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R N Frank

Publications and source records attributed to R N Frank.

At least 37 records · Page 2Linked to original sources
PubMed1993

Cultured ocular cells and extracellular matrices: role of growth factors, retinoic acid and cell polarity.

Culture of various types of cells on gelled, reconstituted extracellular matrices results in decreased cellular proliferation. In the present study, we evaluated several possible mechanisms for this inhibition, as applied to cultured bovine retinal microvascular endothelial cells (EC) or to retinal pigment epithelial (RPE) cells: whether the inhibition might be related to (a) inactivation of fibroblast growth factor (FGF) by binding of the molecules present in the medium to a matrix component; (b) release of an inhibitor by the matrix in culture; or (c) inhibitory properties of the matrix macromolecules themselves. Our results suggest that mechanism (c) is most likely. The reasons are, first, that culture of EC or RPE cells on several different extracellular matrix substrates in the presence of logarithmically increasing concentrations of acidic or basic fibroblast growth factors (aFGF or bFGF) leads to a vertical shift of the plots of cell number after 4 days in culture vs. log growth factor concentration for both types of cells. The same result obtains when cells are cultured with logarithmically increasing concentrations of all-trans retinoic acid, which inhibits EC but not RPE cell proliferation in a dose-dependent fashion. This is consistent with mechanism (b) or (c), but not (a), for which one would expect a horizontal shift. Second, washing the matrices prior to the plating of cells with 1M NaCl, which elutes aFGF and partially elutes bFGF molecules from basement membranes, does not alter the growth of cells plated after the wash. This suggests also that growth factor binding to the matrix is not a likely mechanism for the observed inhibition. Incubation of matrices with culture medium prior to plating cells does not usually alter the ability of the medium thus "conditioned" to support cell growth, arguing against the possibility that the matrices release a soluble activator or inhibitor of such growth. However, in some experiments performed with lots of Matrigel (a commercially available basement membrane extract from a murine tumor) obtained prior to mid-1991, media "conditioned" by incubation with this matrix did show enhanced ability to facilitate EC and RPE cell proliferation. Finally, if RPE cells or EC are plated on various substrates, allowed to attach for 24 hr., and then the same or other substrates are poured over the cells, the effect on proliferation of the matrices plated on the apical surfaces of the cells is often less than that of matrices plated adjacent to their basal surfaces. Although in most cases these differences are not statistically significant, there is an apparent trend.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1993

Mitogenesis and retinal pigment epithelial cell antigen expression in the rat after krypton laser photocoagulation.

PURPOSE: Polypeptide growth factors, such as the acidic and basic fibroblast growth factors (aFGF and bFGF), may be important in the pathogenesis of subretinal neovascularization. The authors studied the relationship of aFGF and bFGF expression to retinal pigment epithelial (RPE) cell and choriocapillary endothelial cell proliferation in krypton-laser-treated regions of the retina, RPE, and choroid of a model of subretinal neovascularization in the pigmented rat they developed. METHODS: Multiple krypton laser burns were applied to the posterior poles of the eyes of pigmented rats according to a protocol described for producing subretinal neovascularization in these animals. At intervals up to 80 days after treatment, the retinas, RPE, and choroid of these animals were examined by [3H]-thymidine autoradiography and electron microscopic immunocytochemistry, using antibodies to aFGF, bFGF, cytoplasmic retinaldehyde-binding protein, opsin, and various basement membrane macromolecules. RESULTS: Nuclear radiolabeling became evident in these layers when the label was injected as late as 75 days after photocoagulation, but the number of labeled nuclei was greatest when isotope was injected 2-5 days after laser treatment. Although there were labeled nuclei in the retina, RPE, and choroid, the frequency of labeling as a fraction of the total number of nuclei appeared to be greatest in the RPE and choriocapillaris. Non-laser-damaged RPE cells were immunocytochemically strongly positive for cytoplasmic retinaldehyde-binding protein, but were negative for aFGF and bFGF. After laser treatment, many RPE cells lost their cytoplasmic retinaldehyde-binding protein positivity but stained strongly for aFGF and bFGF within intracellular structures of variable shape and homogeneous appearance. Although these structures had an appearance suggestive of basement membrane, they did not stain with antibodies to collagens type IV or V, to laminin, or to heparan sulfate proteoglycan core protein. They also did not stain with an antibody to the N-terminal peptide of opsin. Choriocapillary endothelial cells were unreactive with antibodies to aFGF, bFGF, or cytoplasmic retinaldehyde-binding protein either before or after laser treatment. FGF-positive RPE cells persisted for the 80-day duration of the experiment. CONCLUSIONS: Because the presence of FGF-positive RPE cells coincides temporally with increased nuclear thymidine labeling in the RPE and choriocapillaris, aFGF and/or bFGF may be at least partly responsible for initiating the process of cell proliferation and subretinal neovascularization in these animals.

