Identifying the Serine Elastase Elevated in Pulmonary Vascular Disease

描述（由申请人提供）：本R21的主要目的是使用一种新的策略从肺组织中纯化小鼠内源性血管弹性酶（EVE）并鉴定其人类同源物。这很重要，因为我们已经将EVE与实验和临床肺血管改变和肺动脉高压（PAH）的进展联系起来。我们还表明，EVE的抑制足以逆转实验诱导的PAH，我们现在提供的初步数据表明，EVE的抑制可以逆转PAH患者接受肺移植获得的培养肺外植体的晚期新内膜病变。因此，确定EVE的蛋白结构对于确定其在PAH中的作用和开发选择性抑制剂至关重要。我们计划在感染鼠型卡波西肉瘤病毒（MHV-68）一周后，从过表达S100A4的转基因小鼠肺中纯化EVE。感染后以及3个月后病毒再次激活后，肺部丝氨酸弹性酶活性升高与与临床PAH患者相似的严重新内膜病变有关。除了模拟PAH的肺血管病理外，感染MHV-68的S100A4小鼠在其他方面与临床PAH相关。与MHV-68同源的病毒抗原与人类多环芳烃有关。PAH和晚期新生内膜病变患者肺动脉中S100A4表达升高，S100A4刺激培养的人肺动脉平滑肌细胞增殖和迁移。我们的建议有三个具体目标。Aim 1试图使用flag标记的重组elafin（一种高亲和力结合EVE的抑制剂）从S100A4小鼠肺中纯化EVE。我们将利用质谱（MS/MS MALDI-TOF）分析的肽序列信息来鉴定编码EVE的全长cDNA。这些肽和cDNA序列将用于制造抗体和核糖探针来定位肺中的酶。目的2利用小鼠EVE序列信息检索数据库，鉴定或克隆人类同源物，定位PAH患者肺组织中的EVE。该R21的次要目的是观察到与C57BL6相比，S100A4肺中miR -155水平的降低与MHV-68反应时EVE升高之间可能存在联系。这一概念源于这样一个事实，即丝氨酸弹性酶的转录因子PU.1是miR-155的假定靶标。Aim 3旨在确定miR- 155敲除小鼠在接种MHV-68后是否表现出更高的EVE活性，以及这是否反过来导致肺血管疾病。综上所述，这些研究将建立EVE的蛋白鉴定，并为赋予宿主易感性的遗传因子（S100A4基因）如何与环境因子（MHV-68病毒）相互作用导致肺血管疾病提供新的见解。

DESCRIPTION (provided by applicant): The primary objective of this R21 is to use a novel strategy to purify murine endogenous vascular elastase (EVE) from lung tissue and to identify its human homolog. This is important because we have linked EVE to the progression of experimental and clinical pulmonary vascular changes and pulmonary hypertension (PAH). We have also shown that inhibition of EVE is sufficient to reverse experimentally induced PAH and we now present preliminary data indicating that inhibition of EVE can regress advanced neointimal lesions in cultured explants of lung obtained from PAH patients undergoing lung transplant. Thus, determining the protein structure of EVE is essential in defining its role in PAH and in developing selective inhibitors. We plan to purify EVE from the lungs of transgenic mice that overexpress S100A4 one week after these mice have been infected with a murine form of the Kaposi Sarcoma virus (MHV-68). Elevation of serine elastase activity in lung following infection, and after re-activation of the virus three months later, is associated with severe neointimal lesions similar to those seen in patients with clinical PAH. Besides mimicking the pulmonary vascular pathology of PAH, the S100A4 mouse infected with MHV-68 is relevant to clinical PAH in other ways. Viral antigens homologous to MHV-68 have been linked to human PAH. There is heightened S100A4 expression in pulmonary arteries from patients with PAH and advanced neointimal lesions, and S100A4 stimulates proliferation and migration of cultured human pulmonary artery smooth muscle cells. Our proposal has three specific aims. Aim 1 seeks to purify EVE from the S100A4 mouse lung using FLAG-tagged recombinant elafin, the inhibitor that binds EVE with high affinity. We will use peptide sequence information from mass spectrometry (MS/MS MALDI-TOF) analyses to identify the full-length cDNA encoding EVE. The peptide and cDNA sequences will be used to make antibodies and riboprobes to localize the enzyme in the lung. Aim 2 applies sequence information from murine EVE to search databases to identify or clone the human homolog, and to localize EVE in lung tissue from PAH patients. The secondary objective of this R21 pursues the observation that there might be a link between reduced levels of microRNA (miR)-155 in S100A4 compared to C57BL6 lungs, and heightened EVE in response to MHV-68. This notion derives from the fact that PU.1, a transcription factor for serine elastase, is a putative target of miR-155. Aim 3 seeks to determine whether the miR- 155 knockout mouse exhibits heightened EVE activity following MHV-68 inoculation, and if this in turn leads to pulmonary vascular disease. Taken together, these studies will establish the protein identify of EVE, and provide new insight into how a genetic factor that confers host susceptibility (S100A4 gene) interacts with an environmental factor (MHV-68 virus) to cause pulmonary vascular disease. PUBLIC HEALTH RELEVANCE: Our group has shown that an enzyme called endogenous vascular elastase (EVE) plays a pivotal role in the pathogenesis of pulmonary arterial hypertension (PAH) and that inhibition of EVE may be a strategy to reverse the disease. We therefore plan to establish the structure of both the murine form of EVE and of the human enzyme, so that in the future highly targeted therapies can be developed to inhibit its activity. We also propose to determine whether EVE may be regulated by a microRNA and whether the level of this microRNA determines host susceptibility to virus-induced pulmonary vascular disease.