May 2010
Volume 51, Issue 5
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Retinal Cell Biology  |   May 2010
Epithelial-Mesenchymal Transition and Proliferation of Retinal Pigment Epithelial Cells Initiated upon Loss of Cell-Cell Contact
Author Affiliations & Notes
  • Shigeo Tamiya
    From the Departments of Ophthalmology and Visual Sciences and
    Biochemistry and Molecular Biology, School of Medicine, University of Louisville, Louisville, Kentucky.
  • LanHsin Liu
    From the Departments of Ophthalmology and Visual Sciences and
  • Henry J. Kaplan
    From the Departments of Ophthalmology and Visual Sciences and
  • Corresponding author: Shigeo Tamiya, Department of Ophthalmology and Visual Sciences, School of Medicine, University of Louisville, Louisville, KY 40202; shigeo.tamiya@louisville.edu
Investigative Ophthalmology & Visual Science May 2010, Vol.51, 2755-2763. doi:10.1167/iovs.09-4725
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      Shigeo Tamiya, LanHsin Liu, Henry J. Kaplan; Epithelial-Mesenchymal Transition and Proliferation of Retinal Pigment Epithelial Cells Initiated upon Loss of Cell-Cell Contact. Invest. Ophthalmol. Vis. Sci. 2010;51(5):2755-2763. doi: 10.1167/iovs.09-4725.

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      © 2016 Association for Research in Vision and Ophthalmology.

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Abstract

Purpose.: Molecular mechanisms that initiate epithelial-mesenchymal transition (EMT) involved in ocular fibrotic complications remain elusive. Studies were conducted to examine the role of cell-cell contact in regulating EMT and proliferation of retinal pigment epithelial (RPE) cells.

Methods.: Porcine RPE cells were isolated as sheets and cultured in vitro on lens capsules. Cell morphology was examined by microscopy. Western blot analysis and immunostaining were used to follow protein expression. Cell proliferation and RPE function were assessed by BrdU incorporation and phagocytosis assay, respectively.

Results.: RPE cells in the center of each sheet maintained cell-cell contacts and retained a differentiated phenotype. Disruption of cadherin function in these cells resulted in the loss of cell-cell contact and the concomitant induction of mesenchymal marker protein expression and cell proliferation. RPE cells at the edge of the sheet migrated away from the sheet, underwent EMT, and initiated proliferation, which was accompanied by a switch in cadherin expression from P-cadherin to N-cadherin. Although TGF-β is thought to be a classic inducer of EMT, it was unable to initiate EMT in RPE cells maintaining cell-cell contact. However, change to α-SMA–positive myofibroblasts was induced by TGF-β in cells that had already undergone EMT.

Conclusions.: EMT and the onset of proliferation in RPE cells is initiated by loss of cell-cell contact. TGF-β cannot initiate EMT or the proliferation of RPE cells maintaining cell-cell contact but appears to play an important secondary role downstream of EMT in inducing transition to a myofibroblast phenotype—a phenotype linked to the development of fibrotic complications.

