Calpain Activates Intracellular TGF-beta1 in Pulmonary Hypertension

描述（申请人提供）：肺动脉高压（PAH）是一种严重的进行性疾病，其关键特征是肺血管重塑，与细胞外基质（包括胶原蛋白）的积累和血管细胞增殖、中膜和外膜增厚、闭塞性新生内膜和复杂的丛状病变相关，导致毛细血管前肺动脉闭塞和肺动脉压持续升高。PDGF、EGF、VEGF和TGF？1参与肺血管重构过程。我们最近首次报道了钙蛋白酶的整体敲除可预防缺氧诱导的肺动脉高压的肺血管重构，钙蛋白酶抑制剂MDL 28170可预防野百合碱诱导的肺动脉高压的进展。钙蛋白酶是钙依赖性非溶酶体中性半胱氨酸内肽酶家族，其通过哺乳动物细胞（包括肺血管细胞）中底物蛋白的有限蛋白水解起作用。Calpain是PDGF、EGF和VEGF的下游信号转导分子，介导PDGF和EGF诱导的肺动脉平滑肌细胞（PASMCs）胶原合成和增殖以及VEGF诱导的肺动脉内皮细胞（PAECs）血管生成。我们最近发现，钙蛋白酶切割和激活TGF？1和内分泌TGF？PASMCs内存在信号通路。为了验证钙蛋白酶在动物模型肺血管重塑中的作用，我们将利用肺动脉高压突破倡议（PHBI）提供的人肺组织，并测试我们的新假设，钙蛋白酶激活细胞内TGF？1例与IPAH相关的肺血管重构。为了验证这一假设，我们提出了一个多学科的方法，重点放在以下具体目标：#1是确定是否钙蛋白酶抑制预防或减弱胶原合成和增殖的PASMCs和PAECs患者特发性肺动脉高压（IPAH）; #2是确定是否钙蛋白酶激活细胞内TGF？IPAH患者的PASMC和PAEC中有1例。肺血管细胞中钙蛋白酶调节胶原合成和增殖的证据可能会导致新的药理学策略的发展，这些策略将靶向与肺动脉高压相关的肺血管重塑中的钙蛋白酶。 公共卫生相关性：我们最近报道，整体钙蛋白酶敲除可预防缺氧诱导的肺动脉高压中的肺血管重塑，而钙蛋白酶抑制剂MDL 28170可预防野百合碱诱导的已建立的肺动脉高压的进展。该建议是研究一种新的假说，钙蛋白酶激活细胞内TGF？特发性肺动脉高压1例。这项研究对肺动脉高压领域的贡献有望导致新的药理学策略的发展，这些策略将靶向与肺动脉高压相关的肺血管重塑中的钙蛋白酶。该项目的成功完成将有助于为肺动脉高压的防治提供一种新的方法。

DESCRIPTION (provided by applicant): Pulmonary arterial hypertension (PAH) is a severe and progressive disease, a key feature of which is pulmonary vascular remodeling that is associated with accumulation of extracellular matrix, including collagen, and vascular cell proliferation, medial and adventitial thickening, occlusive neointima, and complex plexiform lesions, leading to obliteration of precapillary pulmonary arteries and sustained elevation of pulmonary arterial pressure. PDGF, EGF, VEGF, and TGF?1 participate in the process of pulmonary vascular remodeling. We recently reported for the first time that global knockout of calpain prevents pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension and that the calpain inhibitor MDL28170 prevents the progression of established pulmonary hypertension induced by monocrotaline. Calpain is a family of calcium-dependent non-lysosomal neutral cysteine endopeptidases that act via limited proteolysis of substrate proteins in mammalian cells including pulmonary vascular cells. Calpain is the downstream signal transduction molecule of PDGF, EGF and VEGF, and mediates PDGF- and EGF-induced collagen synthesis and proliferation of pulmonary artery smooth muscle cells (PASMCs) and VEGF-induced angiogenesis of pulmonary artery endothelial cells (PAECs). We have recently found that calpain cleaves and activates TGF?1 and that an intracrine TGF? signal pathway exists in PASMCs. To validate the role of calpain in pulmonary vascular remodeling in animal models, we will take advantage of the human lung tissues provided by the Pulmonary Hypertension Breakthrough Initiative (PHBI) and test our novel hypothesis that calpain activates intracellular TGF?1 in pulmonary vascular remodeling associated with IPAH. To test this hypothesis, we propose a multidisciplinary approach focusing on the following specific aims: #1 is to determine whether calpain inhibition prevents or attenuates collagen synthesis and proliferation of PASMCs and PAECs from patients with idiopathic pulmonary arterial hypertension (IPAH); #2 is to determine whether calpain activates intracellular TGF?1 in PASMCs and PAECs from patients with IPAH. Proof of regulation of collagen synthesis and proliferation by calpain in pulmonary vascular cells may lead to development of new pharmacologic strategies that will target calpain in pulmonary vascular remodeling associated with pulmonary hypertension. PUBLIC HEALTH RELEVANCE: We recently reported that global calpain knockout prevents pulmonary vascular remodeling in hypoxia- induced pulmonary hypertension and the calpain inhibitor MDL28170 prevents the progression of established pulmonary hypertension induced by monocrotaline. This proposal is to study a novel hypothesis that calpain activates intracellular TGF?1 in idiopathic pulmonary arterial hypertension. The contribution of the proposed research to the field of pulmonary hypertension is expected to be leading to development of new pharmacologic strategies that will target calpain in pulmonary vascular remodeling associated with pulmonary hypertension. Successful completion of this project will help provide a new method for the prevention and treatment of pulmonary hypertension.