Evaluation of proliferating cell abundance and phenotypes in proliferative diabetic retinopathy.

BACKGROUND: The aim of this work is to evaluate the abundance, origins, and phenotypes of actively proliferating cells in proliferative diabetic retinopathy (PDR). METHODS: Eleven epiretinal membranes from patients undergoing surgery for PDR were evaluated by indirect immunofluorescence for evidence of cell proliferation using the nuclear cell proliferation marker Ki67 and for cell identities using glial fibrillary acidic protein (GFAP), glutamine synthetase, and α-smooth muscle actin (αSMA). RESULTS: Ki67 positivity was consistently rare in PDR epiretinal membranes at 3.02 ± 1.42 % of the total cell population. The majority of the Ki67-positive cells were also positive for GFAP (74.0 %) with lower proportions positive for αSMA (30.7 %) and glutamine synthetase (1.5 %). Co-localization studies using glial and myoid markers revealed that virtually all (92 %) of the αSMA-positive cells are also GFAP positive and thus derive from glia. CONCLUSIONS: Entry into cell cycle and thus cell proliferation appears to be a rare phenomenon in PDR involving only a small percentage of the total cell population. Glia and/or glial-derived myofibroblasts appear to be the predominate cell types in epiretinal scar tissues and also account for the majority of the actively proliferating cells.

Myofibroblast and extracellular matrix origins in proliferative vitreoretinopathy.

BACKGROUND: To evaluate origins of the fibrocontractive cell populations and their relation to collagens I and II in proliferative vitreoretinopathy (PVR). METHODS: Human PVR membranes were evaluated by indirect immunofluorescence for GFAP, cytokeratin-18 (CK-18), α-smooth muscle actin (αSMA), collagens I and II. Collagen expression by porcine Müller and retinal pigment epithelial cells (RPE) was evaluated using RT-PCR of RNA harvested from freshly isolated primary and proliferating cultures. RESULTS: Collagen I was detected in all PVR samples and was widely distributed in the extracellular matrix. In contrast, collagen II was present in only two of the ten samples and was localized to thin, acellular bands near the border of the tissues. Using cell type-specific markers CK-18 and GFAP, RPE and glia were localized to the collagen I-rich matrices. Cells positive for GFAP and CK-18 can also co-express αSMA. Normal and proliferating RPE express collagen I, but Müller cells show no evidence of collagen I expression until they proliferate in culture. In contrast, normal RPE and Müller cells contain message for collagen II which is lost shortly after introduction into culture. CONCLUSIONS: Collagen I appears to be the predominate fibrillar collagen in human PVR membranes and collagen II a comparatively minor component. Müller cells and RPE are physically associated with the collagen I matrix and are capable of expressing this protein suggesting that they are the origin. It also appears that the majority of myofibroblasts in PVR membranes are derived from either RPE or Müller cells suggesting that they play a major role in membrane development.

Photoreceptor-like cells from reprogramming cultured mammalian RPE cells.

PURPOSE: Previous studies showed that chick retinal pigment epithelium (RPE) cells can be reprogrammed by a specific gene to take on the path of photoreceptor differentiation. In this study, we tested whether this reprogramming scheme could be applied to mammalian RPE cells. METHODS: Human RPE cell lines ARPE-19, a spontaneously transformed line of RPE cells derived from a 19-year-old person, and hTERT-RPE1, a telomerase-immortalized RPE cell line derived from a 1-year-old person, were commercially obtained and cultured as recommended. Primary RPE cell cultures were established using RPE isolated from 3- to 6-month-old pig and postnatal day 5 mouse. Cultured cells were transduced with a virus expressing neuroD, neurogenin1 (ngn1), or ngn3, basic helix-loop-helix (bHLH) genes previously identified as capable of inducing RPE-to-photoreceptor reprogramming in the chick system. Alternatively, cells in the culture were transfected chemically or physically through electroporation with vector DNA expressing one of the three genes. The cultures were then analyzed for RPE-to-photoreceptor reprogramming with in situ hybridization and/or immunostaining for photoreceptor gene expression. RESULTS: Both hTERT-RPE1 and ARPE-19 cultures gave rise to cells bearing markers of photoreceptors after transduction or transfection with vehicles expressing neuroD or ngn1. The new cells expressed genes encoding photoreceptor proteins, including interphotoreceptor retinoid-binding protein IRBP), recoverin, retinal cone arrestin 3, transducin α-subunit, Cone-rod homeobox protein (Crx), and red opsin. They displayed morphologies resembling differentiating photoreceptor cells. In primary porcine and mouse RPE cell cultures, transduction with lenti virus (Lvx-IRES-ZsGreen1) expressing ngn1 or ngn3 resulted in the emergence of ZsGreen1+ cells that exhibited morphologies reminiscent of differentiating photoreceptor cells. Immunochemistry showed that some ZsGreen1+ cells were positive for neural marker microtubule-associated protein 2 (Map2) and photoreceptor hallmark proteins red opsin and rhodopsin. CONCLUSIONS: The results suggest that cells in human RPE cell lines and in primary cultures of porcine and mouse RPE respond to gene-induced reprogramming by giving rise to photoreceptor-like cells. The responsiveness of primary RPE cells, especially those from porcine cells, enhances the biologic feasibility of exploring RPE-to-photoreceptor reprogramming for in situ mammalian photoreceptor replacement without cell transplantation.

