C Increasing D-glucose led to a dose-dependent increase of RUNX1 protein expression in HRMECs (top) and HUVECs (bottom) as determinedby Western blot (n5 3 experiment performed in triplicate) D The increase in RUNX1 protein in HRMECs induced by 30 mmolL D-glucose wasindependent of osmotic forces (n 5 3 experiment performed in triplicate) E Normal retinal vessels showed no staining of RUNX1 (L vessellumen RPE retinal pigment epithelium) (left panel) Scale bars 5 100 mm A subset of vessels in FVM stained positively for RUNX1 (arrows)Asterisks denote nonstaining vessels (left) Scale bar 5 50 mm P lt 005 P lt 001 P lt 0001

diabetesdiabetesjournalsorg Lam and Associates 1953

after transfection indicating RUNX1 also contributes toretinal EC proliferation (Fig 3B)

Ro5ndash3335 RUNX1-Core Binding Factor b InhibitorBlocks EC Tube Formation In Vitro and AberrantAngiogenesis In VivoTo investigate the potential therapeutic relevance ofRUNX1 the small molecule Ro5ndash3335 RUNX1ndashcore bindingfactor b inhibitor II was tested in vitro and in vivo (20)Treatment with RUNX1 inhibitor reduced total tube length(18 reduction) nodes (35 reduction) and meshes (46reduction) supporting RUNX1rsquos role in vascular morpho-genesis (Fig 3C)

Neovascular tufts of P17 C57BL6J mice with OIRidentified by isolectin B4 and CD31 staining showedincreased expression of RUNX1 whereas RUNX1 stainingwas absent from the underlying normal retinal vasculatureindicating a role for RUNX1 in active pathological angio-genesis (Fig 4A) In vivo testing of the effects on aberrantangiogenesis of RUNX1 inhibition was conducted by inject-ing Ro5ndash3335 intravitreally in mice with OIR There was nosignificant change in the extent of vaso-obliteration and noeffect was observed in the contralateral eye (data not shown)

Eyes treated with inhibitor exhibited a 50 reductionin neovascularization compared with vehicle-treated eyes(Fig 4B)

DISCUSSION

Our analyses of CD311 cells from FVMs of patients withPDR in conjunction with in vitro and in vivo validationassays suggest a role for RUNX1 in the proliferation mi-gration and morphogenesis of ECs in vitro as well as inaberrant angiogenesis in vivo RUNX proteins have pleio-tropic functions in vascular development hematopoiesisand cancer through direct transcriptional regulation oftarget genes and complex interactions with fundamentalsignaling mechanisms including Notch and transforminggrowth factor-b pathways (2122) RUNX1 is a major con-tributor to cancer progression and metastasis most notablyin acute myeloid leukemia and also plays a role in angio-genesis (1823ndash26)

Previous studies in model organisms have shown thatglucose levels trigger RUNX1 expression via reactive oxygenspeciesndashmediated upregulation of hypoxia-inducible factor1 and regulate hematopoietic stem cell production by

Figure 3mdashRole of RUNX1 in EC function A Scratch-wound assay using HRMECs at 0 (left column) 6 (middle column) and 12 h (rightcolumn) with scramble siRNA (top) and RUNX1 siRNA (bottom) treatment Dark gray regions denote wound areas Scale bar 5400 mm Quantification of wound closure rates shows that knockdown of RUNX1 effectively inhibits wound closure (n 5 10 experimentperformed in duplicate) B Ki67 staining 48 h posttransfection demonstrates significant reduction in cell number and proliferative capacity ofRUNX1 siRNA-treated cells compared with cells treated with scramble siRNA Scale bar 5 200 mm Quantification of percentage of DAPI-positive nuclei colocalized with Ki67 stain (n 5 6 experiment performed in duplicate) C HRMECs treated with Ro5ndash3335 RUNX1 inhibitorovernight exhibited reduced tube-forming capacity compared with vehicle-treated cells at 6 h after plating There was statistically significantreduction in tube length meshes and nodes Scale bar 5 500 mm n 5 4 experiment performed in duplicate P lt 005 P lt 001 P lt0001

1954 Role of RUNX1 in Aberrant Retinal Angiogenesis Diabetes Volume 66 July 2017

endothelial to hematopoietic transition in a RUNX1-dependentmanner (2728) Consistent with these observations ourresults demonstrate that high glucose regulates RUNX1 andimplicates RUNX1 in aberrant retinal angiogenesis

Previous studies of PDR have mainly focused on geneexpression in FVMs or biomarkers in the vitreous (2930)Although FVMs from PDR may be highly informative path-ological tissues they are largely unstudied The window forprocessing samples postsurgery is narrow and intraopera-tive confounders such as FVM size location and involve-ment of sensory retina further complicate successful sampleacquisition CD311 cells from FVMs do not grow readily inculture making additional experimentation difficult Lim-ited sample size may also skew our gene ontology analysis

We report elevated RUNX1 expression in ECs of patient-derived FVMs from patients with PDR Also we demon-strate a role for RUNX1 in EC migration proliferation andvascular morphogenesis Furthermore we report selectivelyenhanced expression of RUNX1 in neovascular tufts in anexperimental model of OIR and that inhibition of RUNX1function reduced retinal neovascularization These findings

including the high glucosendashdependent expression of RUNX1identify a novel pathway of potential therapeutic interestand implicate RUNX1 in aberrant angiogenesis in multipleconditions

