sb-290157 and Macular-Degeneration

The mechanisms that connect complement system activation and basal deposit formation in early stages of age-related macular degeneration (AMD) are insufficiently understood, which complicates the design of efficient therapies to prevent disease progression. Using human fetal (hf) retinal pigment epithelial (RPE) cells, we have established an in vitro model to investigate the effect of complement C3a on RPE cells and its role in the formation of sub-RPE deposits. The results of these studies revealed that C3a produced after C3 activation is sufficient to induce the formation of sub-RPE deposits via complement-driven proteasome inhibition. C3a binds the C3a receptor (C3aR), stimulates deposition of collagens IV and VI underneath the RPE, and impairs the extracellular matrix (ECM) turnover by increased MMP-2 activity, all mediated by downregulation of the ubiquitin proteasome pathway (UPP). The formation of basal deposits can be prevented by the addition of a C3aR antagonist, which restores the UPP activity and ECM turnover. These findings indicate that the cell-based model can be used to test potential therapeutic agents in vitro. The data suggest that modulation of C3aR-mediated events could be a therapeutic approach for treatment of early AMD.

Topics: Anaphylatoxins; Arginine; Benzhydryl Compounds; Cells, Cultured; Complement Activation; Complement C3a; Cysteine Proteinase Inhibitors; Enzyme-Linked Immunosorbent Assay; Extracellular Matrix; Humans; Leupeptins; Macular Degeneration; Matrix Metalloproteinase 2; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Retinal Pigment Epithelium

Age-related macular degeneration (AMD) is a complex disease caused by genetic and environmental factors, including genetic variants in complement components and smoking. Smoke exposure leads to oxidative stress, complement activation, endoplasmic reticulum (ER) stress, and lipid dysregulation, which have all been proposed to be associated with AMD pathogenesis. Here we examine the effects of smoke exposure on the retinal pigment epithelium (RPE). Mice were exposed to cigarette smoke or filtered air for 6 months. RPE cells grown as stable monolayers were exposed to 5% cigarette smoke extract (CSE). Effects of smoke were determined by biochemical, molecular, and histological measures. Effects of the alternative pathway (AP) of complement and complement C3a anaphylatoxin receptor signaling were analyzed using knock-out mice or specific inhibitors. ER stress markers were elevated after smoke exposure in RPE of intact mice, which was eliminated in AP-deficient mice. To examine this relationship further, RPE monolayers were exposed to CSE. Short term smoke exposure resulted in production and release of complement C3, the generation of C3a, oxidative stress, complement activation on the cell membrane, and ER stress. Long term exposure to CSE resulted in lipid accumulation, and secretion. All measures were reversed by blocking C3a complement receptor (C3aR), alternative complement pathway signaling, and antioxidant therapy. Taken together, our results provide clear evidence that smoke exposure results in oxidative stress and complement activation via the AP, resulting in ER stress-mediated lipid accumulation, and further suggesting that oxidative stress and complement act synergistically in the pathogenesis of AMD.

Topics: Acetylcysteine; Animals; Arginine; Benzhydryl Compounds; Blotting, Western; Cells, Cultured; Complement Activation; Complement Factor B; Complement Pathway, Alternative; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Free Radical Scavengers; Heat-Shock Proteins; Humans; Lipid Metabolism; Lipids; Macular Degeneration; Mice; Mice, Inbred C57BL; Mice, Knockout; Nicotiana; Nicotine; Oxidative Stress; Receptors, Complement; Retinal Pigment Epithelium; Reverse Transcriptase Polymerase Chain Reaction; Smoke; Transcription Factor CHOP