Epithelial-mesenchymal transition (EMT) is a process in which epithelial cells lose their differentiated phenotypes and become mesenchymal-like cells. 1 Various aspects of cell function, including cell adhesion, extracellular matrix turnover, cytoskeletal protein expression, and various cell behaviors, such as cell migration and proliferation, change upon EMT. EMT is crucial during embryogenesis and organ development, and it plays central roles in wound healing and tissue generation. 1 However, EMT is also thought to mediate pathologic processes such as fibrosis and the transition of cancer cells to a fibroblastic, migratory phenotype linked to metastasis. 
One such pathologic process within the eye in which EMT appears to play a central role is proliferative vitreoretinopathy (PVR). 2,3 The hallmark of PVR is the formation of a contractile proliferative membrane on the epiretinal surfaces of the retina composed of several cell types, including the retinal pigment epithelial (RPE) cells. Normally, the retinal pigment epithelium is a monolayer of cells closely apposed to the photoreceptors and plays a vital role in the maintenance of photoreceptor function. During retinal detachment, RPE cells become dislodged from their monolayer into the vitreous cavity or subretinal space, where they can adhere to the detached retina, initiate proliferation, and undergo EMT to a fibrotic phenotype. These fibrotic cells can then contract, creating a tractional retinal detachment and hindering surgical reattachment. 
Various factors have been suggested to play a role in EMT, such as modified growth factor signaling, loss of normal matrix adhesion, or changes in cell-cell adhesion profile. For example, transforming growth factor-beta (TGF-β) has been linked to EMT in vivo, 4,5 and mutation of Smad3, a key downstream signaling molecule in the TGF-β pathway, significantly reduced PVR in a mouse model. 6 Cell-cell contact inhibition mediated by the homotypic adhesion of cadherins on adjacent cells is responsible for defining organ size and enforcing tissue topology from Drosophila to mammals. 7 Recent studies suggest that cell-cell contacts are important in the maintenance of an epithelial phenotype; disruption of such contacts has been linked to EMT. 8,9 When RPE cells are dissociated into single cells and placed in primary culture, they initiate proliferation and undergo EMT, 10 whereas sheets of RPE cells in culture reportedly maintain their morphology for a more extended period. 1113  
We hypothesized that loss of RPE cell-cell contact, as a result of retinal detachment during PVR or as a result of dissociation in cell culture, initiates EMT and proliferation. To begin to address this hypothesis, we used a modified RPE sheet primary culture model. Although cells in the central regions of the sheets retained cell-cell contacts, cells on the edges of the sheets lacked such contacts. We show that cells in the central regions of the sheets maintained RPE differentiation in culture and remained nonproliferative, whereas cells at the edges of the sheets underwent EMT, initiated proliferation, and migrated away from the sheets. We further demonstrate that EMT and initiation of proliferation coincided with a switch in cadherin isoform expression, from P- to N-cadherin. Finally, we examined the role of TGF-β in the EMT of RPE cells. TGF-β was unable to initiate EMT or proliferation in RPE cells in the central regions of the sheets, where cell-cell contacts were maintained. However, TGF-β stimulated the transition of dedifferentiated cells to a myofibroblastic phenotype known to be highly contractile. Taken together, our results suggest that it is the loss of cell-cell contacts that initiates EMT in RPE cells and that, though TGF-β can augment the transition to a myofibroblastic phenotype, it cannot initiate EMT in RPE cells when cell-cell contacts are retained. 
Materials and Methods
Primary Culture of RPE Sheets
All cell culture supplies were from Invitrogen (Carlsbad, CA) unless stated otherwise. The lens posterior capsule was used as a basement membrane for RPE cell attachment and culture. Lens capsules and RPE cells were isolated from fresh porcine eyes, kindly donated by Swift Meat Packing Co. (Louisville, KY). The use of animal tissue was approved by the University of Louisville Institutional Animal Care and Use Committee and conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. 
Porcine lens capsular bags were prepared as previously described. 14 The anterior capsule was cut off using surgical scissors, and the remaining pinned-down posterior capsule was put into sterile deionized water for a minimum of 30 minutes to kill any residual lens epithelial cells. Water was replaced with growth media consisting of DMEM supplemented with 10% FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin and left overnight at 4°C before use. RPE cells were harvested as sheets using dispase, as described previously. 15 Isolated RPE sheets were gently placed on lens capsules. RPE cells were cultured in growth media for up to 8 days. In some cases, 2 mM EGTA or 10 ng/mL TGF-β2 (Millipore, Billerica, MA) was added to the culture media. Cultures were monitored daily using an inverted fluorescence microscope (Axiovert 200; Zeiss Microimaging, Thornwood, NY) equipped with a digital camera (AxioCam; Zeiss Microimaging). 
Immunocytochemical/Histochemical Staining
The following three protocols were used. In all cases, RPE sheets cultured on lens posterior capsule were fixed with 4% paraformaldehyde for 10 minutes, and stained samples were observed using an inverted fluorescence microscope (Axiovert 200; Zeiss Microimaging). 
For immunocytochemical staining of differentiation/EMT markers and cell adhesion molecules, samples were blocked using 5% goat serum in 0.3% Triton X-100/1%BSA-PBS and were incubated with primary antibodies in 1% BSA-PBS either at 4°C overnight or at 37°C for 1 hour. After three washes with PBS, samples were incubated with a secondary antibody in 1% BSA-PBS for 1 hour at room temperature. Samples were washed two times. Some samples were counterstained with phalloidin conjugated to Texas red X or Alexa 488 dye. 
For bromo-2-deoxyuridine (BrdU) staining, 20 μM BrdU was added to the culture medium for the final 4 hours before fixation of RPE cells. Fixed samples were treated with 1% Triton X-100 in 4 N HCl for 10 minutes to denature the nuclear material. Samples were blocked using 5% goat serum in 1% BSA-PBS and were incubated with anti–BrdU antibody in 1% BSA-PBS at 4°C overnight. Samples were washed three times and were incubated with a secondary antibody in 1% BSA-PBS for 1 hour at room temperature. After two washes, nuclei were counterstained with Hoechst dye. 
For Na,K-ATPase staining of paraffin-embedded sections, after fixation, samples were embedded in paraffin, and 5-μm sections were prepared. Sections were deparaffinized and rehydrated using a series of incubations with xylene (15 and 10 minutes) and ethanol (2 minutes each in 100%, 100%, 95%, 80%, and 70%), followed by three washes in PBS. Antigen retrieval was performed by microwaving in citrate buffer (pH 6.0) for 10 minutes. Sections were blocked using 5% goat serum in 1% BSA-PBS, and incubated with anti–Na,K-ATPase α1 subunit antibody in 1% BSA-PBS at 4°C overnight. After three washes, samples were incubated with a secondary antibody for 1 hour at room temperature. Samples were washed two times, and the nucleus was counterstained with Hoechst dye. 
Western Blot Analysis
RPE sheets cultured on lens capsule for 8 days were microdissected under a dissecting microscope to separate the central region with the heavily pigmented RPE cells, where cell-cell contacts were maintained, from the less pigmented migratory cells at the edges of the sheets. Isolated cells were snap frozen and kept in −80°C until use. Frozen cells were lysed in ice-cold RIPA buffer composed of 50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% deoxycholate, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, and protease inhibitor cocktail and phosphatase inhibitor cocktail I and II (Sigma Aldrich, St. Louis, MO), and protein concentration was determination by BCA assay (Pierce, Rockford, IL). For Western blot analysis, proteins were separated by SDS-PAGE and transferred to nitrocellulose membrane. Membranes were blocked with blocking buffer (SEA BLOCK; Pierce, Rockford, IL) for 1 hour at room temperature, followed by overnight incubation in primary antibodies (one raised in mouse and the other raised in rabbit) in a 1:1 mixture of blocking buffer and PBS at 4°C. Nitrocellulose membranes were washed three times in Tween-20 Tris-buffered saline composed of 30 mM Tris, 150 mM NaCl, and 0.1% Tween-20, pH 7.4, and were incubated for 1 hour with anti–rabbit secondary antibody conjugated with dye (IRDye 800; Rockland Immunochemicals, Gilbertsville, PA) and anti–mouse secondary antibody conjugated with Alexa Fluor 680 dye (Invitrogen). After three washes in Tween-20 Tris-buffered saline and two washes in PBS, membranes were scanned with an infrared scanner (Odyssey; LICOR, Lincoln, NE) to detect and quantify bands. 
Antibodies
The following antibodies were used for immunostaining or Western blot analysis: mouse monoclonal anti–Na,K-ATPase α1 subunit (clone M8-P1-A3) and mouse monoclonal anti–α-smooth muscle actin (Sigma Aldrich, St. Louis, MO), rabbit polyclonal anti–vimentin (C-20-R) and rabbit polyclonal anti P-cadherin Ab (H105; Santa Cruz Biotech, Santa Cruz, CA), anti–RPE65 mouse monoclonal Ab (clone 401.8B11.3D9; Millipore, Temecula, CA), mouse monoclonal anti N-cadherin Ab (clone 3B9; Invitrogen, Carlsbad, CA), mouse monoclonal anti–BrdU Ab (Iowa Hybridoma Bank, Iowa City, IA), and rabbit polyclonal anti–cyclin D1 Ab (Cell Signaling Technology). 
Scanning Electron Microscopy
RPE sheets on lens capsule were rinsed in PBS and then fixed in 3% buffered glutaraldehyde for 20 minutes. Samples were washed twice in 0.1 M CAC buffer, dehydrated in increasing concentrations of ethanol from 50% to 100%, and placed in hexamethyldisilazane for 10 minutes and air dried. Dried samples were gold coated and examined using a scanning electron microscope (Vega II; Tescan, Cranberry Township, PA). 
Photoreceptor Outer Segment Phagocytosis Assay
Purified photoreceptor outer segment (POS) was obtained from fresh porcine eyes using a modified method described by Chaitin and Hall. 16 Purified ROS were stored at 4°C in DMEM containing 17% sucrose, as described by Sun et al., 17 and used within 1 week. POS was labeled by FITC, as previously described, 18 and was added to cultured RPE cells for 100 minutes in growth media with 2.5% sucrose. 19 Quenching the fluorescence of surface-bound POS using trypan blue and cellular fixation followed the method described by Finnemann et al. 18 Nuclei were counterstained using Hoechst dye before mounting and were observed under a fluorescence microscope. 
Results
Morphologic Changes
RPE sheets from porcine eyes were isolated and placed in primary culture, as described in Materials and Methods. Initially, all cells in the sheets showed typical cobblestone epithelial morphology and heavy pigmentation (Fig. 1a). After 1 day to 2 days in culture, cells at the edges of these sheets flattened and started to migrate away from the sheets. By day 4, cells at the migrating edge were less pigmented and had lost epithelial morphology (Fig. 1b). This loss of pigment and epithelial morphology appeared to progress with distance as the cells migrated away from the sheets. By day 6, the migrating cells retained little pigment and transitioned to a flattened, elongated fibroblastic morphology (Fig. 1d). In contrast, cells within the central region of the original sheet remained heavily pigmented and retained an epithelial morphology (Fig. 1c). 
Figure 1.
 