Acknowledgments The authors thank Dr Magali Saint-Geniez for insightfuldiscussions and providing mouse retinal samples Dr Eric Ng Mark Graham andDr Arturo Machuca-Parra for helpful discussions and Dr Christina Marko fortechnical contributions (all from Schepens Eye Research Institute Boston MA) Theauthors also thank Kristin Waraska and Andrew Gagne of the Harvard BiopolymersFacility Harvard Medical School and the Ocular Genetics Institute core facilityMassachusetts Eye and Ear (Boston MA) for their insights and workFunding The work performed in the manuscript was supported by NationalInstitutes of Health grants R01-EY-005318 (to PAD) R00-EY-021624 (to JFA-V)UH2NS100121-01 (to JFA-V) R21-EY-027061 (to LAK) K12-EY-16335 (toLAK) and Center Core grant P30-EY-003790 American Diabetes AssociationInnovation Award 7ndash12-IN-11 (to PAD) Massachusetts Lions Eye Research Fund(to JFA-V) E Matilda Ziegler Foundation for the Blind (to LAK) Karl KirchgessnerFoundation (to LAK) Department of Ophthalmology Harvard Medical School(to JFA-V and LAK) the Howard Hughes Medical Institute Medical ResearchFellows Program (to DJO) and the Vascular Biology Program at Boston ChildrenrsquosHospital (to DRB)

Figure 4mdashRUNX1 inhibition reduces neovascularization in the OIR model A Retina of P17 C57BL6J mice with OIR costained for RUNX1 andvessels (IB4 or CD31) showing positive RUNX1 staining conforming to neovascular tufts (arrows) and not to normal underlying vasculature Scalebars5 50 mm B Retinal flat mounts of P17 C57BL6J mice following OIR induction and intravitreal injection of 75 mmolL Ro5ndash3335 or vehicleat P13 and P15 (red overlay identifies the avascular area and neovascularization respectively) Scale bars 5 1 mm There was a nonsignificantdownward trend in avascular area but a significant reduction in neovascularization in the treated group compared with vehicle-treated group(nvehicle 5 7 nRo5ndash3335 5 9) Experiment performed in triplicate P lt 001

diabetesdiabetesjournalsorg Lam and Associates 1955

Duality of Interest PAD LAK and JFA-V have filed a provisional patentapplication on RUNX1 regulation for the treatment of aberrant angiogenesis and PDRNo other potential conflicts of interest relevant to this article were reportedAuthor Contributions JDL and DJO performed the experimentsanalyzed the data and wrote the manuscript LLW DA CP-W AVS andJC-V performed experiments DM AOS-R DE and DRB provided tissuesamples and reviewed and edited the manuscript TvZ LS and XG obtaineddata and reviewed the manuscript PAD LAK and JFA-V conceived theproject analyzed the data and wrote the manuscript PAD LAK and JFA-V arethe guarantors of this work and as such had full access to all the data in the studyand take responsibility for the integrity of the data and the accuracy of the dataanalysis

References1 Chung AS Ferrara N Developmental and pathological angiogenesis Annu RevCell Dev Biol 201127563ndash5842 Kim LA DrsquoAmore PA A brief history of anti-VEGF for the treatment of ocularangiogenesis Am J Pathol 2012181376ndash3793 Hsu YJ Hsieh YT Yeh PT Huang JY Yang CM Combined tractional andrhegmatogenous retinal detachment in proliferative diabetic retinopathy in the anti-VEGF era J Ophthalmol 201420149173754 Kuiper EJ Van Nieuwenhoven FA de Smet MD et al The angio-fibrotic switchof VEGF and CTGF in proliferative diabetic retinopathy PLoS One 20083e26755 Saint-Geniez M Maharaj AS Walshe TE et al Endogenous VEGF is required forvisual function evidence for a survival role on muumlller cells and photoreceptors PLoSOne 20083e35546 Sang DN DrsquoAmore PA Is blockade of vascular endothelial growth factorbeneficial for all types of diabetic retinopathy Diabetologia 2008511570ndash15737 Jo DH Cho CS Kim JH Jun HO Kim JH Animal models of diabetic reti-nopathy doors to investigate pathogenesis and potential therapeutics J Biomed Sci201320388 Kim LA Wong LL Amarnani DS et al Characterization of cells from patient-derived fibrovascular membranes in proliferative diabetic retinopathy Mol Vis 201521673ndash6879 Trapnell C Pachter L Salzberg SL TopHat discovering splice junctions withRNA-Seq Bioinformatics 2009251105ndash111110 Huang da W Sherman BT Lempicki RA Bioinformatics enrichment tools pathstoward the comprehensive functional analysis of large gene lists Nucleic Acids Res2009371ndash1311 Smith LE Wesolowski E McLellan A et al Oxygen-induced retinopathy in themouse Invest Ophthalmol Vis Sci 199435101ndash11112 Connor KM Krah NM Dennison RJ et al Quantification of oxygen-inducedretinopathy in the mouse a model of vessel loss vessel regrowth and pathologicalangiogenesis Nat Protoc 200941565ndash157313 Liang CC Park AY Guan JL In vitro scratch assay a convenient and in-expensive method for analysis of cell migration in vitro Nat Protoc 20072329ndash33314 Gebaumlck T Schulz MM Koumoutsakos P Detmar M TScratch a novel andsimple software tool for automated analysis of monolayer wound healing assaysBiotechniques 200946265ndash274

15 Edgar R Domrachev M Lash AE Gene Expression Omnibus NCBI gene ex-

pression and hybridization array data repository Nucleic Acids Res 200230207ndash