Animals↗
PubMed1993

Effects of a high-galactose diet on the interphotoreceptor matrix of two strains of rat.

PURPOSE: The authors previously reported that a diet containing 30% galactose retards the development of the late-onset photoreceptor dystrophy in the spontaneously hypertensive (SHR) rat. It was suggested that the dystrophy might result from faulty galactosylation of a critical glycoprotein or glycosaminoglycan (GAG) in the interphotoreceptor matrix (IPM). In the current study, this hypothesis was tested by studying IPM protein and GAG composition in SHR and normotensive Wistar-Kyoto (WKy) control strain rats fed a standard or a high-galactose (30%) diet. METHODS: The authors performed biochemical analyses of the IPM of SHR and of WKy control rats fed either the basal diet or a 30% galactose diet for 14 mo and labeled at the termination of the experiment with 3H-glucosamine and 35S-sulfate. Analyses included high-performance liquid chromatography and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) of extracted proteoglycans with identification of the GAG by selective enzymatic degradation and immunoblotting of SDS-PAGE gels for interphotoreceptor retinol-binding protein (IRBP), with slot blots for additional quantitation. RESULTS: Although several differences were detected in GAG radiolabeling between the two strains, only one, a decreased overall synthesis of 3H-glucosamine-labeled GAG in the WKy rats, was altered by the high-galactose diet. There was no apparent difference in the protein patterns of the IPM as evaluated by two-dimensional SDS-PAGE and IEF. However, slot blots of that portion of the IPM extracted from the neural retinas showed less reactivity per microgram of protein for IRBP in the galactose-fed animals of both strains than for the rats fed a basal diet. This was statistically significant, however, only in the WKy rats. CONCLUSIONS: The composition of the IPM of these two strains of rat appears similar to the composition of the IPM of other species that have been studied. Whether the quantitative alteration in IRBP that appears to be produced by galactose feeding has functional significance, with particular relevance to retarding the development of the photoreceptor cell dystrophy of the SHR rat, is unclear at this time.

Animals↗
PubMed1993

Effects of endothelin on cultured bovine retinal microvascular pericytes.

PURPOSE: The endothelins, a recently discovered family of peptides, include endothelin-1, the most potent vasoconstrictor known. Retinal microvascular pericytes are thought to be contractile cells analogous to the smooth muscle cells of larger vessels, but the physiologic stimulus for their contraction is unknown. We hypothesized that the endothelins might serve as such stimuli. METHODS: The intracellular free/bound Ca++ ratio increases rapidly immediately before muscle cell contraction. We evaluated changes in this ratio in cultured bovine retinal microvascular pericytes and, for comparison, three other types of ocular cells. We loaded the cells with the calcium-sensitive, fluorescent dye Indo-1. Using a laser cytometer, we monitored the time course of changes in fluorescence of individual cells in response to several putative vasoactive agents, including, in particular, endothelin-1, 2, and 3. RESULTS: Endothelin-1 (ET-1) produced a rapid rise in the free/bound Ca++ ratio, followed by a slow decline. The response occurred at ET-1 concentrations as low as 1 x 10(-12) mol/l, and was graded in amplitude and concentration dependent. After an initial application of ET-1, repeat applications yielded no response. Endothelin-2 was less effective than ET-1, and ET-3 had no effect at all, but both agents blocked the response to ET-1. Several other agents also raised the free/bound Ca++ ratio, but were substantially less effective than ET-1. When any of these agents, except for histamine, was added after even a submaximal concentration of ET-1, no response was observed, but ET-1 applied after these agents produced a large response. Histamine could elicit a rise in the free/bound Ca++ ratio after application of ET-1 to cultured pericytes. The ET-1 response occurred in Ca(++)-free medium and in medium containing 10(-4) mol/l verapamil or nifedipine, indicating that the results we observed are due primarily to the release of free Ca++ from bound intracellular stores. Endothelin-1 produced a similar change in the free/bound Ca++ ratio in cultured bovine RPE cells, but not in retinal microvascular endothelial cells or lens epithelial cells. CONCLUSIONS: ET-1 is at least three orders of magnitude more effective in producing the release of free intracellular Ca++ than other agents tested. It appears to act through specific cell surface receptors, which can be blocked by prior application of other endothelin isopeptides, but not by structurally dissimilar molecules. However, with the exception of histamine, all of these agents appear to act through a common intracellular pathway, because application of ET-1 blocks the subsequent effect of the other agents tested, except histamine. Alternatively, ET-1 may be capable of desensitizing the receptors for these agents without occupying the receptor sites. Because cultured retinal pericytes are extremely sensitive to ET-1 in a response closely linked to muscle cell contraction, ET-1 must be considered a highly plausible agonist for pericyte contraction in vivo.