The insulin-like growth factor system modulates retinal pigment epithelial cell tractional force generation.

PURPOSE: The goal of this study was to determine the influence, if any, of the insulin-like growth factors (IGFs) on retinal pigment epithelial (RPE) cell tractional force generation and the contributions of vitreous insulin-like growth factor-binding proteins (IGFBPs) toward control of growth factor activity. METHODS: IGF effects on RPE were evaluated in tissue culture assays that involved incubation on three-dimensional collagen matrices with responses measured as progressive reduction in matrix thickness. IGFBP effects were evaluated by using the same system, exposing cells to a non-IGFBP-binding growth factor analogue (R(3)IGF-I) or IGFBPs alone or in combination with native growth factors. RESULTS: RPE cells generated tractional forces in response to IGF-I and -II with IGF-I being the more potent stimulus. Differential RPE responses to R(3)IGF-I reflected minor amounts of endogenous IGFBP production. IGFBP-2, -3, and -5 were effective inhibitors of both ligands, whereas IGFBP-6 reduced cell responses to IGF-II only. IGFBP-direct effects on the cells were binding-protein-specific, in that IGFBP-1 had detectable stimulatory effects, and IGFBP-3, -4, -5, and -6 inhibited RPE responses. CONCLUSIONS: IGF-I and -II are potent promoters of RPE cell tractional force generation in vitro. The effects of the six high-affinity IGFBPs on RPE responses are generally inhibitory and protein-specific. IGF ligands and binding proteins are known to be present in the vitreous, the environment that drives RPE responses in proliferative vitreoretinopathy (PVR), suggesting that the IGF system plays a potentially important role in the pathophysiology of this fibrocontractive disease.

Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model.

Purpose: Extracellular deposits containing hydroxyapatite, lipids, proteins, and trace metals that form between the basal lamina of the RPE and the inner collagenous layer of Bruch's membrane are hallmarks of early AMD. We examined whether cultured RPE cells could produce extracellular deposits containing all of these molecular components. Methods: Retinal pigment epithelium cells isolated from freshly enucleated porcine eyes were cultured on Transwell membranes for up to 6 months. Deposit composition and structure were characterized using light, fluorescence, and electron microscopy; synchrotron x-ray diffraction and x-ray fluorescence; secondary ion mass spectroscopy; and immunohistochemistry. Results: Apparently functional primary RPE cells, when cultured on 10-µm-thick inserts with 0.4-µm-diameter pores, can produce sub-RPE deposits that contain hydroxyapatite, lipids, proteins, and trace elements, without outer segment supplementation, by 12 weeks. Conclusions: The data suggest that sub-RPE deposit formation is initiated, and probably regulated, by the RPE, as well as the loss of permeability of the Bruch's membrane and choriocapillaris complex associated with age and early AMD. This cell culture model of early AMD lesions provides a novel system for testing new therapeutic interventions against sub-RPE deposit formation, an event occurring well in advance of the onset of vision loss.

The role of Müller cells in fibrocontractive retinal disorders.

Despite advances in surgical management of fibrocontractive retinal disorders, proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR) remain major causes of blindness and there is still considerable uncertainty about the origins and roles of the cell types involved. Muller cells and cells identified as retinal glia are consistently identified in epiretinal tissues from both types of disorders. However, their abundance relative to total cell populations is generally low, leaving their role in these disorders uncertain. Studies of Müller cell biology using tissue culture and animal models provide evidence of the remarkable capacity of this cell type for graded responses to environmental insult, the capacity to proliferate, translocate from the retina and alter phenotype and thus, functional characteristics. This review considers the potential roles of Müller cells in fibrocontractive retinal disorders and, in particular, evidence that Müller cells function as an effector cell type in traction retinal detachment associated with PVR and PDR.

