Clyde Guidry

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.

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.

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 Bruchs 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 Bruchs 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.

Myofibroblast and extracellular matrix origins in proliferative vitreoretinopathy.

BackgroundTo evaluate origins of the fibrocontractive cell populations and their relation to collagens I and II in proliferative vitreoretinopathy (PVR).MethodsHuman 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.ResultsCollagen 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.ConclusionsCollagen 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.

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.

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.

Quantitative vitreous collection using a modified infusion fluid during vitrectomy.

PURPOSE To develop a vitreous sampling method that increases yield while reducing risk of harm to the patient. METHODS Potential negative effects of fluoresceinated BSS infusion solution on biologic activity were assessed by monitoring changes in fibroblast contraction of collagen gels in response to serum or vitreous. Paired vitreous samples were collected from ten patients before and during infusion of fluoresceinated BSS solution. The extent to which the vitreous was diluted was calculated by comparing the levels of fluorescence in the samples with that in fluoresceinated BSS. Protein concentrations and levels of contraction-stimulating activity were measured for each sample. RESULTS Fluoresceinated BSS did not alter fibroblast morphology, rate, or extent of gel contraction. Differences in corrected protein concentrations and biologic activities of the undiluted and diluted homogenous vitreous samples were not statistically significant. CONCLUSIONS The use of fluoresceinated BSS infusion yields larger vitreous samples from which the native biochemical characteristics can be determined. Patient safety during collection is enhanced because ocular hypotony and collapse can be avoided.