21016 The Diabetes Control and Complications Trial Research Group The relationship

of glycemic exposure (HbA1c) to the risk of development and progression of reti-

nopathy in the diabetes control and complications trial Diabetes 199544968ndash98317 McLeod DS Hasegawa T Baba T et al From blood islands to blood vessels

morphologic observations and expression of key molecules during hyaloid vascular

system development Invest Ophthalmol Vis Sci 2012537912ndash792718 Iwatsuki K Tanaka K Kaneko T et al Runx1 promotes angiogenesis by

downregulation of insulin-like growth factor-binding protein-3 Oncogene 200524

1129ndash113719 Namba K Abe M Saito S et al Indispensable role of the transcription factor

PEBP2CBF in angiogenic activity of a murine endothelial cell MSS31 Oncogene

200019106ndash11420 Cunningham L Finckbeiner S Hyde RK et al Identification of benzodiazepine

Ro5-3335 as an inhibitor of CBF leukemia through quantitative high throughput

screen against RUNX1-CBFb interaction Proc Natl Acad Sci U S A 201210914592ndash

1459721 Burns CE Traver D Mayhall E Shepard JL Zon LI Hematopoietic stem cell fate

is established by the Notch-Runx pathway Genes Dev 2005192331ndash234222 Ito Y Miyazono K RUNX transcription factors as key targets of TGF-beta su-

perfamily signaling Curr Opin Genet Dev 20031343ndash4723 Doll A Gonzalez M Abal M et al An orthotopic endometrial cancer mouse

model demonstrates a role for RUNX1 in distant metastasis Int J Cancer 2009125

257ndash26324 Scheitz CJ Lee TS McDermitt DJ Tumbar T Defining a tissue stem cell-driven

Runx1Stat3 signalling axis in epithelial cancer EMBO J 2012314124ndash413925 Planagumagrave J Diacuteaz-Fuertes M Gil-Moreno A et al A differential gene ex-

pression profile reveals overexpression of RUNX1AML1 in invasive endometrioid

carcinoma Cancer Res 2004648846ndash885326 Nagamachi A Htun PW Ma F et al A 59 untranslated region containing the

IRES element in the Runx1 gene is required for angiogenesis hematopoiesis and

leukemogenesis in a knock-in mouse model Dev Biol 2010345226ndash23627 Harris JM Esain V Frechette GM et al Glucose metabolism impacts the

spatiotemporal onset and magnitude of HSC induction in vivo Blood 2013121

2483ndash249328 Imanirad P Solaimani Kartalaei P Crisan M et al HIF1a is a regulator of

hematopoietic progenitor and stem cell development in hypoxic sites of the mouse

embryo Stem Cell Res (Amst) 20141224ndash3529 Ishikawa K Yoshida S Kobayashi Y et al Microarray analysis of gene ex-

pression in fibrovascular membranes excised from patients with proliferative diabetic

retinopathy Invest Ophthalmol Vis Sci 201556932ndash94630 McAuley AK Sanfilippo PG Hewitt AW et al Vitreous biomarkers in diabetic

retinopathy a systematic review and meta-analysis J Diabetes Complications 2014

28419ndash425

1956 Role of RUNX1 in Aberrant Retinal Angiogenesis Diabetes Volume 66 July 2017

after transfection indicating RUNX1 also contributes toretinal EC proliferation (Fig 3B)

Ro5ndash3335 RUNX1-Core Binding Factor b InhibitorBlocks EC Tube Formation In Vitro and AberrantAngiogenesis In VivoTo investigate the potential therapeutic relevance ofRUNX1 the small molecule Ro5ndash3335 RUNX1ndashcore bindingfactor b inhibitor II was tested in vitro and in vivo (20)Treatment with RUNX1 inhibitor reduced total tube length(18 reduction) nodes (35 reduction) and meshes (46reduction) supporting RUNX1rsquos role in vascular morpho-genesis (Fig 3C)

Neovascular tufts of P17 C57BL6J mice with OIRidentified by isolectin B4 and CD31 staining showedincreased expression of RUNX1 whereas RUNX1 stainingwas absent from the underlying normal retinal vasculatureindicating a role for RUNX1 in active pathological angio-genesis (Fig 4A) In vivo testing of the effects on aberrantangiogenesis of RUNX1 inhibition was conducted by inject-ing Ro5ndash3335 intravitreally in mice with OIR There was nosignificant change in the extent of vaso-obliteration and noeffect was observed in the contralateral eye (data not shown)

Eyes treated with inhibitor exhibited a 50 reductionin neovascularization compared with vehicle-treated eyes(Fig 4B)

DISCUSSION

Our analyses of CD311 cells from FVMs of patients withPDR in conjunction with in vitro and in vivo validationassays suggest a role for RUNX1 in the proliferation mi-gration and morphogenesis of ECs in vitro as well as inaberrant angiogenesis in vivo RUNX proteins have pleio-tropic functions in vascular development hematopoiesisand cancer through direct transcriptional regulation oftarget genes and complex interactions with fundamentalsignaling mechanisms including Notch and transforminggrowth factor-b pathways (2122) RUNX1 is a major con-tributor to cancer progression and metastasis most notablyin acute myeloid leukemia and also plays a role in angio-genesis (1823ndash26)

Previous studies in model organisms have shown thatglucose levels trigger RUNX1 expression via reactive oxygenspeciesndashmediated upregulation of hypoxia-inducible factor1 and regulate hematopoietic stem cell production by

