Research Article The Role of E-Cadherin in Maintaining the ...downloads.hindawi.com/journals/joph/2016/4805986.pdf · epithelium from underlying Bowman s membrane. Corneal epithelium

Research ArticleThe Role of E-Cadherin in Maintaining the BarrierFunction of Corneal Epithelium after Treatment withCultured Autologous Oral Mucosa Epithelial Cell SheetGrafts for Limbal Stem Deficiency

Fawzia Bardag-Gorce Richard H Hoft Andrew Wood Joan Oliva Hope NiiharaAndrew Makalinao Jacquelyn Thropay Derek Pan Imara Meepe Kumar TigerJulio Garcia Amanda Laporte Samuel W French and Yutaka Niihara

Los Angeles Biomedical Research Institute (LA BioMed) Harbor UCLA Medical Center Torrance CA 90502 USA

Correspondence should be addressed to Fawzia Bardag-Gorce fgorcelabiomedorg

Received 10 May 2016 Revised 29 July 2016 Accepted 1 August 2016

Academic Editor Taras Ardan

Copyright copy 2016 Fawzia Bardag-Gorce 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

The role of E-cadherin in epithelial barrier function of cultured autologous oral mucosa epithelial cell sheet (CAOMECS) graftswas examined CAOMECS were cultured on a temperature-responsive surface and grafted onto rabbit corneas with Limbal StemCell Deficiency (LSCD) E-cadherin levels were significantly higher in CAOMECS compared to normal and LSCD epitheliumBeta-catenin colocalized with E-cadherin in CAOMECS cell membranes while phosphorylated beta-catenin was significantlyincreased ZO-1 occludin and Cnx43 were also strongly expressed in CAOMECS E-cadherin and beta-catenin localization atthe cell membrane was reduced in LSCD corneas while CAOMECS-grafted corneas showed a restoration of E-cadherin and beta-catenin expression LSCD corneas did not show continuous staining for ZO-1 or for Cnx43 while CAOMECS-grafted corneasshowed a positive expression of ZO-1 and Cnx43 Cascade Blue hydrazide did not pass through CAOMECS Because E-cadherininteractions are calcium-dependent EGTA was used to chelate calcium and disrupt cell adhesion EGTA-treated CAOMECScompletely detached from cell culture surface and E-cadherin levels were significantly decreased In conclusion E cadherin highexpression contributed to CAOMECS tight and gap junction protein recruitment at the cell membrane thus promoting cellularadhesion and a functional barrier to protect the ocular surface

1 Introduction

Ocular surface regeneration with cultured oral mucosaepithelial cells has been used for almost a decadeThe efficacyof this technique has been successfully demonstrated byinvestigators who have used various methods for preparingand delivering the graft to the ocular surface [1ndash6] Inves-tigators have also studied the epithelial barrier function ofcultured oral mucosal or limbal epithelial cell sheets [4 67] However these reports are still vague about the defini-tion of an epithelial barrier function Does a barrier func-tion include a barrier to conjunctivalization of the corneaa barrier to external toxic and infectious agents andor

barriers to neo-vascularizationThese are questions that havenot been answered The reports that have investigated oralmucosal epithelial cell sheets barrier function documentedvarious results Satake et al 2008 [4] and Shimazaki et al2009 [7] cultured oral mucosa epithelial cells (OMECS) onamniotic membrane and reported that OMECS tissue waspermeable to fluorescein and that limbal stem cells had abetter barrier function than cultured oral mucosal epithelialcells respectively However Hori et al 2008 [8] and Hayashiet al 2010 [9] cultured OMECS on temperature-responsivesurfaces (CellSeed Inc Tokyo Japan) and using MUC16and ZO-1 staining reported a functional barrier RecentlyDuncan et al 2015 [10] used Corning inserts (Corning Inc

Hindawi Publishing CorporationJournal of OphthalmologyVolume 2016 Article ID 4805986 13 pageshttpdxdoiorg10115520164805986

2 Journal of Ophthalmology

NY) to culture OMECS and used Quantum Dot to report afunctional barrier In the present study we used assays forE-cadherin and other proteins associated with intercellularadhesion to examine the epithelial barrier of CAOMECSgrafts We investigated adhesion proteins and junctionalcomplexes before and after grafting onto corneas with LSCDThe presence of normal cell-to-cell junctional complexesis critical to the efficacy and safety of CAOMECS Cell-to-cell adhesion includes adherens tight and gap junctions(AD TJ and GJ resp) all of which contribute to main-taining epithelial integrity Cadherin proteins are adhesionproteins that control cell contacts and cell motility [11]WhileE-cadherin extracellular domains mediate Ca2+-dependentcell-cell binding their intracellular domains recruit beta-catenin proteins which in turn interact with actin cytoskele-ton filaments promoting adhesiveness [12] thereby limitingdestabilization of cell junctional complexes and contributingto the epithelial barrier function

The temperature-sensitive culture plate developed byCellSeed Inc (Tokyo Japan) allows CAOMECS harvestingin a tissue-like multilayered sheet ready for grafting ontothe recipient cornea [1 13] The harvested cell sheets containintact extracellular matrix (ECM) that increase adhesionsbetween CAOMECS graft and the recipient corneal surfaceOur previous study using microarray analysis of CAOMECSgene expression [14] demonstrated that gap junction genesConnexin 43 (Cnx43) and Connexin 45 were upregulatedTheupregulation of these two gap junction proteins suggestedthat cell-to-cell interactions were at least partially functionalWe also examined E-cadherin signaling as it is essential forcytoskeleton organization cell adhesion and functions as asuppressor of cell proliferationmigration [15 16]The expres-sion of membranous E-cadherin levels favors the formationof TJ and GJ complexes by recruiting beta-catenin which inturn recruit alpha-catenin and cytoskeleton filaments thatinteract with ZO-1 and finally with Cnx43 This E-cadherinsignaling increases cell-to-cell interactions to promote celladhesion and probably improve the epithelial barrier func-tion Part of beta-catenin pool is phosphorylated in thecytoplasm and then degraded by the proteasome pathway[17] If beta-catenin is not phosphorylated it translocates tothe nucleus and stimulates Wnt pathway gene expressionsthat are involved in cell proliferation and migration [18]

In the present study we compared the levels of AJ TJand GJ proteins in CAOMECS graft in healthy and normalrabbit corneal epithelial cells and in corneal epithelial surfacecells present after experimentally induced LSCD The distri-bution of these junctional complexes was also investigatedin rabbit corneas that received CAOMECS for treatment ofexperimentally induced LSCD

2 Materials and Methods

21 Animal Studies New Zealand white rabbits weighingbetween 25 and 3 kg were used They were maintainedaccording to the Guidelines of Animal Care as describedby the National Academy of Sciences and published by theInstitute of Laboratory Animal Resources Commission onLife Sciences National Research Council

22 LSCDModel and CAOMECS Grafting The experimentalprotocol was approved by the IACUC and performed aspreviously reported [13] Briefly the protocol was establishedwith the following schedule (1) anesthesia (2) LSCD creationthat was surgically accomplished by performing a 360-degreesuperficial lamellar dissection of the limbal zone (3) follow-up and corneas exams were performed for up to threemonths (4) oral mucosa biopsy (5) epithelial cells isolationand primary cell culture (6) CAOMECS grafting onto corneasurface that was accomplished using 3 to 5 sutures to securethe sheet onto cornea surface (7) follow-up (8) gradingof corneal opacification and superficial visualization and(9) sacrifice of the experimental animals 6 months afterCAOMECS graft [13]

23 Corneal Epithelial Cell Sampling Control and LSCDrabbits were lightly sedated and corneas were exposed to20 isopropyl alcohol for one minute to remove cornealepithelium from underlying Bowmanrsquos membrane Cornealepithelium was then rinsed with sterile saline Scraping andremoval of all visible corneal epithelial cells were performedusing a Crescent knife 2mm angled double bevel (KatenaProducts Inc Denville NJ) The cells were collected in PBScentrifuged and then resuspended and lysed in protein lysisbuffer for subsequent biochemical analysisThe samples werethen frozen at minus80∘C until all samples healthy and LSCDrsquosepithelial cells were gathered for biochemical analysis

24 Oral Mucosa Epithelial Cell Isolation and Cell CultureA small oral mucosal biopsy was performed on a sedatedanimal using a 6mm diameter disposable punch biopsyinstrument (Biopunch HealthLink Jacksonville FL) Thebiopsy specimen was washed in sterile saline sanitized inpovidone iodine washed again in sterile saline and thenwashed in DMEM cell culture mediaThe specimen was thenused to isolate oral mucosal epithelial cells as previouslydescribed [13] Briefly the specimen was incubated withDispase I for one hour at 37∘C (Roche Diagnostics GmbHMannheim Germany) the epithelium was separated fromthe lamina propria and then subjected to trypsin digestionto separate the epithelial cells The isolated primary epithelialcells were then seeded at 5 times 105 density on UpCell-insert a temperature-responsive culture dish (CellSeed IncTokyo Japan) and cocultured with mitomycin C- (MMC-)treated NIH3T3 feeder cells After 4 days of cell culture themedia were changed and EGF was added at 10 ngmL finalconcentration Cell culture media were changed every twodays After 2 weeks of growth CAOMECS were harvestedby reducing the culture temperature to room temperature for40min Two CAOMECS were produced from each biopsyThe first one was used for grafting onto LSCD corneas aspreviously reported [13] and the second one was used forbiochemical analysis

