Safety and acceptability of an organic light-emitting diode sleep mask as a potential therapy for retinal disease.

PurposeThe purpose of the study was to study the effect of an organic light-emitting diode sleep mask on daytime alertness, wellbeing, and retinal structure/function in healthy volunteers and in diabetic macular oedema (DMO).Patients and methodsHealthy volunteers in two groups, 18-30 yrs (A), 50-70 yrs (B) and people with DMO (C) wore masks (504 nm wavelength; 80 cd/m2 luminance; ≤8 h) nightly for 3 months followed by a 1-month recovery period. Changes from baseline were measured for (means): psychomotor vigilance task (PVT) (number of lapses (NL), response time (RT)), sleep, depression, psychological wellbeing (PW), visual acuity, contrast sensitivity, colour, electrophysiology, microperimetry, and retinal thickness on OCT.ResultsOf 60 participants, 16 (27%) withdrew, 8 (13%) before month 1, due to sleep disturbances and mask intolerance. About 36/55 (65%) who continued beyond month 1 reported ≥1 adverse event. At month 3 mean PVT worsened in Group A (RT (7.65%, P<0.001), NL (43.3%, P=0.005)) and mean PW worsened in all groups (A 28.0%, P=0.01, B 21.2%, P=0.03, C 12.8%, P<0.05). No other clinically significant safety signal was detected. Cysts reduced/resolved in the OCT subfield of maximal pathology in 67% Group C eyes. Thinning was greater at 3 and 4 months for greater baseline thickness (central subfield P<0.001, maximal P<0.05).ConclusionSleep masks showed no major safety signal apart from a small impairment of daytime alertness and a moderate effect on wellbeing. Masks were acceptable apart from in some healthy participants. Preliminary data suggest a beneficial effect on retinal thickness in DMO. This novel therapeutic approach is ready for large clinical trials.

PERP expression stabilizes active p53 via modulation of p53-MDM2 interaction in uveal melanoma cells.

The activation and regulation of target genes by the tumour-suppressor p53 dictates the fate of a cell, with cell cycle arrest or apoptosis being two distinct outcomes. PERP (p53 apoptosis effector related to PMP-22), a p53 transcriptional target, is induced specifically during apoptosis but not cell cycle arrest. Downregulation of PERP is associated with the aggressive, monosomy 3-type of uveal melanoma (UM), the most common primary intraocular tumour in adults, and increased PERP expression has a pro-apoptotic effect in UM cells. Here, we identify a novel effect of PERP expression, as elevated PERP protein positively influences active levels of its own transcriptional regulator, p53. Using fluorescent fusion proteins of PERP, p53 and MDM2, we demonstrate in single living UM cells that PERP expression significantly enhances p53 activity and its nuclear localization, increases p53-dependent transcription (including that of MDM2) while allowing oscillatory nucleo-cytoplasmic shuttling of p53/MDM2 complexes. Phosphorylation of p53 serine residues that interfere with the interaction between p53 and its negative regulator MDM2 and enhance pro-apoptotic gene transcription also occurs subsequent to PERP expression. These results implicate a role for PERP in amplifying functional p53 levels that promote p53-dependent apoptosis, and reveal a potential target for exploitation in enhancing p53 activity.

Expanded polytetrafluoroethylene as a substrate for retinal pigment epithelial cell growth and transplantation in age-related macular degeneration.