Time course of phase-contrast images of RPE cells cultured on the lens capsule. Images of live cells were captured 0, 4, and 6 days after the cell sheet was set. Magnified images of insets (79 μm × 79 μm; a–d) are shown across the bottom. On day 0, all the cells are heavily pigmented with typical epithelial morphology. By day 4, cells at the migrating edge are larger and elongated and have less pigment. By day 6, cells at the migrating edge are even less pigmented (d). In contrast, cells within the original sheet maintain heavy pigmentation and epithelial morphology (c). Scale bar, 100 μm.
Figure 1.
 
Time course of phase-contrast images of RPE cells cultured on the lens capsule. Images of live cells were captured 0, 4, and 6 days after the cell sheet was set. Magnified images of insets (79 μm × 79 μm; a–d) are shown across the bottom. On day 0, all the cells are heavily pigmented with typical epithelial morphology. By day 4, cells at the migrating edge are larger and elongated and have less pigment. By day 6, cells at the migrating edge are even less pigmented (d). In contrast, cells within the original sheet maintain heavy pigmentation and epithelial morphology (c). Scale bar, 100 μm.
Phagocytic Capacity and Expression of the RPE-Specific Marker RPE65 Are Diminished in Migrating RPE Cells
The loss of epithelial morphology and pigment in RPE cells at the edges of the sheets suggested that these cells may be losing their differentiated phenotype. One important property of differentiated RPE cells is the ability to phagocytose POS. Examination of the cell surface using scanning electron microscopy revealed that dense apical microvilli, used for phagocytosis of POS, were evident on the heavily pigmented RPE cells that maintained an epithelial phenotype in the central regions of the sheets (Fig. 2A). In contrast, cells migrating away from the sheets lost most of their apical microvilli—only short microvilli were observed sporadically on these cells. Consistent with the loss of microvilli, POS uptake showed that the migrating cells had diminished phagocytic activity (Figs. 2B, 2C). 
Figure 2.
 
Microvilli and phagocytic activity of cultured RPE cells. (A) Scanning electron micrograph of RPE cells cultured for 6 days. Samples were tilted 40° and observed using a scanning electron microscope. Top: regions indicated by white insets (22 μm × 22 μm) are magnified at the bottom. Dotted lines: cell border. Arrows: nuclei. Note that heavily pigmented differentiated cells retain dense microvilli, whereas the less pigmented cells that have migrated out of the original RPE sheet (dedifferentiated) have lost most of the microvilli. Scale bar, 25 μm. Images are representative of results obtained in five different samples. (B) Fluorescence micrograph showing ingested FITC-labeled POS by RPE cells cultured for 6 days. Cells were incubated in the presence of FITC-labeled POS for 100 minutes. Cells were washed, treated with trypan blue, washed, fixed, and observed using a fluorescence microscope. The high phagocytic activity shown by the heavily pigmented RPE cells (Diff.) is lost in the less pigmented dedifferentiated cells (De-diff.). Scale bar, 20 μm. Images are representative of results obtained in four different samples. (C) Number of labeled POS ingested by cultured RPE cells. For each sample, four different fields of view (79 μm × 107 μm) were counted for heavily pigmented differentiated cells and less pigmented cells close to the migrating edge. Data shown are mean ± SEM of four different samples. Significant difference (P < 0.05) was observed in the number of ingested POS between two different cell phenotypes.
Figure 2.
 
Microvilli and phagocytic activity of cultured RPE cells. (A) Scanning electron micrograph of RPE cells cultured for 6 days. Samples were tilted 40° and observed using a scanning electron microscope. Top: regions indicated by white insets (22 μm × 22 μm) are magnified at the bottom. Dotted lines: cell border. Arrows: nuclei. Note that heavily pigmented differentiated cells retain dense microvilli, whereas the less pigmented cells that have migrated out of the original RPE sheet (dedifferentiated) have lost most of the microvilli. Scale bar, 25 μm. Images are representative of results obtained in five different samples. (B) Fluorescence micrograph showing ingested FITC-labeled POS by RPE cells cultured for 6 days. Cells were incubated in the presence of FITC-labeled POS for 100 minutes. Cells were washed, treated with trypan blue, washed, fixed, and observed using a fluorescence microscope. The high phagocytic activity shown by the heavily pigmented RPE cells (Diff.) is lost in the less pigmented dedifferentiated cells (De-diff.). Scale bar, 20 μm. Images are representative of results obtained in four different samples. (C) Number of labeled POS ingested by cultured RPE cells. For each sample, four different fields of view (79 μm × 107 μm) were counted for heavily pigmented differentiated cells and less pigmented cells close to the migrating edge. Data shown are mean ± SEM of four different samples. Significant difference (P < 0.05) was observed in the number of ingested POS between two different cell phenotypes.
Next, the expression of RPE65, a key enzyme within the visual cycle that is widely used as a retinal pigment epithelium–specific differentiation marker, was examined in the cultured RPE cells. Western blot analysis demonstrated that RPE cells in the central region of each sheet maintained a high level of RPE65 expression comparable to that seen in situ (Fig. 3A). In contrast, cells migrating away from the sheet showed reduced expression of RPE65. Immunocytochemical staining confirmed the Western blot finding with strong staining observed in the central region of the sheet and only weak staining evident in the migrating cells (Fig. 3B). 
Figure 3.
 
