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

富含肉豆蔻酸丙氨酸的 C 激酶底物 (MARCKS) 对血管平滑肌和内皮细胞差异增殖中与 stathmin (KIS) 相互作用的激酶的影响

• 批准号: 10198997
• 负责人: RAJABRATA SARKAR
• 金额: $ 38.63万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-10 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10198997
• 关键词: 26S proteasome; Adhesives; Affect; Alanine; Amputation; Angioplasty; Antiplatelet Drugs; Atherosclerosis; Binding; Biological; Blood Vessels; Bypass; Calcineurin inhibitor; Caliber; Cardiovascular Diseases; Cause of Death; Cell Nucleus; Cell Proliferation; Cells; Cerebrovascular Circulation; Chemosensitization; Complex; Coronary Circulation; Cyclin-Dependent Kinase Inhibitor; Data; Developed Countries; Devices; Endarterectomy; Endothelial Cells; Endothelium; Failure; Fluorescence Resonance Energy Transfer; Goals; Health; Hemorrhage; Hyperplasia; Iatrogenesis; Immunoprecipitation; In Situ; Incidence; Injury; Intervention; Knockout Mice; Knowledge; Laser Microscopy; Lesion; Life; Location; Lower Extremity; Medial; Mediating; Messenger RNA; Metabolism; Modeling; Molecular Target; Mus; Myocardial Infarction; Operative Surgical Procedures; Outcome; Paclitaxel; Patients; Pharmaceutical Preparations; Phenotype; Phosphorylation; Phosphotransferases; Population; Prevention; Procedures; Process; Proliferating; Protein Biosynthesis; Proteins; Publishing; Regulation; Reporting; Risk; Role; Series; Signal Transduction; Sirolimus; Site; Small Interfering RNA; Smooth Muscle Myocytes; Stents; Stroke; Surface; Testing; Therapeutic; Thrombosis; Tissues; Trauma; Ubiquitination; United States; Vascular Smooth Muscle; Work; antiproliferative agents; blood damage; cell type; clopidogrel; differential expression; endothelial regeneration; healing; improved outcome; in vivo; knock-down; mutant; novel; prevent; protein degradation; protein expression; restenosis; restoration; stathmin; targeted treatment; vascular smooth muscle cell proliferation

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.

概括 美国有超过 8000 万人患有心血管疾病，导致超过 700 万人罹患心血管疾病 每年进行血运重建手术。血运重建手术包括血管内手术、血管成形术或 支架置入术，或开放式外科手术、动脉内膜切除术或搭桥术。所有这些程序都会造成创伤 血管并损伤内皮。这种创伤会引起一系列的生物变化，从而导致 内侧血管平滑肌细胞 (VSMC) 迁移至内膜并在那里增殖，从而导致 血管腔内的细胞病变，内径减小，最终导致血管 再狭窄。目前，药物洗脱支架（DES）和药物涂层球囊（DCB）用于预防 再狭窄。这些装置上的药物通常是钙调神经磷酸酶抑制剂（例如西罗莫司）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 VSMC 增殖和内皮细胞 (EC) 增殖。内皮细胞提供抗血栓、抗 血管的粘合表面。当抗增殖药物阻止内皮细胞再生时 药物，患者需要无限期地继续使用强效抗血小板药物（氯吡格雷）。失败的 抗血小板治疗可导致危及生命的血管原位血栓形成。我们之前曾报道过 敲低富含肉豆蔻酸丙氨酸的 C 激酶底物 (MARCKS) 会导致 VSMC 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 p27kip1 依赖性的。在 VSMC 中，p27kip1 表达水平更高，并被捕获在细胞核中，而在 EC 中，p27kip1 表达 减少了。 p27kip1 的表达受 26s 蛋白酶体降解的调节。降解 p27kip1 是一个多步骤过程，从激酶与 Stathmin (KIS) 相互作用的磷酸化开始， 它允许 p27kip1 从细胞核转运。在 VSMC 中，MARCKS 敲低会降低 KIS 蛋白 表达。形成鲜明对比的是，MARCKS 敲低导致 EC 中 KIS 表达增加。此举的目标 提案的目的是定义 MARCKS 差异调节这些细胞中 KIS 表达的机制。 两种细胞类型。总体假设是 MARCKS 与 VSMC 中的 KIS 结合，但不与 EC 结合，从而预防， KIS 的降解。该假设将在三个具体目标中进行检验：1）确定 VSMC 和 EC 中 KIS 表达的调控 2) 确定 MARCKS 和 KIS 的结构域 介导 MARCKS 保护 KIS 免受 VSMC 降解，但不影响 EC 降解，并且 3) 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，以确定其他潜在更好或协同作用 针对内膜增生的转化治疗的目标。