Eicosanoid-Induced Vascular Growth During Injury

描述(由申请人提供)：目前的建议是我们在上一个资助期进行的研究的延伸，旨在调查通过CYP1B1产生的花生四烯酸(AA)代谢物的贡献，以及血管损伤和动脉粥样硬化导致Ang II诱导的新生内膜生长的潜在机制。基于我们新的初步数据：a)选择性的CYP1B1抑制剂2，4，32，52-四甲氧基二苯乙烯(TMS)或腺病毒CyP1B1 shRNA(CYP1B1 ShRNA)阻止Ang II刺激的大鼠颈动脉球囊损伤引起的新生内膜生长；b)Ang II促进野生型(CyP1B1/)线损伤小鼠颈动脉的新生内膜生长，但不促进CyP1B1缺陷(CyP1B1-/-)；c)TMS治疗抑制高脂饮食(HFD)喂养的Apo E基因敲除(apoE/-)小鼠脂肪条纹区的产生和Ang II的加速；TMS或CYP1B1 shRNA可抑制Ang II和AA诱导的大鼠VSMC中NADPH氧化酶的激活，但不能阻止CYP1B1-/-小鼠VSMC中NADPH氧化酶的激活。这些发现导致我们提出如下中心假说：CYP1B1通过AA代谢物/脂质过氧化产物导致NADPH氧化酶的激活和ROS的产生，通过激活一个或多个信号分子，参与血管损伤时Ang II引起的再狭窄和Ang II引起的高胆固醇血症引起的动脉粥样硬化及其加速的动脉粥样硬化的发病机制。为了验证这一假说，我们将针对以下具体目标进行验证：目的1.确定CYP1B1在Ang II刺激的大鼠和线状损伤小鼠颈动脉新生内膜生长中的作用；目的2a)研究CYP1B1在小鼠颈动脉损伤后动脉粥样硬化和新生内膜生长中的作用；目的2b)研究细胞色素P1B1在血管紧张素转换酶II诱导的动脉粥样硬化、动脉瘤和新生内膜生长加速中的作用。3.探讨CYP1B1依赖Ang II和AA诱导高胆固醇血症小鼠VSMC产生NADPH氧化酶和ROS的机制。为了达到这些目标，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和转基因小鼠。这些技术包括：1)腺病毒CyP1B1 shRNA；2)apoE-/-/CyP1B1/和我们实验室构建的双敲除-apoE-/-/CyP1B1-/-和Huckkin-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，以及5)高效液相色谱-电喷雾质谱(LC-ESI-MS)鉴定AA代谢产物。这些拟议的研究将促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，并使我们能够展示CyP1B1作为潜在的新靶点，用于开发更有效的治疗药物，如TMS，用于治疗血管损伤引起的Ang II诱导的再狭窄和高胆固醇血症引起的动脉粥样硬化。

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.