Polarized expression of Na,K-ATPase and expression of retinal pigment epithelium–specific marker RPE65 and vimentin, an intermediate filament protein that is increased on EMT in a variety of epithelial cells, in cultured RPE cells. (A) RPE65 and vimentin expression by cultured and in situ RPE cells. Western blot for RPE65, vimentin, and β-actin (loading control) representative of results obtained in five separate samples are shown. Lane 1, in situ sample; lane 2, heavily pigmented cells from the center of the sheet; lane 3, cells that have migrated out of the sheet. Bottom: RPE65 and vimentin band density normalized for protein loading. Results are mean ± SEM of five different samples. *P < 0.05, significantly different from in situ sample. (B) Immunocytochemical staining of RPE65. RPE cells cultured for 6 days were fixed and stained using an antibody against RPE65. Top: corresponding phase-contrast micrographs for the RPE65 staining shown at the bottom. Scale bar, 20 μm. Images are representative of results obtained in 10 different samples. (C) Immunohistochemical staining of Na,K-ATPase α1 subunit (NKAα1). RPE cells cultured for 6 days were fixed, paraffin embedded, and sectioned. After deparaffinization and antigen retrieval, sections were stained using an antibody against Na,K-ATPase α1 subunit. *Lens capsule. Scale bar, 10 μm. Note that NKAα1 expression is limited to the apical membrane in the heavily pigmented cells (Diff.). (D–F) Immunocytochemical staining of vimentin. RPE cells cultured for 6 days were fixed and stained using an antibody against vimentin. Top: corresponding phase-contrast micrographs for the RPE65 staining shown at the bottom. (D) RPE cells were cultured in normal growth media. (E) RPE cells received 2 mM EGTA for the final 2 days. (F) RPE cells received 10 ng/mL TGF-β2 for the entire 6-day period. Staining for vimentin was observed only in the presence of EGTA in the heavily pigmented cells in the center of the sheet. Scale bar, 20 μm. Images are representative of results obtained in (D) 10, (E) four, and (F) four samples.
Figure 3.
 
Polarized expression of Na,K-ATPase and expression of retinal pigment epithelium–specific marker RPE65 and vimentin, an intermediate filament protein that is increased on EMT in a variety of epithelial cells, in cultured RPE cells. (A) RPE65 and vimentin expression by cultured and in situ RPE cells. Western blot for RPE65, vimentin, and β-actin (loading control) representative of results obtained in five separate samples are shown. Lane 1, in situ sample; lane 2, heavily pigmented cells from the center of the sheet; lane 3, cells that have migrated out of the sheet. Bottom: RPE65 and vimentin band density normalized for protein loading. Results are mean ± SEM of five different samples. *P < 0.05, significantly different from in situ sample. (B) Immunocytochemical staining of RPE65. RPE cells cultured for 6 days were fixed and stained using an antibody against RPE65. Top: corresponding phase-contrast micrographs for the RPE65 staining shown at the bottom. Scale bar, 20 μm. Images are representative of results obtained in 10 different samples. (C) Immunohistochemical staining of Na,K-ATPase α1 subunit (NKAα1). RPE cells cultured for 6 days were fixed, paraffin embedded, and sectioned. After deparaffinization and antigen retrieval, sections were stained using an antibody against Na,K-ATPase α1 subunit. *Lens capsule. Scale bar, 10 μm. Note that NKAα1 expression is limited to the apical membrane in the heavily pigmented cells (Diff.). (D–F) Immunocytochemical staining of vimentin. RPE cells cultured for 6 days were fixed and stained using an antibody against vimentin. Top: corresponding phase-contrast micrographs for the RPE65 staining shown at the bottom. (D) RPE cells were cultured in normal growth media. (E) RPE cells received 2 mM EGTA for the final 2 days. (F) RPE cells received 10 ng/mL TGF-β2 for the entire 6-day period. Staining for vimentin was observed only in the presence of EGTA in the heavily pigmented cells in the center of the sheet. Scale bar, 20 μm. Images are representative of results obtained in (D) 10, (E) four, and (F) four samples.
Migrating RPE Cells Lose Apicobasal Polarity and Undergo EMT
Concomitant with the loss of epithelial morphology, cells undergoing EMT classically lose apicobasal polarity. Na,K-ATPase is restricted to the apical surfaces of RPE cells in situ. Thus, it serves as a marker of RPE polarization. Figure 3C shows that apical expression of Na,K-ATPase is maintained in RPE cells in the central region of the original sheet; however, though Na,K-ATPase continues to be expressed on the migrating RPE cells, it is evenly distributed across the surfaces of these cells, demonstrating that the cells have lost polarity. 
Expression of vimentin, which is significantly increased on EMT in a variety of epithelial cells including RPE, 1,20 was also examined. Western blot analysis showed that vimentin expression was low in cells from the central region of each sheet (comparable to expression levels in retinal pigment epithelium in situ; Fig. 3A). In contrast, vimentin expression was induced in the cells migrating away from the sheet. Additionally, immunocytochemical staining for vimentin showed that vimentin expression was largely confined to the migrating cells (Fig. 3D). Loss of pigment, acquisition of fibroblastic morphology, diminished phagocytic capacity, loss of RPE65 expression, loss of polarity, and induction of vimentin expression indicate that RPE cells that lose cell-cell contact and migrate away from the original sheet are undergoing EMT. 
Migrating RPE Cells Initiate Cell Proliferation
In addition to EMT, the proliferation of RPE cells plays a major role in the development of PVR. We used BrdU incorporation as a measure of RPE cell proliferation in our primary cultures and found that cells in the central regions of the sheets remained nonproliferative whereas the cells migrating away from the sheets initiated proliferation (Fig. 4A). 
Figure 4.
 
Proliferation of cultured RPE cells. (A) Immunocytochemical staining for BrdU and corresponding phase-contrast images. RPE cells were cultured in the presence of 20 μM BrdU for 4 hours. Cells were fixed, treated with acid/detergent mixture to denature nuclear material, and stained using an antibody against BrdU. Top: phase-contrast images. BrdU staining is shown in the second row, and BrdU staining merged with phase-contrast images is shown in the third row. Scale bar, 50 μm. (B) Western blot analysis of cyclin D1 expression. β-Actin was used as a loading control. Lane 1, in situ sample; lane 2, heavily pigmented cells from the center of the sheet; lane 3, cells that have migrated out of the sheet. Bottom: bar graph shows cyclin D1 band density normalized to β-actin expression. Results are mean ± SEM of four different samples. BrdU uptake in the presence of (C) 2 mM EGTA for the final 2 days and (D) 10 ng/mL TGF-β2 for the entire 6-day period. Scale bar, 50 μm. BrdU uptake is observed only in the presence of EGTA in the heavily pigmented cells in the center of the sheet.
Figure 4.
 