Figure 3mdashRole of RUNX1 in EC function A Scratch-wound assay using HRMECs at 0 (left column) 6 (middle column) and 12 h (rightcolumn) with scramble siRNA (top) and RUNX1 siRNA (bottom) treatment Dark gray regions denote wound areas Scale bar 5400 mm Quantification of wound closure rates shows that knockdown of RUNX1 effectively inhibits wound closure (n 5 10 experimentperformed in duplicate) B Ki67 staining 48 h posttransfection demonstrates significant reduction in cell number and proliferative capacity ofRUNX1 siRNA-treated cells compared with cells treated with scramble siRNA Scale bar 5 200 mm Quantification of percentage of DAPI-positive nuclei colocalized with Ki67 stain (n 5 6 experiment performed in duplicate) C HRMECs treated with Ro5ndash3335 RUNX1 inhibitorovernight exhibited reduced tube-forming capacity compared with vehicle-treated cells at 6 h after plating There was statistically significantreduction in tube length meshes and nodes Scale bar 5 500 mm n 5 4 experiment performed in duplicate P lt 005 P lt 001 P lt0001

1954 Role of RUNX1 in Aberrant Retinal Angiogenesis Diabetes Volume 66 July 2017

endothelial to hematopoietic transition in a RUNX1-dependentmanner (2728) Consistent with these observations ourresults demonstrate that high glucose regulates RUNX1 andimplicates RUNX1 in aberrant retinal angiogenesis

Previous studies of PDR have mainly focused on geneexpression in FVMs or biomarkers in the vitreous (2930)Although FVMs from PDR may be highly informative path-ological tissues they are largely unstudied The window forprocessing samples postsurgery is narrow and intraopera-tive confounders such as FVM size location and involve-ment of sensory retina further complicate successful sampleacquisition CD311 cells from FVMs do not grow readily inculture making additional experimentation difficult Lim-ited sample size may also skew our gene ontology analysis

We report elevated RUNX1 expression in ECs of patient-derived FVMs from patients with PDR Also we demon-strate a role for RUNX1 in EC migration proliferation andvascular morphogenesis Furthermore we report selectivelyenhanced expression of RUNX1 in neovascular tufts in anexperimental model of OIR and that inhibition of RUNX1function reduced retinal neovascularization These findings

including the high glucosendashdependent expression of RUNX1identify a novel pathway of potential therapeutic interestand implicate RUNX1 in aberrant angiogenesis in multipleconditions

Acknowledgments The authors thank Dr Magali Saint-Geniez for insightfuldiscussions and providing mouse retinal samples Dr Eric Ng Mark Graham andDr Arturo Machuca-Parra for helpful discussions and Dr Christina Marko fortechnical contributions (all from Schepens Eye Research Institute Boston MA) Theauthors also thank Kristin Waraska and Andrew Gagne of the Harvard BiopolymersFacility Harvard Medical School and the Ocular Genetics Institute core facilityMassachusetts Eye and Ear (Boston MA) for their insights and workFunding The work performed in the manuscript was supported by NationalInstitutes of Health grants R01-EY-005318 (to PAD) R00-EY-021624 (to JFA-V)UH2NS100121-01 (to JFA-V) R21-EY-027061 (to LAK) K12-EY-16335 (toLAK) and Center Core grant P30-EY-003790 American Diabetes AssociationInnovation Award 7ndash12-IN-11 (to PAD) Massachusetts Lions Eye Research Fund(to JFA-V) E Matilda Ziegler Foundation for the Blind (to LAK) Karl KirchgessnerFoundation (to LAK) Department of Ophthalmology Harvard Medical School(to JFA-V and LAK) the Howard Hughes Medical Institute Medical ResearchFellows Program (to DJO) and the Vascular Biology Program at Boston ChildrenrsquosHospital (to DRB)

Figure 4mdashRUNX1 inhibition reduces neovascularization in the OIR model A Retina of P17 C57BL6J mice with OIR costained for RUNX1 andvessels (IB4 or CD31) showing positive RUNX1 staining conforming to neovascular tufts (arrows) and not to normal underlying vasculature Scalebars5 50 mm B Retinal flat mounts of P17 C57BL6J mice following OIR induction and intravitreal injection of 75 mmolL Ro5ndash3335 or vehicleat P13 and P15 (red overlay identifies the avascular area and neovascularization respectively) Scale bars 5 1 mm There was a nonsignificantdownward trend in avascular area but a significant reduction in neovascularization in the treated group compared with vehicle-treated group(nvehicle 5 7 nRo5ndash3335 5 9) Experiment performed in triplicate P lt 001

diabetesdiabetesjournalsorg Lam and Associates 1955

Duality of Interest PAD LAK and JFA-V have filed a provisional patentapplication on RUNX1 regulation for the treatment of aberrant angiogenesis and PDRNo other potential conflicts of interest relevant to this article were reportedAuthor Contributions JDL and DJO performed the experimentsanalyzed the data and wrote the manuscript LLW DA CP-W AVS andJC-V performed experiments DM AOS-R DE and DRB provided tissuesamples and reviewed and edited the manuscript TvZ LS and XG obtaineddata and reviewed the manuscript PAD LAK and JFA-V conceived theproject analyzed the data and wrote the manuscript PAD LAK and JFA-V arethe guarantors of this work and as such had full access to all the data in the studyand take responsibility for the integrity of the data and the accuracy of the dataanalysis

