The effect of Myristolated alanine-rich C Kinase Substrate (MARCKS) on kinase interacting with stathmin (KIS) in differential proliferation of vascular smooth muscle and endothelial cells

总结 美国有超过8000万人患有心血管疾病， 每年都要进行血管重建手术。血运重建手术是血管内、血管成形术或 支架植入术或开放性外科手术、动脉内膜切除术或分流术。所有这些手术都会造成 损伤血管内皮。这种创伤会引起一系列的生物学变化， 中膜血管平滑肌细胞（VSMCs）迁移到内膜，在内膜增殖， 血管管腔中的细胞病变，减小内径并最终导致血管 restenose。目前，药物洗脱支架（DES）和药物涂层球囊（DCB）用于预防 再狭窄这些装置上的药剂通常是钙调磷酸酶抑制剂（例如西罗莫司）或钙调磷酸酶抑制剂。 化疗药物（如紫杉醇）。所有这些药物的共同点是它们都抑制 VSMC增殖和内皮细胞（EC）增殖。内皮提供抗血栓形成，抗- 血管的粘附表面。当内皮再生被抗增殖药物阻止时， 药物，患者需要无限期地保持有效的抗血小板药物（氯吡格雷）。失败 抗血小板方案可导致危及生命的血管原位血栓形成。之前我们已经报道 敲低肉豆蔻酸化富含丙氨酸的C激酶底物（MARCKS）导致VSMC的停滞， 增殖和EC增殖的适度增强，使其成为预防 再狭窄我们进一步证明MARCKS对增殖的影响是p27 kip 1依赖的。在 VSMCs中，p27 kip 1以更高的水平表达，并被困在细胞核中，而在EC中，p27 kip 1表达 减少了。p27 kip 1的表达受26 s蛋白酶体降解的调节。降解 p27 kip 1是一个多步骤过程，始于通过激酶与stathmin相互作用（KIS）的磷酸化， 其允许p27 kip 1从细胞核转运。在VSMC中，MARCKS敲低降低KIS蛋白 表情与此形成鲜明对比的是，MARCKS敲低增加了EC中KIS的表达。这个目标 一个建议是定义MARCKS通过哪些机制差异调节KIS表达，在这些 两种细胞类型。总的假设是MARCKS结合VSMC中的KIS，而不是EC阻止， KIS的退化。这一假设将在三个具体目标进行检验：1）确定 KIS在血管平滑肌细胞和内皮细胞中表达的调控2）确定MARCKS和KIS的结构域， 介导MARCKS保护KIS免于在VSMC中降解，而不是EC中降解，以及3）为了确定体内 组织特异性MARCKS敲低和KIS缺失的影响。拟议工作的理由是 进一步描述MARCKS信号传导的下游效应，以鉴定其他潜在的更好或协同的 靶向内膜增生的转化治疗的靶点。

Summary 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.