NEP and susceptibility to hypoxic pulmonary hypertension

描述(由申请人提供)： 在与人类疾病密切相关的缺氧性肺动脉高压(PHTN)的大型动物模型中，最早的肺动脉(PA)平滑肌细胞(SMC)增殖改变发生在内侧/外膜交界处。SMC和/或肌成纤维细胞向更远端血管的迁移也是一个显著的特征。还没有描述一种能够充分模拟这些缺氧诱导的变化的小鼠模型。中性内肽酶(NEP；或neprilysin)是一种重要的细胞表面多肽酶，可降解血管活性神经肽(如蛙皮素样肽[Blps])，可能促进血管重塑。NEP还被证明通过新的不依赖于多肽酶的机制直接参与细胞内信号传递。最后，NEP最近被认为与减少炎症、癌变和生长有关。这些观察结果支持这样一种可能性，即在肺中(与 NEP对缺氧性PHTN的易感性具有保护作用。我们现在有强有力的初步证据来支持这一概念。我们发现，在小鼠中，NEP的靶向缺失容易导致过度的缺氧性PHTN。由此产生的结构变化比先前描述的小鼠模型和 首次显示内/外膜交界处的近端改变。招聘 去分化的SMC或肌成纤维细胞进入远端循环也是一个主要特征。这种独特的血管重塑模式，以及文献中耐人寻味的观察，表明BLP、BLP受体、特定的蛋白激酶C同工酶(PKCα、Delta和epsilon)、Rho和粘着斑激酶(FAK)发挥着关键作用。将检验以下假设：#1)NEP保护肺血管系统免受缺氧性PHTN的发展，并通过抑制PA SMC的增殖、迁移和收缩来限制血管重构。#2)选定的神经肽(最初的焦点：BLP)是慢性缺氧性NEP基因敲除(KO)中观察到的夸大肺血管重构的主要原因 老鼠。低氧诱导的BLPs和BLP受体上调是其升高的原因之一 内侧/外膜改变。#3)上调BLP受体后信号中间产物(PKC a， Delta，以及epsilon，Rho和FAK)导致了夸张的重建。NEP抑制这些 多肽酶在PA-SMC信号转导及增殖和迁移反应中的作用 依赖和独立的机制。综合实验将在单个和 双KO小鼠，灌流肺，离体肺动脉和PA SMC。这些研究将借鉴一种独特的低氧诱导的肺血管重塑小鼠模型。 为了增加我们对控制缺氧性PHTN易感性的机制的了解，并可以找到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 cell (SMC) proliferative changes occur at the medial/adventitial border. Migration of SMC and/or myofibroblasts to more distal vessels is also a prominent feature. A murine model that adequately mimics these hypoxia-induced changes has not been described. Neutral endopeptidase (NEP; or neprilysin) is an important cell surface peptidase that degrades vasoactive neuropeptides (like the bombesin-like peptides [BLPs]), that may promote vascular remodeling. NEP has also been shown to directly engage in intracellular signaling by novel peptidase independent mechanisms. Finally, NEP has recently been associated with decreased inflammation, carcinogenesis and growth. These observations support the possibility that in the lung (in contrast to the heart and systemic vasculature) NEP could exert a protective effect on susceptibility to hypoxic PHTN. We now have strong preliminary evidence to support this concept. We have found that targeted deletion of NEP in mice predisposes to exaggerated hypoxic PHTN. The resulting structural changes are more substantial than in previously described mouse models and for the first time demonstrate proximal changes at the medial/adventitial border. Recruitment of dedifferentiated SMC or myofibroblasts into the distal circulation is also a major feature. This unique pattern of vascular remodeling, together with intriguing observations in the literature, suggest key roles for BLPs, BLP receptors, selected isozymes of protein kinase C (PKC alpha, delta, and epsilon), Rho, and focal adhesion kinase (FAK). The following hypotheses will be tested: #1) NEP protects the lung vasculature from the development of hypoxic PHTN and limits vascular remodeling by suppressing the proliferation, migration, and contraction of PA SMC. #2) Selected neuropeptides (initial focus: BLPs) are largely responsible for the exaggerated pulmonary vascular remodeling observed in the chronically hypoxic NEP knockout (KO) mouse. Hypoxia- induced upregulation of BLPs and BLP receptors contributes to the increased medial/adventitial changes. #3) Upregulation of BLP post-receptor signaling intermediates (PKC a, delta , and epsilon, Rho, and FAK) contributes to the exaggerated remodeling. NEP inhibits these signaling intermediates as well as proliferation and migratory responses of PA SMC by peptidase dependent and independent mechanisms. Integrated experiments will be performed in single and double KO mice, perfused lungs, isolated pulmonary arteries and PA SMC. These studies will draw on a unique mouse model of hypoxia-induced pulmonary vascular remodeling to increase our understanding of the mechanisms that control susceptibility to hypoxic-PHTN and could identify new therapeutic targets to limit or reverse this important clinical problem.