Isoforms of platelet-derived growth factor and its receptors in epiretinal membranes: Immunolocalization to retinal pigmented epithelial cells

Exp. Eye Res. (1995) 60, 607-619

Isoforms of Platelet-derived Growth Factor and Its Receptors in Epiretinal Membranes" Immunolocalization to Retinal Pigmented Epithelial Cells S T A N L E Y A . VINORES*,JEFFREY D. HENDERER, JEREMYMAHLOW, CHARLIE CHIU, NANCYL. DEREVJANIK, W I L L I A M LAROCHELLE',CARL CSAKY a, AND P E T E R A. C A M P O C H I A R O

The Wilmer Ophthalmological Institute, Johns Hopkins University, School of Medicine, Baltimore, MD 21287 and "The National Institutes of Health, Bethesda, MD 20892, U.S.A. (Received Seattle 18 July 1994 and accepted in revised form 23 January 1995) Epiretinal membranes (ERMs) form on the inner surface of the retina in conjunction with various ocular disease processes, but the factors controlling their development are not understood. The predominant cell types involved are retinal pigmented epithelial (RPE) cells and retinal gila. Cultured RPE cells secrete platelet-derived growth factor (PDGF), which is chemotactic and mitogenic for both RPE ceils and retinal gila and, therefore, could be involved in the development of ERMs. In the present study, we performed immunohistochemical staining for PDGF A chain (PDGF-A), PDGF B chain (PDGF-B), and both types of PDGF receptors (PDGF,= and PDGFrp) on ERMs associated with various disease processes. PDGF-A is detected in most ERMs, regardless of the associated disease process, and it appears to be localized predominantly in RPE cells, recognized by the presence of pigment and the immunohistochemical demonstration of some or all of the following RPE-associated epitopes: class rn/?-tubulin, keratin, the 65kDa microsomal protein recognized by the RPE9 antibody, and cellular retinaldehyde-binding protein. PDGF-B is found only in minor subpopulations of cells in about half of the ERMs evaluated and, with only occasional exceptions, appears to be localized almost entirely in blood-borne cells found in and around vessels in vascularized ERMs. Both PDGFr= and PDGF,p are demonstrated in most ERMs with neither isotype consistently predominating: they are found predominantly on RPE cells with many cells expressing both receptor types. ERMs with little or no RPE cell component contain little or no PDGF and PDGF receptor, whereas those in which the RPE cell represents the major cell type, have widespread PDGF and PDGF receptor positivity. These findings show that RPE cells in ERMs produce PDGF-A and PDGF= and PDGFp receptors and suggest that autocrine and paracrine stimulation with PDGF may be involved in ERM pathogenesis. 1. Introduction Epiretinal m e m b r a n e s (ERMs) are sheets of cells and extracellular matrix that form on the inner surface of the retina. They are frequently located predominantly in the macula, in which case they are referred to as m a c u l a r puckers. Because of their propensity to affect the macula, ERMs are a major cause of decreased v i s u a l acuity and metamorphopsia. ERMs m a y occur in association with several ocular disorders (Clarkson et al., 1977; Kampik et al. 1981), but occur most c o m m o n l y after retinal r e a t t a c h m e n t surgery or in the absence of other identifiable ocular disease, in which case they are referred to as idiopathic ERMs or idiopathic m a c u l a r puckers. ERMs occurring after retinal r e a t t a c h m e n t surgery m a y be located predominantly in the m a c u l a (post retinal detachment m a c u l a r puckers) or m a y be more extensive, resulting in traction retinal detachment or tractionrhegmatogenous detachment, a process referred to as proliferative vitreoretinopathy (PVR).

* For correspondence at: 825 Maumenee Bldg., The Wilmer Ophthalmological Institute, The Johns Hopkins UniversitySchool of Medicine, 600 N., Wolfe St., Baltimore, MD 21287-9289, U.S.A. OO1A. 5835/95/O60683 + 13 $08.00/0

