Over 80 million people in the United States have cardiovascular disease resulting in over 7 million revascularization procedures each year. Revascularization procedures are endovascular, angioplasty or stenting, or open surgical procedures, endarterectomy or bypass. All of these procedures cause trauma to the blood vessel and damage the endothelium. This trauma causes a series of biological changes that result in the medial vascular smooth muscle cells (VSMCs) migrating to the intmal where they proliferate causing a cellular lesion in the lumen of the vessel, reducing the inner diameter and ultimately causing the vessel to restenose. Currently, drug-eluting stents (DES) and drug-coated balloons (DCB) are used to prevent restenosis. The agents on these devices are frequently calcineurin inhibitors (such as sirolimus) or chemotherapeutics (such as paclitaxel). What all these agents have in common is that they all inhibit both VSMC proliferation and endothelial cell (EC) proliferation. The endothelium provides an antithrombotic, anti- adhesive surface for blood vessels. When endothelium regeneration is prevented by the antiproliferative agents, the patient needs to remain on a potent antiplatelet agent (clopidogrel) indefinitely. Failure of the antiplatelet regiment can result in life-threatening in situ thrombosis of the vessel. We have previously reported that knockdown of the myristolated alanine-rich C kinase Substrate (MARCKS) results in arrest of VSMC proliferation and a modest potentiation of EC proliferation, making it an ideal target for the prevention of restenosis. We further demonstrated that the effect of MARCKS on proliferation is p27kip1-dependent. In VSMCs, p27kip1 is expressed at greater levels and is trapped in the nucleus whereas in ECs, p27kip1 expression is decreased. The expression of p27kip1 is regulated by degradation by the 26s proteasome. Degradation of p27kip1 is a multi-step process beginning with phosphorylation by the kinase interaction with stathmin (KIS), which allows p27kip1 to transit from the nucleus. In VSMCs, MARCKS knockdown decreases KIS protein expression. In stark contrast, MARCKS knockdown in increased KIS expression in ECs. The goal of this proposal is to define the mechanism through which MARCKS differentially regulates KIS expression in these two cell types. The overall hypothesis is that MARCKS binds to KIS in VSMCs, but not ECs preventing, degradation of KIS. This hypothesis will be tested in three Specific Aims: 1) To determine the point of regulation of KIS expression in VSMCs and ECs 2) To determine the domains of MARCKS and KIS that mediate MARCKS protection of KIS from degradation in VSMCs but not ECs and 3) To determine the in vivo effect of tissue-specific MARCKS knockdown and KIS deletion. The rationale for the proposed work is to further delineate the downstream effects of MARCKS signaling to identify other potentially better or synergistic targets for translational therapy targeting intimal hyperplasia.
Over 80 million people in the United States have cardiovascular disease resulting in over 7 million revascularization procedures each year. The durability of these procedures is limited by restenosis. We have previously demonstrated that knockdown of MARCKS prevents restenosis and that MARCKS knockdown differentially affects KIS expression in the different cell types in the vessel wall. We seek to understand how MARCKS differentially regulates KIS to provide the necessary knowledge to further MARCKS as a target for therapy to prevent restenosis.
