Paracrine Action of BMP3 in Pulmonary Hypertension

项目摘要/摘要 骨形态发生蛋白3在肺动脉高压中的旁分泌作用 摘要肺动脉高压(PAH)是一种以持续性毛细血管前病变为特征的严重血管疾病。 肺动脉高压，导致右心衰竭和死亡。尽管在过去几十年里进行了密集的研究 PH仍然是一种发病率和死亡率都很高的不治之症。需要改进的新方向和新疗法 对PAH的认识和治疗是迫切需要的。 尽管有证据表明骨形态发生蛋白(BMP)信号通路的重要性 在PAH中，我们目前对这一途径的几个成员的确切病理生理作用的了解 仍然是有限的。在本R01中，我们建议进行详细的功能和机制研究，以了解 多环芳烃中的BMP3。我们假设PASMC来源的BMP3抑制PAEC功能障碍和病理 肺血管重塑。基于可靠的初步研究，我们建议测试这一中心 有三个具体目标的假设。 目的1明确BMP3在PAH中的调控及其在体外的作用。我们将评估肺BMP3的水平 临床型和非临床型PAH患者肺和血清BMP3水平及循环BMP3水平的研究 动物。我们还将确定性别是否影响人类和啮齿动物的肺BMP3表达，以及 我们将研究PASMC来源的BMP3在PASMC和PAEC共培养中对PAEC功能的影响 分离自非PAH和PAH患者。 目的2研究细胞特异性BMP3缺失和BMP3上调的体内效应。我们会 提供SMC和EC特异性BMP3 KO的心肺表型的详细特征 老鼠。我们还将在SUGEN/缺氧模型中检测外源性重组BMP3对PAH的影响 在老鼠身上。使用一种新的方法，我们已经证明这种方法对蛋白质是高度特异和有效的 在肺中过度表达(即化学修饰的mRNAs(ModRNAs))，我们将评估BMP3 在SUGEN/缺氧模型中，modRNA逆转PAH。 目的3将剖析BMP3的分子机制。我们将进行一系列的体外和体内研究 确定骨形态发生蛋白3的调节机制(S)，明确骨形态发生蛋白3的作用机制(S)。推动者 活性检测、免疫共沉淀实验、GST下拉检测、定量聚合酶链式反应测定 蛋白质印迹分析将确定BMP3信号所涉及的通路。PASMC和PASMCs的混合培养 BMP3-KO小鼠和大鼠体内过表达BMP3的PAECs和肺将确定 与BMP3信号有关的通路。

PROJECT ABSTRACT/SUMMARY Paracrine Action of BMP3 in Pulmonary Hypertension Pulmonary arterial hypertension (PAH) is a severe vascular disease characterized by persistent precapillary pulmonary hypertension, leading to right heart failure and death. Despite intense research in the last decades PH remains an incurable disease with a high morbidity and mortality. New directions and therapies to improve understanding and treatment of PAH are desperately needed. Although evidence demonstrates the importance of the Bone Morphogenetic Protein (BMP) signaling pathway in PAH, our current understanding of the exact pathophysiological role of several members of this pathway remains limited. In this R01 we propose detailed functional and mechanistic studies to understand the role of BMP3 in PAH. We hypothesize that PASMC-derived BMP3 inhibits PAEC dysfunction and pathological pulmonary vascular remodeling. Based on robust preliminary studies, we propose testing this central hypothesis with 3 specific aims. Aim 1 will define BMP3 regulation in PAH and its effects in vitro. We will assess the levels of pulmonary BMP3 levels and circulating BMP3 levels in lung and serum of patients with clinical PAH and of PAH-diseased animals. We will also determine whether sex affects pulmonary BMP3 expression in humans and rodents, and we will investigate the effect of PASMC-derived BMP3 on PAEC function in cocultures of PASMCs and PAECs isolated from non-PAH and PAH patients. Aim 2 will characterize the in vivo effects of cell-specific BMP3 deletion and of BMP3 upregulation. We will provide a detailed characterization of the cardiopulmonary phenotypes of SMC- and EC-specific BMP3 KO mice. We will also examine the effects of exogenous recombinant BMP3 on PAH in the Sugen/Hypoxia model in rats. Using a novel approach, which we have demonstrated to be highly specific and efficient for protein overexpression in the lung (i.e. chemically modified mRNAs (modRNAs)), we will assess whether BMP3 modRNA reverses PAH in the Sugen/Hypoxia model in rats. Aim 3 will dissect the molecular mechanisms of BMP3. We will perform a series of in vitro and in vivo studies to determine the mechanism(s) of BMP3 regulation and to define BMP3 mechanism(s) of action. Promoter activity assays, co-immunoprecipitation experiments, GST pull down assays, quantitative PCR measurements and western blot analyses will determine the pathways implicated in BMP3 signals. Cocultures of PASMCs and PAECs and lungs from BMP3-KO mice and from rats overexpressing BMP3 in vivo will determine the pathways implicated in BMP3 signals.