EET-Induced Cardioprotection: Role of Opioids and Nitric Oxide (NO)

描述(申请人提供)：心血管疾病(CVD)是美国头号死亡原因，缺血性心脏病是CVD患者死亡的首要原因。大多数缺血性心脏病患者冠状动脉阻塞，心脏组织缺乏灌流(缺血)，导致心肌损伤和再灌流后的进一步损伤。花生四烯酸(AA)、11，12-和14，15-环氧二十碳三烯酸(EETs)的新型CYP环氧酶代谢产物在缺血过程中增加，特别是在再灌流后。内源性EETs和外源性EETs在狗、大鼠和小鼠心脏中产生显著的心脏保护作用；然而，这些有益作用的机制尚不清楚。根据耐人寻味的初步数据，阿片受体和一氧化氮(NO)释放可能是EET介导的心脏保护的两个主要角色。本研究将利用一氧化氮合酶(NOS)亚型的大鼠和基因敲除小鼠，以及心肌细胞和心脏成纤维细胞的细胞模型，深入阐明EET诱导心肌保护的关键因素。需要检验的假设是，在缺血/再灌注时，心肌细胞和心脏成纤维细胞释放的EETs是有效的心脏保护剂，可以诱导内源性NO的进一步释放，从而产生有益的效果。具体地说，我们将(1)确定一氧化氮(NO)是EET在完整的大鼠和小鼠心脏和心肌细胞中诱导的心肌保护的媒介。大鼠、NOS基因敲除小鼠和心肌细胞(H9c2细胞)将被用来证明EET诱导的心脏保护是通过NO信号通路介导的。将确定一氧化氮合酶的异构体(eNOS、nNOS或iNOS)及其由eETs激活的信号通路。(2)明确心肌成纤维细胞作为心肌细胞缺氧/复氧损伤的保护因子(S)，在释放EETs和可能的NO方面发挥作用。(3)确定两种主要的心脏保护因子EETs和阿片类药物在心脏保护中是否存在串扰。重要的是，EETs很容易被可溶性环氧化物水解酶(SEH)水解成二羟基二十碳三烯酸(DHETs)，对心肌梗死面积没有影响。这一发现表明，具有优越药代动力学的新型合成EET类似物可能比sEH抑制剂和不稳定的EETs具有更好的治疗靶点。新合成的长效EET类似物将用于阐明EET的信号通路和缩小梗塞面积。作为该领域的领导者，寻找新的稳定的内源性EETs类似物将是本项目的主要目标，并在细胞和整个动物心脏损伤模型中展示其强大的抗缺血和抗炎作用。长期目标是更好地描述一种具有多个治疗靶点的新的内源性系统，这可能会建议采用联合疗法来更好地治疗缺血/再灌注损伤。 公共卫生相关性：拟议的研究与国家健康研究所的使命相关，因为这些研究将通过对内源性因素在缺血和再灌流期间保护心脏的机制的新发现，更好地理解和推进缺血/再灌流领域的知识。使用新合成的具有更好药代动力学的化合物将产生新的稳定的EETs，以改善对缺血/再灌流心肌的保护。了解多种调节因子(阿片类药物，NO)的功能可能会为使用多靶点治疗人类缺血性心脏病提供洞察力。

DESCRIPTION (provided by applicant): Cardiovascular disease (CVD) is the number one cause of death in the United States and ischemic heart disease is the leader in mortality among patients with CVD. Most individuals with ischemic heart disease have blocked coronary arteries and cardiac tissue has a lack of perfusion (ischemia), resulting in myocardial damage and further injury following reperfusion. The novel CYP-epoxygenase metabolites of arachidonic acid (AA), 11,12- and 14,15-epoxyeicosatrienoic acids (EETs) are increased during ischemia and particularly following reperfusion. Endogenously produced EETs and exogenously administered EETs produce marked cardioprotective effects in dog, rat and mouse hearts; however, the mechanisms responsible for these beneficial effects remain unclear. Based on intriguing preliminary data, opioid receptors and nitric oxide (NO) release may be 2 major players in EET-mediated cardioprotection. This study will utilize rats and genetic knockout mice of nitric oxide synthase (NOS) isoforms as well as cellular models of cardiomyocytes and cardiac fibroblasts to elucidate in depth, the key contributing factors responsible for EET-induced cardioprotection. The hypothesis to be tested is that upon ischemia/reperfusion, EETs released from cardiomyocytes and cardiac fibroblasts are potent cardioprotective agents that induce the further release of endogenous NO to produce their beneficial effects. Specifically, we will (1) determine that nitric oxide (NO) is a mediator of EET-induced cardioprotection in intact rat and mouse hearts and cardiomyocytes. Both rats and NOS knockout mice and cardiomyocytes (H9c2 cells) will be used to demonstrate that EET-induced cardioprotection is mediated via a NO signaling pathway. NOS isoforms (eNOS, nNOS or iNOS) and their signaling pathways activated by the EETs will be identified. (2) Determine that cardiac fibroblasts play a role in releasing EETs and possibly NO as regulatory factor(s) to protect cardiomyocytes from hypoxia/reoxygenation injury. (3) Determine if cross-talk occurs between the two major cardioprotective factors, EETs and opioids, in cardioprotection. Importantly, EETs are readily hydrolyzed by soluble epoxide hydrolase (sEH) to dihydroxyeicosatrienoic acids (DHETs) which have no effect on myocardial infarct size. This finding suggests that novel synthetic EET analogs with superior pharmacokinetics may represent better therapeutic targets than sEH inhibitors and labile EETs. The newly synthesized longer-acting EET analogs will be used to elucidate signaling pathways of the EETs and to reduce infarct size. As leaders in the this field, identification of novel stable analogs of endogenous EETs will be a major goal of this project as well as demonstrating their powerful anti-ischemic and anti-inflammatory actions in cellular and whole animal models of cardiac injury. The long term goal is to obtain better characterization of a novel endogenous system with multiple therapeutic targets that may suggest combined therapy for better treatment of ischemia/reperfusion injury. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to the National Institute of Health's mission because these studies will lead to a better understanding and advance knowledge in the ischemia/reperfusion field by new discoveries of mechanisms by which endogenous factors protect the heart during ischemia and reperfusion. Using newly synthesized compounds with better pharmacokinetics will lead to new classes of stable EETs for improved protection of the ischemic/reperfused myocardium. Understanding the functions of multiple regulatory factors (opioids, NO) may provide insight into using multiple targets to treat ischemic heart disease in man.