Neprilysin Apoptosis and Hypoxic Pulmonary Hypertension

描述（由申请方提供）：在与人类疾病密切相似的缺氧性肺动脉高压（PHTN）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。血管远端肌化也是一个显著特征。平滑肌细胞在重塑肺血管中的聚集调节机制尚不清楚。细胞凋亡的易感性可能是一个重要的决定因素。脑啡肽酶（NEP）是一种参与血管活性神经肽降解的关键细胞表面肽酶。不太好理解的是新出现的概念，即NEP也可能通过新的肽酶独立机制直接参与细胞内信号传导来影响细胞反应。NEP最近被认为与癌细胞的凋亡有关。这些观察结果支持NEP可能通过增加PA SMC对缺氧诱导的凋亡的敏感性来对缺氧PHTN发挥保护作用。NEP的丧失则通过保护PA SMC免于凋亡而易诱发对缺氧反应的过度肺血管重塑。我们现在有数据支持这一概念。我们已经发现，小鼠中NEP的缺失倾向于过度缺氧PHTN。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敏感性降低。NEP替代增加了NEP缺失的PA SMC对缺氧诱导的凋亡的敏感性。在NEP不存在的情况下，对细胞凋亡的保护与细胞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可能通过其胞质尾区与多种尚未鉴定的蛋白质进行非酶促相互作用。将检验以下假设：#1）NEP通过增加PA SMC对缺氧诱导的细胞凋亡的易感性，至少部分地保护肺血管系统免受缺氧PHTN的发展，并限制血管重塑; #2）NEP通过降低选定激酶的活性，增加PA SMC对缺氧诱导的细胞凋亡的易感性（重点：PI 3激酶、Akt、ERK、p38、jun激酶和PKC-δ）和应激蛋白的表达（重点：热休克蛋白;热休克蛋白）重要的调节细胞凋亡;#3）NEP有助于调节这些选定的激酶和HSP，并通过肽酶依赖性和肽酶非依赖性机制促进缺氧诱导的PA SMC凋亡。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中进行综合实验。野生型和突变形式的NEP的慢病毒构建体将被用于解剖肽酶依赖性与新的肽酶独立的影响，并提供新的信息的机制，调节PA SMC对细胞凋亡的敏感性。这些研究还将利用缺氧诱导的肺血管重塑的独特小鼠模型，以增加我们对控制慢性缺氧PHTN易感性的机制的了解，并可以确定新的治疗靶点来限制或扭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.