Proliferation of cultured RPE cells. (A) Immunocytochemical staining for BrdU and corresponding phase-contrast images. RPE cells were cultured in the presence of 20 μM BrdU for 4 hours. Cells were fixed, treated with acid/detergent mixture to denature nuclear material, and stained using an antibody against BrdU. Top: phase-contrast images. BrdU staining is shown in the second row, and BrdU staining merged with phase-contrast images is shown in the third row. Scale bar, 50 μm. (B) Western blot analysis of cyclin D1 expression. β-Actin was used as a loading control. Lane 1, in situ sample; lane 2, heavily pigmented cells from the center of the sheet; lane 3, cells that have migrated out of the sheet. Bottom: bar graph shows cyclin D1 band density normalized to β-actin expression. Results are mean ± SEM of four different samples. BrdU uptake in the presence of (C) 2 mM EGTA for the final 2 days and (D) 10 ng/mL TGF-β2 for the entire 6-day period. Scale bar, 50 μm. BrdU uptake is observed only in the presence of EGTA in the heavily pigmented cells in the center of the sheet.
Expression of cyclin D1, which is involved in cell cycle progression, was examined by Western blot analysis. Consistent with the notion that RPE cell-cell contact in the central region of each sheet imposes cell cycle arrest (e.g., through the inhibition of cyclin D1 expression), we found that cyclin D1 was low in the quiescent cells in the central region of the sheet, whereas it was increased in the proliferating cells migrating away from the sheet (Fig. 4B). 
Maintenance of RPE Phenotype Is Calcium Dependent, and EMT and Onset of Proliferation in Migrating RPE Cells Are Associated with Switch in Cadherin Expression from P- to N-Cadherin
Addition of 2 mM EGTA, used to disrupt cadherin ligation by chelating calcium, led to loss of RPE cell-cell contacts in the central region of each sheet within 4 hours (data not shown). After 2 days in the presence of EGTA, the cells initiated expression of the EMT marker vimentin and began to incorporate BrdU as a measure of proliferation (Figs. 3E, 4C). 
Mammalian RPE cells in situ have been shown to express P-cadherin, which is required for human RPE viability in vivo, 21 whereas the expression of E-cadherin is species dependent. 22,23 We found that RPE cells in the central region of each sheet expressed abundant P-cadherin, but this expression was lost on the migrating cells at the edges of the sheet (Figs. 5A, 5B). EMT and onset of cell proliferation is classically accompanied by a change in cadherin expression to N-cadherin. 9 In contrast to P-cadherin, N-cadherin expression was low in the central region of the RPE sheet, and it was induced on migrating cells at the edges of the sheet (Figs. 5A, 5C). 
Figure 5.
 
Expression of cadherins by cultured RPE cells. (A) Western blot analyses of P- and N-cadherin expression. Left: Western blot for P- and N-cadherin and for β-actin (loading control) representative of results obtained in five separate samples. Right: cadherin band density normalized for protein loading. Results are mean ± SEM of five different samples. *P < 0.05, significantly different from differentiated cells. Immunocytochemical staining of (B) P-cadherin and (C) N-cadherin. RPE cells cultured for 6 days were fixed and stained using antibodies specific for each protein. Top: phase-contrast images. Middle: corresponding staining for each protein. Regions indicated by white dotted insets (79 μm × 79 μm) are magnified at the bottom. Scale bar, 50 μm.
Figure 5.
 
Expression of cadherins by cultured RPE cells. (A) Western blot analyses of P- and N-cadherin expression. Left: Western blot for P- and N-cadherin and for β-actin (loading control) representative of results obtained in five separate samples. Right: cadherin band density normalized for protein loading. Results are mean ± SEM of five different samples. *P < 0.05, significantly different from differentiated cells. Immunocytochemical staining of (B) P-cadherin and (C) N-cadherin. RPE cells cultured for 6 days were fixed and stained using antibodies specific for each protein. Top: phase-contrast images. Middle: corresponding staining for each protein. Regions indicated by white dotted insets (79 μm × 79 μm) are magnified at the bottom. Scale bar, 50 μm.
EMT or Proliferation Not Initiated by TGF-β2 in RPE Cells Maintaining Cell-Cell Contact
TGF-β is classically involved in EMT and fibrotic changes in a variety of cell types. 5,24 TGF-β2 is the major TGF-β isoform in the eye; therefore, we added 10 ng/mL TGF-β2 to our RPE cultures to determine whether it would initiate EMT and proliferation in the RPE cells. Surprisingly, culture for 6 days in the presence of TGF-β2 had no detectable effect on epithelial morphology, pigmentation, proliferation, or vimentin expression in RPE cells maintaining cell-cell contacts in the central regions of the sheets (Figs. 3F, 4D). 
Past studies have shown that adding TGF-β to cultured RPE cells induces a highly contractile myofibroblastic phenotype that is linked to the expression of α-smooth muscle actin (α-SMA). 2527 Therefore, we wondered whether TGF-β2 may induce the expression of α-SMA. Interestingly, TGF-β2 did not induce α-SMA in cells in the central regions of the RPE sheets, but it did induce expression in cells that had undergone EMT (Fig. 6). Western blot analysis shows that though α-SMA was not induced by TGF-β2 in the differentiated cells, there was strong α-SMA expression in the dedifferentiated cells in the presence of TGF-β2 (Fig. 6A). Phalloidin staining showed prominent stress fibers in the cells expressing α-SMA, and most α-SMA was incorporated into these stress fibers (Fig. 6C). Taken together, these results suggest that though TGF-β2 cannot initiate EMT in the RPE cells in the central region of each sheet where cell-cell contacts are maintained, it induces a myofibroblast-like expression pattern in the migrating cells characterized by the induction of α-SMA and the organization of stress fibers characteristic of highly contractile cells. 
Figure 6.
 
Expression of α-SMA in cultured RPE cells. (A) Western blot analysis of α-SMA expression by RPE cells cultured in the presence or absence of 10 ng/mL exogenous TGF-β2. β-Actin was used as a loading control. Representative results obtained in three separate samples are shown. Bottom: α-SMA band density normalized for protein loading. Results are mean ± SEM of three different samples. Strong expression of α-SMA was observed in the presence of TGF-β2 only in the dedifferentiated cells. *P < 0.05, significantly different from differentiated cells in control condition. (B, C) RPE cells cultured for 6 days were fixed, stained using an antibody against α-SMA, and counterstained for F-actin using Texas-red X-phalloidin. (B) RPE cells cultured in the absence of exogenous TGF-β2. Limited number of α-SMA–positive cells was observed only in dedifferentiated cells close to the original edge of RPE sheet. (C) RPE cells cultured for 6 days in the presence of 10 ng/mL TGF-β2. Strong expression of α-SMA, which is expressed as stress fibers, was observed in numerous dedifferentiated cells. No α-SMA staining was observed in the heavily pigmented differentiated RPE phenotype. Scale bar, 50 μm. Insets measure 79 μm × 79 μm.
Figure 6.
 