Changes in IGF activities in human diabetic vitreous.

Müller cells, the principal glia of the retina, generate tractional forces in response to IGF-I and platelet-derived growth factor and are present in diabetic fibro-vascular scar tissues causing traction retinal detachment. While diabetes-associated increases in vitreous IGFs have been reported, paradoxically high concentrations of these same growth factors in normal vitreous suggest the presence of more complex mechanisms regulating growth factor bioavailability. To define diabetes-associated changes in vitreous biological activity, the stimulatory effects of 68 samples were evaluated using Müller cell tractional force generation as a target bioassay. Dose-response profiles were used to calculate vitreous specific activity (per unit protein) and total vitreous activity (per unit volume). Vitreous samples from patients lacking diabetes or other retinal pathology had undetectable or low activities, whereas diabetic retinopathy was associated with 6.9- and 8.7-fold increases in vitreous specific and total activities, respectively. Secondary analyses revealed no activity differences associated with patient sex, age, or the presence of vitreous hemorrhage. However, compared with diabetes alone, the presence of proliferative diabetic retinopathy was associated with additional 2.3-fold increases in vitreous specific and total activities. Vitreous dose-response assays performed with and without growth factor-neutralizing antibodies enable attribution of vitreous activity to IGFs (53.9%) and, to a lesser extent, platelet-derived growth factors (14.5%). Because the observed increases in vitreous growth factor activity grossly exceed the reported increases in growth factor concentration, these data indicate that diabetes-associated changes in vitreous biological activity involve more complex biochemical changes that ultimately yield increased growth factor bioavailability and/or Müller cell responsiveness.

Acute aminoglycoside retinal toxicity in vivo and in vitro.

PURPOSE: Intentional and inadvertent intraocular administration of aminoglycosides is associated with cases of retinal toxicity. Clinical manifestations resemble a vaso-occlusive event and include edema, intraretinal hemorrhage, and nonperfusion detected by fluorescein angiogram. This study was conducted to measure the retinal function in avascular and isolated perfused retinas to separate vascular and neurologic effects of gentamicin. Enhanced understanding of the mechanism of gentamicin toxicity may lead to development of aminoglycosides that can be used to treat ophthalmic infections without retinal damage. METHODS: Whole animals and isolated rabbit and rat retina preparations were used to study the dose dependence and reversibility of toxicity on the ERG, with a 1- and a 10-mg/mL solution of gentamicin. The amplitude and implicit times of the a-, b-, and c-waves were measured before, during, and after exposure to the drug. RESULTS: In whole-animal ERG studies, intraocular administration of gentamicin eliminated the b-wave but left the a-wave intact. The c-wave was reduced in amplitude. Histopathologic evaluation demonstrated diffuse disruption of the nerve fiber layer and the inner plexiform layers. Isolated retinal studies showed that the b-wave was reduced in amplitude in the presence of low-dose gentamicin (1 mg/mL) and completely eliminated by high-dose gentamicin (10 mg/mL). This effect was reversible for short-term exposure to gentamicin. CONCLUSIONS: The results indicate that the initial loss of function due to exposure to aminoglycoside antibiotics is independent of the vascular supply. Elimination of the b-wave was dose dependent and reversible, indicating that a component of gentamicin toxicity is mediated through pathways other than vascular supply. Short-term effects are reversible, suggesting a receptor-mediated process.

Müller cell production of insulin-like growth factor-binding proteins in vitro: modulation with phenotype and growth factor stimulation.

