Heme Oxygenase Regulation of Eicosanoid Biosynthesis

描述(申请人提供)：血管内皮细胞对氧化应激和损伤刺激具有适应性反应，对血管功能障碍具有强大和持续的保护作用。抵抗内皮功能障碍和损伤是一种重要的保护机制，因为内皮功能保留的患者较不容易患高血压和肥胖症等临床血管疾病。然而，内皮功能障碍和血管疾病之间的联系却知之甚少。以前的工作已经确定了血红素加氧酶(HO-1/HO-2)、内毒素和脂联素是关键的内皮保护分子。然而，每个系统的重要性以及决定内皮功能障碍易感性或抵抗力的确切分子事件尚不清楚。我们已发表的初步数据表明：1)HO-1的诱导或过表达可以消除糖尿病、肥胖和高血压对血管系统的损害后果，而HO活性的抑制会加剧这种损害；2)HO-1活性降低会增加氧化应激、炎症、血管功能障碍和胰岛素抵抗；3)HO-1的表达与EET和脂联素的水平呈正相关；4)EET激动剂治疗HO-2-/-小鼠可挽救明显的内皮功能障碍和炎症表型；5)EET-TG小鼠表现出更高的脂联素水平。因此，我们假设HO-1和EET是层级组织的，并且千丝万缕地联系在一起，形成了一个功能上相互关联的模块，在这个模块中，HO-1和EET协同工作，激活包括脂联素及其下游信号分子(AKT，AMPK)在内的关键保护系统，使血管内皮细胞对损伤性刺激具有抵抗力；因此，其中一个保护系统的缺陷导致肥胖时血管损伤的表现。主要目的是通过重点研究HO-1-EET模块作为对抗损伤介导的血管功能障碍的关键保护机制来确定增强血管阻力的最佳条件。这些都是复杂的研究，需要复杂的方法。因此，我们组装了一组转基因小鼠(HO-1-/-、HO-1、HO-2-/-、EC-SOD-/-、APN-/-、sEHKO、HO-1-TG、EET-TG和APN-TG)，并开发了一种慢病毒基因转移策略来提供功能的丧失和获得；这些结合高度特异的探针(SiRNA)和不同的药理试剂(EET激动剂/拮抗剂、酶抑制剂)将为评估因果关系和进行机制分析提供必要的工具。我们还开发了一种多方面的方法来评估血管内皮细胞的活力和功能。生成的数据应该提供关于HO-1-EET轴如何影响负责血管保护的血管表型控制的可靠信息，以及治疗和预防由内皮功能障碍引起的血管疾病的转化性临床研究的框架。 与公共卫生相关：肥胖和血管功能障碍是心血管疾病的主要原因，心血管疾病仍然是美国发病率和死亡率的主要原因，并给社会带来巨大的经济负担，每年约为1000亿美元。这一建议试图从高脂血症、高血压、肥胖和代谢综合征等危险因素了解血管疾病的发展，但更重要的是，阐明保护血管内皮细胞所需的机制。

DESCRIPTION (provided by applicant): The vascular endothelium has an adaptive response to oxidative stress and injurious stimuli that confers powerful and sustained protection against vascular dysfunction. Resistance to endothelial dysfunction and injury is an important protective mechanism as patients with preserved endothelial function are less predisposed to clinical vascular disease such as hypertension and obesity. However, the link between endothelial dysfunction and vascular disease is poorly understood. Previous work has identified the heme oxygenases (HO-1/HO-2), EETs and adiponectin as key endothelial protective molecules. However, the importance of each system and the precise molecular events that determine susceptibility or resistance to endothelial dysfunction are not known. Our published and preliminary data show that: 1) HO-1 induction or overexpression abrogates the injurious consequences of diabetes, obesity and hypertension on the vasculature whereas inhibition of HO activity exacerbates it; 2) diminished HO activity increases oxidative stress, inflammation, vascular dysfunction and insulin resistance; 3) there is a positive relationship between HO-1 expression and the levels of EET and adiponectin; 4) treatment of HO-2-/- mice with an EET agonist rescues the apparent endothelial dysfunction and the inflammatory phenotype; and 5) EET-Tg mice exhibit higher adiponectin levels. Accordingly, we hypothesize that HO-1 and EET are organized hierarchically and are inextricably linked forming a functionally-inter-related module in which HO-1 and EET work in concert to activate key protective systems, including adiponectin and downstream signaling molecules (AKT, AMPK), rendering the vascular endothelium resistant to injurious stimuli; consequently, a deficiency in one of these protective systems contributes to the manifestation of vascular injury in obesity. The major goal is to determine the optimum conditions for enhanced vascular resistance by focusing on the HO-1-EET module as a critical protective mechanism against injury-mediated vascular dysfunction. These are complex studies that require a sophisticated approach. Therefore, we assembled a battery of genetically modified mice (HO-1-/-, HO-1, HO-2-/-, EC-SOD-/-, APN-/-, sEHKO, HO-1-Tg, EET-Tg, and APN-Tg) and developed a lentiviral gene transfer strategy to provide loss and gain of functions; these together with highly specific probes (siRNAs) and distinct pharmacological agents (EET agonists/antagonists, enzymatic inhibitors) will provide the necessary tools for assessing the cause-and-effect relationship and carrying out a mechanistic analysis. We also developed a multifaceted approach to assess the vitality and functionality of the vascular endothelium. The data generated should provide solid information of how the HO-1-EET axis influences the control of the vascular phenotype that is responsible for vascular protection as well as the framework for translational clinical research to both treat and prevent vascular disease that results from endothelial dysfunction. PUBLIC HEALTH RELEVANCE: Obesity and vascular dysfunction are major contributors to cardiovascular disease which remains a major cause of morbidity and mortality in the United States and places a significant economic burden upon society, about 100 billion dollars a year. This proposal seeks to understand the development of vascular disease from risk factors including hyperlipidemia, hypertension, obesity and the metabolic syndrome, but more importantly, to elucidate the mechanism necessary to preserve the vascular endothelium.