NEP and susceptibility to hypoxic pulmonary hypertension

描述（由申请人提供）： 在与人类疾病密切相似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）增殖变化发生在内/外膜边缘。SMC和/或肌成纤维细胞向更远端血管的迁移也是一个突出特征。尚未描述充分模拟这些缺氧诱导的变化的鼠模型。中性内肽酶（NEP;或脑啡肽酶）是一种重要的细胞表面肽酶，其降解可促进血管重塑的血管活性神经肽（如铃蟾肽样肽[BLP]）。NEP还被证明通过新的肽酶非依赖性机制直接参与细胞内信号传导。最后，NEP最近与减少炎症，致癌和生长有关。这些观察结果支持了在肺中（与肺相比） 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支持这一概念。 我们已经发现，小鼠中NEP的靶向缺失倾向于过度缺氧PHTN。所产生的结构变化比先前描述的小鼠模型中的结构变化更显著，并且 第一次显示内/外膜边界的近端变化。招募 去分化的SMC或肌成纤维细胞进入远端循环也是主要特征。这种独特的血管重塑模式，以及文献中有趣的观察结果，表明BLP、BLP受体、蛋白激酶C（PKC α、δ和β）的选定同工酶、Rho和粘着斑激酶（FAK）的关键作用。 将测试以下假设：#1）NEP通过抑制PA SMC的增殖、迁移和收缩来保护肺脉管系统免受缺氧PHTN的发展并限制血管重塑。#2）选定的神经肽（初始焦点：BLP）主要负责在慢性缺氧NEP敲除（KO）中观察到的夸大的肺血管重塑。 老鼠.缺氧诱导的BLP和BLP受体的上调有助于增加 中膜/外膜变化。#3）BLP受体后信号传导中间体（PKC α， δ，和α，Rho，和FAK）有助于夸大的重塑。NEP抑制这些 信号中间体以及PA SMC对肽酶的增殖和迁移反应 依赖和独立机制。综合实验将在单一和 双KO小鼠、灌注肺、分离的肺动脉和PA SMC。这些研究将利用一种独特的缺氧诱导肺血管重构的小鼠模型 增加我们对控制缺氧性PHTN易感性的机制的理解，并可以确定新的治疗靶点，以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.