PURPOSE: Müller cells are present in diabetic fibrocontractive ocular tissues and generate tractional forces in response to insulin-like growth factors. Recent studies indicate that diabetes-associated increases in vitreous insulin-like growth factor activity are, in part, attributable to changes in insulin-like growth factor binding proteins (IGFBPs). The objectives of this study were to evaluate Müller cells as a source of IGFBPs and characterize changes associated with cell phenotype and growth factor stimuli known to be present in diabetic vitreous. METHODS: Müller cells isolated from normal porcine retina were maintained in culture for 1 and 5 weeks, yielding phenotypes described as proliferative and myofibroblastic. RNA preparations from porcine liver, retina, and Müller cell cultures were evaluated by RT-PCR and Northern blot analysis. IGFBP production was verified by Western ligand and Western blot analysis of Müller-cell-conditioned media and detergent-extracted proteins. RESULTS: Molecular biological analyses of RNA from normal retina and from proliferative and myofibroblastic Müller cells did not detect message for IGFBP-1, but revealed progressive increases in message abundance for IGFBP-2, -3, -4, and -6. IGFBP-5 message was detected in all samples, but was least abundant in myofibroblastic Müller cells. Stimulation of myofibroblastic Müller cells by IGF-I and -II, but not PDGF, further increased message abundance and production of IGFBP-2, -4, -5, and -6, but not IGFBP-3. CONCLUSIONS: Müller cell production of IGFBPs changes with phenotype and, in most cases, is highest in the cells most likely to participate in fibrocontractive retinal disease. IGFBP production by these cells is further increased by IGF-I and -II, growth factors known to be present and active in proliferative vitreoretinal disorders, suggesting that Müller cells represent a potential source of vitreous IGFBPs in disorders involving this cell type.

Insulin-like growth factor binding proteins modulate Müller cell responses to insulin-like growth factors.

PURPOSE: Müller cells are consistently identified in diabetic fibrocontractive ocular tissues and, in response to insulin-like growth factor I, generate tractional forces of the type that cause retinal detachment. Recent studies suggest that diabetes-associated increases in vitreous insulin-like growth factor activity cannot be attributed to simple increases in concentration alone, suggesting that more complex biochemical changes in vitreous growth factor control mechanisms are involved. The goal of this study was to evaluate the contributions of vitreous insulin-like growth factor-binding proteins (IGFBPs) toward control of growth factor activity. METHODS: Native and recombinant IGFBPs effects were evaluated on IGF-I- and -II-stimulated Müller cells in tissue culture assays that involved cell incubation on three-dimensional collagen gels and that monitored progressive matrix condensation. IGFBP degradation by Müller cell-secreted proteases was assessed in Western ligand blots, and direct stimulatory effects were evaluated by incubating cells with IGFBPs alone. RESULTS: IGFBP direct stimulatory effects on Müller cells were significant, but relatively modest, and IGFBP modulation through Müller cell-secreted proteases was undetectable. In contrast, IGFBP inhibitory effects on IGF-I and -II were highly variable and, in some cases, profound. IGFBP-3 effectively inhibited IGF-I and -II stimulation with detectable effects at concentrations equimolar to the growth factor. IGFBP-1, -2, -4, and -5 were of intermediate effectiveness as inhibitors, 3- to 11-fold less active than IGFBP-3. IGFBP-6 had virtually no inhibitory effects on IGF-I, but was moderately effective against IGF-II. CONCLUSIONS: IGFBP effects on IGF-I- and -II-stimulated Müller cells are primarily inhibitory with only modest direct stimulatory effects of limited physiologic relevance. IGFBP-2 and -3, the major binding proteins identified in vitreous, most likely function as the vitreous growth factor sink and control ligand activity through sequestration.

Tractional force generation by human müller cells: growth factor responsiveness and integrin receptor involvement.

PURPOSE: To assess the ability of human Müller cells to generate tractional forces and to determine the role of growth factors and collagen binding integrins in this process. METHODS: Müller cells were isolated from papain-DNase-digested human retina. Cell identity and changes in cell phenotype were confirmed by immunodetection of glial fibrillary acidic protein (GFAP), cellular retinaldehyde-binding protein (CRALBP), vimentin, and alpha-smooth muscle actin (alpha-SMA). Generation of tractional force was assessed with a tissue culture assay involving incubation of cells on three-dimensional collagen gels. The effects of contraction-promoting growth factors were examined by adding these directly to the culture medium. Müller cell expression and the involvement of specific integrin receptors were assessed by immunodetection, RT-PCR, and subunit-specific blocking antibodies. RESULTS: During maintenance in culture, human Müller cells adopted a fibroblast-like phenotype capable of generating tractional forces. Matrix contraction was stimulated in a dose-dependent fashion by insulin-like growth factor I and platelet-derived growth factor. Modest responses were observed with high concentrations of transforming growth factor (TGF)-beta1 and -beta2. Müller cells express all four integrin subunits that comprise the collagen-binding receptors including alpha1, alpha2, alpha3, and beta1. Blocking antibodies against receptor subunits alpha2 and beta1 significantly reduced the overall rate of matrix contraction. Antibodies against the alpha1 subunit were modestly inhibitory, whereas anti-alpha3 was without effect. CONCLUSIONS: Human Müller cells acquire the capacity to generate tractional forces in vitro and the contraction-promoting growth factors insulin-like growth factor (IGF)-I and platelet-derived growth factor (PDGF) are potent stimuli. Generation of tractional force by Müller cells primarily involves integrin receptors containing alpha2 and beta1 subunits.