Cell types that have been identified in epiretinal m e m b r a n e s include retinal pigmented epithelial (RPE) cells, retinal gila, macrophages and fibroblasts Green et al., 1979; Hiscott et al., 1984; Kampik et al., 1981 ; Machemer et al., 1978; Vinores et al., 1990a). Macrophages make up a very small component of the cells and the relative contribution of fibroblasts is unclear, because specific markers for fibroblasts are unavailable and dedifferentiated RPE and glia can resemble fibroblasts. By combining immttnohistochemical data (obtained using a panel of antibodies) with morphological characteristics, m a n y cells in epiretinal m e m b r a n e s can be unequivocally identified as RPE or retinal glia. The pathogenesis of ERM formation is poorly understood, but the frequent association of ERMs with retinal detachment, ocular surgery and ocular t r a u m a suggests a role for exaggerated w o u n d repair analogous to scarring elsewhere in the body (Cowley et al., 1989). Retinal cryopexy and laser treatment, two modalities used to create limiting scarring around retinal tears to seal them, have been demonstrated to stimulate ERM growth (Campochiaro et al., 1987; Cowley et al., 1989). RPE cells play a major role in the repair of cryopexy and laser lesions, and other types of © 1995 Academic Press Limited

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FIG. 1. (a) Pigmented ceils in an ERM from a subfovial choroidal neovascular membrane due to ocular histoplasmosis (case 33) are immunostained with antibodies that recognize both chains of PDGF (blue-violet reaction product). Weak PDGF-positivity is also seen in the extracellular matrix, x 600. (b) PDGF-A positivity (red reaction product) in pigmented cells (arrowhead) from an ERM of a diabetic patient (case 23). x 600. (c) A control section from the same ERM (case 23) in which normal rabbit serum was substituted for the primary antibody is negative. × 600. (d) PDGF-A positivity (red reaction product) in unpigmented cells on the surface (upper right) of an ERM from a diabetic patient (case 24). × 600. (e) Serial section from the same case (case 24) in which normal rabbit serum was substituted for primary antibody is negative (absence of red immunoreaction product). × 600. (f) PDGF-B positivity (red reaction product) is confined to blood-borne cells (arrowheads) found in and around vessels in an ERM from a patient with diabetic retinopathy (case 27). Hematoxylin counterstain, x 600. (g) Positivity for PDGFr~ (red reaction product, arrowheads) is visualized on cells from a subfoveal choroidal neovascular membrane due to ocular histoplasmosis (case 33). x 240. (h) The same cluster of ceils (arrowheads) demonstrates positivity for PDGFrp in this serial

P . D G F A N D ITS RECEPTORS IN ERM

retinal injuries (Cleary and Ryan, 1980; Lincoff et al., 1981; Miller et al., 1986; Wallow et al., 1973). The proliferation and migration of RPE cells after retinal detachment may represent an attempt at wound repair and could set the stage for ERM formation. The RPE is normally quiescent and proliferates only when participating in wound repair or when placed in culture. Understanding the signals responsible for altered growth regulation in the RPE could provide important insights into retinal wound repair and ERM formation. Growth factors have been implicated in the pathogenesis of ERMs. The acidic (Fredj-Reygrobellet et al., 1991 ; Malecaze et al., 1991) and basic (Hanneken et al., 1991) forms of fibroblast growth factor, epidermal growth factor, insulin-like growth factor-1 (FredjReygrobellet et al., 1991) and interleukins 1~ and 2 (Tang et al., 1993) have all been demonstrated in ERMs and may play a role in their formation or growth. We have recently demonstrated by the following findings that platelet-derived growth factor (PDGF) may play an important role in growth regulation of RPE cells in culture and in vivo (Campochiaro et al., 1994). (1) Cultured RPE cells produce PDGF A chain (PDGF-A) and PDGF B chain (PDGF-B), and possess both types of PDGF receptors (~ and//receptors); (2) PDGF receptors on RPE are autophosphorylated in serum-free medium (presumably from RPE-derived PDGF) and the autophosphorylation is blocked by suramin, an agent that displaces PDGF from its receptors; (3) A neutralizing antibody to PDGF blocks RPE cell proliferation in serum-free media. These findings suggest that cultured RPE possess an autocrine loop driven by PDGF; they produce PDGF which stimulates their own growth. The PDGF autocrine loop may be involved in wound repair, because PDGF expression is increased in RPE in an in vitro wound repair model and after retinal laser treatment in mice or retinal detachment in mice and rabbits (Campochiaro et al., 1994; Derevjanik et al., 1994). Ira PDGF autocrine loop is involved in ERM formation, one would predict that RPE cells in epiretinal membranes express one or both isoforms of PDGF and one or both PDGF receptors. The present study was designed to examine this issue. 2. Materials and Methods Epiretinal membranes obtained during vitrectomy were promptly fixed for 1 hr at room temperature in 2% paraformaldehyde in 0.1 ~ phosphate buffer, pH 7.4, with 5 % sucrose. The specimens were cryoprotected by gradually increasing the sucrose concentration to 20% and were then frozen in a 2 : 1 ratio