BACKGROUND: Retinal pigment epithelial (RPE) transplantation presents a potential treatment for age-related macular degeneration (AMD). A suitable transplant membrane that can support an intact functioning RPE monolayer is required. Expanded polytetrafluoroethylene (ePTFE) possesses the physical properties required for a transplanting device; however, cells do not attach and spread on ePTFE. This study investigated the ability of surface-modified ePTFE to optimise the growth and function of healthy RPE monolayers. METHODS: ePTFE discs were modified by ammonia gas plasma treatment. ARPE-19 cells were seeded on the membranes and maintained in media supplemented with retinoic acid and reduced serum. Cell number, morphology and proliferation were analysed. RPE monolayer function was investigated through formation of cell-cell junctions and phagocytosis of photoreceptor outer segments (POS). RESULTS: Ammonia gas plasma treatment resulted in enhanced cell growth and good monolayer formation with evidence of cell-cell junctional proteins. Furthermore, RPE monolayers were able to phagocytose POS in a time-dependent manner. CONCLUSIONS: ePTFE can be surface-modified to support an intact functional monolayer of healthy RPE cells with normal morphology and the ability to perform RPE-specific functions. Following further investigation ePTFE may be considered for use in transplantation.

Cross-linked actin networks (CLANs) in bovine trabecular meshwork cells.

A cytoskeletal feature of human trabecular meshwork (HTM) cells in vitro and ex vivo is the presence of cross-linked actin networks (CLANs) that are abundant in a proportion of TM cells exposed to dexamethasone (DEX) and also in cells from glaucoma patients. We wished to determine whether CLANs were present in the bovine trabecular meshwork (BTM), whether they were similarly induced by dexamethasone and whether the structures were comparable to CLANs in HTM cells. Cultures of HTM and BTM cells and ex vivo dissections of BTM tissue were stained with phalloidin (F-actin) and propidium iodide (nuclei) and imaged by confocal microscopy, thereafter being subjected to image analysis. Some CLAN-like structures were identified in ex vivo BTM tissue cultured with and without DEX. However we found that BTM cells in culture produced abundant CLANs when exposed to DEX; comparable to the best response from HTM cells. The CLANs were of similar dimensions and morphology to those found in human cells and they had a similar half life of 2 or 3 days following the removal of DEX. This work demonstrates that BTM cells provide a suitable model for future investigations of CLAN formation and function. BTM cultures are sufficiently hardy to thrive in low serum and serum-free conditions so we were able to show that aqueous humor stimulates CLAN formation in the target cells. Future research is directed at identifying the aqueous component(s) responsible for CLAN production.

Replacement of the RPE monolayer.

There are numerous scenarios in which replacing the diseased RPE monolayer is an attractive but as yet unrealised goal. The proof of concept that vision can be improved by placing a healthy neuroretina onto a different, healthy, underlying RPE layer is demonstrated in patch graft transplantations. The surgical procedure to relocate the neuroretina is both complex and is hampered by postoperative complications and as such newer replacement procedures are also being investigated including stem cell replacement therapies. Past studies have largely focused on using cell suspensions and have had disappointing outcomes largely due to the lack of control over cellular differentiation, incomplete attachment onto Bruch's membrane and subsequent integration into the existing RPE monolayer. The choice of which cells to transplant is still under investigation and is complicated by factors such as the ease of collection of an adequate sample, rejection following implantation, the age of the cells and ethical issues. In all these situations, however, understanding the mechanisms of cellular differentiation are likely to be prerequisite to future successes.The current research into replacing the RPE monolayer is briefly discussed with reference to our experiences comparing IPE and RPE cells in an in vitro environment.

Monte Carlo simulation of latanoprost induced iris darkening.

The aim of this study was to provide numerical evidence that latanoprost induced iris darkening (LIID) can be caused by changes to the melanin granule size distribution in the anterior segment of the iris. Iridectomies from two patients were used, where both had undergone unilateral treatment with latanoprost and had exhibited LIID. The untreated eye provided the comparative control. Micrographs from the iris samples were analysed to determine the number and size of the mature melanin granules. Monte Carlo (MC) simulation of light propagation in the iris was performed to examine the changes in reflectance and absorption with varying particle size and density. The reflected intensity of light was obtained as a function of wavelength. CIE colour theory was employed in order to estimate a perceived colour from the reflectance data. MC simulations showed that the reflectance was reduced for the LIID irises compared to the control irises for both subjects and for all wavelengths of light. The MC simulated colours were in good agreement with the in vivo photography of the eye colour. Hence, we have demonstrated that increases in melanin granule size causes iris darkening, and can explain LIID.

