Anterior subcapsular (or anterior polar) cataracts are

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1 Alpha-Smooth Muscle Actin Expression in Cultured Lens Epithelial Cells Toshiyuki Nagamoto, 1,2 Goro Eguchi, 1,3 and David C. Beebe 2 PURPOSE. Lens epithelial cells transdifferentiate to myofibroblasts during the formation of anterior subcapsular cataracts and secondary cataracts. One of the defining characteristics of myofibroblasts is the expression of -smooth muscle actin ( -SMA). This study investigated some of the factors that influence -SMA expression in lens epithelial cells. METHODS. Bovine, rabbit, and human lens epithelial explants or cells were cultured with or without serum. Immunohistochemistry and immunoblotting were used to detect and quantitate -SMA expression. RESULTS. Cells from all species studied expressed -SMA in primary explant culture with or without serum. Immunostaining for -SMA first appeared in a diffuse granular pattern, then accumulated at the cell cortex, and eventually was detected along stress fibers. When lens epithelial cells migrated onto cell-free regions of the capsule or were transferred to a plastic culture dish, -SMA expression increased significantly. Expression of -SMA positively correlated with cell size and cell migration. CONCLUSIONS. Expression of -SMA is a common feature of cultured mammalian lens epithelial cells. Because -SMA expression occurred without the addition of exogenous factors, the fibrosis seen in anterior subcapsular cataracts or secondary cataracts may reflect the intrinsic properties of lens epithelial cells. Interaction between lens epithelial cells and their substratum appears to be an important regulator of myofibroblast formation. Understanding the factors that regulate -SMA expression in lens epithelial cells could lead to the development of methods for preventing secondary cataracts and anterior subcapsular cataracts. (Invest Ophthalmol Vis Sci. 2000;41: ) From the 1 Division of Morphogenesis, Department of Developmental Biology, National Institute for Basic Biology, Okazaki, Japan; 2 Cataract Research Center, Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, Missouri; and 3 Kumamoto University, Kumamoto, Japan. Supported in part by National Institutes of Health Grants EY04853 and EY09179, a National Eye Institute Core Grant to the DOVS, a Research to Prevent Blindness Professorship to DCB, and Grants-in-Aid for General and Special Project Researches from the Ministry of Education, Sciences, Sports, and Culture (GE). Submitted for publication August 4, 1999; revised November 1, 1999; accepted November 8, Commercial relationships policy: N. Corresponding author: Toshiyuki Nagamoto, Department of Ophthalmology, Kyorin University School of Medicine, Shinkawa Mitaka, Tokyo , Japan. nagamoto@qc4.so-net.ne.jp Anterior subcapsular (or anterior polar) cataracts are caused by the accumulation of a fibrous mass beneath the anterior capsule. These cataracts share several similarities with the fibrous opacities that often occur in posterior capsular opacification (PCO) after cataract surgery (also called after-cataracts or secondary cataracts). In both cases, lens epithelial cells transdifferentiate to myofibroblast-like cells. 1 4 Myofibroblasts are spindle-shaped cells that express -smooth muscle actin ( -SMA) and secrete large amounts of pericellular extracellular matrix composed of collagens type I, III, and IV Normal lens epithelial cells are not separated from each other by extracellular matrix and do not express -SMA. The factors responsible for the formation of anterior subcapsular cataracts and PCO in vivo are not known. However, the cytokine transforming growth factor-beta (TGF- ) can initiate myofibroblast formation, fibrosis, and cataract formation in cultured rat lenses and in transgenic mice This has led to the suggestion that elevated levels of TGF- in the aqueous humor could contribute to the formation of these cataracts. Anterior subcapsular cataracts are usually well-circumscribed, fibrous opacities occurring near the center of the lens epithelium. They involve a kind of fibrous metaplasia of the lens epithelium. 18 In some cases the fibrous mass may protrude from the anterior of the lens to form a pyramidal cataract. Anterior polar cataracts are most often congenital, but also may occur after inflammation, 19 atopic dermatitis, 20 and ocular trauma. 2 PCO is a common postoperative complication of cataract surgery and is generally classified into four clinical categories: Elschnig s pearls, capsular fibrosis, Soemmerring s ring, and lentoid of Thiel. The most common and clinically significant forms of PCO are capsular fibrosis and Elschnig s pearls. Capsular fibrosis is considered to be a wound healing reaction after surgery. Fibrosis is always prominent around the anterior capsulotomy margin and sometimes extends to the central portion of the posterior capsule, where it is accompanied by capsular wrinkles and folds. The accumulation of large amounts of extracellular matrix appears to be the major cause of opacification in this form of PCO. We recently reported that myofibroblast-like lens epithelial cells express -SMA in the fibrous region around the capsulotomy margin after experimental cataract surgery in rabbits. 6 Lens epithelial cells cultured in serum-containing medium on plastic substrata or in collagen gels can also transform into myofibroblast-like cells. 9,11 13 To our knowledge, Investigative Ophthalmology & Visual Science, April 2000, Vol. 41, No Copyright Association for Research in Vision and Ophthalmology