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of 20% sucrose in phosphate buffer: OCT compound (Miles, Elkhart, IN, U.S.A.) as previously described (Barthel and Raymond, 1990). Normal human eyes were obtained from a local eye bank and processed in the same manner after removal of the cornea and lens. Six micrometers frozen sections were immersed in methanol containing 0-75% H202 for 10rain at -20°C, washed with Tris-buffered saline (TBS), and blocked with 10% normal serum (rabbit serum for mouse or goat antibodies, goat serum for rabbit antibodies). The sections were then immunoreacted overnight at 4°C in a humidified chamber with one of the primary antibodies described in Table I. Fo~ controls, normal rabbit serum (NRS) or normal goat serum (NG8) was substituted for primary antibody (or antiserum) or, additionally, with PDGF and PDGF receptor antibodies, the primary antibody (or antiserum) was incubated for I hr at 4°C with a 10-fold molar excess of purified antigen. The slides were warmed to room temperature, washed twice for 5 min with 1% NRS (for goat or mouse antibodies) or NGS (for rabbit antibodies) and incubated 30 min with a 1:200 dilution of rabbit anti-goat immunoglobulins conjugated to alkaline phosphatase (for goat antiPDGF antibodies; Kirkegaard and Perry, Gaithersburg, MD, U.S.A.), a 1:25 dilution of rabbit anti-mouse immunoglobulins (DAKO) in 1% NRS-TBS (for routine antibodies), or a 1:50 dilution of goat anti-rabbit globulins (Arnel) in 1% NGS-TBS (for rabbit antibodies). Specimens immunoreacted with PDGF antibodies (goat) were then washed twice for 5 rain each with TBS. The cells exposed to other primary antibodies were washed as before and incubated 30 rain with a 1:400 dilution of a mouse peroxidase-antiperoxidase complex (Amel) in 1% NILg-TBS (for mouse monoclonal antibodies) or with a 1:100 dilution of a rabbit peroxidase-anti-peroxidase complex (Arnel) in 1% NGS-TBS (for rabbit antibodies). All slides except those reacted with goat antibodies (PDGF) were washed twice for 5 min with 0.05 ~,i Tris buffer, pH 7-6. Immunoreaction product for PDGF (goat antibodies) was visualized by combining 3 ml TBS with 0-3 ml each of nitroblue tetrazolium (NBT) concentrate and 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) concentrate (Kirkegaard and Perry), which yields a blueviolet reaction product, and covering the slides with this solution for 45 rain. The other antibodies were visualized by immunoperoxidase using freshly prepared 0.178 mg m1-1 3-amino-9-ethylcarbazole (AEC) in 0"89 M sodium acetate, pH 5-1, containing 2.1 x 10-4% H20 z, which was made from a filtered stock solution of 1.67mg AEC (ml dimethylsulfoxide) -1, for 10 rain to yield a red color. Double-labeling was performed by combining

section from the same case (case 33). x 240. (i) PDGFr, immunostaining (red reaction product) on unpigmented ceils from a PVR ERM (case 12). x 120. (j) PDGFrppositivity(red reaction product) is shown on unpigmented cellsfrom an E1LMof a diabetic patient (case 22). x 240.

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k FIG. 2. (a) PDGF-A is demonstrated in a group of cells (arrowheads) on the surface of an ERM from a patient with diabetic retinopathy (case 26). Hematoxylin counterstain, x 240. (b) A serial section from the same case shows positive staining for PDGF,.~ in a group of similar cells (arrowheads). Hematoxylin counterstain, × 240. (c) Another serial section from the same case demonstrates PDGFrfpositivity in a group of similar cells on the same surface (arrowheads). Hematoxylin counterstain, × 240. (d) Another serial section from the same case shows immunoreactivity for the RPE-associated RPE9 epitope in the same group of cells on the surface of the ERM (arrowheads). Hematoxylin counterstain, × 240. (e) PDGFr~ immunopositivity (red, arrowheads) is demonstrated in pigmented cells in an ERM from a patient with Eale's disease (case 32). × 600. (f) PDGFr~ is visualized (red, arrowheads) in pigmented cells from the same ERM (case 32). × 600. (g) PDGFr~ is also demonstrated (red reaction product, arrowhead) in a pigmented cell from a PVR ERM (case 13). × 600. (h) A control section from case 32 [for comparison with 2(e) and 2(t)] in which normal rabbit serum was substituted for primary antibody is negative (absence of red immunoreaction product), x 600. (i) Double-labeling immunohistochemistry shows simultaneous staining for PDGF-A (red reaction product) and keratin (blue reaction product) within the same cells (arrowheads) in an ERM from a patient with a macular hole (case 31). Note that the color is intermediate to the red and blue reaction products seen in the previous figures.