Phenotypic variation of retinal pigment epithelium in age-related macular degeneration.

PURPOSE: To determine whether retinal pigment epithelium (RPE) in eyes with age-related macular degeneration (ARMD) express vimentin and alpha smooth muscle actin (alphaSMA), two cytoskeletal proteins associated with phenotypic variation in culture. METHODS: Six eyes with late ARMD and three age-matched control eyes were preserved in buffered 4% paraformaldehyde and cryosectioned at 10 microm. Stages of RPE morphology and pigmentation were assessed by the Alabama Age-Related Macular Degeneration Grading System. Vimentin, alphaSMA, and glial fibrillary acidic protein (GFAP) expression was detected by indirect immunofluorescence. These results were compared with regional variations in disease severity. RESULTS: RPE changes in ARMD included acquired expression of vimentin, but alphaSMA-positive cells were rare. GFAP expression increased in Müller cells in the neural retina in association with RPE changes and photoreceptor degeneration. CONCLUSIONS: The initial stages of RPE changes in eyes with ARMD mimic those reported for cultured RPE cells. The absence of alphaSMA-positive cells in regions of RPE atrophy suggests that RPE are lost rather than persist in a dedifferentiated state.

Vitreous IGFBP-3 effects on Müller cell proliferation and tractional force generation.

PURPOSE: Previous studies from this laboratory revealed that vitreous insulin-like growth factor binding protein-3 (IGFBP-3) is a biologically active fragment of the intact protein. The goal of this study was to characterize its effects on Müller cell proliferation and tractional force generation, activities relevant to proliferative diabetic retinopathy (PDR). METHODS: Müller cells were isolated from normal porcine retina. The vitreous-type IGFBP-3 fragment was isolated from normal human plasma and compared with intact recombinant protein for the ability to modulate Müller cell proliferation and tractional force generation in tissue culture models. RESULTS: Müller cells were stimulated to proliferate by serum and platelet-derived growth factor (PDGF), but not insulin-like growth factor (IGF)-I or IGF-II. The cells were similarly unresponsive to IGFBP-3 or the IGFBP-3 fragment alone or in combination with IGF-I or IGF-II. In contrast, Müller cells demonstrated robust extracellular matrix contraction in response to IGF-I, IGF-II, and PDGF. Intact IGFBP-3 attenuated extracellular matrix contraction in response to IGF-I and IGF-II while the IGFBP-3 fragment modulated cell responses to IGF-II only. Neither binding protein altered cell responses to PDGF. CONCLUSIONS: Intact IGFBP-3 modulates Müller cell tractional force generation stimulated by IGF-I and IGF-II while the effects of the vitreous-type fragment are limited to IGF-II. Porcine Müller cells proliferate in response to PDGF, but not IGF-I or IGF-II. Both forms of IGFBP-3 are also without mitogenic effects alone or in combination with IGFs. It appears that Müller cell tractional force generation in PDR is driven by vitreous IGF activity and proliferation is stimulated by growth factors outside of the IGF system.

Isolation and characterization of vitreous insulin-like growth factor binding proteins.