2 IOVS, April 2000, Vol. 41, No. 5 -Smooth Muscle Actin Expression in Lens Cells 1123 expression of -SMA in lens epithelial cells cultured without serum has not been reported. In the present studies we examined the temporal and spatial patterns of -SMA expression in bovine, rabbit, and human lens epithelial cells cultured with and without serum. Lens epithelial explants and lens epithelial cells cultured on plastic culture dishes were compared to determine whether the substrate affected -SMA expression. Lens epithelial cells of all species studied expressed -SMA in primary explant culture in serum-free medium. This suggests that -SMA expression is a common feature of cultured mammalian lens epithelial cells and that capsular fibrosis after cataract surgery may result from an intrinsic property of lens epithelial cells. Our results also suggest that cell substratum interactions may be more important in regulating myofibroblast transdifferentiation than are cytokines and growth factors. MATERIALS AND METHODS Specimens Bovine eyes were obtained from an abattoir soon after death and were returned to the laboratory on ice. Japanese white rabbits weighing 2 to 3 kg were used in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Rabbit eyes were enucleated after they were killed with intravenous overdose of pentobarbital sodium. Human eyes were obtained from Mid-America Transplant Services (St. Louis, MO). Use of human tissues was approved by the Institutional Review Board at Washington University and conformed to the Declaration of Helsinki on the use of human subjects in research. For studies of -SMA expression and cell migration in bovine lens epithelia a minimum of two lenses were cultured in serum-free medium, fixed, and examined immediately after dissection or after culture for 1, 2, 3, 4, 5, 7, 10, 14, or 28 days. Another series of lenses was examined at the same time points after culture in serum-supplemented medium. In a similar manner, at least two rabbit lenses were studied immediately after dissection or after culture in serum-containing or serum-free medium for 3, 4, 5, 7, 10, and 24 days after dissection. For studies on human lens epithelia, specimens obtained from one more lenses were examined immediately after dissection or after culture in serum-free or serum-supplemented medium for 3, 4, 5, 7, 10, and 14 days. In experiments to examine the effect of serum on -SMA expression, three lens epithelia were cultured in serum-containing medium and three in serum-free medium. One sample from each treatment was used for immunohistochemistry and two were used for immunoblotting. Isolation of Explants and Tissue Culture To prepare primary lens explants, eyes were cut in half 3 to 5 mm posterior to the limbus. The entire vitreous was removed. After making a circular posterior capsulotomy, the zonules were cut, the peripheral capsule was radially incised at eight locations, and the lens was placed in a culture dish with the anterior capsule downward. After the addition of a few drops of culture medium, the capsule with attached epithelial cells was peeled off the posterior fiber mass and pinned down on the dish, and the lens fibers were gently removed. Sufficient culture medium was then added to cover the explant (Fig. 1). Explants were cultured in a humidified atmosphere of 5% CO 2 FIGURE 1. Illustration of the dissection procedures used in this study. Lens epithelial explants were pinned down on a culture dish with cells facing upward. P, posterior; A, anterior; LECs, lens epithelial cells. and 95% air at 37 C in medium with or without 10% fetal bovine serum (FBS). Two culture media were used: minimal essential medium or Ham s F-12 with the addition of 0.15% sodium bicarbonate, 100 U/ml penicillin, 100 g/ml of streptomycin, and 0.25 g/ml of amphotericin B. Results with these two media were similar and are treated together throughout the article. The medium was changed on day 1 and thereafter every 2 days. Lens epithelial cells were dissociated from primary cultures with 0.1% trypsin and 0.02% EDTA in phosphatebuffered saline (PBS) for 10 to 15 minutes at 37 C, harvested after neutralization of trypsin with 0.1% soy bean trypsin inhibitor or 10% FBS in culture medium, and counted using a hemocytometer chamber at least four times for each sample. Immunohistochemistry for -SMA Expression of -SMA was determined using a horseradish peroxidase-streptavidin-biotin method (Histostain SP kit; Zymed, San Francisco, CA). A mouse monoclonal