Role of Skeletal Muscle in Pulmonary Vascular Remodeling

项目总结 肺动脉高压伴心衰并保留射血分数(PH-HFpEF)是最多的 PH是世界范围内的共同病因，目前还没有被证明有效的治疗方法。此前，我们有 发现令人信服的证据表明，PH-HFpEF调节sirtuin-3(SIRT3)在 骨骼肌。在过去的资助期间，我们发现了一个新的内分泌信号轴，它连接着抑郁 骨骼肌SIRT3对与肺血管系统相互作用的信号分子的产生/分泌 PH-HFpEF组。然而，骨骼肌SIRT3介导的Scretome调节的潜在机制 肺血管的病理改变目前还不清楚。 基于基于质谱学的血浆蛋白质组学方法，我们的新数据显示2-微球蛋白 (B2M)在PH-HFpEF患者中水平较高。循环和骨骼肌中的B2M水平 在骨骼肌SIRT3缺乏的小鼠中显著增加。血浆和肌肉活组织检查 PH-HFpEF患者的验证队列也显示，随着疾病严重程度的增加，B2M水平也更高。 经B2M重组蛋白处理后WW结构域的氧化还原酶表达降低 (WWOX)，它在PASMC中起到抑制细胞增殖的作用。B2M治疗也增加了 冠层成纤维细胞生长因子信号调节因子2(CNPY2)的表达--一个未折叠的蛋白应答 (UPR)启动剂，已知可在PAECs中诱导过度的血管生成和增殖。而B2M治疗 增加PAECs的迁移/增殖和CNPY2过表达的PAECs调节的介质减少 WWOX在PASMCs中，PAECs CNPY2在PH-HFpEF中的作用仍不清楚。因此，我们假设 骨骼肌分泌的B2M作为内分泌信号分子诱导远隔肺 PAECs CNPY2和PASMCs WWOX失调导致的血管重塑。 特异性目标1将确定骨骼肌B2M是否是影响多个 肺血管病理表型。使用野生型和骨骼肌特异性B2M KO小鼠，具有或 没有高脂喂养(HFD)，导致PH-HFpEF表型，我们将表征 PH-HFpEF中的骨骼肌B2M。在目标2中，我们将确定PAECs CNPY2在肺血管中的作用 EC特异性Cnpy2KO小鼠的重塑和PH-HFpEF。《特定目标3》将使用血浆和肌肉 从生物库和II期临床试验收集的PH-HFpEF或单独患有HFpEF的患者的活检 评估B2M是否可以作为PH-HFpEF严重程度和预后的相关生物标志物。可诱导的全能 PH-HFpEF患者的干细胞(IPSC)来源的内皮细胞也将用于评估CNPY2的临床相关性。 我们预计，拟议研究计划的成功执行将提供新的机制 了解PH-HFpEF的发病机制，有助于为指导未来的研究提供重要信息 对于看起来无法治愈的PH-HFpEF的治疗方法的发展。

PROJECT SUMMARY Pulmonary hypertension associated with heart failure with preserved ejection fraction (PH-HFpEF) is the most common cause of PH worldwide, and at present, it has no proven effective therapies. Previously, we have found convincing evidence that PH-HFpEF-regulating sirtuin-3 (SIRT3) is predominantly decreased in the skeletal muscle. During the past grant period, we found a new endocrine signaling axis that links depressed skeletal muscle SIRT3 to production/secretion of signaling molecules that interact with pulmonary vasculature in PH-HFpEF. However, underlying mechanisms by which skeletal muscle SIRT3-mediated scretome regulates pathological changes in pulmonary vasculature are not well-understood at present. Based on a mass spectrometry-based plasma proteomics approach, our new data show that 2-microglobulin (B2M) is present at higher levels in patients with PH-HFpEF. Both circulating and skeletal muscle levels of B2M are markedly increased in mice with skeletal muscle SIRT3 deficiency. Plasma and muscle biopsies from a validation cohort of PH-HFpEF patients also show higher B2M levels with increased disease severity. Treatment with B2M recombinant protein reduces expression of WW domain-containing oxidoreductase (WWOX), which plays a role in repressing cell proliferation, in PASMCs. B2M treatment also increases expression of canopy fibroblast growth factor signaling regulator 2 (CNPY2), an unfolded protein response (UPR) initiator known to induce excessive angiogenesis and proliferation, in PAECs. While B2M treatment increases PAECs migration/proliferation and media conditioned by CNPY2-overexpressed PAECs reduces WWOX in PASMCs, the role of PAECs CNPY2 in PH-HFpEF remains elusive. Thus, we hypothesize that skeletal muscle-secreted B2M acts as an endocrine signaling molecule which induces remote pulmonary vascular remodeling via dysregulating PAECs CNPY2 and PASMCs WWOX. Specific Aim 1 will determine whether skeletal muscle B2M is an important signaling molecule affecting multiple pulmonary vascular pathophenotypes. Using wild-type and skeletal muscle-specific B2m KO mice, with or without high-fat feeding (HFD), which induces a PH-HFpEF phenotype, we will characterize the contribution of skeletal muscle B2M in PH-HFpEF. In Aim 2, we will determine the role of PAECs CNPY2 in pulmonary vascular remodeling and PH-HFpEF with EC-specific Cnpy2 KO mice. Specific Aim 3 will use plasma and muscle biopsies of patients with PH-HFpEF or HFpEF alone collected from biobanks and Phase II clinical trials to evaluate whether B2M can be a relevant biomarker of PH-HFpEF severity and prognosis. Inducible pluripotent stem cell (iPSC)-derived ECs of PH-HFpEF patients will also be used to evaluate clinical relevance of CNPY2. We expect that successful execution of the proposed research plan would provide new mechanistic understanding of PH-HFpEF pathogenesis and aid in providing important information to guide future research towards development of therapeutic approaches for the presentably untreatable PH-HFpEF.