25 Functional Barrier of CAOMECS

(1) Calcium rigidifies the extracellular E-cadherindomain In the absence of calcium E-cadherin losesits affinity for the facing E-cadherin which causes

Journal of Ophthalmology 3

cells to lose their adhesion to each other EGTA wasused to chelate calcium and destabilize E-cadherinanchoring bridges between cells Fully grownCAOMECS were incubated after two weeks withEGTA from sigma at 25mM and 5mM dissolvedin cell culture media for 24 hours CAOMECS werethen harvested and cell lysates were analyzed forE-cadherin levels

(2) Rabbit oral mucosa epithelial cell sheets were cocul-tured for two weeks in Transwell insert perme-able support (24mm insert 04 120583m Corning Incor-porated Kennebunk ME) with NIH-3T3 feedercells at the bottom of the 6-multiplate well WhenCAOMECS were fully grown they were incubatedwith Cascade Blue at 20120583M for 24 hours CAOMECSwere then harvested and fluorescence was measuredin 1 120583g of total protein from cell media and fromCAOMECS cell lysates Controls were CAOMECScultured and harvested without Cascade Blue addi-tion Fluorescence was measured using a PerkinElmer LS 30 spectrofluorometer at 120582 excitation370 nm and 120582 emission 430 nm

26 Impression Cytology Millicell culture inserts 12mmdiameter 04 120583m pore size with mixed cellulose membrane(Millipore Billerica MA) were used to sample cornealsurface cells The inserts were placed on the cornea for fewseconds and then slowly removed The inserts were thenallowed to dry for 1 h at room temperature The inserts werethen fixed in 10 neutral buffered formalin overnight andconserved in 70 ethanol prior to immune-histochemicalstaining analysis

27 Immunohistochemistry Harvested CAOMECS werefixed in 10 neutral buffered formalin processed andparaffin embedded sectioned and stained with HampE orused for immunofluorescent staining using E-cadherinN-cadherin beta-catenin from (BD Bioscience San JoseCA Lot are 65490 25721 and 63115 resp) ZO-1 andCK4 (Santa Cruz Biotech Santa Cruz CA Lot are K0413 and G1514 resp) K6 (Abcam Cambridge MALot GR112707-1) Cnx43 (Abcam Cambridge MA Lot GR1969-5) Ki-67 (Santa Cruz Biotech Santa Cruz CA Lot E1403) Muc5AC (LifeSpan Biosciences Inc Seattle WA Lot 60020) K13 (Santa Cruz Biotechnology Santa Cruz Lot E1711) and deltaNp63 (Biocare Medical Concord CA Lot 120815) antibodies Normal sham and grafted rabbit corneaspecimens were fixed in 10 neutral buffered formalinThe processed tissues were sectioned slides were stainedwith HampE and immunofluorescent staining was performedusing the same antibodies listed above Alexa Fluor 488donkey anti-mousegoat fluorophore conjugated secondaryantibodies were used Propidium iodide (Invitrogen EugeneOR) or 410158406-diamidino-2-phenylindole (DAPI) (ThermoScientific Waltham MA) was also used for staining nuclearDNA A Nikon 400 fluorescent microscope was used toanalyze the slides Picture processing and analysis wereperformed using Adobe Photoshop CS5

28Western Blot Protein concentration wasmeasured usinga Bio-Rad protein assay Five micrograms of total proteinsfrom harvested and lysed CAOMECS was used and com-pared to 5 micrograms of healthy normal rabbit cornealepithelium harvested from the cornea surface with scrapingFive micrograms of protein scraped and removed from thecorneal surface of a rabbit eye with experimentally inducedLSCD was also processed for western blot analysis Cellhomogenates were separated by SDS-PAGE electrophoresisusing 4ndash20 gradient polyacrylamide gels Proteins weretransferred to a PVDF membrane (Bio-Rad Hercules CA)for 1 h in 25mM Tris-HCl (pH = 83) glycine 192mM and20 methanol Antibodies against Phospho-Akt Ser473 andGAPDH (Millipore Temecula CA Lot 2089910) phosphobeta-catenin (Ser33-Ser37-Th41) (Cell Signaling TechnologyBD Bioscience San Jose CA Lot 3) and occludin (Invit-rogen Life technology Grand Island NY Lot 1578827A)were used Goat anti-mouse and sheep anti-goat antibodies(Bio-Rad Hercules CA) were used as secondary antibodiesImmunodetection was done using ECL plus (AmershamBioscience Corp Piscataway NJ) Densitometric measure-ments of the bands were done using the GS-800 imagingdensitometer (Bio-Rad Hercules CA)

29 Statistics Analysis Data were obtained from at least threeseparate experiments Bars represent mean values plusmn SEM119901 values are determined by one-way ANOVA and Student-Newman Keuls for multiple group comparisons (Sigma-StatSoftware San Francisco CA) Statistical significance was setat 119901 = or lt to 005 Bar graphs were shown as mean plusmn SEM119899 = 3-4

3 Results

To test our hypothesis that CAOMECS cultured on atemperature-responsive cell culture surface has the char-acteristics of functional and safe corneal epithelium wemeasured and histologically analyzed the levels and distri-bution of epithelial markers (E-cadherin N-cadherin beta-catenin phosphorylated beta-catenin phosphorylated AktZO-1 occludin and Cnx43)

E-cadherin was significantly more expressed inCAOMECS graft compared to the corneal epitheliumof a healthy and normal eye (NE) The level of expressionof N-cadherin was much lower in both tissues (Figure 1(a))Total beta-catenin levels measurements did not show asignificant difference while phosphorylated beta-cateninwas increased in CAOMECS compared to NE indicatinggreater turnover of beta-catenin in CAOMECS (Figure 1(b))We thenmeasured the levels of Cnx43 and occluding both ofwhich are important proteins in the junctional complexes ofmany cell types Results showed that CAOMECS expressedhigh levels of these two junctional proteins (Figures 1(d) and1(e))

The isolated oral mucosa epithelial cells (starting mate-rial) were not used as a control in the Western blot anal-ysis because their junctional complexes were damaged byenzymatic treatment in the isolation procedures Thereforewe used normal cornea epithelial cells instead and controls


Cadherins

NECAOMECS

Den

sitom

etric

uni

ts120583

g of

tota

l

0

5

10

15

20

25

prot

eins

N-cadherinE-cadherin

p = 0255

p = 0013

(a)

NECAOMECS

Beta-catenin

Den

sitom

etric

uni

ts120583

g of

tota

l

0

5

10

15

20

25

30

prot

eins

P Beta-cateninBeta-catenin

p = 0012

p = 0061

(b)

Beta-actin

Den

sitom

etric

uni

ts120583

g of

tota

l

0

2

4

6

8

10

12

prot

eins

CAOMECSNE

p = 07

(c)

Cnx43

0

05

1

15

2

Ratio

Cnx

43b

eta-

actin

pro

tein

s

CAOMECSNE

p = 0001

(d)

Occludin

0

02

04

06

08

Ratio

occ

ludi

nG

APD

H p

rote

ins

CAOMECSNE

p = 0023

(e)

GAPDH

Beta-actin

NE CAOMECS

Cnx43

Occludin

N-cadherin

E-cadherin

Phospho beta-catenin

Beta-catenin

(f)

Figure 1 Western blot analysis of E-cadherin and N-cadherin (a) beta-catenin and phosphorylated beta-catenin (b) beta-actin levels (c)Cnx43 (d) and occludin (e) levels (f) Pictures of targeted protein CAOMECS cultured autologous oral mucosa epithelial cell sheet NErabbit healthy normal epithalial cells

normal oral mucosa tissue sections were used in histolog-ical analysis CAOMECS was histologically analyzed for E-cadherin N-cadherin beta-catenin ZO-1 CK4 DeltaNp63Cnx43 and Ki-67 (Figures 2(a)ndash2(h) arrows) Control oralmucosa sections stained positive for E-cadherin in squamousepithelial cells and stained negative in basal cells that stainedpositive for Cnx43 deltaNp63 and Ki-67 (Figures 2(i)ndash2(l)arrows) Histological analysis also showed that CAOMECSmarkedly stained for E-cadherin in the cell membranevisualizing bridges that solidify adhesion between adjacentepithelial cells (Figure 2(a) arrow) CAOMECS did notstain positive for N-cadherin (Figure 2(b)) Our analysisalso showed that beta-catenin an E-cadherin downstreamsignaling protein was located in the cytoplasmic membranecolocalizing with E-cadherin which further documents theformation of AJ in CAOMECS (Figure 2(c)) TJ protein ZO-1plays a major role in epithelial cell adhesion by connectingbeta-catenin and intermediate filaments to gap junctionproteins Cnx43 and 45 [19] ZO-1 highlighted the intercellular

bridges in CAOMECS (green and in a spotty staining Fig-ure 2(d) arrows) Cnx43 also stained CAOMECS intercellularspace in green around the basal cells (Figure 2(e) arrow)almost similar to deltaNp63 and Ki-67 staining (Figures 2(g)and 2(h) arrows) CAOMECS epithelial differentiation wasanalyzed by Cytokeratin 4 (CK4) staining showing that onlythe apical squamous cells were positive for CK4 (Figure 2(f)arrow) Figure 2(m) shows an HampE of a CAOMECS

