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

摘要 美国有8000多万人患有心血管疾病，导致700多万人 每年都会进行血管重建手术。血管重建术包括血管内、血管成形术或 支架植入术，或开放手术，动脉内膜切除术或搭桥术。所有这些程序都会对 血管和内皮细胞受损。这种创伤会引起一系列生物变化，从而导致 内侧血管平滑肌细胞(VSMCs)迁移到内膜，在那里它们增殖，导致 血管管腔内的细胞病变，内径缩小，最终导致血管 重新收紧。目前，药物洗脱支架(DES)和药物涂层球囊(DCB)用于预防 血管再狭窄。这些装置上的药物通常是钙调神经磷酸酶抑制剂(如西罗莫司)或 化疗药物(如紫杉醇)。所有这些药物的共同点是，它们都抑制这两种 VSMC增殖和内皮细胞(EC)增殖。血管内皮细胞提供了一种抗血栓、抗血栓的 用于血管的粘合剂表面。当血管内皮细胞再生被抗增殖药物阻止时 药物，患者需要无限期地使用一种有效的抗血小板药物(氯吡格雷)。失灵了 抗血小板药物可导致危及生命的血管原位血栓形成。我们之前已经报道过 肉豆蔻化富含丙氨酸的C激酶底物(MARCKS)的敲除导致VSMC停滞 扩散和适度加强EC的扩散，使其成为预防疟疾的理想目标 血管再狭窄。我们进一步证明Marcks对细胞增殖的影响是依赖于p27kip1的。在……里面 VSMC中p27kip1表达水平较高，主要分布在细胞核内，而内皮细胞中p27kip1表达水平较高 是减少的。P27kip1的表达受26S蛋白酶体降解的调节。降级 P27kip1是一个多步骤的过程，开始于通过激酶与stathmin(KIS)相互作用而磷酸化， 这使得p27kip1可以从细胞核通过。在VSMC中，Marcks基因敲除降低了KIS蛋白 表情。与此形成鲜明对比的是，Marcks基因敲除了内皮细胞中KIS的表达。这样做的目的是 建议定义Marcks通过不同方式调节KIS在这些 两种细胞类型。总体假设是Marcks与VSMC中的KIS结合，而不是ECs阻止， KIS的退化。这一假设将在三个具体目标中进行检验：1)确定 调节VSMC和ECs中KIS的表达2)确定MARCKS和KIS的结构域 介导KIS在VSMCs而不是ECs中降解的保护作用和3)体内测定 组织特异性MARCKS基因敲除和KIS缺失的影响。拟议工作的基本原理是 进一步描述Marcks信号的下游影响，以确定其他潜在的更好或协同作用 针对内膜增生的转化性治疗靶点。

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.