antibody directed against -SMA (clone 1A4, IgG2a; Dako, Glostrup, Denmark) was used as the primary antibody. Samples were immersed in 4% paraformaldehyde or 10% neutral buffered formalin for 15 to 30 minutes, permeabilized with 0.1% Triton X-100 for 15 minutes, blocked with normal goat serum for 15 minutes, and incubated with the primary antibody (dilution 1:50 1:100 with PBS) in a humidified chamber for 30 to 60 minutes. As a negative control mouse IgG2a (X943; Dako) was applied instead of the primary antibody. After treatment with primary antibody, explants were incubated with biotinylated secondary antibody (goat anti-mouse IgG) for 15 to 30 minutes, followed by streptavidin peroxidase conjugate for 15 minutes. Peroxidase was visualized by addition of 3-amino-9-ethyl carbazole (AEC) and hydrogen peroxide. Cells were rinsed with PBS three times after each treatment. Cell nuclei were counterstained with hematoxylin. In some samples, streptavidin fluorescein or streptavidin Texas Red (1:500 in PBS; Amersham Life Science, Arlington Heights, IL) were used instead of streptavidin peroxidase AEC system. Double labeling for -SMA and filamentous actin was performed using the antibody to -SMA and Texas Red phalloidin (Molecular Probes, Eugene, OR). Fluorescent-stained samples were visualized with a Zeiss 410 confocal microscope (Carl Zeiss, Thornwood, NY). Immunoblotting for -SMA Cell lysates were prepared from the cultured cells using lysis buffer consisting of 50 mm Tris-HCl (ph 8.0), 150 mm NaCl,

3 1124 Nagamoto et al. IOVS, April 2000, Vol. 41, No. 5 FIGURE 2. Immunostaining for -SMA in a bovine lens epithelial explant immediately after dissection. (A) Central area; (B) peripheral area. Nuclei were counterstained with hematoxylin. All cells were negative for -SMA. Bar, 25 m. 100 g/ml phenylmethyl sulfonyl fluoride, 1 g/ml aprotinin, and 1% Triton X-100. Lysates were mixed with the equal volume of 2 sample buffer consisting of 100 mm Tris-HCl (ph 6.8), 200 mm dithiothreitol, 4% sodium dodecyl sulfate, 0.2% bromophenol blue, and 20% glycerol and were separated in 7.5% or 10% SDS polyacrylamide gels (5 or 10 g cellular protein/lane). Proteins were blotted to nitrocellulose membranes, and the blots were blocked with 5% nonfat dry milk in PBS containing 0.1% Tween 20 (PBS-T) and incubated with the antibody against -SMA (clone 1A4, 1:500) for 1 hour. After washing three times with PBS-T, the membrane was incubated peroxidase-conjugated goat anti-mouse IgG (cat. no ; Cappel, Organon Teknika, West Chester, PA) for 1 hour and washed three times with PBS-T. Antibody was visualized on luminescence detection film (Hyperfilm-ECL; Amersham, Amersham, England) after the peroxidase hydrogen peroxidase catalyzed oxidation of luminol (ECL Western blotting detection reagents; Amersham) according to the manufacture s instructions. The mean density of bands was measured using NIH Image software, version 1.57 (NIH Image is available on the Internet at A linear increase in band density was obtained when different amounts of total cellular protein were applied in each lane (data not shown). of the capsule and, eventually, to the reverse side of the capsule. Only when cultured with serum did cells migrate from the capsule onto the plastic culture dish. Although most lens epithelial cells remained as a monolayer, multilayered regions were sometimes detected. Expression of -SMA was detected in characteristic spatial and temporal patterns in cultured lens epithelial cells from the three species studied. Staining for -SMA was always detected earlier in cells located at the peripheral zone of bovine and rabbit explants, but appeared at approximately the same time throughout human epithelial explants. In general, -SMA was first detected in primary explants on day 1 in bovine, on day 2 or 3 in rabbit, and on day 4 or 5 in human explants. In bovine and rabbit explants, the expression of -SMA in the central region of the explant was related to the presence or absence of cell-free zones that were often found in this region immediately after dissection. Cell-free areas occurred in the center of the explant with variable frequency. In some cases the presence of these areas appeared to be associated with a delay in receiving the lenses after death, but this could not explain all cases in which this occurred. If nearly all central cells were present after dissection, -SMA was first expressed 4 to 5 days after culture. However, if there was a cell-free area in the central zone, -SMA was first detected in this region 2 to 3 days after culture. The percentage of cells expressing -SMA appeared to be higher 5 to 10 days after culture if a large cell-free region was present immediately after dissection. When cells first expressed -SMA, staining was seen in a diffuse, granular pattern or occasionally at the cell cortex (Fig. 3A). Later, -SMA immunoreactivity was consistently localized Percentage of Cells Expressing -SMA Accurate cell counting was not possible in whole mount specimens because cells often overlapped