The broad objective of this proposal is to elucidate molecular mechanisms underlying the proliferative response of vascular smooth muscle cells (VSMC) to arterial injury, a process that is thought to contribute significantly to restenosis. Recent studies performed in my laboratory have identified positive regulators of cell growth that are induced at early time points following angioplasty in the rat carotid artery, including cdk2, cyclin E, cyclin A, cdk4 and cdc2. Our results show that overexpression of a cdk2 dominant-negative mutant inhibits entry of VSMCs into S-phase. We also have evidence suggesting that inhibition of cdk2 activity by cdk inhibitor p27 may contribute to the reestablishment of the quiescent state at late time points post-angioplasty. To begin to elucidate the mechanisms that control the expression cell cycle regulatory genes in VSMCs, we have demonstrated that elements within the -209 to +79 cyclin E and -79 to +100 cyclin A promoter regions confer cell cycle-dependent transcriptional control of cyclin gene expression in VSMCs. This Proposal represents an extension, as well as a departure from this previous work. The investigations outlined in Specific Aim 1 will further characterize the cell-cycle regulatory networks underlying injury-induced VSMC proliferation. The studies outlined in Specific Aim 2 will identify the cis-elements and cognate binding proteins underlying growth factor- and injury-induced regulation of cyclin E and cyclin A gene expression in VSMCs. Initially, regulatory sequences will be identified by transient transfection of reporter constructs in cultured VSMCs; subsequently, the relevance of cis-elements to injury-induced regulation of cyclin E and A gene expression will be tested in organ cultures of injured arteries. Investigations outlined in Specific Aim 3 will determine the relevance of p27 with respect to intimal thickening in animal models of vascular injury. These studies will include gain-of function experiments using an adenovirus vector directing the overproduction of p27, and loss-of function studies using p27-deficient mice. Finally, to assess the role of p27 on spontaneous atherosclerosis, the kinetics of atheromatous lesion formation will be examined in different genetic backgrounds obtained by crossbreeding p27 null mice and the atherosclerosis susceptible apoE-deficient mice. These studies should yield valuable mechanistic insights into the regulation of VSMC proliferation and may therefore provide a framework for novel therapeutic interventions.