Expression of α-SMA in cultured RPE cells. (A) Western blot analysis of α-SMA expression by RPE cells cultured in the presence or absence of 10 ng/mL exogenous TGF-β2. β-Actin was used as a loading control. Representative results obtained in three separate samples are shown. Bottom: α-SMA band density normalized for protein loading. Results are mean ± SEM of three different samples. Strong expression of α-SMA was observed in the presence of TGF-β2 only in the dedifferentiated cells. *P < 0.05, significantly different from differentiated cells in control condition. (B, C) RPE cells cultured for 6 days were fixed, stained using an antibody against α-SMA, and counterstained for F-actin using Texas-red X-phalloidin. (B) RPE cells cultured in the absence of exogenous TGF-β2. Limited number of α-SMA–positive cells was observed only in dedifferentiated cells close to the original edge of RPE sheet. (C) RPE cells cultured for 6 days in the presence of 10 ng/mL TGF-β2. Strong expression of α-SMA, which is expressed as stress fibers, was observed in numerous dedifferentiated cells. No α-SMA staining was observed in the heavily pigmented differentiated RPE phenotype. Scale bar, 50 μm. Insets measure 79 μm × 79 μm.
Discussion
In addition to playing a crucial role in developmental processes, EMT is associated with fibrotic diseases and with cancer metastasis. EMT and ectopic proliferation of RPE cells have been suggested to contribute to the development of PVR. 2,3,28 When RPE cells become dislodged into the vitreous cavity or beneath the neurosensory retina, they experience an environmental change with regard to exposure to cytokines and growth factors, and their normal cell-cell and cell-matrix interactions are disrupted. Any of these changes may contribute to the subsequent initiation of proliferation and EMT leading to PVR. Here, we have used cultured sheets of retinal pigment epithelium to examine factors contributing to EMT. In agreement with previous studies, 1113 cells in the center of each sheet, where original cell-cell contact is maintained, retained RPE morphology and pigmentation. In addition, apicobasal polarity and RPE function (phagocytic activity and RPE65 expression) were also retained. Expression of cyclin D1, which is required for the transition of cells into the S phase, 29 was low in these cells maintaining cell-cell contact, and BrdU incorporation confirmed that these cells were nonproliferative. This is in agreement with past studies that have shown cell-cell contacts in epithelial cells prevents transactivation of the cyclin D1 regulatory subunit by sequestering β-catenin. 29,30 Loss of cell-cell contact at the edges of the sheets led to EMT and the initiation of proliferation. In addition, the disruption of cell-cell contact by EGTA treatment, which has previously been shown to disturb cadherin ligation of RPE cells, 31,32 induced vimentin expression and proliferation in the central region of the sheet. These data strongly suggest that loss of cell-cell contact is responsible for initiating EMT and the proliferation of RPE cells. The importance of cell-cell adhesion in the maintenance of RPE phenotype is supported by a recent study 33 demonstrating that the disruption of RPE cell adhesion by inactivation of PTEN resulted in RPE cell EMT. 
Cell adhesion molecules, especially cadherins, have been shown to play a major role in regulating cell fate. 7 Homotypic ligation of cadherins on the surfaces of epithelial cells has been shown to be important in maintaining epithelial differentiation and cell cycle arrest. 3436 Therefore, we hypothesized that cadherin ligation was responsible for preventing EMT and proliferation in RPE cells in the central region of each sheet. P-cadherin has been consistently detected in mammalian retinal pigment epithelium in situ, whereas the expression of E-cadherin, the well-characterized cadherin isoform expressed by most epithelial cell types, in RPE cells is species dependent. 22,23 Mutation of P-cadherin in humans results in RPE atrophy, suggesting the importance of this protein in RPE cell function and maintenance. 21 A recent study using a systematic siRNA array demonstrated that P-cadherin is a primary regulator of cell-cell adhesion and, thus, an inhibitor of migration in breast epithelial cells. 37 Furthermore, Sarrio et al. 38 have shown that the transfection of P-cadherin mRNA into an invasive breast cancer cell line inhibited cell migration and invasion. Thus, the loss of P-cadherin that we observed on cells at the edges of the RPE sheets may be responsible for initiating the migratory phenotype of these cells. Future studies will examine the molecular details of P-cadherin–mediated cell-cell contact in RPE cells, its potential role in preventing EMT, and P-cadherin downregulation during EMT. 
Interestingly, we found that RPE cell migration and EMT coincided with a switch from P- to N-cadherin. A similar cadherin switch from E-cadherin to N-cadherin is known to occur during EMT in a variety of cell types. 9 Increased expression of N-cadherin has been shown to induce a motile and invasive phenotype in epithelial cells, possibly by modulating growth factor signaling. 3942 In the RPE cells, Van Aken et al. 43 demonstrated that N-cadherin is involved in the signal initiated by c-met, the HGF receptor, which plays a role in RPE invasion of the collagen matrix. HGF, which has been suggested to play a role in EMT of several epithelial cell types, including RPE cells, 44 is also known as scatter factor for its ability to dissociate or “scatter” tightly growing epithelial colonies to form elongated fibroblastic cells. Taken together, data showing that N-cadherin plays a role in c-met–initiated signaling that is normally involved in dissociating cell-cell contact, and possibly EMT, agrees with our findings. It should be noted, however, that like E-cadherin, N-cadherin can mediate cell-cell contact and bind to β-catenin. 22,31,43,45,46 Thus, the molecular details of how the induction of N-cadherin contributes to EMT in RPE cells specifically, and epithelial cells generally, is still unclear, and an understanding of the molecular details of how N-cadherin leads to EMT will be a crucial avenue of future investigation. 
TGF-β is classically linked to EMT. 5,24 Thus, we were surprised to find that TGF-β2 was unable to initiate either EMT or proliferation in RPE cells maintaining cell-cell contact. However, once cell-cell contacts were lost, TGF-β2 treatment led to the induction of α-SMA and the formation of stress fibers characteristic of highly contractile myofibroblasts. Such results then imply a secondary effect for TGF-β2—downstream of the initial phase of EMT in RPE cells—during transition to a myofibroblastic phenotype. Such a transition to a contractile phenotype may provide for the development of a tractional retinal detachment, which is the primary event leading to the failure of retinal reattachment surgery in PVR. Indeed, knockout of the key TGF-β downstream signaling molecule, Smad3, significantly reduced PVR-like pathology in a mouse model. 6 It will be of interest to determine whether the role for TGF-β is similarly cell-cell contact–dependent in other cells undergoing EMT and, thus, whether its role is also secondary to the initiation of EMT triggered by loss of cell-cell contact. 
Footnotes
 Supported by University of Louisville Intramural Research Incentive Grant (ST), the Kentucky Research Challenge Trust Fund (HJK), an unrestricted grant from Research to Prevent Blindness Inc., and the Kentucky Lions Eye Foundation.
Footnotes
 Disclosure: S. Tamiya, None; L. Liu, None; H.J. Kaplan, None
The authors thank Douglas Dean for constructive advice on interpreting part of the data and for critical reading of the manuscript; Qiling Z. Ruley for technical contributions during the early stages of establishment of the culture model; and Jon Klein for providing access to the LICOR Odyssey infrared scanner that was used for Western blot analysis. The BrdU antibody (clone G3G4) developed by Stephen J. Kaufman was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the Department of Biology, University of Iowa. 
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Figure 1.
 
Time course of phase-contrast images of RPE cells cultured on the lens capsule. Images of live cells were captured 0, 4, and 6 days after the cell sheet was set. Magnified images of insets (79 μm × 79 μm; a–d) are shown across the bottom. On day 0, all the cells are heavily pigmented with typical epithelial morphology. By day 4, cells at the migrating edge are larger and elongated and have less pigment. By day 6, cells at the migrating edge are even less pigmented (d). In contrast, cells within the original sheet maintain heavy pigmentation and epithelial morphology (c). Scale bar, 100 μm.
Figure 1.
 