and some multinuclear cells were present. Therefore, to calculate the percentage of cells expressing -SMA, cells were dissociated with 0.1% trypsin and 0.02% EDTA in PBS, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, seeded in drops on slides coated with aminopropyl-triethoxysilane, dried, and stained for -SMA using biotin streptavidin peroxidase AEC system mentioned above. At least five counts were performed for each specimen. RESULTS Lens Epithelial Cell Behavior, Morphology, and -SMA Expression Immunostaining for -SMA was negative in lens explants of all species immediately after dissection (Fig. 2). Cultured lens epithelial cells migrated into cell-free areas on the cellular side FIGURE 3. Distribution of -SMA in the cytoplasm of bovine lens epithelial cells cultured without serum for 4 days. Cells were double labeled with an antibody to -SMA (green) and with Texas Red phalloidin to detect filamentous actin (red). (A) Cells within the epithelial monolayer. Although actin filaments were most abundant at the cell cortex, -SMA was diffusely distributed in a granular pattern (arrow). (B) Cells migrating over the epithelial monolayer contained stress fibers, some of which expressed -SMA (arrow). Bar, 25 m.

4 IOVS, April 2000, Vol. 41, No. 5 -Smooth Muscle Actin Expression in Lens Cells 1125 FIGURE 4. Immunohistochemical detection of -SMA in a human lens epithelial explant cultured with 10% FBS for 12 days. (A) A few cells in the epithelial sheet in the central area of the explant showed granular staining for -SMA. (B) Some cells that migrated over the epithelial cells at the periphery of the capsule expressed -SMA along stress fibers. (C) Cells that migrated on the reverse (aqueous) side of the capsule expressed -SMA along stress fibers. (D) Cells that grew onto the dish demonstrated cytoplasmic enlargement and prominent -SMA expression along well-developed stress fibers. Bar, 25 m. to stress fibers. Many cells that formed stress fibers did not stain for -SMA. The period needed for the cells to become confluent on the cellular side of the lens capsule differed according to the number of cells that survived after dissection and whether or not serum was included in the medium. When most cells survived and the explant was cultured without serum, epithelial cells covered the capsule on days 3 to 5 in bovine, days 4 to 7 in rabbit, and days 7 to 10 in human lens epithelial explants. When explants were cultured with 10% FBS, epithelial cells covered the capsule 2 to 4 days after culture in bovine and rabbit explants and in 5 to 7 days after culture in explants of human epithelia. Bovine lens epithelial cells often formed disorganized, multilayered regions on the cellular side of the capsule. These included round aggregates of what appeared to be degenerating cells at the boundary area between peripheral and central zones and stellate-shaped aggregates of spindle-shaped cells near the periphery of the capsule. In addition, some cells migrated on top of the monolayer, eventually forming a multilayered region. Cells migrating on the surface of the epithelial sheet were broad and flat and contained numerous well-developed stress fibers that often stained prominently for -SMA (Figs. 3B, 4C). The formation of multilayered patches was faster and more frequent in cultures with serum than without serum and faster at the periphery than the center of the explants (Figs. 5A 5D). Cells migrated to cover cell-free regions of the capsule in all species, but migration was more extensive and rapid when explants were cultured in serum-containing medium. Bovine cells began to migrate over the edge of the capsule from 1 to 2 days after culture with or without serum and become confluent on the reverse side of the capsule 1 week after culture with 10% fetal bovine serum and 3 weeks after culture without serum. In contrast, migration onto the reverse side of the capsule was seen infrequently in rabbit and human explants cultured without serum, but was frequently observed when explants were cultured with serum. Cells migrating on the reverse (aqueous) side of the capsule were broad and flat and contained numerous well-developed stress fibers, many of which stained prominently for -SMA (Figs. 4D, 5F). The frequency with which these cells stained for -SMA was greatest FIGURE 5. Expression of -SMA in bovine explants cultured for 11 days with or without serum. Antibody staining was visualized with Texas Red-labeled secondary antibodies. (A), (C), and (E) are from samples cultured with 10% FBS, and (B), (D), and (F) without serum. (A, B) Cells at the central area of the capsule; (C, D) cells at the periphery; (E) cells that migrated onto the dish; (F) cells that migrated onto the reverse side of the capsule. Multilayer formation was faster and more frequent in cultures with serum than without serum and at the periphery than at the center (A through D). The cells on the dish and under the capsule demonstrated prominent -SMA expression along stress fibers (E, F). Bar, (A) 50 m; (B through F) 25 m.