Polyurethanes as potential substrates for sub-retinal retinal pigment epithelial cell transplantation.

Transplantation of cultured retinal pigment epithelial (RPE) cells under the failing macular is a potential treatment for age related macular degeneration. An important step in the development of this procedure is the identification of a suitable membrane on which to grow and transplant the cells. This paper evaluates the potential of using polyurethanes in this application since they possess several of the required properties, such as, flexibility, robustness, biostability and good biocompatiblilty although their hydrophobicity can limit cell adhesion. Three commercially available polyether urethanes (Pellethane, Tecoflex and Zytar) were evaluated in terms of their wettability using dynamic contact angle analysis and their ability to support a monolayer of functioning RPE cells (ARPE-19) . Furthermore Pellethane and Tecoflex were treated with a simple air plasma treatment and analysed as above. In the "as received condition" only a few RPE cells attached to the Pellethane and Tecoflex and remained clumped. RPE cells grew to confluence on the Zytar substrate by 7 days without further surface modification. Air gas plasma treatment of both Pellethane and Tecoflex increased the wettability of the surfaces and this resulted in the growth of a monolayer of well-spread RPE cells on both materials. Morphologically these cells grew with a normal 'cobblestone' phenotype. These results demonstrate the potential of these polyurethanes for this application.

Edible mushroom (Agaricus bisporus) lectin modulates human retinal pigment epithelial cell behaviour in vitro.

The retinal pigment epithelium (RPE) plays a major role in the development of proliferative vitreoretinopathy (PVR). In particular, RPE cells are implicated in generating the contraction forces seen. The present study was undertaken to investigate whether human RPE binds a lectin from the common edible mushroom, Agaricus bisporus, and to evaluate the effect of any binding on RPE-mediated matrix contraction in an in vitro model of PVR. Fluorescein isothiocyanate (FITC)-labelled Agaricus bisporus lectin (ABL) was used to study binding of lectin to normal retina, PVR scar tissue specimens and cultured human RPE. The effect of a 3-day exposure of ABL on human RPE-mediated contraction was evaluated using 2- and 3D RPE-populated collagen matrices. Effect of ABL on cell adhesion was measured using a collagen type I adhesion assay and determining the relative cellular attachment using absorbance readings. The normal RPE monolayer did not stain with FITC-ABL while PVR scar tissue stained intensely. Staining of in vitro RPE was characteristic but time-dependent. ABL caused a dose-dependent inhibition of RPE-mediated contraction of both 2D (one-way ANOVA, F = 7.94, p < 0.008) and 3D collagen matrices (one-way ANOVA, F = 164.955, p < 0.001). Pre-incubation of ABL with RPE in the 2D model caused a dramatic arrest of contraction (one-way ANOVA, F = 20.1, p < 0.001) that was due to a dose-dependent inhibition of adhesion (one-way ANOVA, F = 15.603, p < 0.001). Recovery of contraction was partially reversible on removal of ABL and was dependent on initial concentration of the lectin. ABL inhibits contraction and adhesion of human RPE cells in vitro without apparent cytotoxicity. It therefore deserves consideration as a potential therapeutic agent in the prevention and treatment of PVR and other non-ocular anomalous wound-healing processes.

Pathobiology of epiretinal and subretinal membranes: possible roles for the matricellular proteins thrombospondin 1 and osteonectin (SPARC).