Animals↗
PubMed1992

Ultrastructural immunocytochemistry of subretinal neovascular membranes in age-related macular degeneration.

BACKGROUND: The authors studied various cellular and extracellular matrix components of subretinal neovascular membranes (SRNVM) from patients with age-related macular degeneration (ARMD). METHODS: Electron microscopic immunocytochemistry was used on the subfoveal neovascular membranes surgically removed from three patients with disciform lesions due to ARMD. FINDINGS: The SRNVMs always contained large "feeder" vessels along with many new capillaries in different stages of maturation. Capillaries were sparse and embedded in an abundant stroma. The majority of the nonvascular cells were either retinal pigment epithelial (RPE) cells or fibroblast-like cells. The RPE cells formed single or multiple layers on one side of the membranes. The stroma was composed mainly of collagen types I and IV and fibronectin, with small amounts of collagen types III, V, and VI. The absence of Bruch's membrane suggests that a splitting may occur between the RPE cells and Bruch's membrane with the new vessels growing into this cleft. A thickened layer of collagen type IV was often present under the RPE cells. The basement membranes of the newly formed capillaries were morphologically ill-defined, and contained substantial amounts of collagen type IV and fibronectin, but, unlike the basement membranes of normal capillaries, they lacked laminin or heparan sulfate proteoglycan. CONCLUSION: These results on the ultrastructural components of the SRNVMs may be useful in clarifying the nature of the disciform process in ARMD.

Aged↗
PubMed1992

Risk factors for retinal vein occlusions. A case-control study.

The authors conducted a case-control study of risk factors for retinal vein occlusions using 87 patients with vein occlusions, chosen randomly from photographic files from their institution between 1985 and 1990, and a control group of 85 subjects 38 years of age (the youngest individual in the vein occlusion group) or older, who were randomly selected from the records of two general ophthalmologists in the authors' department. Certain risk factors for retinal vein occlusion were highly significant when subjects with retinal vein occlusion were compared with the control group. These risk factors included systemic hypertension (odds ratio [OR], 3.86; 95% confidence interval [Cl], 2.08 to 7.16), open-angle glaucoma (OR, 2.89; 95% Cl, 1.38 to 6.05), and male sex (OR, 2.61; 95% Cl, 1.43 to 4.79). Race, presence of diabetes mellitus, history of coronary artery disease or stroke, and family history of diabetes, glaucoma, coronary artery disease, or stroke were not significant risk factors in the population studied. Logistic analysis of the risk factors showed no interactions. Risk factors for branch retinal vein occlusion and central retinal vein occlusion were identical.

Adult↗
PubMed1991

On the pathogenesis of diabetic retinopathy. A 1990 update.

Although most investigators now agree that chronic hyperglycemia is the basis for diabetic retinopathy, this has not been proven definitively. Even if chronic hyperglycemia is the initial common pathway leading to retinopathy and other complications of diabetes, it appears to act by different mechanisms in different tissues. The enzyme, aldose reductase, may play a major role in the development of diabetic retinopathy, but contradictory evidence exists. At the present time, results of the only study of aldose reductase inhibition and diabetic retinopathy reported in humans were negative. Another mechanism worthy of consideration is nonenzymatic glycation (glycosylation) of proteins, but there is no direct evidence of a causal role in diabetic retinopathy. Several growth factors have been identified in the retina that may promote neovascularization, and at least two inhibitors may prevent the process. There is evidence to support a role for basic and, perhaps, acidic fibroblast growth factors in retinal vasoproliferation. Transforming growth-factor beta, a peptide produced by capillary pericytes and smooth muscle cells and activated by the interaction of these cells with vascular endothelial cells, appears to be an important inhibitor of neovascularization, as is the vascular basement membrane.