Time course of phase-contrast images of RPE cells cultured on the lens capsule. Images of live cells were captured 0, 4, and 6 days after the cell sheet was set. Magnified images of insets (79 μm × 79 μm; a–d) are shown across the bottom. On day 0, all the cells are heavily pigmented with typical epithelial morphology. By day 4, cells at the migrating edge are larger and elongated and have less pigment. By day 6, cells at the migrating edge are even less pigmented (d). In contrast, cells within the original sheet maintain heavy pigmentation and epithelial morphology (c). Scale bar, 100 μm.
Figure 2.
 
Microvilli and phagocytic activity of cultured RPE cells. (A) Scanning electron micrograph of RPE cells cultured for 6 days. Samples were tilted 40° and observed using a scanning electron microscope. Top: regions indicated by white insets (22 μm × 22 μm) are magnified at the bottom. Dotted lines: cell border. Arrows: nuclei. Note that heavily pigmented differentiated cells retain dense microvilli, whereas the less pigmented cells that have migrated out of the original RPE sheet (dedifferentiated) have lost most of the microvilli. Scale bar, 25 μm. Images are representative of results obtained in five different samples. (B) Fluorescence micrograph showing ingested FITC-labeled POS by RPE cells cultured for 6 days. Cells were incubated in the presence of FITC-labeled POS for 100 minutes. Cells were washed, treated with trypan blue, washed, fixed, and observed using a fluorescence microscope. The high phagocytic activity shown by the heavily pigmented RPE cells (Diff.) is lost in the less pigmented dedifferentiated cells (De-diff.). Scale bar, 20 μm. Images are representative of results obtained in four different samples. (C) Number of labeled POS ingested by cultured RPE cells. For each sample, four different fields of view (79 μm × 107 μm) were counted for heavily pigmented differentiated cells and less pigmented cells close to the migrating edge. Data shown are mean ± SEM of four different samples. Significant difference (P < 0.05) was observed in the number of ingested POS between two different cell phenotypes.
Figure 2.
 
Microvilli and phagocytic activity of cultured RPE cells. (A) Scanning electron micrograph of RPE cells cultured for 6 days. Samples were tilted 40° and observed using a scanning electron microscope. Top: regions indicated by white insets (22 μm × 22 μm) are magnified at the bottom. Dotted lines: cell border. Arrows: nuclei. Note that heavily pigmented differentiated cells retain dense microvilli, whereas the less pigmented cells that have migrated out of the original RPE sheet (dedifferentiated) have lost most of the microvilli. Scale bar, 25 μm. Images are representative of results obtained in five different samples. (B) Fluorescence micrograph showing ingested FITC-labeled POS by RPE cells cultured for 6 days. Cells were incubated in the presence of FITC-labeled POS for 100 minutes. Cells were washed, treated with trypan blue, washed, fixed, and observed using a fluorescence microscope. The high phagocytic activity shown by the heavily pigmented RPE cells (Diff.) is lost in the less pigmented dedifferentiated cells (De-diff.). Scale bar, 20 μm. Images are representative of results obtained in four different samples. (C) Number of labeled POS ingested by cultured RPE cells. For each sample, four different fields of view (79 μm × 107 μm) were counted for heavily pigmented differentiated cells and less pigmented cells close to the migrating edge. Data shown are mean ± SEM of four different samples. Significant difference (P < 0.05) was observed in the number of ingested POS between two different cell phenotypes.
Figure 3.
 
Polarized expression of Na,K-ATPase and expression of retinal pigment epithelium–specific marker RPE65 and vimentin, an intermediate filament protein that is increased on EMT in a variety of epithelial cells, in cultured RPE cells. (A) RPE65 and vimentin expression by cultured and in situ RPE cells. Western blot for RPE65, vimentin, and β-actin (loading control) representative of results obtained in five separate samples are shown. Lane 1, in situ sample; lane 2, heavily pigmented cells from the center of the sheet; lane 3, cells that have migrated out of the sheet. Bottom: RPE65 and vimentin band density normalized for protein loading. Results are mean ± SEM of five different samples. *P < 0.05, significantly different from in situ sample. (B) Immunocytochemical staining of RPE65. RPE cells cultured for 6 days were fixed and stained using an antibody against RPE65. Top: corresponding phase-contrast micrographs for the RPE65 staining shown at the bottom. Scale bar, 20 μm. Images are representative of results obtained in 10 different samples. (C) Immunohistochemical staining of Na,K-ATPase α1 subunit (NKAα1). RPE cells cultured for 6 days were fixed, paraffin embedded, and sectioned. After deparaffinization and antigen retrieval, sections were stained using an antibody against Na,K-ATPase α1 subunit. *Lens capsule. Scale bar, 10 μm. Note that NKAα1 expression is limited to the apical membrane in the heavily pigmented cells (Diff.). (D–F) Immunocytochemical staining of vimentin. RPE cells cultured for 6 days were fixed and stained using an antibody against vimentin. Top: corresponding phase-contrast micrographs for the RPE65 staining shown at the bottom. (D) RPE cells were cultured in normal growth media. (E) RPE cells received 2 mM EGTA for the final 2 days. (F) RPE cells received 10 ng/mL TGF-β2 for the entire 6-day period. Staining for vimentin was observed only in the presence of EGTA in the heavily pigmented cells in the center of the sheet. Scale bar, 20 μm. Images are representative of results obtained in (D) 10, (E) four, and (F) four samples.
Figure 3.
 
Polarized expression of Na,K-ATPase and expression of retinal pigment epithelium–specific marker RPE65 and vimentin, an intermediate filament protein that is increased on EMT in a variety of epithelial cells, in cultured RPE cells. (A) RPE65 and vimentin expression by cultured and in situ RPE cells. Western blot for RPE65, vimentin, and β-actin (loading control) representative of results obtained in five separate samples are shown. Lane 1, in situ sample; lane 2, heavily pigmented cells from the center of the sheet; lane 3, cells that have migrated out of the sheet. Bottom: RPE65 and vimentin band density normalized for protein loading. Results are mean ± SEM of five different samples. *P < 0.05, significantly different from in situ sample. (B) Immunocytochemical staining of RPE65. RPE cells cultured for 6 days were fixed and stained using an antibody against RPE65. Top: corresponding phase-contrast micrographs for the RPE65 staining shown at the bottom. Scale bar, 20 μm. Images are representative of results obtained in 10 different samples. (C) Immunohistochemical staining of Na,K-ATPase α1 subunit (NKAα1). RPE cells cultured for 6 days were fixed, paraffin embedded, and sectioned. After deparaffinization and antigen retrieval, sections were stained using an antibody against Na,K-ATPase α1 subunit. *Lens capsule. Scale bar, 10 μm. Note that NKAα1 expression is limited to the apical membrane in the heavily pigmented cells (Diff.). (D–F) Immunocytochemical staining of vimentin. RPE cells cultured for 6 days were fixed and stained using an antibody against vimentin. Top: corresponding phase-contrast micrographs for the RPE65 staining shown at the bottom. (D) RPE cells were cultured in normal growth media. (E) RPE cells received 2 mM EGTA for the final 2 days. (F) RPE cells received 10 ng/mL TGF-β2 for the entire 6-day period. Staining for vimentin was observed only in the presence of EGTA in the heavily pigmented cells in the center of the sheet. Scale bar, 20 μm. Images are representative of results obtained in (D) 10, (E) four, and (F) four samples.
Figure 4.
 