An LSCD rabbit model was then created as previouslyreported [13] All superficial limbal tissue and other corneaepithelium were surgically removed Follow-up exams weredone monthly for 3 months after surgical limbectomy Stabi-lized LSCDwas evidenced by the neovascularization coveringcentral cornea (Figure 3(b)) opaque corneal surface (con-juntivalization) and low presence of inflammatory cells asindicated by impression cytology-based histological analysis(Figure 3(e)) Goblet cells were detected in central corneawith Muc5AC staining as well as CK13 positive staining(Figures 3(d) and 3(f) resp) reflecting the invasion of the


(a) E-cadherin (b) N-cadherin

(c) Beta-catenin (d) ZO-1

(e) Cnx43 (f) CK4

(g) DeltaNp63 (h) Ki-67

(i) E-cadherin (j) Cnx43 (k) DeltaNp63

(l) Ki-67 (m) CAOMECS HampE

Figure 2 Immunofluorescent staining analyzing epithelial barrier and proliferation markers of CAOMECS (a to h) and control oral mucosaltissue sections (i to l) In green (magnification times40) (a) and (i) are E-cadherin (arrow) (b) is N-cadherin (c) is beta-catenin and (d) is ZO-1Nuclear staining is achieved using DAPI (blue) or propidium iodide (red) In green (magnification times40) (e) and (j) are Cnx43 (arrow) (g)and (k) (magnification times20) are DeltaNp63 and (h) and (l) are Ki-67 (arrow) (f) A staining for CK4 in green (magnification times40) (m) is anHampE staining of CAOMECS

naked stroma by the conjunctival epithelium resulting indevelopment of total LSCD

Once LSCD was stable 3 months after limbectomy allof the corneal surface cells (including most pannus tissue)were removed with scraping and collected in lysis buffer forbiochemical analysis Results showed a significantly lower

expression of E-cadherin in LSCDrsquos epithelium and nosignificant changes in beta-catenin (Figure 4(a)) HoweverAkt was more phosphorylated in the LSCDrsquos epithelial cellssuggesting a decrease in epithelial adhesion (Figure 4(b))Upregulated E-cadherin exerts a negative control on phos-phoinositide 3-kinase (PI3K)Akt signaling activation by


(a) (c)

(f)(d)

(b)

(e)

Figure 3 Rabbit LSCD model (a) is a normal rabbit cornea (b) and (c) are LSCD cornea 3 months after limbectomy without and withfluorescein staining respectively (d) and (f) are corneal epithelium collected by impression cytology from the surface of the LSCD centralcorneal epithelium and subjected to immunofluorescent staining in green for Muc5AC and CK13 respectively (e) is pannus tissue harvestedfrom the LSCD cornea and processed for HampE staining Blue is DAPI for nuclei staining Note that LSCD cornea surface was invaded byconjunctival cells that stained positive for goblet cells (d) and for CK13 (f) indicating invasion of conjunctival epithelium over the cornea(magnification times20)

inhibiting beta-catenin downstream signaling thus prevent-ing epithelial cell decreased adhesion The levels of Cnx43were not found to be significantly different in normal rabbitcorneal epithelium and LSCDrsquos epithelia (Figure 4(c)) How-ever occludin levels were significantly decreased in LSCDcornea compared to normal epithelium cornea (Figure 4(e))The targeted protein bands are shown in Figure 4(f)

Three months after limbectomy rabbitrsquos oral mucosawas biopsied and used to engineer CAOMECS graft TheCAOMECS graft was then used for grafting onto a rabbitcornea with LSCD After 6 months of follow-up and eyeexams all corneas were analyzed for junctional complexesIn our recently published study [13] we reported the successof CAOMECS grafting and showed reepithelialization ofcorneas with no epithelial defect Grafted corneas showed anE-cadherin expression pattern (Figure 5(a)) similar to thatof normal healthy corneas (Figure 5(a)) However sham-operated corneas showed a disrupted E-cadherin stainingand a decrease of its expression mainly at the basal cellmembrane (Figure 5(a)) indicating an unhealthy epithe-lium CAOMECS grafted corneas (Figure 5(b)) also showedbeta-catenin staining concentrated at the cell membranesimilar to healthy corneas (Figure 5(b)) while the sham-operated cornea did not show well-defined beta-catenin cell

membrane staining (Figure 5(b)) This result was expected asthe sham eyes showed cornea epithelial thinning and erosionas well as significant goblet cell invasion (Figures 5(a) and5(b))

The corneal epithelium is the main barrier protectingthe eye from harmful external agents such as microbesand chemicals and contributes to the transparency of thecornea This barrier function depends on TJs that sealthe intercellular space TJ molecule ZO-1 stained positivebetween adjacent CAOMECS epithelial cells (Figures 2(d)and 2(e)) CAOMECS grafted cornea also showed a positivestaining of ZO-1 at the cell junctions (Figure 6(a)) while thesham eye tissue section showed an uneven and discontinuousZO-1 staining (Figure 6(a)) As expected these interactionswere stabilized by intercellular tethering forces generated bythe AJs which are proximal to TJs thus reflecting a normalfunctional epithelial barrier

Cnx43 was mainly found in the basal cell layer of normalcorneal epithelium as well as CAOMECS grafted corneas(Figure 6(b)) while the sham corneas did not show anyCnx43 staining (Figure 6(b)) These results reflected thebeneficial effects of CAOMECS grafting onto corneas ofrabbit with LSCD Evidence of oral epithelial cells existencein CAOMECS grafted corneas six months after the grafting


E-cadherin and beta-catenin

NEDE

Den

sitom

etric

uni

ts120583

g of

p = 0913

p = 0025

0

5

10

15

20

25

tota

l pro

tein

s

Beta-cateninE-cad

(a)

Phospho-Akt

Den

sitom

etric

uni

ts120583

g of

p = 0033

0

5

10

15

20

25

tota

l pro

tein

s

DENE

(b)

Connexin 43

Den

sitom

etric

uni

ts120583

g of

p lt 0001

0

1

2

3

4

5

6

tota

l pro

tein

s

DENE

(c)

Beta-actin

Den

sitom

etric

uni

ts120583

g of

p = 0093

0

2

4

6

8

10

tota

l pro

tein

s

DENE(d)

Ratio

occ

ludi

nG

APD

H

Occludin

p = 0079

0

01

02

03

04

05

prot

ein

DENE(e)

GAPDH

Phospho-Akt

Cnx43

Beta-actin

Occludin

Beta-catenin

E-cadherinDENE

(f)

Figure 4Western blot analysis of E-cadherin and beta-catenin (a) phospho-Akt (b) Cnx43 (c) beta-actin (d) and occludin (e) levels Beta-actin and GAPDH (f) were used for loading control NE rabbit healthy epithalial cells DE LSCDrsquos epithelium 3 months after limbectomyE-cadherin levels decreased significantly in the LSCD cornea while phosph-Akt levels were increased

was also investigated Figure 7 documents the existence ofthese cells using Cytokeratin 6 (K6) biomarker expressedin oral mucosal epithelial cells but not on the cornealepithelium CAOMECS grafted corneas showed scattered orspotted positive staining of K6 indicating the presence of oralmucosal epithelial cells on the ocular surface even 6 monthsafter grafting (Figure 7)

Calcium covalently binds to Ca2+ binding domainpresent in the N-Terminal of E-cadherin allowing bridgingbetween neighboring cells When calcium is removed E-cadherin molecule becomes disordered and easily dena-tured causing E-cadherinmolecules to dissociate EGTAwasused to chelate extracellular calcium and demonstrate E-cadherin role in epithelial integrity and thus epithelial barrierCAOMECS were cultured for two weeks and treated withEGTA for 24 hours before harvest CAOMECS commencedto detach in 25mM EGTA treatment (Figure 8(a) whitearrow) and was completely detached in 5mM EGTA treat-ment (Figure 8(a) black arrow) A semiquantitative analysisof E-cadherin levels showed a significant decrease of E-cadherin after Ca2+ chelation by EGTA (Figure 8(b)) In

addition the epithelial barrier function of CAOMECS graftwas investigated using a small fluorescent probe CascadeBlue hydrazide that does not penetrate cells unless thereis a destabilization of cell membrane (Figure 8(c)) Resultsshowed a high concentration of fluorescence in CAOMECSculture media and no fluorescence in the cells Moreoverthere was no fluorescence in the culture media of the 3T3feeder cells or in the 3T3 cells by themselves indicatingthat CAOMECS exerted a barrier to Cascade Blue molecule(Figure 8(d))

4 Discussion

CAOMECS is a successful technology for ocular surfacereconstruction in cases of preexisting limbal stem cell defi-ciency (LSCD) its efficacy has been demonstrated by severalgroups of investigators [1 20] Since E-cadherin signalingis important for establishing all cell-to-cell junctional com-plexes the present study investigated E-cadherin signalingin CAOMECS before and after grafting on corneas of rabbitwith experimentally induced LSCDWe examined junctional