References1 Chung AS Ferrara N Developmental and pathological angiogenesis Annu RevCell Dev Biol 201127563ndash5842 Kim LA DrsquoAmore PA A brief history of anti-VEGF for the treatment of ocularangiogenesis Am J Pathol 2012181376ndash3793 Hsu YJ Hsieh YT Yeh PT Huang JY Yang CM Combined tractional andrhegmatogenous retinal detachment in proliferative diabetic retinopathy in the anti-VEGF era J Ophthalmol 201420149173754 Kuiper EJ Van Nieuwenhoven FA de Smet MD et al The angio-fibrotic switchof VEGF and CTGF in proliferative diabetic retinopathy PLoS One 20083e26755 Saint-Geniez M Maharaj AS Walshe TE et al Endogenous VEGF is required forvisual function evidence for a survival role on muumlller cells and photoreceptors PLoSOne 20083e35546 Sang DN DrsquoAmore PA Is blockade of vascular endothelial growth factorbeneficial for all types of diabetic retinopathy Diabetologia 2008511570ndash15737 Jo DH Cho CS Kim JH Jun HO Kim JH Animal models of diabetic reti-nopathy doors to investigate pathogenesis and potential therapeutics J Biomed Sci201320388 Kim LA Wong LL Amarnani DS et al Characterization of cells from patient-derived fibrovascular membranes in proliferative diabetic retinopathy Mol Vis 201521673ndash6879 Trapnell C Pachter L Salzberg SL TopHat discovering splice junctions withRNA-Seq Bioinformatics 2009251105ndash111110 Huang da W Sherman BT Lempicki RA Bioinformatics enrichment tools pathstoward the comprehensive functional analysis of large gene lists Nucleic Acids Res2009371ndash1311 Smith LE Wesolowski E McLellan A et al Oxygen-induced retinopathy in themouse Invest Ophthalmol Vis Sci 199435101ndash11112 Connor KM Krah NM Dennison RJ et al Quantification of oxygen-inducedretinopathy in the mouse a model of vessel loss vessel regrowth and pathologicalangiogenesis Nat Protoc 200941565ndash157313 Liang CC Park AY Guan JL In vitro scratch assay a convenient and in-expensive method for analysis of cell migration in vitro Nat Protoc 20072329ndash33314 Gebaumlck T Schulz MM Koumoutsakos P Detmar M TScratch a novel andsimple software tool for automated analysis of monolayer wound healing assaysBiotechniques 200946265ndash274

15 Edgar R Domrachev M Lash AE Gene Expression Omnibus NCBI gene ex-

pression and hybridization array data repository Nucleic Acids Res 200230207ndash

21016 The Diabetes Control and Complications Trial Research Group The relationship

of glycemic exposure (HbA1c) to the risk of development and progression of reti-

nopathy in the diabetes control and complications trial Diabetes 199544968ndash98317 McLeod DS Hasegawa T Baba T et al From blood islands to blood vessels

morphologic observations and expression of key molecules during hyaloid vascular

system development Invest Ophthalmol Vis Sci 2012537912ndash792718 Iwatsuki K Tanaka K Kaneko T et al Runx1 promotes angiogenesis by

downregulation of insulin-like growth factor-binding protein-3 Oncogene 200524

1129ndash113719 Namba K Abe M Saito S et al Indispensable role of the transcription factor

PEBP2CBF in angiogenic activity of a murine endothelial cell MSS31 Oncogene

200019106ndash11420 Cunningham L Finckbeiner S Hyde RK et al Identification of benzodiazepine

Ro5-3335 as an inhibitor of CBF leukemia through quantitative high throughput

screen against RUNX1-CBFb interaction Proc Natl Acad Sci U S A 201210914592ndash

1459721 Burns CE Traver D Mayhall E Shepard JL Zon LI Hematopoietic stem cell fate

is established by the Notch-Runx pathway Genes Dev 2005192331ndash234222 Ito Y Miyazono K RUNX transcription factors as key targets of TGF-beta su-

perfamily signaling Curr Opin Genet Dev 20031343ndash4723 Doll A Gonzalez M Abal M et al An orthotopic endometrial cancer mouse

model demonstrates a role for RUNX1 in distant metastasis Int J Cancer 2009125

257ndash26324 Scheitz CJ Lee TS McDermitt DJ Tumbar T Defining a tissue stem cell-driven

Runx1Stat3 signalling axis in epithelial cancer EMBO J 2012314124ndash413925 Planagumagrave J Diacuteaz-Fuertes M Gil-Moreno A et al A differential gene ex-

pression profile reveals overexpression of RUNX1AML1 in invasive endometrioid

carcinoma Cancer Res 2004648846ndash885326 Nagamachi A Htun PW Ma F et al A 59 untranslated region containing the

IRES element in the Runx1 gene is required for angiogenesis hematopoiesis and

leukemogenesis in a knock-in mouse model Dev Biol 2010345226ndash23627 Harris JM Esain V Frechette GM et al Glucose metabolism impacts the

spatiotemporal onset and magnitude of HSC induction in vivo Blood 2013121

2483ndash249328 Imanirad P Solaimani Kartalaei P Crisan M et al HIF1a is a regulator of

hematopoietic progenitor and stem cell development in hypoxic sites of the mouse

embryo Stem Cell Res (Amst) 20141224ndash3529 Ishikawa K Yoshida S Kobayashi Y et al Microarray analysis of gene ex-

pression in fibrovascular membranes excised from patients with proliferative diabetic

retinopathy Invest Ophthalmol Vis Sci 201556932ndash94630 McAuley AK Sanfilippo PG Hewitt AW et al Vitreous biomarkers in diabetic

retinopathy a systematic review and meta-analysis J Diabetes Complications 2014

28419ndash425

1956 Role of RUNX1 in Aberrant Retinal Angiogenesis Diabetes Volume 66 July 2017

endothelial to hematopoietic transition in a RUNX1-dependentmanner (2728) Consistent with these observations ourresults demonstrate that high glucose regulates RUNX1 andimplicates RUNX1 in aberrant retinal angiogenesis