PURPOSE: Previous studies from this laboratory revealed that vitreous insulin-like growth factor (IGF) biological activity increases in proliferative diabetic retinopathy and that this activity is normally attenuated by IGFBPs. The goal of this study was to identify and characterize the species involved. METHODS: Human and porcine vitreous, plasma, recombinant IGFBP-2, and IGFBP-3 were separated by gel electrophoresis. Functional IGFBPs were detected in Western ligand blots with biotinylated IGF-II. IGFBPs were identified using IGFBP-specific antibodies. RESULTS: Western ligand blots of normal vitreous and plasma detected two major proteins at ∼35 kDa and ∼29 kDa. Western blot analysis of human and porcine vitreous and plasma confirmed the identity of the ∼35-kDa band as IGFBP-2 and the ∼29-kDa band as a fragment of IGFBP-3. Western blot and Western ligand blot analyses of vitreous and plasma proteins separated by two-dimensional gel electrophoresis revealed that the IGFBP-3 fragments in vitreous and plasma have virtually identical profiles. Lyase digestion revealed that the ∼29-kDa IGFBP-3 fragment is a glycoprotein with a peptide core of ∼25 kDa. N-terminal sequence data obtained from vitreous IGFBP-3 revealed that the protein is proteolytically truncated at the C terminus. CONCLUSIONS. Normal human and porcine vitreous contain two major IGFBPs, IGFBP-2 and an ∼29-kDa fragment of IGFBP-3. Both IGFBPs retain biological activity, and IGFBP-3 has one or more glycosylation sites with a protein core of ∼25 kDa. Systematic comparisons indicate that the vitreous IGFBP-3 is similar to and perhaps identical with a previously described IGFBP-3 fragment in plasma with reduced growth factor affinities.

The influence of alloxan-induced diabetes on Müller cell contraction-promoting activities in vitreous.

PURPOSE: Previous studies from this laboratory revealed that vitreous insulin-like growth factor biological activity increases in diabetes and that this change can precede the onset of proliferative diabetic retinopathy. The goal of this study was to characterize this phenomenon in an animal model of alloxan-induced diabetes. METHODS: Swine made diabetic with intravenous alloxan were euthanized at times varying from 0 to 90 days. Vitreous samples from normal and diabetic swine were evaluated for changes in Müller cell contraction-promoting activity, the presence of insulin-like growth factor binding protein (IGFBP), and carbonic anhydrase-I and -II. Ocular tissues from these animals were also evaluated for changes in contraction-promoting growth factors and IGFBP message levels. RESULTS: Alloxan-induced diabetes is associated with significant increases in vitreous Müller cell contraction-promoting activity that are present in as few as 30 days and are sustained for at least 90 days. Biochemical studies revealed that the increases cannot be attributed to loss of growth factor-attenuating IGFBPs, changes in local expression of contraction-promoting growth factors, or vitreous hemorrhage. CONCLUSIONS: The previously reported increases in Müller cell contraction-promoting activity detected in human diabetic vitreous are present in diabetic swine within 30 days of chemical induction. The increase does not appear to be attributable to loss of growth factor control, increases in local growth factor expression, or vitreous hemorrhage, suggesting that other mechanisms are involved. It is the authors' speculation that diabetes induces blood-vitreous barrier changes that allow a different subset of plasma proteins to enter vitreous fluids.

Fibrocontractive Müller cell phenotypes in proliferative diabetic retinopathy.

PURPOSE: To evaluate Müller cells as a potential source of fibrocontractive cells in proliferative diabetic retinopathy. METHODS: Temporal changes in glial fibrillary acidic protein (GFAP), vimentin, glutamine synthetase, and alpha smooth muscle actin (alphaSMA) expression in cultures of freshly isolated porcine Müller cells were evaluated by indirect immunofluorescence and Western blotting. A similar evaluation was performed on freshly isolated Müller cells maintained in high- and low-glucose culture. Cryosections of six diabetic epiretinal tissues were evaluated for the same antigens. RESULTS: Müller cell changes in culture included loss of glutamine synthetase and GFAP, with coincident gains in alphaSMA immunoreactivity. Vimentin immunoreactivity persisted without obvious change. Similar changes were observed when the cells were maintained in high- or low-glucose culture medium. All six diabetic epiretinal membranes contained positively identified Müller cells with vimentin, GFAP, and glutamine synthetase immunoreactivities. There was a progressive loss of glutamine synthetase and GFAP content and a coincident increase in alphaSMA content as the cells assumed an elongated, fibroblastlike morphology. CONCLUSIONS: Continuous culture in high- versus low-glucose medium does not influence Müller cell phenotype changes. Positively identified Müller cells are present in diabetic epiretinal tissues and appear to undergo the same progression of phenotype changes observed in culture. Cells capable of generating tractional forces associated with proliferative diabetic retinopathy can arise from Müller cells.

Phenotype-associated changes in retinal pigment epithelial cell expression of insulin-like growth factor binding proteins.