Aldehyde Reductase↗
PubMed1990

Ultrastructural localization of extracellular matrix components in human retinal vessels and Bruch's membrane.

We have used the electron microscopic immunogold technique to localize precisely various extracellular matrix components in human retinal vessels and Bruch's membrane. Collagen types IV and V, laminin, and heparan sulfate proteoglycan core protein were localized in basement membranes of retinal capillaries. In addition to the capillary basement membrane components, collagen types I and III and fibronectin were found in the basement membranes of retinal arterioles and venules. The basement membranes of choriocapillaries and retinal pigment epithelial cells in Bruch's membrane also showed a similar distribution of the retinal capillary basement membrane components. Both the inner and outer collagenous layers of Bruch's membrane contained collagen types I and III along with fibronectin, whereas collagen type VI was mostly limited to the central elastic lamina. The precise localization and distribution of various extracellular matrix components in human retinal vessels and Bruch's membrane may be important for understanding their normal function as well as their alteration in disease.

Aged↗
PubMed1990

Sorbinil does not prevent galactose-induced glomerular capillary basement membrane thickening in the rat.

We investigated the role of the polyol pathway in the pathogenesis of glomerular basement membrane thickening in galactosaemic rats, an animal model that develops basement membrane lesions comparable to those of human diabetic subjects. Normotensive Wistar-Kyoto rats fed a 30% galactose diet for nine months developed significant glomerular basement membrane thickening by comparison with rats on a control test diet (p = 0.008). However, addition of an aldose reductase inhibitor, sorbinil (250 mg/kg diet), to the galactose diet did not prevent the increase in glomerular basement membrane thickness. Furthermore, by using a quantitative electron microscopic immunogold technique, we examined biochemical alterations in the composition of glomerular basement membranes in this animal model. The labelling density (comparable to relative concentration) of collagen type IV in thickened glomerular basement membranes of galactosaemic animals was significantly increased by comparison to those of control rats (p = 0.015). However, there was no significant difference in labelling densities of laminin and heparan sulfate proteoglycan core protein of these animals. Thus, our results indicate that an increase in glomerular basement membrane thickness accompanied by an increase in the labelling density of collagen type IV occurs in the galactosaemic rats, but this thickening is not prevented by sorbinil at the dose used in this experiment. Our study raises the strong possibility that glomerular basement membrane thickening in galactosaemic rats may not be due to excessive polyol pathway activity.

Aldehyde Reductase↗
PubMed1990

Increases in collagen type IV and laminin in galactose-induced retinal capillary basement membrane thickening--prevention by an aldose reductase inhibitor.

Biochemical alterations in the composition of retinal capillary basement membrane components were investigated in galactosemic rats, an animal model that develops basement membrane lesions comparable to those of diabetic retinopathy. Normotensive Wistar-Kyoto rats fed a 30% galactose diet for 9 months developed significant thickening of retinal capillary basement membranes by comparison with animals fed a control test diet (P less than 0.001), or animals on a diet containing 30% galactose and 250 mg kg-1 of the aldose reductase inhibitor sorbinil (P less than 0.001). A quantitative electron microscopic immunogold technique applied on ultrathin sections of the retinas of these animals showed that the labeling densities of collagen type IV and laminin per unit cross-sectional area (which is presumably proportional to the concentrations of these molecules) were significantly increased in the retinal capillary basement membranes of galactose-fed rats, compared with animals on the control test diet. Increases in these two components of basement membranes were prevented by addition of sorbinil to the diet. However, there was no significant change in the labeling density of heparan sulfate proteoglycan (HSPG) core protein in the basement membranes of galactose-fed rats in comparison to animals on either the control diet or galactose-sorbinil diet. Two types of striated fibrillar materials were frequently found in areas of focal thickening of basement membranes of galactose fed rats only. Thinner fibrils reacted strongly with collagen type III antibody, whereas thicker fibrils reacted weakly with collagen type I antibody. Our results indicate that there is an increase in labeling densities of collagen type IV and laminin in thickened basement membranes of retinal capillaries of galactosemic rats along with the expression of interstitial collagens like collagen type III and an abnormal collagen that weakly cross-reacts with antibody to collagen type I, and these effects of galactosemia on the basement membranes are preventable by an aldose reductase inhibitor.

Aldehyde Reductase↗
PubMed1990

Proliferative response and macromolecular synthesis by ocular cells cultured on extracellular matrix materials.

