Vascular proliferative diseases are characterized by abnormal smooth muscle cell (vsmc) proliferation, leading to early closure of vascular grafts or angioplasty and a recurrence of clinical iscemia. Our research is focused on understanding the molecular and cellular mechanisms of vsmc growth regulation by cell cycle proteins. Vsmcs are normally quiescent, proliferate at low indices, and reside in G0/G1 phase of the cell cycle. Following vascular injury, vsmcs are stimulated to divide in the presence of mitogens. Progression through G1 and entry into S phase is regulated by the binding and phosphorylation of cyclin/cyclin-dependent kinase (cdks) complexes, predominantly cyclinD/cdk4/6 and cyclin E/cdk2. Cyclin-dependent kinase inhibitors (CKIs) are naturally occurring gene products that inhibit cyclin/cdk activity leading to G1 arrest. The CKIs, p27Kip1 and p21Cip1, inactivate G1 cyclin/Cdk complexes in vsmcs. For example, p27Kip1 is constitutively expressed in normal arteries, is downregulated after arterial injury, becomes upregulated during the latter phases of arterial repair and is inversely correlated with vsmc proliferation. In vsmcs, p27Kip1 and p21Cip1 inactivate cdk2 and cdk4 while p16Ink4 inhibits only cdk4. These molecular mechanisms account for the different effects of the CKIs on vsmc proliferation. The molecular mechanisms of growth regulation have also been tested in p27Kip1, p21Cip1, and p27Kip1/p21Cip1 knockout mice. The absence of p27Kip1 leads to chronic hyperplasia of vsmcs, accelerated lesion formation within arteries, and vessel occlusion. Lesion development is accelerated and more pronounced in p27Kip1 compared to p21Cip1 null mice. The phenotype of the double null p27Kip1/p21Cip1 accentuates the p27Kip1 phenotype, suggesting some degree of synergy between the two CKIs.Using a yeast two-hybrid approach, we have cloned and characterized novel p27Kip1 binding proteins that regulate p27Kip1 function. Three proteins have been sequenced, antibodies have been raised, and biochemical studies are in progress to characterize their function. Knock-out mice deficient in these proteins are being generated. Taken together, these studies define the molecular function and regulation of p27Kip1 and p21Cip1 in vascular tissue. On the basis of these molecular studies, novel therapeutic approaches to vascular proliferative diseases are being developed.
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