Epiretinal and subretinal membranes are fibrocellular proliferations which form on the surfaces of the neuroretina as a sequel to a variety of ocular diseases. When these proliferations complicate rhegmatogenous retinal detachment (a condition known as proliferative vitreoretinopathy or PVR), the membranes often contain numerous retinal pigment epithelial (RPE) cells and a variety of extracellular proteins. The extracellular proteins include adhesive proteins like collagen, laminin and fibronectin. In addition, several matricellular proteins with potential counter-adhesive functions are present in the membranes. Two such matricellular proteins, thrombospondin 1 and osteonectin (or SPARC: Secreted Protein Acidic and Rich in Cysteine), tend to be co-distributed with the RPE cells in PVR membranes. By virtue of their counter-adhesive properties, thrombospondin 1 and SPARC may reduce RPE cell-matrix adhesion and so permit key RPE cellular activities (for example, migration or shape change) in periretinal membrane development. Furthermore, within a 'cocktail' containing other proteins such as the metalloproteinases and growth factors like the scatter factor/hepatocyte growth factor family, matricellular proteins may play a role in the RPE cell dissociation from Bruch's membrane, which characterises early PVR.

Agaricus bisporus (edible mushroom lectin) inhibits ocular fibroblast proliferation and collagen lattice contraction.

The edible mushroom lectin from Agaricus bisporus (ABL) has antiproliferative effects on a range of cell types. This investigation was undertaken to test whether it might have inhibitory activity on Tenon's capsule fibroblasts in in vitro models of wound healing and therefore have a use in the modification of scar formation after glaucoma surgery.Human ocular fibroblasts in monolayers and in three-dimensional collagen lattices were exposed to ABL (0-100 microg ml(-1)). Proliferation was studied by the MTS assay and by counting haematoxylin-stained cells; contraction was measured as a change in the diameter of three-dimensional collagen lattices. Toxicity was investigated using a fluorescent viability assay. FITC-labelled lectin was used to study cell binding and internalization of ABL.ABL caused a dose-dependent inhibition of proliferation and lattice contraction without significant toxicity. Proliferation was inhibited by 5-40% in the dose range 20-100 microg ml(-1) Significant inhibition of lattice contraction was achieved with 40 microg ml(-1) ABL, and at 100 microg ml(-1) contraction was completely prevented. FITC-ABL binds to the cell surface and accumulates around the nuclear envelope when internalized. These experiments have shown that ABL possesses key features required of an agent that might control scarring processes and suggest that ABL might be especially useful where subtle modification of healing is needed. Further evaluation is warranted.

Loss of heterozygosity on chromosomes 3, 9, 13, and 17, including the retinoblastoma locus, in uveal melanoma.

PURPOSE: To identify tumor-suppressor loci that may contribute to the pathogenesis of uveal melanoma. METHODS: Multiplex fluorescence microsatellite assays were performed on 27 uveal melanomas using markers at 3p25-p26, 3p14.2, 9p21-p23, 13q14, 13q12.3-q13, and 17p13, close to or within the von Hippel Lindau (VHL), fragile histidine triad (FHIT), p16/cyclin-dependent kinase inhibitor 2 (CDKN2A), retinoblastoma (RB1), breast cancer 2 (BRCA2), and p53 tumor suppressor loci, respectively. Further markers on chromosomes 3 and 9 were analyzed individually. RESULTS: Loss of heterozygosity (LOH) was identified in 63% of tumors, most frequently on chromosome 3 (52%), in association with epithelioid cells (P = 0.0002) and microvascular loops (P = 0.0008). In the majority of cases, LOH on chromosome 3 was detected at all informative markers. The second most common alteration was LOH at an RB1 intragenic marker (21% tumors), with retention of a more centromeric 13q marker (near BRCA2). The pattern of LOH on chromosome 9p was consistent with the involvement of a region telomeric to CDKN2A. LOH at TP53 was infrequent. CONCLUSIONS: In the majority of cases, chromosome 3 LOH involves an entire chromosome homologue, which hampers identification of the relevant suppressor loci. This LOH correlates with the presence of microvascular loops and epithelioid cells, two of the recognized histologic indicators of poor prognosis. Data for chromosomes 13 and 9 support a role for RB1 in the pathogenesis of uveal melanoma but also raise the possibility of the involvement of additional loci close to RB1 and CDKN2A.

Precursor cystatin C in cultured retinal pigment epithelium cells: evidence for processing through the secretory pathway.