To investigate the effects of extracellular matrix components on cellular function, we cultured several types of ocular cells on substrates composed of extracellular matrix materials that were layered on culture dishes either as dried films or as gels. We measured cellular proliferation on these substrates and on a series of gels composed of varying proportions of rat tail tendon type I collagen and Matrigel, a commercially available extract of a basement membrane-producing murine tumor. In addition, we studied the biosynthesis of collagens and of proteoglycans by these cultured cells using [3H]-L-proline and [35S]-sulfate. The proliferative abilities of the various types of ocular cells on the dried film substrates, on uncoated plastic culture vessels, and on pure type I collagen gel, were similar. However, proliferation of ocular cells cultured on gels composed of greater than or equal to 90% Matrigel was markedly reduced. There was little or no inhibition of growth of two types of non-ocular cells: rat C6 astrocytoma cells, and human dermal fibroblasts. Histologic studies showed that the ocular cells tested often formed long strands and capillary-like tubes, and tended to "burrow" beneath the surface of substrates containing high percentages of Matrigel. Fibroblasts infrequently formed tubes, and exhibited the burrowing property also on gels containing primarily type I collagen, while C6 cells showed neither of these behaviors on any of the matrices tested. The elution pattern of newly synthesized [3H]-labeled and [35S]-labeled macromolecules produced by all of the cultured cell types, and detected by Sepharose CL-4B chromatography in the medium and in the cell layer plus matrix fractions did not vary following culture on the different substrates. Approximately twofold more of the newly synthesized collagens and proteoglycans were deposited in the cell layer plus matrix, and proportionately less appeared in the medium, when cells were cultured on type I collagen gels and on Matrigel than on the dried film substrates. These experiments demonstrate the influence of the extracellular matrix on several aspects of cell behavior, and provide further evidence that modification of the composition of the extracellular matrix may be an important determinant of normal or pathological cell function.

Animals↗
PubMed1990

Pericyte coverage of retinal and cerebral capillaries.

We performed electron microscopic morphometric analyses on capillaries from macular and peripheral retinas of five adult cynomolgous monkeys and three elderly human subjects. Measurements from the monkey retinal capillaries were compared to those made on capillaries from frontal, temporal, parietal, and occipital cerebral cortex of the same animals. We measured the percent coverage of the endothelial lining of the capillaries by pericyte processes, as well as the ratio of the cytoplasmic areas of pericytes and endothelial cells. In addition, we compared the thickness of the capillary basement membranes in three regions: overlying pericytes; overlying endothelial cells; and interposed between pericytes and endothelial cells. In both monkey and human retinas, pericyte processes covered greater than 85% of the circumference of the capillary endothelial tube, whereas pericyte coverage of monkey cerebral capillaries was highly significantly (P less than 0.001) less than that of capillaries in either the macular or peripheral retina. The ratio of pericyte to endothelial cell cytoplasmic areas also was lower in the monkey cerebral cortex than in the retina, though the statistical significance was less than that of the length measurements. In all tissues measured, both from monkeys and humans, the portions of the capillary basement membranes interposed between pericytes and endothelial cells were highly significantly (P less than 0.0001) thinner than the regions of capillary basement membranes covering pericytes and endothelial cells. Considering functions that have been proposed for pericytes, these measurements suggest that regional control of microcirculatory flow and of blood-tissue barrier integrity, as well as control of endothelial cell proliferation, should be much greater in the retina than in the cerebral cortex. Thinner basement membranes between pericytes and endothelial cells may permit more cell membrane contacts between these cells, thus facilitating such control.

Aged↗
PubMed1990

Diabetic retinopathy in pregnancy: a review.

Diabetic retinopathy is the leading cause of blindness between the ages of 24-64 years. The first half of this period corresponds to peak fertility and the childbearing years. The effects of pregnancy on diabetic retinopathy are unclear, but recent studies suggest that pregnancy may be less harmful to the retina of the diabetic subject than was thought previously. Nevertheless, there is reason to believe that at least some women experience a worsening of their retinopathy as a result of pregnancy. Thus, careful ophthalmic evaluation and follow-up are essential for the pregnant woman with diabetes. This should include a minimum of one complete eye examination every trimester and within 3 months postpartum. Color fundus photography and laser treatment are safe, whereas fluorescein angiography, although commonly used to evaluate retinal vascular disease, can usually be avoided during pregnancy.