5 1126 Nagamoto et al. IOVS, April 2000, Vol. 41, No. 5 FIGURE 6. Temporal alteration of cell number and percentage of cells expressing -SMA in bovine lens epithelial cells cultured with or without serum. Each point and bar on the graph indicates the mean and SD, respectively, of at least three samples. (A) Cell number in primary explants. Cells cultured with serum increased in number, reached a plateau at 2 weeks, and decreased after 3 weeks of culture. There was no appreciable increase in cell number in explants cultured without serum. Cell number decreased after 3 weeks of culture. (B) Expression of -SMA in primary explant culture. Approximately 50% of bovine lens epithelial cells expressed -SMA 1 week after culture regardless of the presence of serum. More than 90% of cells expressed -SMA after 10 days and 3 weeks of culture without and with serum, respectively. In (C) and (D), cells were transferred from the primary explants cultured for 4 days without serum and seeded at 728 cells/mm 2 without serum and 260 cells/mm 2 with 10% FBS, respectively. (C) Cell proliferation was much better in cultures with serum compared to those without serum. (D) The percentage of cells expressing -SMA rapidly increased in cells cultured on plastic. Nearly 100% of cells expressed -SMA after 3 days of culture with or without serum. *P 0.05, **P 0.01 (Student s t-test). in explants of bovine lens epithelia ( 70%), intermediate in human ( 30 40%), and least in rabbit ( 20%). When explants were cultured with serum, cells often spread from the capsule to the plastic culture dish. However, no or little outgrowth onto the dish occurred in cultures without serum. This may reflect the requirement for serumderived factors to support the adhesion of lens epithelial cells to plastic. Outgrowth onto the dish in serum-containing medium was typically observed by day 1 in bovine, day 2 in rabbit, and day 3 in human epithelia cultured with 10% FBS. Bovine cells migrating on tissue culture plastic were broad and flat and contained numerous well-developed stress fibers, most of which stained prominently for -SMA (Figs. 4E, 5E), similar to cells migrating on the surface of the epithelial sheet or on the reverse (aqueous) side of the capsule. The size of cells migrating on the dish or the reverse side of the capsule was larger than cells migrating on the surface of the epithelial sheet (Figs. 4C 4E, 5C 5F). Cell Proliferation and -SMA Expression At the time of explantation, lens epithelial cells did not contain detectable amounts of -SMA. As expected, lens cell number increased when bovine epithelial explants or dissociated lens epithelial cells were cultured in serum-containing medium. Cell number did not increase appreciably in serum-free culture conditions (Figs. 6A, 6C). In spite of the marked difference in cell accumulation in these two culture conditions, culture in serum had little effect on the rate or extent of -SMA expression (Figs. 6B, 6D). Bovine lens epithelial cells became positive for -SMA more rapidly when cultured on plastic than when cultured on the lens capsule, but the extent of -SMA expression eventually approached 100% in both cases (Figs. 6B, 6D). Western blotting was used to verify the specificity of the immunocytochemistry and to further quantitate the effect of serum on -SMA expression (Fig. 7). A single band of the