Evidence was recently reported that the cysteine proteinase inhibitor, cystatin C, is highly expressed by cultured human retinal pigment epithelial (RPE) cells. As a step towards understanding possible functions of this protein associated with the RPE, the localization, targetting and trafficking of cystatin C were investigated. Constructs encoding an enhanced variant of green fluorescent protein (EGFP) fused to precursor cystatin C and to mature cystatin C were made and transfected into cultured human RPE cells. Expression of fusion proteins was monitored in vivo by fluorescence confocal microscopy. In cells transfected with precursor cystatin C-EGFP, fluorescence was initially targetted to the perinuclear zone, co-localizing with the Golgi apparatus. Transfected cells were observed at intervals over a period of up to 3 weeks, during which time fluorescent vesicles developed peripherally and basally while fluorescence continued to be detected in the Golgi region. Immunochemical analysis of cell lysates confirmed the expression of a fusion protein recognized by antibodies to both cystatin C and EGFP. Cells transfected with the construct lacking the leader peptide of precursor cystatin C presented a diffuse and weak fluorescence. Together, these results imply a leader sequence-dependent processing of cystatin C through the secretory pathway of RPE cells. This was confirmed by the detection, by Western blotting, of the chimaeric protein alongside endogenous cystatin C in the medium of transfected RPE cells.

Matrix metalloproteinases: a role in the contraction of vitreo-retinal scar tissue.

The most common cause of failure of retinal reattachment surgery is formation of fibrocellular contractile membranes on both surfaces of the neuroretina. This intraocular fibrosis, known as proliferative vitreoretinopathy, results in a blinding tractional retinal detachment because of the contractile nature of the membrane. Contractility is a cell-mediated event that is thought to be dependent on locomotion and adhesion to the extracellular matrix. Interactions between cells and the extracellular matrix can be influenced by matrix metalloproteinases (MMPs) and we investigated the role of MMPs in two in vitro models (two- and three-dimensional) of human retinal pigment epithelial (RPE) cell-mediated contraction. MMP activity was detected using enzyme-linked immunosorbent assays and zymography techniques that revealed MMP-1, -2, -3, and -9 positivity during the collagen matrix contraction assays. RPE-populated collagen matrix contraction (three-dimensional) was inhibited using a cocktail of anti-MMP antibodies and with Galardin (a broad-spectrum MMP inhibitor). Galardin inhibition was dose-dependent, reversible, and dependent on cell number. MMP inhibitors had no effect on contraction when RPEs were seeded on two-dimensional collagen matrices or on cellular adhesion to collagen type I. Our results suggest that MMP activity may be required for three-dimensional but not two-dimensional RPE-collagen matrix contraction.

Overexpression of epidermal growth factor receptor restricted to macrophages in uveal melanoma.

OBJECTIVE: To determine whether expression of the epidermal growth factor receptor (EGFR) is of prognostic value in uveal melanoma. METHODS: Thirty consecutive patients treated for primary posterior uveal melanoma by enucleation or local resection were studied. Tumors were examined for EGFR and CD68 expression by immunohistochemistry on formalin-fixed, paraffin-embedded sections. Extracted DNA from paired frozen tumor and blood samples was examined for loss of heterozygosity on chromosome 3 using polymerase chain reaction-based microsatellite analysis. Immunoreactivity for EGFR was correlated with clinicopathological, chromosome 3, and follow-up data. RESULTS: Immunoreactivity for EGFR was observed in 7 (23%) of 30 uveal melanomas, but was restricted to solitary or small groups of cells with macrophage-like morphology. Immunoreactive cells were confirmed as macrophages using an antibody to the macrophage marker CD68. Chromosome 3 loss, epithelioid cells, and microvascular loops were detected in 17 (57%), 22 (73%) and 19 (63%) of the 30 tumors, respectively. Metastatic disease was detected in 5 patients (17%). No correlation was found between any of these variables and EGFR positivity. CONCLUSIONS: The absence of EGFR immunoreactivity in tumor cells does not support the use of EGFR expression as a prognostic indicator in patients with uveal melanoma. Future EGFR studies in uveal melanoma should be interpreted with caution in view of our findings that tumor-associated macrophages can express this receptor.