NE

GE

SE

(a)

NE

GE

SE

(b)

Figure 5 E-cadherin (a) and beta-catenin (b) expression is shown in green Red is nuclear staining with propidium iodide NE normalcornea GE CAOMECS grafted cornea and SE sham cornea Note that E-cadherin and beta-catenin stained markedly on the cell membraneof normal cornea and CAOMECS grafted corneas compared to that of the sham cornea (magnification times40)

complexes and compared the expression levels of these pro-teins between CAOMECS normal corneas LSCD corneasand CAOMECS grafted corneas

E-cadherin is an AJ calcium-dependent protein thatplays an important role in cell adhesion binding cellstogether and regulating intracellular downstream signalingThese bindings increase cytoskeleton interaction with tightand gap junction proteins thus increasing cell adhesionAbundant literature shows that E-cadherin is involved incell polarity [21ndash23] cell differentiation [24 25] and cellproliferation [26] In the present study we found that E-cadherin was significantly expressed in CAOMECS at higherlevels than normal healthy epithelial cells This upregulationwas consistently reproducible among all the CAOMECSgrafts we engineered and suggests that there are relativelystrong adhesions between cells in CAOMECS grafts Notethat cell culture conditions and more specifically cell culturemedia containing calcium are responsible for E-cadherinstabilization at the cell membrane Indeed when calciumwaschelated with EGTA CAOMECS completely lost adhesionto cell culture surface and the levels of E-cadherin weresignificantly reduced This finding indicated that calciumsupplementation in the cell culture media promoted E-cadherin expression and bridging neighboring cells whichalmost certainly increases the epithelial barrier function ofCAOMECS graft

E-cadherin expression is also regulated at the transcrip-tional level and by cytoplasmic proteasome degradation [27ndash29] Our previous study showed that CAOMECS proteasomeactivity levels were higher than those of healthy corneaepithelial cells [30] leading to E-cadherin stabilization at thecell membrane [31]

E-cadherinbeta-catenin complex plays an important rolein cell adhesion and in the formation of the cell skeleton Lossof E-cadherinbeta-catenin complex from the cell membraneis commonly observed in hyperproliferative epithelial cells[29] Wnt pathway acts through the beta-catenin signalingpathway to promote angiogenesis and tumorigenesis if thepathway is unregulated [32 33] This occurs through beta-catenin translocation to the nucleus and activation of T-cellfactorlymphoid enhancer factor (TCFLEF) transcriptionfactors gene expression consequently increasing cell pro-liferation [34] When E-cadherin intracellular downstreamsignaling was analyzed by comparing the levels and dis-tribution of beta-catenin results showed that CAOMECShad similar levels to those of healthy corneal epithelialcells Moreover we found phosphorylated beta-catenin levelshigher in CAOMECS when compared to healthy corneaepithelial cells indicating that beta-catenin was targeted forproteasome degradation [35] Beta-catenin phosphorylationleads to its ubiquitination followed by degradation by theproteasome thus preventing nuclear translocation and TCF


NE

GE

SE

(a)

NE

GE

SE

(b)

Figure 6 ZO-1 (a) and Cnx43 (b) expression is shown in green Red is nuclear staining with propidium iodide NE normal cornea GECAOMECS grafted cornea and SE sham cornea (magnification times40)

genes activation for cell prosurvival behavior [36] An impor-tant function ofmembranous beta-catenin is to recruit alpha-catenin which in turn recruits cytoskeleton filaments as wellas TJ proteins ZO-1 and occludin The AJ proteins togetherwith TJrsquos form the apical junction complex that controlsepithelial cell-to-cell adherence barrier function and reg-ulation of the actin cytoskeleton as well TJs are the mostapical junction complex and provide a functional barrier [37]They are composed of transmembrane proteins occludin andclaudin which anchor scaffolding proteins including ZO-1ZO-2 and ZO-3 Both AJs and TJs have intricate connectionsand play a major role in maintaining the integrity of theepithelial barrier [38] The staining of ZO-1 showed resultssimilar to the results of E-cadherin staining at the cell mem-brane We showed positive staining of ZO-1 at the plasmamembrane of CAOMECS grafted corneas indicating thatCAOMECS grafting reepithelized the cornea surface withcells containing expressed tight junction proteins Moreoveroccludin was expressed in CAOMECS indicating that TJproteins were formed in CAOMECS in a quantity and patternof distribution similar to normal cornea epithelial cells Bybinding to occludin ZO-1 can bind actin filaments and could

possibly serve as a cadherin-associated protein to link nascentadhesions to the actin cytoskeleton [39 40]

Our results showed high levels of E-cadherin at theplasma membrane of CAOMECS and subsequent down-stream signaling of recruited beta-catenin actin filamentsTJ proteins and gap junction proteins These results suggestthat CAOMECS grafting allowed the corneas to be reepithe-lialized with a sheet of multilayered cells that may have abarrier function similar to other types of normal epithelialtissue However the literature reports various results fororal mucosal epithelial cells functional barrier [4 7ndash9] It ispossible that the novel cell culture technique we used resultedin a different outcome than these reports It is also possiblethat oral mucosal epithelial cells are permeable to fluoresceinsodiumHowever fluorescein has a relatively smallmolecularweight of 332306 Daltons Testing the epithelial barrier func-tion with a low molecular weight molecule does not alwaysgive the same result as when the barrier function is testedwith larger molecular weight molecules [4] TJrsquos paracellulardiffusion barrier is semipermeable as it allows the passageof small size hydrophilic molecule and ions [41] Otherinvestigators have demonstrated that oral mucosal epithelial


CAOMECS

OM

NE

GE

SE

Figure 7 Cytokeratin 6 (K6) staining is shown in green Red is nuclear staining with propidium iodide NE normal cornea GE CAOMECSgrafted cornea and SE sham cornea OM is oral mucosa (corneas andOM tissue sectionsmagnification is times20 and CAOMECSmagnificationis times40)

cell sheets grown in a cell culture plate with a temperature-responsive surface temperature-responsive surface have afunctional barrier [7 9 13] Our present study showed thatCAOMECS graft had significantly higher levels of TJ proteinswhen compared to control corneal epithelium These resultssuggest that CAOMECS graftmay have the properties to pre-vent epithelial defects and ulceration and create a functionalbarrier that accelerates and improves corneal surface healing[13] Similar to skin oral mucosal epithelium is an exampleof the toughest and most protective epithelium In this studywe also used Cascade Blue hydrazide fromMolecular Probesin our mechanistic approach to confirm the barrier functionof CAOMECS graft This fluorescent dye is a water solublefluorescent probe with lowmolecular weight (lt1000Daltons)and is too polar to passively diffuse through cell membranesThe barrier that CAOMECS graft exerted on Cascade Bluepassage through the multilayered CAOMECS grafts and thebarrier against Cascade Blue diffusion into CAOMECS cellsconfirm the barrier function of CAOMECS In our previousstudy [13] we showed that CAOMECS reduced the numberof blood vessels and prevented the regrow of pannus tissuein the central cornea of rabbits with experimentally inducedLSCD However whether CAOMECS constitutes a barrier toexternal toxic and infectious agents is not known and remainsto be determined

It is well known that the E-cadherinbeta-catenin com-plex regulates gap junction proteins such as Cnx43 becauseE-cadherinbeta-catenin complex loss was found associatedwith Cnx43 loss at the cell membranes [42 43] Cnx43 levels

and expression in CAOMECS grafts were similar to normalcorneal epithelium Histological analysis of CAOMECS alsodemonstrated the location of these gap junction proteins attheir appropriate intercellular sites and showed the expectedexpressions of epithelial marker Corneal epithelial cells fromrabbits with LSCD showed low expression in Cnx43 E-cadherin and beta-catenin levels reflecting a disruptionof cell adhesion probably caused by inflammation woundhealing and fibrosis In addition phosphorylated Akt wassignificantly upregulated indicating phosphoinositide 3-kinase (PI3K)Akt signaling activation for cell migration[44 45] Because of the potential for cell migration andto further document CAOMECS safety we plan to trackpossible migration of individual cells in CAOMECS with adirect labelling approach [46]

CAOMECS grafted corneas showed the staining of E-cadherin and beta-catenin at the cell membrane while shamcorneas showed a weaker staining of this complex Ourmorphological analysis also showed a positive staining ofCnx43 in CAOMECS grafted corneas similar to that ofnormal healthy corneas The sham corneas (with inducedLSCD but no CAOMECS graft) did not stain positive forCnx43 suggesting relatively weak cell-to-cell adhesion anda lack of cell-cell communication [43] In healthy cornealepithelium Cnx43 was mainly found in the limbus andcornea epithelial basal cells in a pattern very similar todeltaNp63 staining which suggests there might be a role forCnx43 in progenitor stem cells [47 48]We also found Cnx43in CAOMECS grafted cornea basal cells In conclusion we


(a)