Previous studies of PDR have mainly focused on geneexpression in FVMs or biomarkers in the vitreous (2930)Although FVMs from PDR may be highly informative path-ological tissues they are largely unstudied The window forprocessing samples postsurgery is narrow and intraopera-tive confounders such as FVM size location and involve-ment of sensory retina further complicate successful sampleacquisition CD311 cells from FVMs do not grow readily inculture making additional experimentation difficult Lim-ited sample size may also skew our gene ontology analysis

We report elevated RUNX1 expression in ECs of patient-derived FVMs from patients with PDR Also we demon-strate a role for RUNX1 in EC migration proliferation andvascular morphogenesis Furthermore we report selectivelyenhanced expression of RUNX1 in neovascular tufts in anexperimental model of OIR and that inhibition of RUNX1function reduced retinal neovascularization These findings

including the high glucosendashdependent expression of RUNX1identify a novel pathway of potential therapeutic interestand implicate RUNX1 in aberrant angiogenesis in multipleconditions

Acknowledgments The authors thank Dr Magali Saint-Geniez for insightfuldiscussions and providing mouse retinal samples Dr Eric Ng Mark Graham andDr Arturo Machuca-Parra for helpful discussions and Dr Christina Marko fortechnical contributions (all from Schepens Eye Research Institute Boston MA) Theauthors also thank Kristin Waraska and Andrew Gagne of the Harvard BiopolymersFacility Harvard Medical School and the Ocular Genetics Institute core facilityMassachusetts Eye and Ear (Boston MA) for their insights and workFunding The work performed in the manuscript was supported by NationalInstitutes of Health grants R01-EY-005318 (to PAD) R00-EY-021624 (to JFA-V)UH2NS100121-01 (to JFA-V) R21-EY-027061 (to LAK) K12-EY-16335 (toLAK) and Center Core grant P30-EY-003790 American Diabetes AssociationInnovation Award 7ndash12-IN-11 (to PAD) Massachusetts Lions Eye Research Fund(to JFA-V) E Matilda Ziegler Foundation for the Blind (to LAK) Karl KirchgessnerFoundation (to LAK) Department of Ophthalmology Harvard Medical School(to JFA-V and LAK) the Howard Hughes Medical Institute Medical ResearchFellows Program (to DJO) and the Vascular Biology Program at Boston ChildrenrsquosHospital (to DRB)

Figure 4mdashRUNX1 inhibition reduces neovascularization in the OIR model A Retina of P17 C57BL6J mice with OIR costained for RUNX1 andvessels (IB4 or CD31) showing positive RUNX1 staining conforming to neovascular tufts (arrows) and not to normal underlying vasculature Scalebars5 50 mm B Retinal flat mounts of P17 C57BL6J mice following OIR induction and intravitreal injection of 75 mmolL Ro5ndash3335 or vehicleat P13 and P15 (red overlay identifies the avascular area and neovascularization respectively) Scale bars 5 1 mm There was a nonsignificantdownward trend in avascular area but a significant reduction in neovascularization in the treated group compared with vehicle-treated group(nvehicle 5 7 nRo5ndash3335 5 9) Experiment performed in triplicate P lt 001

diabetesdiabetesjournalsorg Lam and Associates 1955

Duality of Interest PAD LAK and JFA-V have filed a provisional patentapplication on RUNX1 regulation for the treatment of aberrant angiogenesis and PDRNo other potential conflicts of interest relevant to this article were reportedAuthor Contributions JDL and DJO performed the experimentsanalyzed the data and wrote the manuscript LLW DA CP-W AVS andJC-V performed experiments DM AOS-R DE and DRB provided tissuesamples and reviewed and edited the manuscript TvZ LS and XG obtaineddata and reviewed the manuscript PAD LAK and JFA-V conceived theproject analyzed the data and wrote the manuscript PAD LAK and JFA-V arethe guarantors of this work and as such had full access to all the data in the studyand take responsibility for the integrity of the data and the accuracy of the dataanalysis

References1 Chung AS Ferrara N Developmental and pathological angiogenesis Annu RevCell Dev Biol 201127563ndash5842 Kim LA DrsquoAmore PA A brief history of anti-VEGF for the treatment of ocularangiogenesis Am J Pathol 2012181376ndash3793 Hsu YJ Hsieh YT Yeh PT Huang JY Yang CM Combined tractional andrhegmatogenous retinal detachment in proliferative diabetic retinopathy in the anti-VEGF era J Ophthalmol 201420149173754 Kuiper EJ Van Nieuwenhoven FA de Smet MD et al The angio-fibrotic switchof VEGF and CTGF in proliferative diabetic retinopathy PLoS One 20083e26755 Saint-Geniez M Maharaj AS Walshe TE et al Endogenous VEGF is required forvisual function evidence for a survival role on muumlller cells and photoreceptors PLoSOne 20083e35546 Sang DN DrsquoAmore PA Is blockade of vascular endothelial growth factorbeneficial for all types of diabetic retinopathy Diabetologia 2008511570ndash15737 Jo DH Cho CS Kim JH Jun HO Kim JH Animal models of diabetic reti-nopathy doors to investigate pathogenesis and potential therapeutics J Biomed Sci201320388 Kim LA Wong LL Amarnani DS et al Characterization of cells from patient-derived fibrovascular membranes in proliferative diabetic retinopathy Mol Vis 201521673ndash6879 Trapnell C Pachter L Salzberg SL TopHat discovering splice junctions withRNA-Seq Bioinformatics 2009251105ndash111110 Huang da W Sherman BT Lempicki RA Bioinformatics enrichment tools pathstoward the comprehensive functional analysis of large gene lists Nucleic Acids Res2009371ndash1311 Smith LE Wesolowski E McLellan A et al Oxygen-induced retinopathy in themouse Invest Ophthalmol Vis Sci 199435101ndash11112 Connor KM Krah NM Dennison RJ et al Quantification of oxygen-inducedretinopathy in the mouse a model of vessel loss vessel regrowth and pathologicalangiogenesis Nat Protoc 200941565ndash157313 Liang CC Park AY Guan JL In vitro scratch assay a convenient and in-expensive method for analysis of cell migration in vitro Nat Protoc 20072329ndash33314 Gebaumlck T Schulz MM Koumoutsakos P Detmar M TScratch a novel andsimple software tool for automated analysis of monolayer wound healing assaysBiotechniques 200946265ndash274

