In studying the injury response to adenoviral vectors in human RPE cells, we made the observations that these cells can """"""""bleb"""""""" in response to various noxious stimuli. Blebbing is the process of pinching off and extrusion of small pieces of the cell membrane. The laboratory is studying the hypothesis that this blebbing may play a role in age-related macular degeneration. In reviewing the literature on AMD, various scientists have noted that both in human pathologic specimens from AMD patients and in some animal models of drusen formation, the RPE was noted to """"""""bud"""""""" at its basal surface. Since many of the studies on the composition of subRPE deposits appear to indicate a cell source, the laboratory has been examining the hypothesis that RPE blebbing may be a process by which these deposits are formed. While blebbing is thought by contemporary scientists to be part of the programmed cell death pathway, we hypothesized that blebbing can also occur in the context of nonlethal cell injury. To explore this concept, focus was placed on the expression and function of heat shock protein 27 (Hsp27), a molecule known to be involved in cell injury and blebbing, in RPE cells. Data was obtained which demonstrated high expression of Hsp27 in RPE cells. Additionally, this molecule was shown to be upregulated during injury and differentiation in RPE cells. Interestingly, the higher Hsp27 expression seen in differentiated versus dividing RPE cells also correlated with increased viability in response to an injury stimulus. To precisely characterize the blebbing response, a retrovirus construct was generated which expresses green fluorescent protein (GFP) targeted to the inner leaflet of the cell membrane via a c-terminus palmitoylation sequence from inactive rRas. GFP-expressing RPE cells, purified by FACS-sorting, uniformly expressed this membrane marker and these cells were used to study injury induced blebbing. Recent observations confirmed the hypothesis that RPE can bleb following exposure to nonlethal injury stimuli and that blebbing is quantitatively more and qualitatively different in differentiated cells than in dividing cells. To mimic possible injury effects present in AMD, combinations of myeloperoxidase and hydrogen peroxide and as well as the oxidant producer menadione were used. Further evidence to indicate the role of these blebs in extracellular deposit formation was obtained by demonstrating that the blebs are actually released from the cell surface. Additionally, when RPE cells are grown on collagen IV and laminin coated cell culture insert transwells, GFP-labeled blebs were found embedded in the substrate underlying injured RPE cells indicating that extracellular matrix trapping of these blebs can occur. If the injury response of the RPE plays a crucial role in AMD pathogenesis then the disease might result from either an injury stimulus or an exaggerated injury response of the RPE occurring in AMD patients. To explore this question, a clinical trial is underway using microarray analysis to compare skin fibroblasts from patients with various stages of AMD and age-matched controls. Skin fibroblasts have been shown to harbor several protein abnormalities seen in central nervous system disorders, such as abnormal amyloid deposition in Alzheimer's patients or expression of the EFEMP1 protein determined to harbor the genetic mutation in patients with Doyne's Honeycomb Retinal Dystrophy. Therefore, it is hypothesized that if exaggerated injury response genes are responsible for subretinal deposits and progression to the later stages of AMD, this gene pattern might also be detected in the mRNA of biopsied skin fibroblasts exposed to sublethal injury in-vitro. The utilization of microarray technology will allow for rapid screening of thousands of genes in affected patients versus age-matched controls. To date, nine patients with various stages of the disease have been enrolled with successful and uncomplicated dermal biopsies performed in all patients. Analysis of these cells is now underway. Further development of the injury hypothesis has also involved examining the process of cell death in RPE cells. Preliminary work has shown that RPE cells contain various proteins, such as caspse 9, CPP32 and PARP, which are involved in the programmed cell death pathway. But surprisingly, unlike control U937 cells, in RPE cells no activation of these proteins or DNA fragmentation has been demonstrated, in the setting of induced cell death, despite the use of multiple cytotoxic stimuli. These results suggest that RPE cells may die utilizing a non-caspase dependent pathway and further indicates their unique response to noxious stimuli. Publications:Sullivan DM, Chung DC, Anglade E, Nussenblatt RB, Csaky KG. Adenovirus-mediated gene transfer of ornithine aminotransferase in cultured human retinal pigment epithelium. Invest Ophthalmol Vis Sci 1996; 37:766-774.Strunnikova N, Baffi J, Silk W, Gonzalez A, Cousins SW, Csaky KG. Regulated Heat Shock Protein 27 Expression in Human Retinal Pigment Epithelium. Invest Ophthalmol Vis Sci 2000; Submitted.Csaky KG, Strunnikova N. A Pilot Study Examining Gene Expression Patterns in Circulating Monocytes and Dermal Fibroblasts in Patients with Age-Related Macular Degeneration. Clinical Trial 2000.