E-cadherin levels

E-cadherin

GAPDH

0

05

1

15

2

25

3

35

E-ca

dher

inG

APD

H p

rote

ins

25 mM5mMCont

p = 0004p = 0005

5mM EGTA 25 mM EGTAControl(b)

(molecular probes)

CAOMECS

3T3 feeder cells

Cascade Blue

(c)

Cascade Blue levels

ControlBlue

p = 0658

p = 0275

p lt 0001

0

50

100

150

200

250

300

350

400

450

Fluo

resc

ent u

nits

120583g

prot

eins

Media 3T3 CAOMECSMedia CAOMECS

(d)

Figure 8 E-cadherin role in CAOMECS epithelial barrier (a) Picture of CAOMECS commencing to detach when treated with 25mMEGTA (white arrow) and completely lost adhesion when treated with 5mM EGTA (black arrow) (b) is a semiquantitative measurement ofE-cadherin Note that E-cadherin levels were significantly reduced when CAOMECS grafts were treated with EGTA (c) is an illustration ofCascade Blue experiment design where the probe was only added in cell culture media of CAOMECS The graph in (d) showed the levelsof Cascade Blue in media and in CAOMECS cells compared to controls (CAOMECS culture without Cascade Blue addition) Note thatfluorescence did not traverse the barrier of CAOMECS

used E-cadherin expression as an indicator of a functionalepithelial barrier because E-cadherin facilitates intercellularadhesion and recruits catenin proteins and actin filamentsto cell borders to increase barrier function CAOMECS graftengineered on temperature-responsive surface allowed theproduction of multilayered epithelial cell sheets with theexpression of epithelial markers necessary for normal cornealepithelial barrier function

Abbreviations

CAOMECS Cultured autologous oral mucosaepithelial cell sheet

LSCD Limbal stem cell deficiencyZo-1 Zonula occludens protein 1Cnx43 Connexin 43EGTA Ethylene glycol-bis (120573-aminoethyl

ether)-NNN1015840N1015840-tetraacetic acid

Competing Interests

The authors declare that they have no competing interests

Acknowledgments

This work is supported by Emmaus Life Science Inc (Tor-rance CA) and CellSeed Inc (Tokyo Japan) Dr Y Niiharais the President and CEO of Emmaus Life Sciences Inc

References

[1] KNishidaMYamato YHayashida et al ldquoCorneal reconstruc-tion with tissue-engineered cell sheets composed of autologousoral mucosal epitheliumrdquoTheNew England Journal ofMedicinevol 351 no 12 pp 1187ndash1196 2004

[2] S C G Tseng ldquoMove stem cells from the mouth to the eyerdquoAmerican Journal of Ophthalmology vol 141 no 2 pp 356ndash3572006


[3] T Inatomi T Nakamura M Kojyo N Koizumi C Soto-zono and S Kinoshita ldquoOcular surface reconstruction withcombination of cultivated autologous oral mucosal epithelialtransplantation and penetrating keratoplastyrdquoAmerican Journalof Ophthalmology vol 142 no 5 pp 757ndash764e1 2006

[4] Y Satake M Dogru G-Y Yamane S Kinoshita K Tsubotaand J Shimazaki ldquoBarrier function and cytologic features ofthe ocular surface epithelium after autologous cultivated oralmucosal epithelial transplantationrdquo Archives of Ophthalmologyvol 126 no 1 pp 23ndash28 2008

[5] S Krishnan G K Iyer and S Krishnakumar ldquoCulture ampcharacterization of limbal epithelial cells amp oral mucosal cellsrdquoIndian Journal of Medical Research vol 131 no 3 pp 422ndash4282010

[6] T Nakamura K Takeda T Inatomi C Sotozono and SKinoshita ldquoLong-term results of autologous cultivated oralmucosal epithelial transplantation in the scar phase of severeocular surface disordersrdquo British Journal of Ophthalmology vol95 no 7 pp 942ndash946 2011

[7] J Shimazaki K Higa N Kato and Y Satake ldquoBarrier functionof cultivated limbal and oral mucosal epithelial cell sheetsrdquoInvestigative Ophthalmology and Visual Science vol 50 no 12pp 5672ndash5680 2009

[8] Y Hori K Nishida M Yamato et al ldquoDifferential expressionof MUC16 in human oral mucosal epithelium and cultivatedepithelial sheetsrdquo Experimental Eye Research vol 87 no 3 pp191ndash196 2008

[9] R Hayashi M Yamato H Takayanagi et al ldquoValidationsystem of tissue-engineered epithelial cell sheets for cornealregenerative medicinerdquo Tissue Engineering Part CMethods vol16 no 4 pp 553ndash560 2010

[10] T J Duncan K Baba Y Oie and K Nishida ldquoA novel methodusing quantum dots for testing the barrier function of culturedepithelial cell sheetsrdquo Investigative Ophthalmology amp VisualScience vol 56 no 4 pp 2215ndash2223 2015

[11] T J C Harris and U Tepass ldquoAdherens junctions frommolecules to morphogenesisrdquo Nature Reviews Molecular CellBiology vol 11 no 7 pp 502ndash514 2010

[12] L G D R Bastos P G deMarcondes J CM de-Freitas-Junioret al ldquoProgeny from irradiated colorectal cancer cells acquirean EMT-like phenotype and activate Wnt120573-catenin pathwayrdquoJournal of Cellular Biochemistry vol 115 no 12 pp 2175ndash21872014

[13] F Bardag-Gorce J Oliva A Wood et al ldquoCarrier-free culturedautologous oral mucosa epithelial cell sheet (CAOMECS) forcorneal epithelium reconstruction a histological studyrdquo OcularSurface vol 13 no 2 pp 150ndash163 2015

[14] F Bardag-Gorce J Oliva A Wood et al ldquoMicroarray analysisof oral mucosal epithelial cell sheetrdquo Tissue Engineering andRegenerative Medicine vol 10 no 6 pp 362ndash370 2013

[15] A Chen H Beetham M A Black et al ldquoE-cadherin loss alterscytoskeletal organization and adhesion in non-malignant breastcells but is insufficient to induce an epithelial-mesenchymaltransitionrdquo BMC Cancer vol 14 article 552 2014

[16] A M Hendley E Provost J M Bailey et al ldquoP120 Catenin isrequired for normal tubulogenesis but not epithelial integrity indeveloping mouse pancreasrdquo Developmental Biology vol 399no 1 pp 41ndash53 2015

[17] V SW Li S S Ng P J Boersema et al ldquoWnt signaling throughinhibition of120573-catenin degradation in an intact Axin1 complexrdquoCell vol 149 no 6 pp 1245ndash1256 2012

[18] R L Daugherty and C J Gottardi ldquoPhospho-regulation of 120573-catenin adhesion and signaling functionsrdquo Physiology vol 22no 5 pp 303ndash309 2007

[19] K Umeda T Matsui M Nakayama et al ldquoEstablishment andcharacterization of cultured epithelial cells lacking expressionof ZO-1rdquo The Journal of Biological Chemistry vol 279 no 43pp 44785ndash44794 2004

[20] C Burillon L Huot V Justin et al ldquoCultured Autologous OralMucosal Epithelial Cell Sheet (CAOMECS) transplantation forthe treatment of corneal limbal epithelial stem cell deficiencyrdquoInvestigative Ophthalmology and Visual Science vol 53 no 3pp 1325ndash1331 2012

[21] R A Desai L Gao S RaghavanW F Liu and C S Chen ldquoCellpolarity triggered by cell-cell adhesion via E-cadherinrdquo Journalof Cell Science vol 122 no 7 pp 905ndash911 2009

[22] A Z Wang G K Ojakian and W J Nelson ldquoSteps in themorphogenesis of a polarized epithelium I Uncoupling theroles of cell-cell and cell-substratum contact in establishingplasma membrane polarity in multicellular epithelial (MDCK)cystsrdquo Journal of Cell Science vol 95 part 1 pp 137ndash151 1990

[23] L N Nejsum and W J Nelson ldquoA molecular mechanismdirectly linking E-cadherin adhesion to initiation of epithelialcell surface polarityrdquo Journal of Cell Biology vol 178 no 2 pp323ndash335 2007

[24] K Hawkins M Keramari F Soncin et al ldquoNovel cell linesisolated from mouse embryonic stem cells exhibiting de novomethylation of the E-cadherin promoterrdquo StemCells vol 32 no11 pp 2869ndash2879 2014

[25] L-T Wang J-P Liou Y-H Li Y-M Liu S-L Pan and C-MTeng ldquoA novel class I HDAC inhibitor MPT0G030 induces cellapoptosis and differentiation in human colorectal cancer cellsvia HDAC1PKC120575 and E-cadherinrdquo Oncotarget vol 5 no 14pp 5651ndash5662 2014

[26] S Zhang X Zhou BWang et al ldquoLoss of VHL expression con-tributes to epithelial-mesenchymal transition in oral squamouscell carcinomardquoOral Oncology vol 50 no 9 pp 809ndash817 2014

[27] J-Y Yang C S Zong W Xia et al ldquoMDM2 promotes cellmotility and invasiveness by regulating E-cadherin degrada-tionrdquo Molecular and Cellular Biology vol 26 no 19 pp 7269ndash7282 2006