The iris after prostanoid treatment.

Morphologic studies to date show that prostanoid-induced iris color change is not associated with any major pathologic process in the tissue. There is no evidence of melanocyte proliferation. The most likely mechanism for iris color darkening is increased melanogenesis, but this is not so marked as to cause any extensive release of melanin granules that might cause iris inflammation or even a pigmentary-type of glaucoma. Some patients, but not all, with color darkening have an apparent thickening of the anterior border zone; it remains to be established whether this is true thickening or merely if the anterior border is emphasized because of increased pigmentation.

Histological effects in the iris after 3 months of latanoprost therapy: the Mainz 1 study.

OBJECTIVE: To determine whether 3 months of topical latanoprost treatment caused proliferative or degenerative effects on the peripheral iris of patients with glaucoma. METHODS: Seventeen patients requiring filtering surgery for primary open-angle glaucoma or pseudoexfoliation glaucoma were randomized to receive topical latanoprost for 3 months (n = 8) or alternative medication (n = 9) before surgery. A trabeculectomy and a peripheral iridectomy specimen was obtained from each patient during surgery. The tissue was subjected to histological and immunohistochemical evaluation using 2 cell cycle markers: proliferating cell nuclear antigen and nuclear-associated protein (Ki-67). RESULTS: No degenerative or pathological changes were seen in the latanoprost-treated irides, including the one specimen in this series in which there was an eye color change. Proliferating cell nuclear antigen and nuclear-associated protein markers were negative for changes in all the test specimens. CONCLUSION: Short-term treatment with latanoprost does not produce morphological changes or cellular proliferation changes in the iris.

Repair in the rabbit outflow system.

An in vivo study was conducted to study repair processes in the injured rabbit outflow system. A uniform injury was produced by raising intraocular pressure (IOP) manometrically to 70 mmHg for 1 h. The recovery process, which was followed clinically for 8 weeks and morphologically for 6 weeks, led to the re-establishment of normal meshwork architecture within this period. The morphological studies included light microscopy, autoradiography and electron microscopy. The initial lesion consisted of large deficits in the meshwork with breakdown of cell-to-cell connections, loss of extracellular materials and disruption of the vessels of the aqueous plexus. There was a significant lowering of IOP in the first week of recovery, which thereafter climbed back to normal. Also in the first week the meshwork became infiltrated with inflammatory cells which cleared by 4 weeks. There was some meshwork cell death by either necrosis or apoptosis. The majority of meshwork cells became activated within the first few days and remained activated for at least the first 2 weeks. Tritiated proline incorporation was maximal between 1 and 2 weeks. Tritiated thymidine labelling was seen throughout, but only after the inflammation subsided was it clear that meshwork cells in all regions of the meshwork were proliferating. Our study provided no evidence that normal meshwork cells have a basal proliferative turnover level. Our injury model involved complete repair of the outflow tissues and that required meshwork cells to become activated, mobilise, undertake synthetic activity and proliferate. This is the first example, other than argon laser trabeculoplasty, where meshwork cells in vivo have been induced to divide. Possible therapeutic implications for glaucoma are discussed.

Challenges in ophthalmic pathology: the vitreoretinal membrane biopsy.

The introduction of vitreoretinal microsurgery has produced a new type of biopsy; that of the vitreoretinal membrane. This review investigates methods by which these scar-like tissues are handled in the laboratory and explores the implications of the results of such evaluations. The study of vitreoretinal membrane biopsies has provided much information concerning the pathobiology of the various conditions which may give rise to the tissue as well as insights into how membranes themselves develop. Moreover, the application of new laboratory techniques is expected to enhance our understanding of the formation of vitreoretinal membranes, and lead to further advances in their surgical and medical management.

