Pulmonary vascular glutamine metabolism at the intersection of hemodynamic forces and smooth muscle proliferation in congenital heart disease

项目摘要/摘要 在患有先天性心脏病(CHD)的儿童中，有一种未知的生物开关驱动着生物 向自持性进行性肺动脉高压(PAH)转变。在高级 CHD继发的PAH与其他PAH组在生物学上有相似之处。然而，信令和 导致早期肺血管疾病的代谢紊乱仍不清楚，目前已有研究。 治疗方法在很大程度上未能解决疾病的潜在病理根源。我的长期研究目标是 明确异常肺血管血流动力学启动和驱动的生化机制 冠心病患者的肺血管功能障碍。本申请的具体目的是描述早期新陈代谢 肺损伤后肺血管平滑肌的力学转导信号紊乱 循环过度，并确定它们对异常血管生长表型的贡献。基于之前的 已发表的工作和与本申请一起提交的新颖的初步数据，我们假设曝光 肺血管平滑肌向过度压力和血流过度导致持续性异常 维持细胞谷氨酰胺和胆固醇代谢变化的机械转导信号， 促进失调的增殖性表型。为了检验这一假设，我们使用了一个唯一的和 临床相关的CHD动物模型，概括了疾病的早期和进展阶段，即 在其他疾病模型中表现不佳。我们的具体目标是：1)确定改变的谷氨酰胺的作用 和胆固醇代谢促进分流性肺动脉平滑的异常增殖表型 肌肉细胞(PASMC)；2)定义促进YAP增加的启动和维持机制 分流PASMC中的信号转导，并描述YAP在改变细胞代谢和增殖中的作用； 在冠心病临床前转化模型中评估代谢生物标记物和治疗靶点。我们会 平行进行C13稳定同位素分解谷氨酰胺和葡萄糖的通量，并进行靶向操作 谷氨酰胺代谢和胆固醇生物合成途径的研究，以评估对细胞增殖的影响。 我们将使用一种新型的微流控细胞培养生物反应器来评估机械刺激的启动和持续。 在分流的平滑肌中诱导机械敏感的转录调节因子YAP。我们还将描绘出 YAP在靶向基因敲除改变血管平滑肌代谢和增殖中的作用 YAP和芯片分析。最后，我们将对已识别的代谢生物标志物进行翻译评估 在我们的CHD模型中以代谢为重点的治疗。本计划书中概述的结构化体验将 巩固我所需的知识和技能，以过渡到独立的研究生涯并实现我的长期目标 科学和职业目标。此外，所获得的科学认识将帮助我们走向更多 明确定义的儿科PAH患者亚群的特殊治疗，并将揭示重要的生物学意义 一种疾病过程的特征，人们主要研究其最高级和最严重的形式。

PROJECT SUMMARY/ABSTRACT In children with congenital heart disease (CHD) there is an unidentified biological switch that drives a biologic transformation towards self-sustaining and progressive pulmonary arterial hypertension (PAH). In advanced disease, PAH secondary to CHD shares biologic similarities with other PAH groups. However, the signaling and metabolic derangements that drive early pulmonary vascular disease remain obscure, and currently available therapies largely fail to address the underlying pathologic origins of disease. My long-term research objective is to define the biochemical mechanisms by which aberrant pulmonary vascular hemodynamics initiate and drive pulmonary vascular dysfunction in CHD. The specific purpose of this application is to describe early metabolic and mechanotransductive signaling derangements in pulmonary vascular smooth muscle exposed to pulmonary overcirculation, and define their contributions to an abnormal vascular growth phenotype. Based on previously published work and novel preliminary data presented with this application, we hypothesize that exposure of pulmonary vascular smooth muscle to excessive pressure and blood flow results in sustained abnormalities of mechanotransductive signaling that perpetuate changes in cellular glutamine and cholesterol metabolism, promoting a dysregulated proliferative phenotype. In order to test this hypothesis, we are using a unique and clinically relevant animal model of CHD that recapitulates an early and progressive phase of disease that is poorly represented in other disease models. Our specific aims are to: 1) determine the role of altered glutamine and cholesterol metabolism in facilitating an abnormal proliferative phenotype in shunt pulmonary artery smooth muscle cells (PASMCs); 2) define the initiating and sustaining mechanisms that promote increased YAP signaling in shunt PASMCs and delineate the role of YAP in altered cellular metabolism and proliferation; and 3) evaluate metabolic biomarkers and therapeutic targets in a pre-clinical translational model of CHD. We will perform parallel C13 stable isotope resolved flux of glutamine and glucose, and conduct targeted manipulation of glutamine metabolism and cholesterol biosynthetic pathways to evaluate the impact on cellular proliferation. We will use a novel microfluidic cell culture bioreactor to assess the initiating and sustaining mechanical stimuli that induce the mechanosensitive transcriptional regulator YAP in shunt smooth muscle. We will also delineate the role of YAP in altered vascular smooth muscle metabolism and proliferation by targeted genetic knockdown of YAP and ChiP analysis. Finally, we will conduct translational evaluations of identified metabolic biomarkers and metabolic focused therapies in our model of CHD. The structured experience outlined in this proposal will solidify the knowledge and skills I require to transition to an independent research career and attain my long term scientific and career goals. Furthermore, the scientific understanding acquired will help us move towards more specific therapies for a clearly defined subset of pediatric patients with PAH, and will reveal important biologic features of a disease process that is largely studied in only its most advanced and severe forms.