6 IOVS, April 2000, Vol. 41, No. 5 -Smooth Muscle Actin Expression in Lens Cells 1127 DISCUSSION FIGURE 7. The effect of culture in serum-containing medium on -SMA expression detected by immunoblotting. Bovine lens epithelial cells were first cultured in serum-free medium and transferred to cleaned lens capsules and cultured for 48 hours in the presence or absence of serum. Cells were lysed, cellular proteins were separated by electrophoresis and blotted onto nitrocellulose, and the blots probed with a monoclonal antibody to -SMA. Each lane included 10 g of total cellular proteins. Lanes 1 and 3: samples treated with FBS; lanes 2 and 4: controls. FBS modestly increased the expression of -SMA. Expression of -SMA by Mammalian Lens Epithelial Cells and Exogenous Growth Factors or Cytokines Lens epithelial cells from each of the three mammalian species studied expressed -SMA when cultured in serum-free, primary explant culture. Immunoblotting studies confirmed the presence of -SMA. Previous studies showed that a small percentage of lens epithelial cells from young rats also expressed -SMA when cultured in serum-free medium. 14 These observations indicate that expression of -SMA is a common response of cultured mammalian lens epithelial cells. Because the expression of -SMA is characteristic of myofibroblasts, these results indicate that exogenous growth factors or cytokines are not required for the transdifferentiation of a substantial fraction of lens epithelial cells to the myofibroblast phenotype. Several studies have shown that myofibroblasts are the characteristic cell type in anterior subcapsular cataracts and the fibrotic plaques found in PCO. 1 4,6,9 In experimental models, the cytokine TGF- can initiate myofibroblast differentiation and the formation of fibrotic plaques resembling those seen in anterior polar cataracts and PCO. 12,14 17 This has led to the suggestion that alterations in TGF- activity in the eye may expected size was observed in both culture conditions. Scanning the lumigrams showed that FBS increased the amount of -SMA 1.3-fold compared to that in the controls. In these cultures the percentages of cells expressing -SMA were 61.5% with 10% FBS and 49.3% without serum (a 1.2-fold difference). Therefore, the effect of serum was to modestly increase the percentage of cells expressing -SMA without an appreciable increase in the amount of -SMA per cell. Rabbit lens epithelial cells also showed a marked increase in cell number when explants were cultured in serum-containing medium. Culture on a plastic substratum increased the rate and extent of cell proliferation and the percentage of cells that expressed -SMA (Figs. 8A, 8B). Overall, the percentage of cells expressing -SMA was lower than that seen in bovine lens epithelial cells, although all cultured explants contained cells that stained positively for -SMA. Variations in -SMA Expression in Different Species The percentage of cells expressing -SMA was examined in bovine, rabbit, and human primary explants cultured for 7 days without serum (Fig. 9). The difference in -SMA expression between these three species was large and statistically significant (Kruskal Wallis test, P 0.001; Post hoc test, Scheffé s F test). Therefore, the regulation of -SMA expression and, presumably, myofibroblast differentiation must vary greatly in the lens epithelial cells of these species. Identifying differences in the biochemical characteristics of the lens epithelial cells from these species could reveal the control points that regulate myofibroblast formation and capsular fibrosis. FIGURE 8. Time course of cell growth and -SMA expression in rabbit lens epithelial cells cultured with 10% FBS. (A) Cell proliferation and -SMA expression in primary explants. Each point and bar on the graph indicates the mean and SD, respectively, of at least three samples. Cell number and -SMA expression peaked after 2 weeks of culture. (B) Cell proliferation and -SMA expression in cells cultured on a plastic dish. Cells were transferred from primary explants cultured for 3 weeks with 10% FBS and seeded at 53 cells/mm 2 with 10% FBS. Each point includes the data from one sample. The number of cells and the percentage of cells expressing -SMA peaked on day 7 and decreased thereafter.