[28] Y Shen D S Hirsch C A Sasiela and J W Wen ldquoCdc42regulates E-cadherin ubiquitination and degradation throughan epidermal growth factor receptor to Src-mediated pathwayrdquoThe Journal of Biological Chemistry vol 283 no 8 pp 5127ndash51372008

[29] M Saitoh T Shirakihara and K Miyazono ldquoRegulation ofthe stability of cell surface E-cadherin by the proteasomerdquoBiochemical and Biophysical Research Communications vol 381no 4 pp 560ndash565 2009

[30] F Bardag-Gorce I Meepe A Wood et al ldquoUbiquitin-prote-asome pathway activity in cultured oral mucosal epithelial cellsheet grafts used for treatment of experimental limbal stemcell deficiency in rabbits The American Society of Cell BiologyAnnual Meetingrdquo Molecular Biology of the Cell vol 2377 pp770ndash1077 2014

[31] J Y Kim J K Nam S-A Lee et al ldquoProteasome inhibi-tion causes epithelial-mesenchymal transition upon TM4SF5expressionrdquo Journal of Cellular Biochemistry vol 112 no 3 pp782ndash792 2011


[32] T N H Masckauchan C J Shawber Y Funahashi C-M Liand J Kitajewski ldquoWnt120573-catenin signaling induces prolifer-ation survival and interleukin-8 in human endothelial cellsrdquoAngiogenesis vol 8 no 1 pp 43ndash51 2005

[33] J Mao S Fan W Ma et al ldquoRoles of Wnt120573-catenin signalingin the gastric cancer stem cells proliferation and salinomycintreatmentrdquo Cell Death amp Disease vol 5 Article ID e1039 2014

[34] W Holnthoner M Pillinger M Groger et al ldquoFibroblastgrowth factor-2 induces LefTcf-dependent transcription inhuman endothelial cellsrdquo The Journal of Biological Chemistryvol 277 no 48 pp 45847ndash45853 2002

[35] X Li C Chen FWang et al ldquoKCTD1 suppresses canonical wntsignaling pathway by enhancing 120573-catenin degradationrdquo PLoSONE vol 9 no 4 Article ID e94343 2014

[36] J L Stamos and W I Weis ldquoThe 120573-catenin destructioncomplexrdquo Cold Spring Harbor Perspectives in Biology vol 5 no1 2013

[37] KMatter andM S Balda ldquoSnapShot epithelial tight junctionsrdquoCell vol 157 no 4 pp 992ndash992e1 2014

[38] A S Yap and E M Kovacs ldquoDirect cadherin-activated cellsignaling a view from the plasma membranerdquo The Journal ofCell Biology vol 160 no 1 pp 11ndash16 2003

[39] A S YapM S Crampton and J Hardin ldquoMaking and breakingcontacts the cellular biology of cadherin regulationrdquo CurrentOpinion in Cell Biology vol 19 no 5 pp 508ndash514 2007

[40] A S Fanning T Y Ma and J M Anderson ldquoIsolation andfunctional characterization of the actin binding region in thetight junction protein ZO-1rdquoThe FASEB Journal vol 16 no 13pp 1835ndash1837 2002

[41] S Michlig S Damak and J Le Coutre ldquoClaudin-based perme-ability barriers in taste budsrdquo Journal of Comparative Neurologyvol 502 no 6 pp 1003ndash1011 2007

[42] L Kanczuga-Koda A Wincewicz A Fudala et al ldquoE-cadherinand 120573-catenin adhesion proteins correlate positively with con-nexins in colorectal cancerrdquo Oncology Letters vol 7 no 6 pp1863ndash1870 2014

[43] P-J Hsiao J-C Jao J-L Tsai W-T Chang K-S Jeng and K-K Kuo ldquoInorganic arsenic trioxide induces gap junction lossin association with the downregulation of connexin43 and E-cadherin in rat hepatic lsquostem-likersquo cellsrdquo Kaohsiung Journal ofMedical Sciences vol 30 no 2 pp 57ndash67 2014

[44] G B Park D Kim Y S Kim et al ldquoThe epstein-barr viruscauses epithelial-mesenchymal transition in human cornealepithelial cells Via SykSrc and AktErk signaling pathwaysrdquoInvestigative Ophthalmology amp Visual Science vol 55 no 3 pp1770ndash1779 2014

[45] W Li J Ma Q Ma et al ldquoResveratrol inhibits the epithelial-mesenchymal transition of pancreatic cancer cells via sup-pression of the PI-3KAktNF-120581B pathwayrdquo Current MedicinalChemistry vol 20 no 33 pp 4185ndash4194 2013

[46] J Oliva F Bardag-Gorce A Wood H Sota and Y NiiharaldquoDirect labeling of 19F-perfluorocarbon onto multilayered cellsheet for MRI-based non-invasive cell trackingrdquo Tissue Engi-neering and Regenerative Medicine vol 12 no 5 pp 371ndash3782015

[47] M Nubile C Curcio H S Dua et al ldquoPathological changesof the anatomical structure and markers of the limbal stem cellniche due to inflammationrdquo Molecular Vision vol 19 pp 516ndash525 2013

[48] D-Q Li Z Wang K-C Yoon and F Bian ldquoCharacterizationisolation expansion and clinical therapy of human corneal

epithelial stemprogenitor cellsrdquo Journal of StemCells vol 9 no2 pp 79ndash91 2014

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


NY) to culture OMECS and used Quantum Dot to report afunctional barrier In the present study we used assays forE-cadherin and other proteins associated with intercellularadhesion to examine the epithelial barrier of CAOMECSgrafts We investigated adhesion proteins and junctionalcomplexes before and after grafting onto corneas with LSCDThe presence of normal cell-to-cell junctional complexesis critical to the efficacy and safety of CAOMECS Cell-to-cell adhesion includes adherens tight and gap junctions(AD TJ and GJ resp) all of which contribute to main-taining epithelial integrity Cadherin proteins are adhesionproteins that control cell contacts and cell motility [11]WhileE-cadherin extracellular domains mediate Ca2+-dependentcell-cell binding their intracellular domains recruit beta-catenin proteins which in turn interact with actin cytoskele-ton filaments promoting adhesiveness [12] thereby limitingdestabilization of cell junctional complexes and contributingto the epithelial barrier function

The temperature-sensitive culture plate developed byCellSeed Inc (Tokyo Japan) allows CAOMECS harvestingin a tissue-like multilayered sheet ready for grafting ontothe recipient cornea [1 13] The harvested cell sheets containintact extracellular matrix (ECM) that increase adhesionsbetween CAOMECS graft and the recipient corneal surfaceOur previous study using microarray analysis of CAOMECSgene expression [14] demonstrated that gap junction genesConnexin 43 (Cnx43) and Connexin 45 were upregulatedTheupregulation of these two gap junction proteins suggestedthat cell-to-cell interactions were at least partially functionalWe also examined E-cadherin signaling as it is essential forcytoskeleton organization cell adhesion and functions as asuppressor of cell proliferationmigration [15 16]The expres-sion of membranous E-cadherin levels favors the formationof TJ and GJ complexes by recruiting beta-catenin which inturn recruit alpha-catenin and cytoskeleton filaments thatinteract with ZO-1 and finally with Cnx43 This E-cadherinsignaling increases cell-to-cell interactions to promote celladhesion and probably improve the epithelial barrier func-tion Part of beta-catenin pool is phosphorylated in thecytoplasm and then degraded by the proteasome pathway[17] If beta-catenin is not phosphorylated it translocates tothe nucleus and stimulates Wnt pathway gene expressionsthat are involved in cell proliferation and migration [18]

In the present study we compared the levels of AJ TJand GJ proteins in CAOMECS graft in healthy and normalrabbit corneal epithelial cells and in corneal epithelial surfacecells present after experimentally induced LSCD The distri-bution of these junctional complexes was also investigatedin rabbit corneas that received CAOMECS for treatment ofexperimentally induced LSCD

2 Materials and Methods

21 Animal Studies New Zealand white rabbits weighingbetween 25 and 3 kg were used They were maintainedaccording to the Guidelines of Animal Care as describedby the National Academy of Sciences and published by theInstitute of Laboratory Animal Resources Commission onLife Sciences National Research Council

22 LSCDModel and CAOMECS Grafting The experimentalprotocol was approved by the IACUC and performed aspreviously reported [13] Briefly the protocol was establishedwith the following schedule (1) anesthesia (2) LSCD creationthat was surgically accomplished by performing a 360-degreesuperficial lamellar dissection of the limbal zone (3) follow-up and corneas exams were performed for up to threemonths (4) oral mucosa biopsy (5) epithelial cells isolationand primary cell culture (6) CAOMECS grafting onto corneasurface that was accomplished using 3 to 5 sutures to securethe sheet onto cornea surface (7) follow-up (8) gradingof corneal opacification and superficial visualization and(9) sacrifice of the experimental animals 6 months afterCAOMECS graft [13]

