Blebbing is the process of pinching off and extrusion of small pieces of the cell membrane and has recently been shown to be associated with programmed cell death. However, cell membrane blebbing is also associated with sublethal cell injury. The laboratory is studying the hypothesis that this blebbing and other cellular responses of the retinal pigment epithelium (RPE) to sublethal injury may play a role in age-related macular degeneration. Interesting when examining human pathologic specimens from AMD patients and in some animal models of drusen formation, the RPE was noted to have cell membrane blebs forming on 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. 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. The laboratory has published its observation 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 further substantiate this finding, the laboratory also demonstrated that RPE express high levels of intracellular heat shock protein 27 (Hsp27), a molecule known to be involved in cell injury and blebbing. Additionally, the molecular events associated with sublethal injury are further being characterized by examining microarrays from RPE injury at various time points following sublethal injury. Results include an upregulation of survival and anti-oxidative molecules and a downregulation of pro-apoptotic genes. Evidence has also been collected on the programmed cell death pathway in RPE cells. We have been able to demonstrate that unlike the conventional programmed cell death pathways which utilize cytochrome c, caspase 3, 9 and PARP to initiate cell death, RPE cells have protective mechanisms which do not allow this pathway to become activated. Instead, the novel programmed cell death molecule, apoptosis induced factor (AIF) appears to be involved in RPE cell death. Additionally, growth factors which might be found in the retina, such as hepatocyte growth factor, appear to also protect the RPE cells from oxidative cell death. These findings indicate that the injury and cell death pathway may be unique in the RPE, that the growth factor microenvironment of the subretinal space may affects these steps and that these pathways may play a crucial role in AMD pathogenesis.
