Eicosanoid-Induced Vascular Growth During Injury

受伤期间类二十烷酸诱导的血管生长

• 批准号: 8583336
• 负责人: KAFAIT U MALIK
• 金额: $ 37.85万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-01-01 至 2015-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8583336
• 关键词: Acceleration; Address; Adenoviruses; Aneurysm; Angiotensin II; Animals; Aortic Injury; Apolipoprotein E; Arachidonic Acids; Area; Arterial Fatty Streak; Arteries; Atherosclerosis; Biochemical; Blood Vessels; CYP1B1 gene; Carotid Arteries; Cells; Cytochrome P450; Data; Development; Diet; Eicosanoids; Fatty acid glycerol esters; Fluorescence; Generations; Grant; Growth; High Pressure Liquid Chromatography; Hypertrophy; In Vitro; Infiltration; Inflammation; Inflammatory; Injury; Knock-out; Knockout Mice; Knowledge; Laboratories; Lesion; Leukocytes; Lipid Peroxides; Lipids; MAPK3 gene; Mass Spectrum Analysis; Measures; Metabolism; Molecular; Molecular Biology Techniques; Mus; NADP; NADPH Oxidase; Oxidases; Pathogenesis; Pharmaceutical Preparations; Plasma; Production; Protein Kinase; Rattus; Reactive Oxygen Species; Research; Research Design; Role; SRC gene; Signaling Molecule; Techniques; Testing; Therapeutic; Transgenic Animals; Transgenic Mice; Vascular Diseases; apolipoprotein E-2; base; feeding; hypercholesterolemia; in vivo; inhibitor/antagonist; injured; macrophage; migration; novel; prevent; restenosis; small hairpin RNA

DESCRIPTION (provided by applicant): The current proposal is an extension of our studies conducted over the previous grant period and is aimed to investigate the contribution of arachidonic acid (AA) metabolites generated via CYP1B1 and the underlying mechanism involved in Ang II-induced neointimal growth caused by vascular injury and atherosclerosis. It is based on our novel preliminary data that: a) the selective CYP1B1 inhibitor 2,4,32,52-tetramethoxystilbene (TMS) or adenovirus CYP1B1 shRNA (CYP1B1 shRNA) prevents Ang II-stimulated neointimal growth caused by balloon injury of rat carotid artery; b) Ang II increases neointimal growth in wire-injured carotid artery of wild type (Cyp1b1+/+),but not CYP1B1 deficient (Cyp1b1-/-), mice; c) treatment with TMS inhibits generation of fatty streak areas and their acceleration by Ang II in Apo E knockout (apoE-/-) mice fed a high fat diet (HFD); and d) Ang II and AA-induced NADPH oxidase activation in rat VSMCs is prevented by TMS or CYP1B1 shRNA, and in VSMC from Cyp1b1-/-, but not Cyp1b1+/+, mice. These findings have led us to the following central hypothesis: CYP1B1, through AA metabolites/lipid peroxides, results in activation of NADPH oxidase and production of ROS, which, by activating one or more signaling molecules, contribute to the pathogenesis of restenosis caused by Ang II during vascular injury and to atherosclerosis produced by hypercholesterolemia and its acceleration by Ang II. To test this hypothesis, we will address the following specific aims: Aim 1. Determine the contribution of CYP1B1 to neointimal growth stimulated by Ang II in balloon-injured rat and wire- injured mouse carotid artery; Aim 2a) Investigate the contribution of CYP1B1 to atherosclerosis and neointimal growth after wire injury of mouse carotid artery; Aim 2b) Examine the contribution of CYP1B1 to Ang II-induced acceleration of atherosclerosis, aneurysm, and neointimal growth; Aim. 3. Investigate the mechanism of CYP1B1-dependent Ang II- and AA-induced NADPH oxidase and ROS production in VSMCs from hypercholesterolemia in mice. To address these aims, we plan to use numerous state-of-the-art in vitro and in vivo cellular and molecular biology techniques and transgenic mice. These include using 1) adenovirus CYP1B1 shRNA; 2) apoE-/-/Cyp1b1+/+ and double knockout-apoE-/-/Cyp1b1-/- and knockin-apoE+/+/CYp1b1+/+ mice that we have generated in our laboratory; 3) isolated VSMCs from these transgenic animals; 4) morphological, histological, immunohistochemical and fluorescence, and biochemical techniques, and 5) HPLC-LC-ESI-MS for the identification of AA metabolites. The proposed studies should advance our current knowledge of the cellular and molecular mechanisms involved in the pathogenesis of restenosis and atherosclerosis and allow us to demonstrate CYP1B1 as a potential novel target for the development of more effective therapeutic drugs such as TMS for treating Ang II-induced restenosis caused by vascular injury and atherosclerosis by hypercholesterolemia.