7 1128 Nagamoto et al. IOVS, April 2000, Vol. 41, No. 5 FIGURE 9. -SMA expression in cultured lens epithelial cells derived from different species. After primary lens epithelial explants were cultured without serum for 7 days, the percentage of cells expressing -SMA was determined. There was a significant variation between species. Approximately 50% of bovine cells, 10% of human cells, and 2% of rabbit cells expressed -SMA (Kruskal Wallis test, P 0.001; post hoc test, Scheffé s F test). be an initiating factor in these pathologic conditions. Our results show that mammalian lens epithelial cells express -SMA and form multilayered aggregates without the addition of TGF- or other growth factors or modulators. Therefore, the fibrosis that is characteristic of anterior polar cataracts and is common in PCO may reflect the intrinsic properties of lens epithelial cells and need not be regulated by growth factors or cytokines in the aqueous or vitreous humors. This result also raises the possibility that the damage to the lens epithelial cell sheet that occurs during cataract surgery may be responsible for the fibrosis that occurs in PCO. Our results on the relationship between cell migration and -SMA expression, discussed below, are consistent with this view. Cell Migration, Cell Shape, and -SMA Expression When lens epithelial cells from the three species studied were cultured under different conditions, there was a consistent positive association between increased cell migration, increased cell size, and the expression of -SMA. This suggests that changes in the interaction between lens cells and their substrate may influence the expression of the myofibroblast phenotype. The expression of -SMA always occurred earlier in cells locating at the periphery of bovine and rabbit epithelial explants. Removal of fiber cells during explantation created a cell-free region at the epithelial periphery. Cells at the margin of the epithelial sheet migrated into these cell-free zones and expressed -SMA. Expression of -SMA was most prominent in cells migrating on plastic and the reverse side of the capsule. These cells were larger and more flattened than cells migrating on the cellular side of the capsule. These observations support the possibility that expression of -SMA is associated with changes in cell shape or increased cell size, presumably associated with an increase in cell substrate adhesion. It is also possible that the increase in the percentage of cells expressing -SMA was somehow a function of increased cell migration. When lens epithelial explants were cultured without serum, the percentage of cells expressing -SMA varied significantly between the three species studied. Bovine lens epithelial cells demonstrated much more prominent expression of -SMA than rabbit or human cells. The behavior of bovine cells also was different from that of rabbit and human cells. In the absence of serum bovine cells rapidly covered the cellular side of the capsule and vigorously migrated onto the reverse side of the capsule. Under these conditions rabbit and human cells rarely migrated onto the reverse side of the capsule. During the period of rapid migration, there was no increase in cell number (Fig. 6A). Accordingly, cell density decreased, and cells became larger and more flattened. This difference in cell migration, size, and shape might explain the variation in -SMA expression between the species. Primary bovine lens epithelial cells began to express -SMA more rapidly when cultured in serum than when cultured without serum (Fig. 6B, 3-day time point). In contrast, at later times the percentage of cells expressing -SMA was significantly lower in cells cultured in serum than in cells cultured without serum (Fig. 6B, 9- and 14-day time points). These observations might be explained by the correlation between cell shape and the expression of -SMA described above. Serum increased the rate of cell migration and cell proliferation; however, the effect of serum on migration occurred more rapidly than its effect on cell number. Migrating cells were generally larger and flatter than cells in the confluent region of the epithelial sheet. These peripheral cells were the first to express -SMA. As cell numbers increased and the bare areas of the capsule became covered with cells, average cell size decreased, and the rate at which cells became positive for -SMA slowed. In contrast, cells in explants cultured without serum migrated more slowly and did not increase in number. Once these cells began to migrate, cell density continually decreased and the percentage of cells expressing -SMA increased steadily. Therefore, the timing of the effects of serum on cell shape and cell number could explain why a larger percentage of serum-treated cells expressed -SMA at earlier culture periods and why a smaller percentage expressed -SMA at later times. TGF- and -SMA Expression Several investigators have shown that TGF- is an inducing or promoting factor for the expression of -SMA in lens epithelial cells. 12,14 17 However, the mechanism by which TGF- promotes -SMA expression is not known. TGF- has multiple biological functions and elicits diverse cellular responses. For example, TGF- inhibits the proliferation of many cell types, including lens epithelial cells, 21 increases the production of extracellular matrix in epithelial cells, 22 leads to increased cell adhesion, 22 and promotes the migration of fibroblasts and tumor cells It is not yet clear whether TGF- directly induces the expression of -SMA in lens epithelial cells or increases -SMA expression secondary to its other biological functions. For example, it is possible that extracellular material secreted in response to TGF- promotes expression of -SMA by stimulating the interaction between extracellular matrix molecules and cell surface integrins. Alternatively, inhibition of cell proliferation and stimulation of cell locomotion by TGF- would tend to decrease cell density. Decreased cell density would lead to changes in cell shape and increased cell size, which could then promote increased expression of -SMA.