15 Edgar R Domrachev M Lash AE Gene Expression Omnibus NCBI gene ex-

pression and hybridization array data repository Nucleic Acids Res 200230207ndash

21016 The Diabetes Control and Complications Trial Research Group The relationship

of glycemic exposure (HbA1c) to the risk of development and progression of reti-

nopathy in the diabetes control and complications trial Diabetes 199544968ndash98317 McLeod DS Hasegawa T Baba T et al From blood islands to blood vessels

morphologic observations and expression of key molecules during hyaloid vascular

system development Invest Ophthalmol Vis Sci 2012537912ndash792718 Iwatsuki K Tanaka K Kaneko T et al Runx1 promotes angiogenesis by

downregulation of insulin-like growth factor-binding protein-3 Oncogene 200524

1129ndash113719 Namba K Abe M Saito S et al Indispensable role of the transcription factor

PEBP2CBF in angiogenic activity of a murine endothelial cell MSS31 Oncogene

200019106ndash11420 Cunningham L Finckbeiner S Hyde RK et al Identification of benzodiazepine

Ro5-3335 as an inhibitor of CBF leukemia through quantitative high throughput

screen against RUNX1-CBFb interaction Proc Natl Acad Sci U S A 201210914592ndash

1459721 Burns CE Traver D Mayhall E Shepard JL Zon LI Hematopoietic stem cell fate

is established by the Notch-Runx pathway Genes Dev 2005192331ndash234222 Ito Y Miyazono K RUNX transcription factors as key targets of TGF-beta su-

perfamily signaling Curr Opin Genet Dev 20031343ndash4723 Doll A Gonzalez M Abal M et al An orthotopic endometrial cancer mouse

model demonstrates a role for RUNX1 in distant metastasis Int J Cancer 2009125

257ndash26324 Scheitz CJ Lee TS McDermitt DJ Tumbar T Defining a tissue stem cell-driven

Runx1Stat3 signalling axis in epithelial cancer EMBO J 2012314124ndash413925 Planagumagrave J Diacuteaz-Fuertes M Gil-Moreno A et al A differential gene ex-

pression profile reveals overexpression of RUNX1AML1 in invasive endometrioid

carcinoma Cancer Res 2004648846ndash885326 Nagamachi A Htun PW Ma F et al A 59 untranslated region containing the

IRES element in the Runx1 gene is required for angiogenesis hematopoiesis and

leukemogenesis in a knock-in mouse model Dev Biol 2010345226ndash23627 Harris JM Esain V Frechette GM et al Glucose metabolism impacts the

spatiotemporal onset and magnitude of HSC induction in vivo Blood 2013121

2483ndash249328 Imanirad P Solaimani Kartalaei P Crisan M et al HIF1a is a regulator of

hematopoietic progenitor and stem cell development in hypoxic sites of the mouse

embryo Stem Cell Res (Amst) 20141224ndash3529 Ishikawa K Yoshida S Kobayashi Y et al Microarray analysis of gene ex-

pression in fibrovascular membranes excised from patients with proliferative diabetic

retinopathy Invest Ophthalmol Vis Sci 201556932ndash94630 McAuley AK Sanfilippo PG Hewitt AW et al Vitreous biomarkers in diabetic

retinopathy a systematic review and meta-analysis J Diabetes Complications 2014

28419ndash425

1956 Role of RUNX1 in Aberrant Retinal Angiogenesis Diabetes Volume 66 July 2017

Duality of Interest PAD LAK and JFA-V have filed a provisional patentapplication on RUNX1 regulation for the treatment of aberrant angiogenesis and PDRNo other potential conflicts of interest relevant to this article were reportedAuthor Contributions JDL and DJO performed the experimentsanalyzed the data and wrote the manuscript LLW DA CP-W AVS andJC-V performed experiments DM AOS-R DE and DRB provided tissuesamples and reviewed and edited the manuscript TvZ LS and XG obtaineddata and reviewed the manuscript PAD LAK and JFA-V conceived theproject analyzed the data and wrote the manuscript PAD LAK and JFA-V arethe guarantors of this work and as such had full access to all the data in the studyand take responsibility for the integrity of the data and the accuracy of the dataanalysis