描述（由申请人提供）：目前的提案是我们在先前资助期进行的研究的延伸，旨在研究通过CYP1B1产生的花生四烯酸（AA）代谢物的贡献，以及Ang ii诱导的血管损伤和动脉粥样硬化引起的新生内膜生长的潜在机制。基于我们新的初步数据：a)选择性CYP1B1抑制剂2,4,32,52-四甲基二苯乙烯（TMS）或腺病毒CYP1B1 shRNA （CYP1B1 shRNA）可阻止Ang ii刺激的大鼠颈动脉球囊损伤引起的新内膜生长；b) Ang II促进野生型（Cyp1b1+/+）颈动脉钢丝损伤小鼠的新内膜生长，但对Cyp1b1缺失（Cyp1b1-/-）小鼠无促进作用；c)经TMS治疗可抑制高脂饮食（HFD） Apo E敲除（apoE-/-）小鼠的脂肪条纹区产生和Ang II的加速；d) TMS或CYP1B1 shRNA可阻止Ang II和aa诱导的大鼠VSMCs中NADPH氧化酶的激活，并且在CYP1B1 -/-小鼠的VSMC中，而不是CYP1B1 +/+小鼠。这些发现使我们提出了以下中心假设：CYP1B1通过AA代谢物/脂质过氧化物，导致NADPH氧化酶的激活和ROS的产生，ROS通过激活一个或多个信号分子，参与血管损伤期间由Ang II引起的再狭窄的发病机制和由高胆固醇血症引起的动脉粥样硬化，并由Ang II加速其发生。为了验证这一假设，我们将解决以下具体目标：目标1。测定CYP1B1在球囊损伤大鼠和钢丝损伤小鼠颈动脉中对Angⅱ刺激新内膜生长的作用；目的2a)探讨CYP1B1在小鼠颈动脉丝损伤后动脉粥样硬化及新生内膜生长中的作用；目的2b)检测CYP1B1在Ang ii诱导的动脉粥样硬化、动脉瘤和新生内膜加速生长中的作用；目标。3。探讨高胆固醇血症小鼠VSMCs中cyp1b1依赖性Ang II-和aa诱导的NADPH氧化酶和ROS产生的机制。为了实现这些目标，我们计划使用许多最先进的体外和体内细胞和分子生物学技术以及转基因小鼠。这些方法包括：1)使用腺病毒CYP1B1 shRNA；2)实验室培养的apoE-/-/Cyp1b1+/+和双敲除apoE-/-/Cyp1b1-/-和敲除apoE+/+/ Cyp1b1+/+小鼠；3)从这些转基因动物中分离VSMCs；4)形态学、组织学、免疫组织化学、荧光和生化技术；5)HPLC-LC-ESI-MS鉴定AA代谢物。拟议的研究将推进我们目前对再狭窄和动脉粥样硬化发病机制的细胞和分子机制的了解，并使我们能够证明CYP1B1是开发更有效的治疗药物（如TMS）的潜在新靶点，用于治疗由血管损伤和高胆固醇血症引起的动脉粥样硬化引起的Ang ii诱导的再狭窄。