23 Corneal Epithelial Cell Sampling Control and LSCDrabbits were lightly sedated and corneas were exposed to20 isopropyl alcohol for one minute to remove cornealepithelium from underlying Bowmanrsquos membrane Cornealepithelium was then rinsed with sterile saline Scraping andremoval of all visible corneal epithelial cells were performedusing a Crescent knife 2mm angled double bevel (KatenaProducts Inc Denville NJ) The cells were collected in PBScentrifuged and then resuspended and lysed in protein lysisbuffer for subsequent biochemical analysisThe samples werethen frozen at minus80∘C until all samples healthy and LSCDrsquosepithelial cells were gathered for biochemical analysis

24 Oral Mucosa Epithelial Cell Isolation and Cell CultureA small oral mucosal biopsy was performed on a sedatedanimal using a 6mm diameter disposable punch biopsyinstrument (Biopunch HealthLink Jacksonville FL) Thebiopsy specimen was washed in sterile saline sanitized inpovidone iodine washed again in sterile saline and thenwashed in DMEM cell culture mediaThe specimen was thenused to isolate oral mucosal epithelial cells as previouslydescribed [13] Briefly the specimen was incubated withDispase I for one hour at 37∘C (Roche Diagnostics GmbHMannheim Germany) the epithelium was separated fromthe lamina propria and then subjected to trypsin digestionto separate the epithelial cells The isolated primary epithelialcells were then seeded at 5 times 105 density on UpCell-insert a temperature-responsive culture dish (CellSeed IncTokyo Japan) and cocultured with mitomycin C- (MMC-)treated NIH3T3 feeder cells After 4 days of cell culture themedia were changed and EGF was added at 10 ngmL finalconcentration Cell culture media were changed every twodays After 2 weeks of growth CAOMECS were harvestedby reducing the culture temperature to room temperature for40min Two CAOMECS were produced from each biopsyThe first one was used for grafting onto LSCD corneas aspreviously reported [13] and the second one was used forbiochemical analysis

25 Functional Barrier of CAOMECS

(1) Calcium rigidifies the extracellular E-cadherindomain In the absence of calcium E-cadherin losesits affinity for the facing E-cadherin which causes








3 Results





Cadherins

NECAOMECS

Den

sitom

etric

uni

ts120583

g of

tota

l

0

5

10

15

20

25

prot

eins


p = 0255

p = 0013

(a)

NECAOMECS

Beta-catenin

Den

sitom

etric

uni

ts120583

g of

tota

l

0

5

10

15

20

25

30

prot

eins


p = 0012

p = 0061

(b)

Beta-actin

Den

sitom

etric

uni

ts120583

g of

tota

l

0

2

4

6

8

10

12

prot

eins

CAOMECSNE

p = 07

(c)

Cnx43

0

05

1

15

2

Ratio

Cnx

43b

eta-

actin

pro

tein

s

CAOMECSNE

p = 0001

(d)

Occludin

0

02

04

06

08

Ratio

occ

ludi

nG

APD

H p

rote

ins

CAOMECSNE

p = 0023

(e)

GAPDH

Beta-actin

NE CAOMECS

Cnx43

Occludin

N-cadherin

E-cadherin


Beta-catenin

(f)








(e) Cnx43 (f) CK4









(a) (c)

(f)(d)

(b)

(e)









NEDE

Den

sitom

etric

uni

ts120583

g of

p = 0913

p = 0025

0

5

10

15

20

25

tota

l pro

tein

s

Beta-cateninE-cad

(a)

Phospho-Akt

Den

sitom

etric

uni

ts120583

g of

p = 0033

0

5

10

15

20

25

tota

l pro

tein

s

DENE

(b)

Connexin 43

Den

sitom

etric

uni

ts120583

g of

p lt 0001

0

1

2

3

4

5

6

tota

l pro

tein

s

DENE

(c)

Beta-actin

Den

sitom

etric

uni

ts120583

g of

p = 0093

0

2

4

6

8

10

tota

l pro

tein

s

DENE(d)

Ratio

occ

ludi

nG

APD

H

Occludin

p = 0079

0

01

02

03

04

05

prot

ein

DENE(e)

GAPDH

Phospho-Akt

Cnx43

Beta-actin

Occludin

Beta-catenin

E-cadherinDENE

(f)





4 Discussion



NE

GE

SE

(a)

NE

GE

SE

(b)







NE

GE

SE

(a)

NE

GE

SE

(b)






CAOMECS

OM

NE

GE

SE







(a)

E-cadherin levels

E-cadherin

GAPDH

0

05

1

15

2

25

3

35

E-ca

dher

inG

APD

H p

rote

ins

25 mM5mMCont

p = 0004p = 0005


(molecular probes)

CAOMECS

3T3 feeder cells

Cascade Blue

(c)

Cascade Blue levels

ControlBlue

p = 0658

p = 0275

p lt 0001

0

50

100

150

200

250

300

350

400

450

Fluo

resc

ent u

nits

120583g

prot

eins


(d)



Abbreviations




Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























3 Results





Cadherins

NECAOMECS

Den

sitom

etric

uni

ts120583

g of

tota

l

0

5

10

15

20

25

prot

eins


p = 0255

p = 0013

(a)

NECAOMECS

Beta-catenin

Den

sitom

etric

uni

ts120583

g of

tota

l

0

5

10

15

20

25

30

prot

eins


p = 0012

p = 0061

(b)

Beta-actin

Den

sitom

etric

uni

ts120583

g of

tota

l

0

2

4

6

8

10

12

prot

eins

CAOMECSNE

p = 07

(c)

Cnx43

0

05

1

15

2

Ratio

Cnx

43b

eta-

actin

pro

tein

s

CAOMECSNE

p = 0001

(d)

Occludin

0

02

04

06

08

Ratio

occ

ludi

nG

APD

H p

rote

ins

CAOMECSNE

p = 0023

(e)

GAPDH

Beta-actin

NE CAOMECS

Cnx43

Occludin

N-cadherin

E-cadherin


Beta-catenin

(f)








(e) Cnx43 (f) CK4









(a) (c)

(f)(d)

(b)

(e)









NEDE

Den

sitom

etric

uni

ts120583

g of

p = 0913

p = 0025

0

5

10

15

20

25

tota

l pro

tein

s

Beta-cateninE-cad

(a)

Phospho-Akt

Den

sitom

etric

uni

ts120583

g of

p = 0033

0

5

10

15

20

25

tota

l pro

tein

s

DENE

(b)

Connexin 43

Den

sitom

etric

uni

ts120583

g of

p lt 0001

0

1

2

3

4

5

6

tota

l pro

tein

s

DENE

(c)

Beta-actin

Den

sitom

etric

uni

ts120583

g of

p = 0093

0

2

4

6

8

10

tota

l pro

tein

s

DENE(d)

Ratio

occ

ludi

nG

APD

H

Occludin

p = 0079

0

01

02

03

04

05

prot

ein

DENE(e)

GAPDH

Phospho-Akt

Cnx43

Beta-actin

Occludin

Beta-catenin

E-cadherinDENE

(f)





4 Discussion



NE

GE

SE

(a)

NE

GE

SE

(b)







NE

GE

SE

(a)

NE

GE

SE

(b)






CAOMECS

OM

NE

GE

SE







(a)

E-cadherin levels

E-cadherin

GAPDH

0

05

1

15

2

25

3

35

E-ca

dher

inG

APD

H p

rote

ins

25 mM5mMCont

p = 0004p = 0005


(molecular probes)

CAOMECS

3T3 feeder cells

Cascade Blue

(c)

Cascade Blue levels

ControlBlue

p = 0658

p = 0275

p lt 0001

0

50

100

150

200

250

300

350

400

450

Fluo

resc

ent u

nits

120583g

prot

eins


(d)



Abbreviations




Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















Cadherins

NECAOMECS

Den

sitom

etric

uni

ts120583

g of

tota

l

0

5

10

15

20

25

prot

eins


p = 0255

p = 0013

(a)

NECAOMECS

Beta-catenin

Den

sitom

etric

uni

ts120583

g of

tota

l

0

5

10

15

20

25

30

prot

eins


p = 0012

p = 0061

(b)

Beta-actin

Den

sitom

etric

uni

ts120583

g of

tota

l

0

2

4

6

8

10

12

prot

eins

CAOMECSNE

p = 07

(c)

Cnx43

0

05

1

15

2

Ratio

Cnx

43b

eta-

actin

pro

tein

s

CAOMECSNE

p = 0001

(d)

Occludin

0

02

04

06

08

Ratio

occ

ludi

nG

APD

H p

rote

ins

CAOMECSNE

p = 0023

(e)

GAPDH

Beta-actin

NE CAOMECS

Cnx43

Occludin

N-cadherin

E-cadherin


Beta-catenin

(f)








(e) Cnx43 (f) CK4









(a) (c)

(f)(d)

(b)

(e)









NEDE

Den

sitom

etric

uni

ts120583

g of

p = 0913

p = 0025

0

5

10

15

20

25

tota

l pro

tein

s

Beta-cateninE-cad

(a)

Phospho-Akt

Den

sitom

etric

uni

ts120583

g of

p = 0033

0

5

10

15

20

25

tota

l pro

tein

s

DENE

(b)

Connexin 43

Den

sitom

etric

uni

ts120583

g of

p lt 0001

0

1

2

3

4

5

6

tota

l pro

tein

s

DENE

(c)

