Peri-arteriolar Myofibroblast Differentiation in the Pathobiology of IPAH

描述(由申请人提供)：特发性和家族性肺动脉高压综合征(IPAH/FPAH)通常与肺内肌化和肺动脉微灌流回路阻塞有关。我们认为，PAH的病理生物学代表了一种功能障碍的血管周围伤口愈合反应，其基础是最近发现的Pur？DNA结合蛋白抑制肺中的转化生长因子？1信号。由于血清反应因子(SRF)和转化生长因子β1调节的Smad蛋白2和3之间的协同作用，创伤修复基因的过度转录激活导致动脉周围肌成纤维细胞(MFB)分化加速和外膜纤维化，从而丧失肺动脉顺应性，最终导致右心衰竭。Smads 2和Smads 3通常会解离基因抑制基因SRF-pur？作为MFB分化过程的第一步，蛋白质复合体允许激活平滑肌肌动蛋白(SM？A)和I型胶原？2亚单位启动子。我们将检验SRF-PUR？在多环芳烃衍生的MFBs中，抑制复合体是不稳定的，这是由于PI3K/Akt前馈信号通路过度活跃和/或次优MEK1/Erk1，2/Egr-1信号介导的反馈抑制减弱所致。在目标1中，我们建议用免疫细胞化学方法来表征参与IPAH/FPAH综合征小动脉周围肌成纤维细胞分化和重塑的转录激活物和抑制物的亚细胞区划。对于目标2，我们将使用以SRF-Pur？为靶点的表观遗传/代谢方法来定义导致IPAH/FPAH综合征小动脉周围肌成纤维细胞过度分化的生化功能障碍。分离自正常或疾病供者的肺动脉外膜成纤维细胞的物理相互作用。我们已经开发了固相ELISA工具来定量评估蛋白质：蛋白质和蛋白质：DNA相互作用，这种相互作用独特地调节外膜MFB的分化过程。能够在MFBs中触发原型基因反应的专门转录调控复合体的组装代表了复杂的血管疾病信号的汇聚点，包括多个代偿层和患者特定的控制层。我们预计，所获得的知识将进一步加深对限速相互作用的基本了解，这些相互作用导致动脉顺应性丧失，通常与最具破坏性的IPAH/FPAH疾病综合征有关。未来对激活物-抑制物动态相互作用的蛋白质生物化学的详细分析可能会揭示肺动脉疾病和右心衰竭的治疗管理的新靶点，最终可能提高患者的长期存活率。 公共卫生相关性：血管壁中的基因激活由蛋白质：蛋白质和蛋白质：DNA复合体控制，这些复合体是感染或物理创伤后伤口快速愈合和组织修复所必需的。当这些过程在心肺系统中变得无法手术或功能障碍时，多余的疤痕组织会积聚并使非常小的血管僵硬，最终减缓正常血流，导致血栓形成、中风、组织梗塞(降解)和猝死。控制肺部伤口愈合的蛋白质复合体可能在肺动脉高压患者中出现故障，如果不进行治疗，最终将损害心脏的右侧，心脏的功能是通往肺的血泵。这项拟议的研究将检查这些调节复合体的蛋白质成分，并确定与诊断为终末期肺动脉高压的患者相比，正常个体的它们是否不同。这项拟议的研究可能会导致为这种毁灭性的肺血管疾病设计新的治疗策略，这种疾病无法治愈，只能通过进行肺移植手术来治疗。(摘要结束)

DESCRIPTION (provided by applicant): Idiopathic and familial syndromes of pulmonary arterial hypertension (IPAH/FPAH) typically are associated with muscularization and obstruction of pulmonary arterial microperfusion circuits in the lung. We propose that the pathobiology of PAH represents a dysfunctional, peri-vascular wound healing response based on a functional deficit in the ability of the recently discovered Pur ? DNA-binding protein to repress TGF?1 signaling in the lung. Excessive transcriptional activation of wound-healing genes due to unchecked collaborative interaction between serum response factor (SRF) and TGF?1-regulated Smad proteins 2 and 3 results in accelerated peri-arteriolar myofibroblast (MFB) differentiation and adventitial fibrosis with loss of pulmonary arterial compliance and eventual right heart failure. Smads 2 and 3 normally dissociate gene-inhibitory SRF-Pur ? protein complexes to allow activation of the smooth muscle ?-actin (SM?A) and type I collagen ?2-subunit promoters as a first step in the MFB differentiation process. We will test the hypothesis that the SRF-Pur? inhibitory complex is unstable in PAH-derived MFBs due to over-active PI3K/Akt feed-forward signaling kinases and/or impaired feed-back inhibition mediated by sub-optimal MEK1/Erk1,2/Egr-1 signaling. In Aim 1, we propose to characterize the sub-cellular compartmentalization of transcriptional activators and repressors implicated in peri-arteriolar myofibroblast differentiation and remodeling in IPAH/FPAH syndromes using an immunocytochemistry approach. For Aim 2, we will define the biochemical dysfunction that causes excess peri-arteriolar myofibroblast differentiation in IPAH/FPAH syndromes using epigenetic/metabolic approaches that target SRF-Pur ? physical interplay in pulmonary artery adventitial fibroblasts isolated from normal or disease-affected donors. We have developed solid-phase ELISA tools to quantitatively evaluate protein:protein and protein:DNA interactions that uniquely regulate the process of adventitial MFB differentiation. The assembly of a specialized transcriptional regulatory complex capable of triggering prototypical gene responses in MFBs represents a convergence point for complex vascular-disease signaling consisting of multiple compensatory and patient-specific layers of control. We expect that knowledge gained could further basic understanding of rate-limiting interactions that foster loss of arterial compliance typically associated with the most devastating IPAH/FPAH disease syndromes. Future detailed analysis of the protein biochemistry of activator-repressor dynamic interplay could reveal novel targets for therapeutic management of pulmonary arterial disease and right heart failure that may ultimately improve patient long-term survival. PUBLIC HEALTH RELEVANCE: Gene activation in the blood vessel wall is controlled by protein:protein and protein:DNA complexes that are necessary for rapid wound healing and tissue repair after infection or physical trauma. When these processes become inoperative or dysfunctional in the cardiopulmonary system, excess scar tissue can accumulate and stiffen very small blood vessels that eventually slows normal blood flow leading to clot formation, stroke, tissue infarction (degradation), and sudden death. Protein complexes that control wound healing in the lung may be faulty in patients with pulmonary hypertension which if untreated will eventually damage the right side of the heart that functions as the blood pump to the lung. The proposed research will examine the protein constituents of these regulatory complexes and determine if they are different in normal individuals compared to patients diagnosed with end-stage pulmonary hypertension. The proposed research may lead to the design of new treatment strategies for this devastating lung vascular disease that has no cure and only treatable by performing lung transplantation surgery. (End of Abstract)