P D G F A N D ITS R E C E P T O R S IN ERM

monoclonal antibodies with polyclonal antiserum (final dilutions the same as above) and performing the immunoreaction procedure as carried out above. The bound polyclonal antibodies were detected first with AEC and the bound monoclonal antibodies were subsequently visualized by incubating the sections with a freshly made and filtered solution containing 0.38mgm1-1 4-chloro-l-napthol, 0"3% H202, and 1.5% ethanol in 0"05 M Tris buffer, pH 7.6, to yield a blue-violet color. Cells coexpressing both antigens assume a reddish-violet color that is distinct from that obtained with either chromogen alone. Sections were washed with Tris buffer and mounted with Aqua Poly/Mount (Polysciences, Warrington, PA, U.S.A.). Micrographs were taken on a Zeiss Axioskop (Carl Zeiss, Thornwood, NY, U.S.A.) with Nomarski optics using 64T tungsten film (Kodak, Rochester, NY, U.S.A.). 3. Results

In normal h u m a n retina, immunohistochemical staining for PDGF was primarily localized around retinal vessels. Some positivity for PDGFr= and PDGFrp was also demonstrated around vessels, as well as in scattered cells in the inner retina, and weak staining was seen in the RPE. PDGFrp-positivity appeared stronger in vessels than in RPE, whereas the converse was true for PDGF~=.Hyalocyte-like cells in the vitreous were intensely positive for both types of PDGF receptors. PDGF was demonstrable to varying extents in nearly all ERMs, regardless of the associated disease process (Table II). Positivity was associated with the extracellular matrix and with subpopulations of cells within the ERM [Fig. l(a)]. When cellular positivity for PDGF was seen, positivity for PDGF-A always coincided. PDGF-A was often visualized in pigmented cells [Fig. l(b) and (c)] showing morphological features of RPE cells, but was not limited exclusively to these cells [Fig. l(d) and (e)]. Comparing serial sections stained for RPE-associated antigens such as class-HI fl-tubulin, keratin, the RPE9 epitope, and CRALBP shows that PDGF-A-positive cells frequently express these markers, suggesting that they are RPE cells. PDGF-B was much less common in ERMs and appeared to be almost entirely localized to blood-borne cells (probably macrophages), found in and around vessels, which were recognized by their morphological appearance and the absence of RPE-or gila-associated antigens [Fig. 1(t)]. One exception was case 12, which appeared

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to be predominantly composed of RPE cells based on the following morphological and immunohistochemical characteristics. There were large numbers of pigmented cells with positive staining of cell clusters (including pigmented cells) for the RPE-associated markers, class HI/%tubulin (Vinores et al., in press), and the RPE9 epitope (Hooks et al., 1989). The majority of cells (including pigmented cells) were positive for CRALBP, which also labels RPE cells as well as Mfiller cells (Bunt-Milam and Saari, 1983). Only a single cell demonstrated GFAP, which colocalizes with CRALBP in M/iller cells (Vinores et al., 1990a), suggesting that the CRALBP-positive cells are RPE cells. Some of these cells, which do not resemble macrophages and are probably RPE cells, weakly express PDGF-B. Another exception was case 16, a PVR ERM in which all RPE-like pigmented cells contained class III fl-tubulin and m a n y contained keratin and CRALBP, suggesting that they are derived from RPE. About half of these cells contained PDGF-A, providing evidence that RPE cells are a source of this factor, and occasional pigmented cells were positive for PDGF-B. PDGF receptors were also demonstrated in variable numbers of cells in nearly all ERMs associated with different disease processes. Neither receptor type predominated; five ERMs contained more PDGFr=positive cells, five ERMs contained more PDGFrf positive cells and 14 ERMs had approximately equal numbers of cells expressing ~ and t-receptors. Serial sections revealed that m a n y cell populations possess both isotypes of PDGF receptors [Fig. l(g) and (h)]. Based on the presence of pigment granules, morphological characteristics, and RPE-associated antigens, PDGF ~ and fl receptors, in addition to PDGF, appear to be expressed by RPE cells [Fig. 2(a)-(h)], as well as macrophages and some unidentified cells in ERMs [Fig. 1(i) and (j)]. Two examples providing evidence that RPE cells contain PDGF receptors follow. In PVR ERM 16, most RPE-like cells by morphological criteria, which also express the RPE-associated markers: class HI fl-tubulin, keratin and CRALBP, are positive for PDGFr~ and clusters of the same cells also demonstrate PDGFr=. In another PVR ERM, case 14, the glia-like, GFAP-positive cells were confined to the surface of the ERM. CRALBP antibodies label RPE cells and M~ller cells (Bunt-Milam and Saari, 1983); Mfiller cells are also positive for GFAP (Vinores et al., 1990a). Cells which were not on the surface, were GFAPnegative and were CRALBP-positive (providing evidence for their RPE identity) contained PDGFr= and