Diabetic Retinopathy↗
PubMed1989

A model of subretinal neovascularization in the pigmented rat.

We produced krypton laser photocoagulation lesions of mild to moderate whiteness in the posterior retinas of one eye of 23 pigmented rats, and identical appearing argon laser burns in the fellow eyes. We observed foci of subretinal neovascularization, histopathologically markedly similar to that which occurs in several human retinal diseases, in the krypton laser treated eyes of 6 of the 14 rats that were followed for one to three months after photocoagulation. No such lesions were observed in the argon laser treated fellow eyes, nor in krypton or argon laser treated eyes examined earlier than one month after photocoagulation. The photocoagulation damaged only the choriocapillaris, the retinal pigment epithelium (RPE), and the photoreceptor layer. In the acute lesions, we did not observe ruptures in Bruch's membrane. The neovascularization was surrounded by multiple layers of RPE cells, a histopathologic finding that has also been reported in some human eyes with subretinal neovascularization in age-related macular degeneration. These observations suggest that the RPE cells may be modifying the proliferative behavior of adjacent choroidal capillaries. This model differs from previous models of subretinal neovascularization in primates, and may be useful for additional studies of this important pathological process.

Animals↗
PubMed1989

Retinovitreal neovascularization in the Royal College of Surgeons rat.

Retinovitreal blood vessels, which we have previously reported in the dystrophic retinas of approximately 20% of older (greater than 15 months of age) spontaneously hypertensive (SHR) rats, also occur in about 20% of retinal dystrophic Royal College of Surgeons (RCS) rats greater than 1 year of age. We have demonstrated previously that these vessels, in the SHR rat, have the anatomic characteristics of retinovitreal neovascularization as it has been described in vasoproliferative retinopathies in humans (1). We now provide strong evidence that the endothelial cells and pericytes of the retinovitreal vessels that occur in RCS rats are proliferating, since they demonstrate by autoradiography significantly increased nuclear labeling with [3H]-thymidine over intra-retinal or choroidal vessels, or the abnormal vessels that grow within the retinal pigment epithelium in these dystrophic retinas. One important difference between the neovascularization that occurs in these rats and that which is observed in various human retinal vascular diseases is the frequent association of the new vessels in dystrophic rat retinas with surrounding cords of proliferating retinal pigment epithelium. The retinovitreal vessels in these strains of dystrophic rats represent a new animal model that may be useful for studying the fundamental processes underlying new blood vessel growth in the retina.

Animals↗
PubMed1989

A cell line derived from non-neoplastic human neuroretinal cells.

We have derived a cell line from an epiretinal membrane excised surgically from a premature female infant born at a gestational age of 25 weeks, and who developed stage 5 retinopathy of prematurity. The cell line, which in early passages appeared immunocytochemically to contain cells with both neuronal and glial characteristics, has been maintained in culture for 14 months at the time this manuscript was submitted, and has survived 20 passages. The cells have a diploid, human karyotype, with most cells possessing 46 normal appearing chromosomes, including 44 autosomes and two X-chromosomes. Morphologically, the cell line at early passages consisted of polygonal cells and also of cells possessing long, spindly branching processes. These two cell types were cloned. Nearly 100% of the cells of both morphologic types in mixed cultures stained immunocytochemically for neuron-specific enolase (NSE), a neuronal marker, and approximately 5-10% of the cells in mixed cultures (including about 50% of the cells with the spindly morphology, that were less prevalent in mixed cultures) stained for glial fibrillary acid protein (GFAP), a glial marker. We have not performed "double-label" immunocytochemistry, but it was evident from the proportion of cells that stained with each marker that many cells must contain both GFAP and NSE. At least 50% of the cells in most of the early cultures were positive for keratin, while all were (and remain) negative for muscle actin. No cells are found that are immunocytochemically positive for factor VIII, a vascular endothelial cell marker. These cultured cells have also been studied immunocytochemically for their production of extracellular matrix substances. The cultures are immunocytochemically positive for type IV (but not type I) collagen, laminin and fibronectin. In later passages, cells of both clones lost their immunocytochemical positivity for GFAP and NSE, and all became positive for keratin. Cells of both clones also developed a similar, polygonal morphology, lacking long processes. By electron microscopy, many of the cells were seen to possess nonmotile cilia, with a 9 + 0 pattern of microtubule doublets. This cell line may be useful for studies of human retinal cell development and metabolism, and responses to pathological processes.

Cell Division↗