Neprilysin Apoptosis and Hypoxic Pulmonary Hypertension

描述（由申请人提供）：在与人类疾病非常相似的低氧性肺动脉高压（PHTN）的大型动物模型中，最早的肺动脉（PA）平滑肌（SMC）结构变化发生在内侧/外膜边界。远端血管肌肉化也是一个显著特征。在重建的肺血管中调节SMCs积累的机制尚不清楚。对细胞凋亡的易感性可能是一个重要的决定因素。Neprilysin （NEP）是参与血管活性神经肽降解的关键细胞表面肽酶。不太受重视的是新兴的概念，即NEP也可能通过新的肽酶非依赖性机制直接参与细胞内信号传导，从而影响细胞反应。NEP最近被认为与癌细胞的凋亡有关。这些观察结果支持了NEP可能通过增加PA SMC对缺氧诱导的凋亡的敏感性来发挥对缺氧PHTN的保护作用的可能性。NEP的缺失会通过保护PA SMC免于凋亡而导致缺氧时肺血管重构的加剧。我们现在有数据来支持这个概念。我们发现小鼠中NEP的缺失易导致缺氧PHTN的过度发生。与野生型对照组相比，NEP缺失小鼠的肺和分离的PA SMC对缺氧诱导的细胞凋亡的易感性降低。NEP替代增加NEP无效PA SMC对缺氧诱导的细胞凋亡的易感性。在缺乏NEP的情况下，对凋亡的保护与凋亡相关激酶和应激蛋白的上调有关。我们也有证据表明NEP可能通过其细胞质尾部与多种尚未确定的蛋白质进行非酶性相互作用。以下假设将被验证：#1)NEP保护肺血管免受缺氧PHTN的发展，并至少部分地通过增加PA SMC对缺氧诱导的细胞凋亡的易感性来限制血管重塑；#2) NEP通过降低在凋亡调控中重要的激酶（重点：PI3激酶、Akt、ERK、p38、jun激酶和PKC-delta）的活性和应激蛋白（重点：热休克蛋白；HSPs）的表达，增加PA SMC对缺氧诱导的凋亡的易感性；#3) NEP通过肽酶依赖和肽酶不依赖的机制参与这些选定的激酶和热休克蛋白的调控，并促进缺氧诱导的PA SMC凋亡。综合实验将在野生型、NEP缺失和靶向NEP过表达小鼠、肺组织和分离的PA SMC中进行。野生型和突变型NEP的慢病毒构建体将用于分析肽酶依赖性和新型肽酶非依赖性作用，并提供有关调节PA SMC对凋亡敏感性的机制的新信息。这些研究还将利用缺氧诱导的肺血管重构的独特小鼠模型来增加我们对控制慢性缺氧PHTN易感性的机制的理解，并可以确定新的治疗靶点来限制或逆转这一重要的临床问题。

DESCRIPTION (provided by applicant): In large animal models of hypoxic pulmonary hypertension (PHTN) that closely parallel human disease, the earliest pulmonary artery (PA) smooth muscle (SMC) structural changes occur at the medial/adventitial border. Distal muscularization of vessels is also a prominent feature. Mechanisms that regulate the accumulation of SMCs in remodeled pulmonary vessels are still unclear. Susceptibility to apoptosis is likely an important determinant. Neprilysin (NEP) is a key cell surface peptidase involved in the degradation of vasoactive neuropeptides. Less well appreciated is the emerging concept that NEP may also influence cell responses by directly engaging in intracellular signaling through novel peptidase-independent mechanisms. NEP has recently been implicated in apoptosis of cancer cells. These observations support the possibility that NEP could exert a protective effect against hypoxic PHTN by increasing susceptibility of PA SMC to hypoxia-induced apoptosis. Loss of NEP would then predispose to exaggerated pulmonary vascular remodeling in response to hypoxia by protecting PA SMC from apoptosis. We now have data to support this concept. We have found that deletion of NEP in mice predisposes to exaggerated hypoxic PHTN. Lungs and isolated PA SMC from NEP null mice have decreased susceptibility to hypoxia-induced apoptosis compared to wild type controls. NEP replacement increases susceptibility of NEP null PA SMC to hypoxia-induced apoptosis. Protection from apoptosis in the absence of NEP is associated with up-regulation of apoptosis-associated kinases and stress proteins. We also have evidence that NEP may be interacting nonenzymatically through its cytoplasmic tail with multiple as yet unidentified proteins. The following Hypotheses will be tested: #1) NEP protects the lung vasculature from the development of hypoxic PHTN and limits vascular remodeling at least in part by increasing susceptibility of PA SMC to hypoxia-induced apoptosis; #2) NEP increases susceptibility of PA SMC to hypoxia-induced apoptosis by decreasing activity of selected kinases (focus: PI3 kinase, Akt, ERK, p38, jun kinase and PKC-delta) and expression of stress proteins (focus: heat shock proteins; HSPs) important in the regulation of apoptosis; #3) NEP contributes to the regulation of these selected kinases and HSPs and promotes hypoxia-induced apoptosis of PA SMC by both peptidase-dependent and peptidase-independent mechanisms. Integrated experiments will be performed in wild type, NEP null and targeted NEP overexpressor mice, lung tissue and isolated PA SMC. Lentiviral constructs for wild type and mutant forms of NEP will be used to dissect peptidase-dependent vs novel peptidase-independent effects and provide new information on mechanisms that regulate susceptibility of PA SMC to apoptosis. These studies will also draw on unique mouse models of hypoxia-induced pulmonary vascular remodeling to increase our understanding of mechanisms that control susceptibility to chronic hypoxic PHTN and could identify new therapeutic targets to limit or reverse this important clinical problem.