Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, also known as statins, are potent inhibitors of cholesterol biosynthesis. Recent experimental and clinical data, however, indicate that the overall benefits of statin therapy may exceed its cholesterol-lowering properties. In this proposal, we hypothesize that statins may attenuate the detrimental effects of vascular glomerular endothelial growth factor (VEGF) in diabetic nephropathy. This hypothesis is based on our recent observations indicating that 1) VEGF increases glomerular endothelial cell (GEC) hyperpermeability, 2) Rho GTPase proteins play a pivotal role in VEGF signaling pathway, and 3) Statins have multiple modulatory effects on Rho-mediated cell signaling pathways (Danesh et al. Proc Natl Acad Sci U S A. 2002, 99:8301-5, and Appendix #4). Based upon those initial observations, the investigators now propose an integrated in vitro and in vivo study to characterize the molecular mechanism of VEGF-induced GEC hyperpermeability, and to examine the modulatory effects of statins on VEGF-induced signaling pathway. The three specific aims of this proposal are:
Aim 1 : To delineate VEGF-induced GEC hyperpermeability: We hypothesize that VEGF stimulation elicits an increase in Rho GTPase activity and myosin regulatory light chain phosphorylation. This sequence of events causes significant actin-myosin cytoskeletal remodeling leading to increased GEC hyperpermeability. Using rat GECs as the experimental model, we will establish the role of Rho family of small GTPases in the VEGF-induced endothelial cell hyperpermeability. The effect of VEGF on cytoskeletal remodeling and myosin II contraction will be assessed using high resolution fluorescent live cell imaging.
Aim 2 : To determine the modulatory effects ofstatins on VEGF-induced signaling pathway: We hypothesize that statins, by preventing geranylgeranylation of small Rho GTPases, will attenuate VEGF-induced hyperpermeability in GECs. The modulatory effects of statins on Rho GTPase activation will be investigated. The effects of statins on myosin regulatory light chain phosphorylation and actin cytoskeletal remodeling will be assessed dynamically by measuring myosin light chain kinase activity in situ using a newly developed fluorescent resonant energy transfer-based biosensor.
Aim 3 : To assess renoprotective effects of statins in animal models of diabetes: Functional and biochemical analyses will determine whether renoprotective effects of statins in animal models of diabetic nephropathy correlate with their modulatory effects on VEGF-induced Rho-regulated signaling cascade. The broad and long term goal of this project is to investigate the role of small G proteins in the pathogenesis of diabetic nephropathy. The proposed study will impact the current management of this disease by identifying VEGF and small Rho GTPases as potential therapeutic targets, and by providing a new rationale for the use of statins in the early stages of diabetic nephropathy.