References1 Chung AS Ferrara N Developmental and pathological angiogenesis Annu RevCell Dev Biol 201127563ndash5842 Kim LA DrsquoAmore PA A brief history of anti-VEGF for the treatment of ocularangiogenesis Am J Pathol 2012181376ndash3793 Hsu YJ Hsieh YT Yeh PT Huang JY Yang CM Combined tractional andrhegmatogenous retinal detachment in proliferative diabetic retinopathy in the anti-VEGF era J Ophthalmol 201420149173754 Kuiper EJ Van Nieuwenhoven FA de Smet MD et al The angio-fibrotic switchof VEGF and CTGF in proliferative diabetic retinopathy PLoS One 20083e26755 Saint-Geniez M Maharaj AS Walshe TE et al Endogenous VEGF is required forvisual function evidence for a survival role on muumlller cells and photoreceptors PLoSOne 20083e35546 Sang DN DrsquoAmore PA Is blockade of vascular endothelial growth factorbeneficial for all types of diabetic retinopathy Diabetologia 2008511570ndash15737 Jo DH Cho CS Kim JH Jun HO Kim JH Animal models of diabetic reti-nopathy doors to investigate pathogenesis and potential therapeutics J Biomed Sci201320388 Kim LA Wong LL Amarnani DS et al Characterization of cells from patient-derived fibrovascular membranes in proliferative diabetic retinopathy Mol Vis 201521673ndash6879 Trapnell C Pachter L Salzberg SL TopHat discovering splice junctions withRNA-Seq Bioinformatics 2009251105ndash111110 Huang da W Sherman BT Lempicki RA Bioinformatics enrichment tools pathstoward the comprehensive functional analysis of large gene lists Nucleic Acids Res2009371ndash1311 Smith LE Wesolowski E McLellan A et al Oxygen-induced retinopathy in themouse Invest Ophthalmol Vis Sci 199435101ndash11112 Connor KM Krah NM Dennison RJ et al Quantification of oxygen-inducedretinopathy in the mouse a model of vessel loss vessel regrowth and pathologicalangiogenesis Nat Protoc 200941565ndash157313 Liang CC Park AY Guan JL In vitro scratch assay a convenient and in-expensive method for analysis of cell migration in vitro Nat Protoc 20072329ndash33314 Gebaumlck T Schulz MM Koumoutsakos P Detmar M TScratch a novel andsimple software tool for automated analysis of monolayer wound healing assaysBiotechniques 200946265ndash274

15 Edgar R Domrachev M Lash AE Gene Expression Omnibus NCBI gene ex-

pression and hybridization array data repository Nucleic Acids Res 200230207ndash

21016 The Diabetes Control and Complications Trial Research Group The relationship

of glycemic exposure (HbA1c) to the risk of development and progression of reti-

nopathy in the diabetes control and complications trial Diabetes 199544968ndash98317 McLeod DS Hasegawa T Baba T et al From blood islands to blood vessels

morphologic observations and expression of key molecules during hyaloid vascular

system development Invest Ophthalmol Vis Sci 2012537912ndash792718 Iwatsuki K Tanaka K Kaneko T et al Runx1 promotes angiogenesis by

downregulation of insulin-like growth factor-binding protein-3 Oncogene 200524

1129ndash113719 Namba K Abe M Saito S et al Indispensable role of the transcription factor

PEBP2CBF in angiogenic activity of a murine endothelial cell MSS31 Oncogene

200019106ndash11420 Cunningham L Finckbeiner S Hyde RK et al Identification of benzodiazepine

Ro5-3335 as an inhibitor of CBF leukemia through quantitative high throughput

screen against RUNX1-CBFb interaction Proc Natl Acad Sci U S A 201210914592ndash

1459721 Burns CE Traver D Mayhall E Shepard JL Zon LI Hematopoietic stem cell fate

is established by the Notch-Runx pathway Genes Dev 2005192331ndash234222 Ito Y Miyazono K RUNX transcription factors as key targets of TGF-beta su-

perfamily signaling Curr Opin Genet Dev 20031343ndash4723 Doll A Gonzalez M Abal M et al An orthotopic endometrial cancer mouse

model demonstrates a role for RUNX1 in distant metastasis Int J Cancer 2009125

257ndash26324 Scheitz CJ Lee TS McDermitt DJ Tumbar T Defining a tissue stem cell-driven

Runx1Stat3 signalling axis in epithelial cancer EMBO J 2012314124ndash413925 Planagumagrave J Diacuteaz-Fuertes M Gil-Moreno A et al A differential gene ex-

pression profile reveals overexpression of RUNX1AML1 in invasive endometrioid

carcinoma Cancer Res 2004648846ndash885326 Nagamachi A Htun PW Ma F et al A 59 untranslated region containing the

IRES element in the Runx1 gene is required for angiogenesis hematopoiesis and

leukemogenesis in a knock-in mouse model Dev Biol 2010345226ndash23627 Harris JM Esain V Frechette GM et al Glucose metabolism impacts the

spatiotemporal onset and magnitude of HSC induction in vivo Blood 2013121

2483ndash249328 Imanirad P Solaimani Kartalaei P Crisan M et al HIF1a is a regulator of

hematopoietic progenitor and stem cell development in hypoxic sites of the mouse

embryo Stem Cell Res (Amst) 20141224ndash3529 Ishikawa K Yoshida S Kobayashi Y et al Microarray analysis of gene ex-

pression in fibrovascular membranes excised from patients with proliferative diabetic

retinopathy Invest Ophthalmol Vis Sci 201556932ndash94630 McAuley AK Sanfilippo PG Hewitt AW et al Vitreous biomarkers in diabetic

retinopathy a systematic review and meta-analysis J Diabetes Complications 2014

28419ndash425

1956 Role of RUNX1 in Aberrant Retinal Angiogenesis Diabetes Volume 66 July 2017