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; 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 美国超过8000万人患有心血管疾病，导致超过700万 每年血运重建程序。血运重建程序是血管内，血管成形术或 支架或开放手术程序，内膜切除术或旁路。所有这些程序会导致创伤 血管并损坏内皮。这种创伤导致一系列生物学变化，导致 内侧血管平滑肌细胞（VSMC）迁移到Intmal，在那里它们增殖导致 血管内腔中的细胞病变，减少内径，并最终导致血管 reten索。目前，使用药物洗脱支架（DES）和涂有药物的气球（DCB）来防止 再狭窄。这些设备上的试剂经常是钙调神经酶抑制剂（例如西洛里木斯）或 化学治疗药（例如紫杉醇）。所有这些代理人的共同点是它们都抑制了 VSMC增殖和内皮细胞（EC）增殖。内皮提供了抗血栓形成的抗 血管的粘合表面。当抗增生性预防内皮再生时 药物，患者需要无限期保留在有效的抗血小板剂（氯吡格雷）上。失败 抗血小板团可以导致威胁生命的容器原位血栓形成。我们以前报道了 肉豆蔻碱富含丙氨酸的C激酶底物（MARCKS）的敲低导致VSMC逮捕 EC增殖的增殖和适度的增强，使其成为预防的理想目标 再狭窄。我们进一步证明，马克克人对增殖的影响是p27kip1依赖性的。在 VSMC，p27KIP1在较高的水平上表达，被困在核中，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。退化 P27KIP1是一个多步骤过程，从与Stathmin（KIS）的磷酸化开始， 这使P27KIP1可以从核中转运。在VSMC中，Marcks敲低降低KIS蛋白 表达。与之形成鲜明对比的是，马克克斯在EC中的KIS表达增加中击倒。目标的目标 建议是定义Marcks在这些机制中差异调节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，但没有阻止EC， KIS的退化。该假设将以三个特定目的进行检验：1）确定点 调节VSMC和ECS中KIS表达的调节2）确定Marcks和Kis的域 介导MARCKS对KIS的保护免受VSMC中的降解，而不是ECS和3）确定体内 组织特异性马克克斯敲低和KIS缺失的影响。拟议工作的理由是 进一步描述了马克克斯信号的下游效应，以识别其他潜在的或协同作用 转化治疗靶向内膜增生的靶标。