Abstract 345: Overexpression of Actin-Binding Rho Activating Protein Triggers Collateral Growth in the Rabbit Hind Limb Ischemic Model
Introduction: In patients with ischemic vascular diseases collateral vessels grow thereby bypassing the occlusion site - a process termed arteriogenesis. This natural collateral growth is often insufficient to compensate for flow deficits. By combining a hind limb ischemic model (femoral artery occlusion) with an arterio-venous shunt in rabbits and rats we could chronically increase fluid shear stress (FSS) in the collateral system. This enhanced collateral growth with a complete restoration of physiological blood flow after 7 days and enabled us to investigate underlying molecular mechanisms. Methods and Results: RNA from growing collaterals 5d after shunt surgery (n=3) was isolated and subjected to rat whole genome microarrays (Agilent). A set of 354 differentially expressed genes (4-fold up/down-regulated, p<0.05) was identified. Relevant targets were validated by quantitative real-time PCR. The increased transcription of actin-binding Rho activating protein (ABRA) correlates tightly with strength and exposure time of FSS. This was confirmed by analysing RNA from growing collaterals at different time points (1, 3, 5, 7 and 14d). Using a MTT cell proliferation assay (ATCC) metabolic activity of porcine endothelial cells in culture was not affected after transfection with ABRA whereas cultivated porcine smooth muscle cells showed a 2fold (p<0,05) increase of dehydrogenase enzyme activity. In a multi-well cell culture secretion experiment ABRA-transfected endothelial cells triggered proliferation activity of smooth muscle cells via paracrine signaling by a factor of 3.4 (p<0.01). To investigate functional consequences on collateral growth we applied a local intra-collateral adenoviral gene transfer of ABRA after occlusion of the femoral artery in rabbits without an arterio-venous shunt. This increased collateral conductance of more than 70 % (n=5, p<0.05) after 7 days compared to controls. Conclusion: We conclude that complete restoration of arterial function of an occluded artery is possible. The physical trigger (FSS) leading to collateral remodelling and growth can be substituted by overexpression of ABRA suggesting that this gene may turn out as a key regulator of arteriogenesis.