Beta-actin

Den

sitom

etric

uni

ts120583

g of

p = 0093

0

2

4

6

8

10

tota

l pro

tein

s

DENE(d)

Ratio

occ

ludi

nG

APD

H

Occludin

p = 0079

0

01

02

03

04

05

prot

ein

DENE(e)

GAPDH

Phospho-Akt

Cnx43

Beta-actin

Occludin

Beta-catenin

E-cadherinDENE

(f)





4 Discussion



NE

GE

SE

(a)

NE

GE

SE

(b)







NE

GE

SE

(a)

NE

GE

SE

(b)






CAOMECS

OM

NE

GE

SE







(a)

E-cadherin levels

E-cadherin

GAPDH

0

05

1

15

2

25

3

35

E-ca

dher

inG

APD

H p

rote

ins

25 mM5mMCont

p = 0004p = 0005


(molecular probes)

CAOMECS

3T3 feeder cells

Cascade Blue

(c)

Cascade Blue levels

ControlBlue

p = 0658

p = 0275

p lt 0001

0

50

100

150

200

250

300

350

400

450

Fluo

resc

ent u

nits

120583g

prot

eins


(d)



Abbreviations




Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of



















(e) Cnx43 (f) CK4









(a) (c)

(f)(d)

(b)

(e)









NEDE

Den

sitom

etric

uni

ts120583

g of

p = 0913

p = 0025

0

5

10

15

20

25

tota

l pro

tein

s

Beta-cateninE-cad

(a)

Phospho-Akt

Den

sitom

etric

uni

ts120583

g of

p = 0033

0

5

10

15

20

25

tota

l pro

tein

s

DENE

(b)

Connexin 43

Den

sitom

etric

uni

ts120583

g of

p lt 0001

0

1

2

3

4

5

6

tota

l pro

tein

s

DENE

(c)

Beta-actin

Den

sitom

etric

uni

ts120583

g of

p = 0093

0

2

4

6

8

10

tota

l pro

tein

s

DENE(d)

Ratio

occ

ludi

nG

APD

H

Occludin

p = 0079

0

01

02

03

04

05

prot

ein

DENE(e)

GAPDH

Phospho-Akt

Cnx43

Beta-actin

Occludin

Beta-catenin

E-cadherinDENE

(f)





4 Discussion



NE

GE

SE

(a)

NE

GE

SE

(b)







NE

GE

SE

(a)

NE

GE

SE

(b)






CAOMECS

OM

NE

GE

SE







(a)

E-cadherin levels

E-cadherin

GAPDH

0

05

1

15

2

25

3

35

E-ca

dher

inG

APD

H p

rote

ins

25 mM5mMCont

p = 0004p = 0005


(molecular probes)

CAOMECS

3T3 feeder cells

Cascade Blue

(c)

Cascade Blue levels

ControlBlue

p = 0658

p = 0275

p lt 0001

0

50

100

150

200

250

300

350

400

450

Fluo

resc

ent u

nits

120583g

prot

eins


(d)



Abbreviations




Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a) (c)

(f)(d)

(b)

(e)









NEDE

Den

sitom

etric

uni

ts120583

g of

p = 0913

p = 0025

0

5

10

15

20

25

tota

l pro

tein

s

Beta-cateninE-cad

(a)

Phospho-Akt

Den

sitom

etric

uni

ts120583

g of

p = 0033

0

5

10

15

20

25

tota

l pro

tein

s

DENE

(b)

Connexin 43

Den

sitom

etric

uni

ts120583

g of

p lt 0001

0

1

2

3

4

5

6

tota

l pro

tein

s

DENE

(c)

Beta-actin

Den

sitom

etric

uni

ts120583

g of

p = 0093

0

2

4

6

8

10

tota

l pro

tein

s

DENE(d)

Ratio

occ

ludi

nG

APD

H

Occludin

p = 0079

0

01

02

03

04

05

prot

ein

DENE(e)

GAPDH

Phospho-Akt

Cnx43

Beta-actin

Occludin

Beta-catenin

E-cadherinDENE

(f)





4 Discussion



NE

GE

SE

(a)

NE

GE

SE

(b)







NE

GE

SE

(a)

NE

GE

SE

(b)






CAOMECS

OM

NE

GE

SE







(a)

E-cadherin levels

E-cadherin

GAPDH

0

05

1

15

2

25

3

35

E-ca

dher

inG

APD

H p

rote

ins

25 mM5mMCont

p = 0004p = 0005


(molecular probes)

CAOMECS

3T3 feeder cells

Cascade Blue

(c)

Cascade Blue levels

ControlBlue

p = 0658

p = 0275

p lt 0001

0

50

100

150

200

250

300

350

400

450

Fluo

resc

ent u

nits

120583g

prot

eins


(d)



Abbreviations




Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















NEDE

Den

sitom

etric

uni

ts120583

g of

p = 0913

p = 0025

0

5

10

15

20

25

tota

l pro

tein

s

Beta-cateninE-cad

(a)

Phospho-Akt

Den

sitom

etric

uni

ts120583

g of

p = 0033

0

5

10

15

20

25

tota

l pro

tein

s

DENE

(b)

Connexin 43

Den

sitom

etric

uni

ts120583

g of

p lt 0001

0

1

2

3

4

5

6

tota

l pro

tein

s

DENE

(c)

Beta-actin

Den

sitom

etric

uni

ts120583

g of

p = 0093

0

2

4

6

8

10

tota

l pro

tein

s

DENE(d)

Ratio

occ

ludi

nG

APD

H

Occludin

p = 0079

0

01

02

03

04

05

prot

ein

DENE(e)

GAPDH

Phospho-Akt

Cnx43

Beta-actin

Occludin

Beta-catenin

E-cadherinDENE

(f)





4 Discussion



NE

GE

SE

(a)

NE

GE

SE

(b)







NE

GE

SE

(a)

NE

GE

SE

(b)






CAOMECS

OM

NE

GE

SE







(a)

E-cadherin levels

E-cadherin

GAPDH

0

05

1

15

2

25

3

35

E-ca

dher

inG

APD

H p

rote

ins

25 mM5mMCont

p = 0004p = 0005


(molecular probes)

CAOMECS

3T3 feeder cells

Cascade Blue

(c)

Cascade Blue levels

ControlBlue

p = 0658

p = 0275

p lt 0001

0

50

100

150

200

250

300

350

400

450

Fluo

resc

ent u

nits

120583g

prot

eins


(d)



Abbreviations




Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NE

GE

SE

(a)

NE

GE

SE

(b)







NE

GE

SE

(a)

NE

GE

SE

(b)






CAOMECS

OM

NE

GE

SE







(a)

E-cadherin levels

E-cadherin

GAPDH

0

05

1

15

2

25

3

35

E-ca

dher

inG

APD

H p

rote

ins

25 mM5mMCont

p = 0004p = 0005


(molecular probes)

CAOMECS

3T3 feeder cells

Cascade Blue

(c)

Cascade Blue levels

ControlBlue

p = 0658

p = 0275

p lt 0001

0

50

100

150

200

250

300

350

400

450

Fluo

resc

ent u

nits

120583g

prot

eins


(d)



Abbreviations




Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















NE

GE

SE

(a)

NE

GE

SE

(b)






CAOMECS

OM

NE

GE

SE







(a)

E-cadherin levels

E-cadherin

GAPDH

0

05

1

15

2

25

3

35

E-ca

dher

inG

APD

H p

rote

ins

25 mM5mMCont

p = 0004p = 0005


(molecular probes)

CAOMECS

3T3 feeder cells

Cascade Blue

(c)

Cascade Blue levels

ControlBlue

p = 0658

p = 0275

p lt 0001

0

50

100

150

200

250

300

350

400

450

Fluo

resc

ent u

nits

120583g

prot

eins


(d)



Abbreviations




Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















CAOMECS

OM

NE

GE

SE







(a)

E-cadherin levels

E-cadherin

GAPDH

0

05

1

15

2

25

3

35

E-ca

dher

inG

APD

H p

rote

ins

25 mM5mMCont

p = 0004p = 0005


(molecular probes)

CAOMECS

3T3 feeder cells

Cascade Blue

(c)

Cascade Blue levels

ControlBlue

p = 0658

p = 0275

p lt 0001

0

50

100

150

200

250

300

350

400

450

Fluo

resc

ent u

nits

120583g

prot

eins


(d)



Abbreviations




Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















(a)

E-cadherin levels

E-cadherin

GAPDH

0

05

1

15

2

25

3

35

E-ca

dher

inG

APD

H p

rote

ins

25 mM5mMCont

p = 0004p = 0005


(molecular probes)

CAOMECS

3T3 feeder cells

Cascade Blue

(c)

Cascade Blue levels

ControlBlue

p = 0658

p = 0275

p lt 0001

0

50

100

150

200

250

300

350

400

450

Fluo

resc

ent u

nits

120583g

prot

eins


(d)



Abbreviations




Competing Interests


Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of






































































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of








































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of





















of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















Documents

Research Article The Role of E-Cadherin in Maintaining the ...downloads.hindawi.com/journals/joph/2016/4805986.pdf · epithelium from underlying Bowman s membrane. Corneal epithelium