x 240. (j) Double-labeling shows co-expression of PDGF-A (red reaction product) and class III fl-tubulin (blue reaction product) within the same cells (arrowheads) in an ERM from a patient with proliferative diabetic retinopathy. Color is intermediate between the red and blue reaction products shown in the previous figures, x 240. (k) Double-labeling demonstrates coexpression of PDGFr~(red reaction product) and class III fl-tubulin (blue reaction product) within the same cells (arrowheads) from a PVR ERM (case 12). Note the color is intermediate between the red and blue reaction products illustrated in the previous figures, x 600.

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FIG. 3. (a) Positive staining for PDGF in a PVR ERM (case 12) using an antiserum that recognizes both the A and B chains. × 200 (b) Preincubation of the PDGF antiserum with purified PDGF (A and B chains) eliminates the immunostaining for PDGF in the same ERM. × 400. (c) Positivity for PDGF,./in a PVR ERM (case 10). x 400. (d) Pre-incubation of the PDGFr~ antiserum with purified fl-receptor eliminates the immunostaining for PDGFr/~in the same ERM. x 400. some also contained PDGF,./. Double-labeling studies provide further support for the expression of PDGF and its receptors by RPE cells by showing the co-expression of PDGF-A with the RPE-associated antigens, keratin [Fig. 2(i)] or class III fl-tubulin [Fig. 2(j)], and the coexpression of PDGFr/ with class III fl-tubulin [Fig. 2(k)]. ERMs that appeared to contain few or no RPE cells (few or no pigmented cells and little or no positivity for RPE-associated antigens), such as cases 2, 6, 15, 29 and 34, had little or no positivity for PDGF and its receptors. Contrastingly, ERMs in which the major cell type appeared to be the RPE cell, such as cases 12, 16, and 22 demonstrated cellular positivity for PDGF and positivity for PDGRr~ and PDGFr~ in the majority of cells. Normal serum controls were consistently negative (Figs l(c), l(e) and 2(h)]. Preincubation of PDGF or PDGF receptor antibodies (antisera) with purified antigen resulted in obliteration or marked reduction of immunohistochemical staining (Fig. 3). 4. Discussion

PDGF was first isolated from platelets (from which it derives its name), but it has been shown to be produced by several cell types including RPE cells (Campochiaro et al., 1989: Mudhar et al., 1993: Yoshida et al., 1992). In the preset study, we have demonstrated immunohistochemical staining for PDGF in most ERMs associated with several different disease processes. The cellular localization of PDGF suggests that the cells within ERMs m a y be its source, but some PDGF is also located in the extracellular matrix. Extracellular matrix localization of PDGF has

been previously demonstrated in other settings: it has been postulated to serve as a storage pool that can be released by appropriate enzymes from damaged or activated cells (Kelly et al., 1993). We also demonstrated PDGFa and fl receptors on cells in most ERMs, suggesting that cells in ERMs are responsive to both isoforms of PDGF (Hart et al., 1988). Many of the cells expressing PDGF receptors can be identified as RPE cells by immunohistochemical and morphological criteria. Our findings contrast, in part, with a recent report stating that only the fl receptors are present on RPE cells in ERMs (Robbins et al., 1994). The different conclusions m a y be attributed to differences in antibody titer and specificity, since both studies used antibodies from different sources, or to difficulties in identifying pathological RPE cells. The report by Robbins et al. (1994) that both types of PDGF receptors are absent from normal RPE cells, while we were able to localize both types on normal RPE cells, supports the former explanation. It is likely that m a n y other ceils expressing PDGF receptors in ERMs that could not be identified m a y also be RPE cells. The identification of RPE cells in ERMs is often difficult because they can assume a variety of morphologies w h e n they are removed from their normal microenvironment and they come in contact with vitreous (Forrester et al., 1986; Vidaurri-Leal et al., 1984: Vinores et al., 1990a, 199b). In addition to morphologic changes, RPE cells can undergo i m m u n o histochemical alterations under pathologic conditions, making them difficult to recognize in ERMs (Vinores et al., 1990a, 1990b, 1992a, 1992b, 1993b). Although several antigens such as keratin, the RPE9 epitope, class III fl-tubulin, and CRALBP have been used as RPE markers (Bunt-Milam and Saari, 1983;