Proliferation of cultured RPE cells. (A) Immunocytochemical staining for BrdU and corresponding phase-contrast images. RPE cells were cultured in the presence of 20 μM BrdU for 4 hours. Cells were fixed, treated with acid/detergent mixture to denature nuclear material, and stained using an antibody against BrdU. Top: phase-contrast images. BrdU staining is shown in the second row, and BrdU staining merged with phase-contrast images is shown in the third row. Scale bar, 50 μm. (B) Western blot analysis of cyclin D1 expression. β-Actin was used as a loading control. Lane 1, in situ sample; lane 2, heavily pigmented cells from the center of the sheet; lane 3, cells that have migrated out of the sheet. Bottom: bar graph shows cyclin D1 band density normalized to β-actin expression. Results are mean ± SEM of four different samples. BrdU uptake in the presence of (C) 2 mM EGTA for the final 2 days and (D) 10 ng/mL TGF-β2 for the entire 6-day period. Scale bar, 50 μm. BrdU uptake is observed only in the presence of EGTA in the heavily pigmented cells in the center of the sheet.
Figure 4.
 
Proliferation of cultured RPE cells. (A) Immunocytochemical staining for BrdU and corresponding phase-contrast images. RPE cells were cultured in the presence of 20 μM BrdU for 4 hours. Cells were fixed, treated with acid/detergent mixture to denature nuclear material, and stained using an antibody against BrdU. Top: phase-contrast images. BrdU staining is shown in the second row, and BrdU staining merged with phase-contrast images is shown in the third row. Scale bar, 50 μm. (B) Western blot analysis of cyclin D1 expression. β-Actin was used as a loading control. Lane 1, in situ sample; lane 2, heavily pigmented cells from the center of the sheet; lane 3, cells that have migrated out of the sheet. Bottom: bar graph shows cyclin D1 band density normalized to β-actin expression. Results are mean ± SEM of four different samples. BrdU uptake in the presence of (C) 2 mM EGTA for the final 2 days and (D) 10 ng/mL TGF-β2 for the entire 6-day period. Scale bar, 50 μm. BrdU uptake is observed only in the presence of EGTA in the heavily pigmented cells in the center of the sheet.
Figure 5.
 
Expression of cadherins by cultured RPE cells. (A) Western blot analyses of P- and N-cadherin expression. Left: Western blot for P- and N-cadherin and for β-actin (loading control) representative of results obtained in five separate samples. Right: cadherin band density normalized for protein loading. Results are mean ± SEM of five different samples. *P < 0.05, significantly different from differentiated cells. Immunocytochemical staining of (B) P-cadherin and (C) N-cadherin. RPE cells cultured for 6 days were fixed and stained using antibodies specific for each protein. Top: phase-contrast images. Middle: corresponding staining for each protein. Regions indicated by white dotted insets (79 μm × 79 μm) are magnified at the bottom. Scale bar, 50 μm.
Figure 5.
 
Expression of cadherins by cultured RPE cells. (A) Western blot analyses of P- and N-cadherin expression. Left: Western blot for P- and N-cadherin and for β-actin (loading control) representative of results obtained in five separate samples. Right: cadherin band density normalized for protein loading. Results are mean ± SEM of five different samples. *P < 0.05, significantly different from differentiated cells. Immunocytochemical staining of (B) P-cadherin and (C) N-cadherin. RPE cells cultured for 6 days were fixed and stained using antibodies specific for each protein. Top: phase-contrast images. Middle: corresponding staining for each protein. Regions indicated by white dotted insets (79 μm × 79 μm) are magnified at the bottom. Scale bar, 50 μm.
Figure 6.
 
Expression of α-SMA in cultured RPE cells. (A) Western blot analysis of α-SMA expression by RPE cells cultured in the presence or absence of 10 ng/mL exogenous TGF-β2. β-Actin was used as a loading control. Representative results obtained in three separate samples are shown. Bottom: α-SMA band density normalized for protein loading. Results are mean ± SEM of three different samples. Strong expression of α-SMA was observed in the presence of TGF-β2 only in the dedifferentiated cells. *P < 0.05, significantly different from differentiated cells in control condition. (B, C) RPE cells cultured for 6 days were fixed, stained using an antibody against α-SMA, and counterstained for F-actin using Texas-red X-phalloidin. (B) RPE cells cultured in the absence of exogenous TGF-β2. Limited number of α-SMA–positive cells was observed only in dedifferentiated cells close to the original edge of RPE sheet. (C) RPE cells cultured for 6 days in the presence of 10 ng/mL TGF-β2. Strong expression of α-SMA, which is expressed as stress fibers, was observed in numerous dedifferentiated cells. No α-SMA staining was observed in the heavily pigmented differentiated RPE phenotype. Scale bar, 50 μm. Insets measure 79 μm × 79 μm.
Figure 6.
 
Expression of α-SMA in cultured RPE cells. (A) Western blot analysis of α-SMA expression by RPE cells cultured in the presence or absence of 10 ng/mL exogenous TGF-β2. β-Actin was used as a loading control. Representative results obtained in three separate samples are shown. Bottom: α-SMA band density normalized for protein loading. Results are mean ± SEM of three different samples. Strong expression of α-SMA was observed in the presence of TGF-β2 only in the dedifferentiated cells. *P < 0.05, significantly different from differentiated cells in control condition. (B, C) RPE cells cultured for 6 days were fixed, stained using an antibody against α-SMA, and counterstained for F-actin using Texas-red X-phalloidin. (B) RPE cells cultured in the absence of exogenous TGF-β2. Limited number of α-SMA–positive cells was observed only in dedifferentiated cells close to the original edge of RPE sheet. (C) RPE cells cultured for 6 days in the presence of 10 ng/mL TGF-β2. Strong expression of α-SMA, which is expressed as stress fibers, was observed in numerous dedifferentiated cells. No α-SMA staining was observed in the heavily pigmented differentiated RPE phenotype. Scale bar, 50 μm. Insets measure 79 μm × 79 μm.
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