PURPOSE: The objectives of this study were to evaluate retinal pigment epithelial (RPE) cells as a source of insulin-like growth factor binding proteins (IGFBPs) and to characterize biosynthetic changes associated with the cell phenotype and vitreous growth factor stimuli known to be present in fibrocontractive diseases. METHODS: Early culture-associated changes in RPE phenotype were characterized by indirect immunofluorescence localization and Western blot analysis of cell lysates. IGFBP expression was evaluated by RT-PCR and Northern blot analysis of total RNA preparations. RESULTS: Normal unperturbed RPE are immunoreactive for cytokeratin 18 and negative for cytokeratin 19, vimentin, and alpha-smooth muscle actin (alphaSMA). Early reactive RPE (7 days in culture) express cytokeratin 18, cytokeratin 19, and vimentin. Myofibroblastic RPE (35 days in culture) express cytokeratin 19, vimentin, and alphaSMA. RT-PCR studies revealed that normal RPE can produce IGFBP-2, -3, -4, -5, and -6 but not IGFBP-1. Early reactive and myofibroblastic RPE have detectable levels of message for IGFBP-3, -5, and -6. However, Northern blot analysis suggests that IGFBP-5 is the predominant binding protein produced. Finally, stimulation with biologically relevant quantities of IGF-I and IGF-II had no detectable effects on IGFBP expression. CONCLUSIONS: Changes in RPE phenotype are accompanied by dramatic changes in IGFBP expression profile, with IGFBP-5 the predominant binding protein produced by myofibroblastic RPE cells.

Retina expresses microsomal triglyceride transfer protein: implications for age-related maculopathy.

The principal extracellular lesions of age-related maculopathy (ARM), the leading cause of vision loss in the elderly, involve Bruch's membrane (BrM), a thin vascular intima between the retinal pigment epithelium (RPE) and its blood supply. With age, 80-100 nm solid particles containing esterified cholesterol (EC) accumulate in normal BrM, and apolipoprotein B (apoB) immunoreactivity is detectable in BrM- and ARM-associated lesions. Yet little evidence indicates that increased plasma cholesterol is a risk factor for ARM. To determine if RPE is capable of assembling its own apoB-containing lipoprotein, we examined RPE for the expression of microsomal triglyceride transfer protein (MTP), which is required for this process. Consistent with previous evidence for apoB expression, MTP is expressed in RPE, the ARPE-19 cell line, and, unexpectedly, retinal ganglion cells, which are neurons of the central nervous system. De novo synthesis and secretion of neutral lipid by ARPE-19 was supported by high levels of radiolabeled EC and triglyceride in medium after supplementation with oleate. Lipoprotein assembly and secretion is implicated as a constitutive retinal function and a plausible candidate mechanism involved in forming extracellular cholesterol-containing lesions in ARM. The pigmentary retinopathy and neuropathy of abetalipoproteinemia (Mendelian Inheritance of Man 200100; Bassen-Kornzwieg disease), which is caused by mutations in the MTP gene, may involve loss of function at the retina.

Apolipoprotein B in cholesterol-containing drusen and basal deposits of human eyes with age-related maculopathy.

Lipids accumulate in Bruch's membrane (BrM), a specialized vascular intima of the eye, and in extracellular lesions associated with aging and age-related maculopathy (ARM). We tested the hypothesis that ARM and atherosclerotic cardiovascular disease share molecules and mechanisms pertaining to extracellular lipid accumulation by localizing cholesterol and apolipoprotein B (apo B) in BrM, basal deposits, and drusen. Human donor eyes were preserved <4 hours postmortem and cryosectioned. Sections were stained with traditional lipid stains and filipin for esterified and unesterified cholesterol or probed with antibodies to apo B, apo E, and apo C-III. Normal adult retinal pigment epithelium (RPE) was subjected to RT-PCR and Western blot analysis for apolipoprotein mRNA and protein. Esterified and unesterified cholesterol was present in all drusen and basal deposits of ARM and normal eyes. Both apo B and apo E but not apo C-III were found in BrM, drusen, and basal deposits. Fewer macular drusen were stained by traditional lipid stains and apolipoprotein antibodies than peripheral drusen. RPE contained apo B and apo E mRNA and protein. Finding cholesterol and apo B in sub-RPE deposits links ARM with important molecules and mechanisms in atherosclerosis initiation and progression. The combination of apo B mRNA and protein in RPE raises the possibility that intraocular assembly of apo B-containing lipoproteins is a pathway involved in forming cholesterol-enriched lesions in ARM.