Hepatocyte growth factor/scatter factor in the eye.

Hepatocyte growth factor, also known as scatter factor (HGF/SF) is a multipotential cytokine which can produce a range of responses in target cells and its influence in the eye in health and disease is just beginning to be appreciated. Usually HGF/SF is synthesised by mesenchymally derived cells and targets and signals epithelial cells in a paracrine manner via their c-Met surface receptor. However, there is growing evidence for the existence of autocrine loops in a number of cell systems prominent among which are ocular cells such as the corneal endothelium, the lens epithelium, the retinal pigment epithelium (RPE) and others. Marked cellular proliferation is stimulated when activated HGF/SF is exposed to hepatocytes, renal epithelium, melanocytes and vascular endothelial cells but it is often a poor mitogen for other cell types. In target cells the cytokine promotes other bioactions such as junctional breakdown, shape change, cell scattering, directional and nondirectional migration, cell survival, invasive behaviour and/or tubule formation. These activities seem to depend on HGF/SF linking with the c-Met receptor and pathways to stimulate the various types of cytokine/receptor response are being unravelled at the present time. In corneal wound healing, HGF/SF is produced by stromal keratocytes and targets the repairing epithelium. HGF/SF is a constituent of tears, aqueous humour and vitreous humour at levels above that found in plasma although it is not clear how much is activated. Aqueous HGF/SF may well influence lens epithelial, corneal endothelial and trabecular meshwork cell survival. Vitreous levels of HGF/SF are elevated in proliferative vitreoretinopathy (PVR), where a target cell is the RPE and in proliferative diabetic retinopathy (PDR) where HGF/SF has been shown to be a major angiogenesis factor. Finally HGF/SF may be involved in the metastatic spread of tumour cells from uveal melanomata and in the formation of vascular channels in these tumours.

Active scatter factor (HGF/SF) in proliferative vitreoretinal disease.

PURPOSE: Hepatocyte growth factor/scatter factor (HGF/SF) possesses mitogenic, motogenic, and morphogenic properties and has recently been implicated in various retinal diseases. The role of HGF/SF in proliferative vitreoretinal disease was investigated. METHODS: Sections of epiretinal membranes were stained immunohistochemically for cytokeratins, to identify HRPE cells, and for HGF/SF receptor (c-Met). Cultured HRPE cells were stained for c-Met and investigated for shape change in response to HGF/SF, by using image analysis. The dose-response relationship for HRPE cells to HGF/SF was investigated by a cell migration assay and the specificity of this response evaluated by a neutralization experiment. Subretinal fluid (SRF) and vitreous from patients with retinal detachment and proliferative vitreoretinopathy (PVR) plus vitreous from eyes obtained after death, eyes with macular hole, and eyes with proliferative diabetic retinopathy (PDR) were investigated for the presence of HGF/SF using an enzyme-linked immunosorbent assay (ELISA). HGF/SF activity was measured using an MDCK cell scatter assay. RESULTS: HRPE cells in epiretinal membranes and in culture expressed c-Met. Cultured HRPE cells responded to HGF/SF by an epithelial-to-mesenchymal shape change and by cell migration, a response that increased with increasing concentrations of HGF/SF. This response was reduced in the presence of neutralizing antibody. There was evidence of HGF/SF in increasing concentrations in more severe PVR and in PDR when measured by ELISA, and, conversely, there was evidence of correspondingly decreasing HGF/SF activity when measured by MDCK cell scatter assay in these diseases. CONCLUSIONS: HGF/SF is present in normal and pathologic vitreous. HRPE cells respond by shape change and cell migration to HGF/SF. Concentrations of HGF/SF increase in proliferative vitreoretinal disease and increase in turn with increased severity of the disease, but HGF/SF bioactivity decreases (consistent with activator depletion). These findings are consistent with the hypothesis that HGF/SF may play a role in the HRPE mesenchymal transformation that typifies PVR.