Dedifferentiation of vascular smooth muscle cells to the synthetic phenotype is of central importance in a broad range of vascular disease processes. SMC phenotype regulation is a basic process that controls the development of restenosis, atherosclerosis, angiogenesis, and collateral blood vessel formation. The intracellular signaling processes that regulate SMC phenotype are at present, incompletely understood. Endothelial cells have been shown to direct vascular smooth muscle cells toward a more differentiated phenotype. Preliminary data from our laboratory suggests that the mechanism by which this occurs is through the PI3-kinase / AKT pathway. Using a co-culture system endothelial cell controlled intracellular signaling pathways that regulate smooth muscle cell phenotype will be defined. Western blots and real time PCR will be used to demonstrate that EC promote a differentiated smooth muscle phenotype as determined by differentiation protein marker expression. Smooth muscle cell differentiation will also be evaluated by cell morphology and cell size. To determine if this is mediated through the IRS-1/PI3-kinase/AKT pathway western blots will be performed for these proteins as well as their down stream targets in smooth muscle cells cultured alone and co-cultured with endothelial cells. These experiments will be repeated using the PI3-kinase inhibitors wortmannin, LY294002, a dominant negative AKT adenovirus, and a myristoylated constitutively active AKT adenovirus. A downstream target of AKT is GSK3B, a known regulator of the GATA transcription factors. GATA-6 has been shown to promote smooth muscle cell differentiation. To determine if endothelial cells regulate SMC differentiation through AKT activation of the transcription factor GATA-6, electromobility shift assays will be performed using probes generated from the GATA-6 binding region of the differentiation protein markers smooth muscle myosin heavy chain and alpha-1 integrin. Promoter reporter assays will be performed using promoter regions from the same differentiation protein markers. Additional studies will be performed for transcription factors and co-activators known to act synergistically with GATA-6. Data obtained from these studies will provide new insight into the intracellular signaling processes that control smooth muscle cell phenotype, allowing for potential enhanced treatment of a wide range of vascular diseases.
