iPLA2 as a Target in Vascular Inflammation, Neointima Formation, and Restenosis

描述（由申请人提供）： 冠心病是美国男性和女性死亡的唯一主要原因。冠状动脉血运重建术包括冠状动脉旁路移植术（CABG）和经皮冠状动脉介入治疗（PCI），是冠心病患者最常见的治疗方法。然而，它也是最昂贵的，并且通常与再狭窄的高发生率相关。虽然再狭窄率显著降低， 裸金属支架（BMS），特别是药物洗脱支架（DES），BMS后持续的高再狭窄率和DES支架内血栓形成的风险增加已被视为冠状动脉血运重建长期疗效的显著限制。以鉴定冠心病的新型治疗靶标为长期目标，本申请具体研究了钙非依赖性磷脂酶A2 b（iPLA 2b）在小鼠股动脉导丝损伤模型中的血管炎症、再内皮化和新生内膜形成中的潜在关键作用。iPLA 2b是磷脂酶A2超家族的成员，其作用于磷脂以产生游离脂肪酸和溶血磷脂。iPLA 2b在血管平滑肌细胞（VSMC）中表达，并与许多人类疾病有关。尽管对其他PLA 2在血管损伤诱导的新生内膜形成中的作用知之甚少，但包括药理学抑制、反义寡核苷酸下调、基因缺失和平滑肌特异性过表达在内的大量证据一致地证明iPLA 2b在小鼠颈动脉结扎模型中的血管炎症和新生内膜形成中起关键作用。为了研究靶向平滑肌特异性iPLA 2b是否足以减少血管炎症和新生内膜形成，建立了更好地模拟PCI的股动脉损伤模型，即新的他莫昔芬诱导的平滑肌特异性iPLA 2b敲除小鼠模型（SM-iPLA 2b-iKO）的开发，目前的建议假设血管损伤引起的VSMC中iPLA 2b的激活在金属丝损伤诱导的血管炎症，再内皮化，和新生内膜形成，从而显著促进冠状动脉血管重建后再狭窄的发展。三个具体目标是：1）检验平滑肌特异性iPLA 2b在血管炎症、再内皮化和新生内膜形成的起始中起关键作用的假设; 2）确定导丝损伤诱导的iPLA 2b活化、血管炎症和新生内膜形成的分子机制; 3）研究导丝损伤后分子和遗传靶向iPLA 2b是否对再内皮化、血管炎症和新生内膜形成的进展有影响。为了实现这一目标，将使用完善的和最生理相关的股动脉导丝损伤模型与几种新型和已建立的遗传动物模型（SM-iPLA 2b-iKO、SM-HIF-1a-iKO和SM-iPLA 2b-Tg/12/15-LO）组合，以在导丝损伤前后选择性抑制或激活iPLA 2b、HIF-1a和12/15-LO。将分离损伤和未损伤的股动脉，然后进行分子、免疫组织学和生物化学研究，以确定iPLA 2b在血管炎症和新生内膜形成中的作用。拟议研究的结果可能会修改目前的范式，即血管炎症的起始和早期进展在很大程度上归因于单核细胞/巨噬细胞，白细胞和内皮细胞之间的协调相互作用，提供了第一个直接的实验证据，表明VSMC在这些早期关键事件中也起着关键作用。重要的是，来自所提出的研究的结果将阐明可能导致将iPLA 2b鉴定为用于预防和治疗血管炎症、再内皮化、新生内膜形成和再狭窄的新型潜在治疗靶点的特定机制。

DESCRIPTION (provided by applicant): Coronary heart disease is the single leading cause of death among men and women in the United States. Coronary revascularization, including coronary artery bypass graft (CABG) and percutaneous coronary intervention (PCI) is the most common modality in patients with coronary diseases. However, it is also among the most costly, and is often associated with a high incidence of restenosis. Although the rate of restenosis is significantly reduced with the use of bare-metal stent (BMS) and particularly with drug-eluting stent (DES), a persistently high rate of restenosis after BMS and an increased risk of in-stent thrombosis with DES have been encountered as significant limitations to the long-term efficacy of coronary revascularization. With a long-term goal of identifying novel therapeutic targets for coronary heart disease, the current application specifically investigates a potentially critical role of calcium independent phospholipase A2b (iPLA2b) in vascular inflammation, re-endothelialization, and neointima formation in a mouse femoral artery wire injury model. iPLA2b is a member of the phospholipase A2 superfamily that acts on phospholipids to produce a free fatty acid and a lysophospholipid. iPLA2b is expressed in vascular smooth muscle cells (VSMC) and is implicated in many human diseases. Whereas little is known about the role of other PLA2 in vascular injury-induced neointima formation, substantial evidence, including pharmacological inhibition, antisense oligonucleotide down- rgulation, genetic deletion, and smooth muscle-specific overexpression, consistently demonstrated that iPLA2b plays a critical role in vascular inflammation and neointima formation in a mouse carotid artery ligation model. To investigate whether targeting smooth muscle-specific iPLA2b is sufficient to reduce vascular inflammation and neointima formation, a femoral artery injury model that better mimics PCI was established, a novel tamoxifen-inducible smooth muscle-specific iPLA2b knockout mouse model (SM-iPLA2b-iKO) was developed, and the current proposal hypothesizes that activation of iPLA2b in VSMC by vascular injury plays a key role in wire injury-induced vascular inflammation, re-endothelialization, and neointima formation, thereby significantly contributing to the development of restenosis after coronary revascularization. Three Specific Aims are: 1) To test the hypothesis that smooth muscle-specific iPLA2b plays a critical role in the initiation of vascular inflammation, re-endothelialization, and neointima formation; 2) To determine the molecular mechanism that underlies wire injury-induced iPLA2b activation, vascular inflammation, and neointima formation; 3) To investigate whether post-wire injury molecular and genetic targeting iPLA2b has an effect on the progression of re-endothelialization, vascular inflammation, and neointima formation. To achieve this goal, a well established and most physiological relevant femoral artery wire injury model will be used in combination with several novel and established genetic animal models (SM- iPLA2b-iKO, SM-HIF-1a-iKO, and SM-iPLA2b-Tg/12/15-LO) to selectively inhibit or activate iPLA2b, HIF-1a, and 12/15-LO before and after wire injury. Injured and non-injured femoral arteries will be isolated and then subjected to molecular, immunohistological, and biochemical studies to determine the role of iPLA2b in vascular inflammation and neointima formation. Results from the proposed studies may modify the current paradigm that the initiation and early progression of vascular inflammation are largely attributed to coordinated interactions among monocytes/macrophages, leukocytes, and endothelial cells by providing the first direct experimental evidence that VSMC also play a critical role in these early key events. Importantly, results from the proposed studies will elucidate specific mechanisms that could lead to the identification of iPLA2b as a novel potential therapeutic target for the prevention and treatment of vascular inflammation, re- endothelialization, neointima formation, and restenosis.