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）通常与肺部肌肉化和肺动脉微灌注回路阻塞有关。我们认为PAH的病理生物学代表了一种功能失调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TGF？1在肺中的信号。由于血清反应因子（SRF）和TGF之间不受控制的协同相互作用，伤口愈合基因的过度转录激活？1调节的Smad蛋白2和3导致动脉周围肌成纤维细胞（MFB）分化加速，并伴有肺动脉顺应性丧失和最终的右心衰。Smads 2和3通常解离基因抑制SRF-Pur ？蛋白质复合物能激活平滑肌吗？肌动蛋白(SM ?A)和I型胶原蛋白？2亚基启动子作为MFB分化过程的第一步。我们将验证SRF-Pur？由于PI3K/Akt前馈信号激酶过度活跃和/或由次优MEK1/Erk1,2/Egr-1信号介导的反馈抑制受损，pah衍生的mfb抑制复合物不稳定。在Aim 1中，我们建议使用免疫细胞化学方法表征与IPAH/FPAH综合征中动脉周围肌成纤维细胞分化和重塑相关的转录激活因子和抑制因子的亚细胞区隔化。在Aim 2中，我们将使用针对SRF-Pur的表观遗传/代谢方法，定义导致IPAH/FPAH综合征中动脉周围肌成纤维细胞过度分化的生化功能障碍。从正常或患病供体分离的肺动脉内皮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，这些相互作用独特地调节了外源性MFB分化过程。在MFBs中，能够触发原型基因反应的特殊转录调控复合物的组装代表了复杂血管疾病信号的汇聚点，该信号由多个代偿性和患者特异性控制层组成。我们期望获得的知识可以进一步了解限速相互作用，这种相互作用促进动脉顺应性丧失，通常与最具破坏性的IPAH/FPAH疾病综合征相关。未来对激活因子-抑制因子动态相互作用的蛋白质生化详细分析可能揭示肺动脉疾病和右心衰治疗管理的新靶点，最终可能提高患者的长期生存率。

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)