P D G F A N D ITS R E C E P T O R S IN ERM

Hooks et al., 1989; Vinores et al., 1990b, 1993a, 1993b, in press), no single marker consistently labels all RPE cells under all circumstances (Vinores et al., 1992a, 1993b). It is, therefore, likely that m a n y RPE cells were not recognized within the ERMs we examined using morphologic and immunohistochemical parameters, and these phenotypically altered RPE cells may be positive for PDGF and its receptors. But since m a n y of the cells positive for PDGF-A and PDGF,= can be identified as RPE cells with a reasonable degree of confidence, it is likely that an autocrine loop exists for RPE cells in ERMs, as we have recently demonstrated for RPE in culture (Campochiaro et al., 1994) and as has been suggested for other cell types (Behi et al., 1993 ; Eccleston et al., 1990; Hermansson et al., 1988; Koyama et al., 1994; LaRochelle et al., 1993). The mRNA for PDGF-A can be detected in quiescent RPE in situ (Mudhar et al., 1993), but is increased after laser treatment or retinal detachment (Campochiaro et al., 1994). Therefore, upregulation of the PDGF autocrine loop may be involved in the proliferative response that occurs in normal wound repair and exaggerated wound repair involved in epiretinal membrane formation. RPE cells are not the only source of PDGF in ERMs, nor are they the only cell capable of responding to PDGF. Macrophages secrete PDGF (Barrett and Benditt, 1988; Martinet et al., 1986; Nakaoka et al., 1990; Ross et al., 1990) and some cells in ERMs that stained for PDGF-A and PDGF-B appeared to be macrophages based upon morphologic criteria; in fact, most PDGF-B staining was in macrophage-like cells. PDGFr: is expressed by astrocytes and M/iller cells in the retina (Mudhar et al., 1993); therefore, retinal glia in ERMs are likely to be targets of paracrine growth stimulation for RPE- and macrophage-derived PDGF (de Juan et al., 1988 ; Harvey et al., 1987; Mudhar et al., 1993; Uchihori and Puro, 1991). Fibroblasts are also very responsive to PDGF and therefore any fibroblasts present in ERMs would likely be stimulated to proliferate by PDGF (Clark et al., 1988; Deuel, 1987; Habenicht et al., 1990; Kingsworth and Slavin, 1991; Lynch and Colvin, 1989; Ross et al., 1986). The data reported in this study, when viewed in the context of previous findings using cultured RPE and animal models of retinal wound repair, support a role for PDGF in ERM formation. Our data also suggest that PDGF-A may play a relatively more important role than PDGF-B. An alternative explanation for the greater immunopositivity for PDGF-A than for PDGF-B in ERMs is that the antibody to PDGF-B is weaker than that for PDGF-A. Some of the ERMs in this series showed little or no positivity for PDGF or its receptors, suggesting that PDGF may not be implicated in the development of all ERMs. Several other growth factors have been identified in ERMs (Fredj-Reygrobellet et al., 1991 ; Hanneken et al., 1991 ; Malecaze et al., 1991 ; Tang et al., 1993) and they also could play a role in ERM formation, but there are m a n y situations in

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which other growth factors exert their proliferative effects indirectly through PDGF (Ikeda et al., 1991; Raines et al., 1989). Even if there are several growth factors that act independently, interruption of the action of one may be sufficient to substantially alter the scarring process. Therefore, the identification of PDGF-A (and to a lesser extent PDGF-B) as a contributer to retinal scarring has important clinical implications, because it suggests that development of ways to neutralize PDGF-A in vivo (e.g. antisense RNA or soluble receptors) may have potential usefulness for the treatment and prevention of proliferative retinal diseases.

Acknowledgements This study was supported by PHS grants EY05951, EYIO017 and core grant P30EYO1765 from the National Eye Institute, Bethesda, MD, U.S.A., and by an unrestricted grant from Research to Prevent Blindness.

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