Identifying the Serine Elastase Elevated in Pulmonary Vascular Disease

描述（由申请人提供）：该R21的主要目的是使用新策略从肺组织中纯化鼠内源性血管弹性蛋白酶（EVE）并鉴定其人同系物。这一点很重要，因为我们已经将EVE与实验和临床肺血管变化和肺动脉高压（PAH）的进展联系起来。我们还表明，抑制EVE足以逆转实验诱导的PAH，我们现在提供的初步数据表明，抑制EVE可以使从接受肺移植的PAH患者中获得的肺培养外植体中的晚期新生内膜病变消退。因此，确定EVE的蛋白质结构对于确定其在PAH中的作用和开发选择性抑制剂至关重要。我们计划从过表达S100 A4的转基因小鼠的肺中纯化EVE，这些小鼠在感染鼠型卡波西肉瘤病毒（MHV-68）一周后。感染后肺中丝氨酸弹性蛋白酶活性的升高，以及3个月后病毒再活化后，与重度新生内膜病变相关，与临床PAH患者中观察到的病变相似。除了模拟PAH的肺血管病理学，感染MHV-68的S100 A4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PAH相关。在PAH和晚期新生内膜病变患者的肺动脉中，S100 A4表达增加，并且S100 A4刺激培养的人肺动脉平滑肌细胞的增殖和迁移。我们的建议有三个具体目标。目的1寻求使用FLAG标记的重组弹力素（elafin）从S100 A4小鼠肺中纯化EVE，弹力素是以高亲和力结合EVE的抑制剂。我们将使用质谱（MS/MS MALDI-TOF）分析的肽序列信息来鉴定编码EVE的全长cDNA。肽和cDNA序列将用于制备抗体和核糖核酸探针，以定位肺中的酶。目的2利用小鼠EVE的序列信息检索数据库，鉴定或克隆人EVE同源物，并在肺动脉高压患者肺组织中定位EVE。该R21的次要目的是观察到与C57 BL 6肺相比，S100 A4中microRNA（miR）-155水平降低与MHV-68应答EVE升高之间可能存在联系。这一概念源于这样的事实，即PU.1是丝氨酸弹性蛋白酶的转录因子，是miR-155的推定靶点。目的3旨在确定miR- 155敲除小鼠在接种MHV-68后是否表现出增强的EVE活性，以及这是否反过来导致肺血管疾病。总之，这些研究将建立EVE的蛋白质鉴定，并提供新的见解，赋予宿主易感性的遗传因素（S100 A4基因）如何与环境因素（MHV-68病毒）相互作用，导致肺血管疾病。 公共卫生相关性：我们的小组已经表明，一种称为内源性血管弹性蛋白酶（EVE）的酶在肺动脉高压（PAH）的发病机制中起着关键作用，抑制EVE可能是逆转该疾病的策略。因此，我们计划建立小鼠形式的EVE和人类酶的结构，以便将来可以开发高度靶向的治疗方法来抑制其活性。我们还建议确定EVE是否可能受到microRNA的调节，以及这种microRNA的水平是否决定宿主对病毒诱导的肺血管疾病的易感性。

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.