8 IOVS, April 2000, Vol. 41, No. 5 -Smooth Muscle Actin Expression in Lens Cells 1129 Additional studies are needed to define the sequence of events that leads to myofibroblast differentiation from lens epithelial cells and to define the mechanism by which TGF- might influence this process. References 1. Novotny GEK, Pau H. Myofibroblast-like cells in human anterior capsular cataract. Virchows Arch A Pathol Anat Histopathol. 1984;404: Pau H, Novotny GEK. Ultrastructural investigations on anterior capsular cataract. Cellular elements and their relationship to basement membrane and collagen synthesis. Graefes Arch Clin Exp Ophthalmol. 1985;223: McDonnell PJ, Zarbin MA, Green WR. Posterior capsule opacification in pseudophakic eyes. Ophthalmology. 1983;90: Cobo LM, Ohsawa E, Chandler D, Arguello R, George G. Pathogenesis of capsular opacification after extracapsular cataract extraction: an animal model. Ophthalmology. 1984;91: Ishibashi T, Hatae T, Inomata H. Collagen types in human posterior capsule opacification. J Cataract Refract Surg. 1994;20: Kurosaka D, Kato K, Nagamoto T. Presence of -smooth muscle actin in lens epithelial cells of aphakic rabbit eyes. Br J Ophthalmol. 1996;80: Uusitalo M, Kivela T. Cell types of secondary cataract: an immunohistochemical analysis with antibodies to cytoskeletal elements and macrophages. Graefes Arch Clin Exp Ophthalmol. 1997;235: Hatae T, Ishibashi T, Yoshitomi F, Shibata Y. Immunocytochemistry of types I-IV collagen in human anterior subcapsular cataracts. Graefes Arch Clin Exp Ophthalmol. 1993;231: Schmitt-Graff A, Pau H, Spahr R, et al. Appearance of alpha-smooth muscle actin in human eye lens cells of anterior capsular cataract and in cultured bovine lens-forming cells. Differentiation. 1990; 43: Sappino AP, Schurch W, Gabbiani G. Biology of disease. Differentiation repertoire of fibroblastic cells: expression of cytoskeletal proteins as marker of phenotypic modulations. Lab Invest. 1990; 63: Greenburg G, Hay ED. Cytodifferentiation and tissue phenotype change during transformation of embryonic lens epithelium to mesenchyme-like cells in vitro. Dev Biol. 1986;115: Kurosaka D, Kato K, Nagamoto T, Negishi K. Growth factors influence contractility and -smooth muscle actin expression in bovine lens epithelial cells. Invest Ophthalmol Vis Sci. 1995;36: Veroman S, Viikmaa M. Frequency of occurrence of abnormal cell types in organ-cultured fetal crystalline lenses and its dependence upon the culture medium used. Ophthalmic Res. 1989;21: Liu J, Hales AM, Chamberlain CG, McAvoy JM. Induction of cataract-like changes in rat lens epithelial explants by transforming growth factor. Invest Ophthalmol Vis Sci. 1994;35: Hales AM, Schulz MW, Chamberlain CG, McAvoy JW. TGF- 1 induces lens cells to accumulate -smooth muscle actin, a marker for subcapsular cataracts. Curr Eye Res. 1994;13: Hales AM, Chamberlain CG, McAvoy JW. Cataract induction in lenses cultured with transforming growth factor-. Invest Ophthalmol Vis Sci. 1995;36: Srinivasan Y, Lovicu FJ, Overbeek PA. Lens-specific expression of transforming growth factor 1 in transgenic mice causes anterior subcapsular cataracts. J Clin Invest. 1998;101: Henkind P, Prose P. Anterior polar cataract. Electron-microscopic evidence of collagen. Am J Ophthalmol. 1967;63: Jampol LM, Kass M, Dueker D, Albert DM. Anterior polar cataracts. Am J Ophthalmol. 1974;78: Garrity JA, Liesegang TJ. Ocular complications of atopic dermatitis. Can J Ophthalmol. 1984;19: Kurosaka D, Nagamoto T. Inhibitory effect of TGF-beta 2 in human aqueous humor on bovine lens epithelial cell proliferation. Invest Ophthalmol Vis Sci. 1994;35: Massague J. The transforming growth factor- family. Annu Rev Cell Biol. 1990;6: Samuel SK, Hurta RAR, Spearman MA, et al. TGF- 1 stimulation of cell locomotion utilizes the hyaluronan receptor RHAMM and hyaluronan. J Cell Biol. 1993;123: Postlethaite AE, Keski-Oja J, Moses HL, Kang AH. Stimulation of the chemotactic migration of human fibroblasts by transforming growth factor beta. J Exp Med. 1987;165: Samuel SK, Hurta RAR, Kondaiah P, et al. Autocrine induction of tumor protease production and invasion by a methallothioneinregulated TGF- 1. EMBO J. 1992;11: