PROJECT SUMMARY Heart failure is a chronic degenerative disease that is a significant health burden in both the developed world and emerging nations. Current treatments primarily slow the progression of this syndrome. There is a need to develop novel preventative and reparative approaches for treatment of heart failure. The focus of this application is to elucidate the mechanisms that regulate angiogenesis under normal conditions and in heart failure. Our hypothesis is that in the damaged heart, angiogenesis is inhibited via a specific signaling pathway initiated by increased production of transforming growth factor-beta (TGF-?). Phosphorylation of Smad3 by TGF-? then alters gene expression leading to endothelial defects. To address this, we will utilize an in vivo mouse model of doxorubicin induced heart failure in Smad3 knockout mice and angiogenesis assays using isolated murine endothelial cells and human cardiac endothelial cells. The hypothesis to be tested is that the inhibition of Smad3 activation in endothelial cells will increase their angiogenic potential, making Smad3 a novel target for enhancing angiogenesis in the diseased heart. Specific Aims designed to test the hypothesis are as follows: 1) Smad3 is critical for the development of cardiac microvascular defects in a mouse model of cardiomyopathy. Smad3 knockout mice will be exposed to doxorubicin. The progression of cardiomyopathy and cardiac microvascular defects in Smad3 knockout mice will be assessed via capillary density evaluation, histopathology, echocardiography and ex vivo aortic ring sprouting; and 2) Smad3 is critical in regulating vascular network formation by murine and human cardiac endothelial cells in vitro. Endothelial cells isolated from the Smad3 knockout mice and Smad3 deficient human endothelial cells will be cultured alone, in a co-culture with cardiac fibroblasts, and in 3-dimensional matrices. In both mouse and human endothelial cell assays, we will assess the role of Smad3 in angiogenesis. We anticipate that this project will help define novel drug targets to stimulate angiogenesis in failing hearts.