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

项目总结/摘要 在患有先天性心脏病（CHD）的儿童中，存在一种未知的生物开关， 向自我维持和进行性肺动脉高压（PAH）转变。在先进 疾病，继发于CHD的PAH与其他PAH组具有生物学相似性。然而，信号和 导致早期肺血管疾病的代谢紊乱仍然不清楚， 这些疗法在很大程度上不能解决疾病的潜在病理学起源。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， 冠心病肺血管功能障碍本申请的具体目的是描述早期代谢 暴露于肺动脉高压时肺血管平滑肌的机械传导信号紊乱 过度循环，并确定其对异常血管生长表型的贡献。基于先前 已发表的工作和本申请提供的新的初步数据，我们假设暴露于 肺血管平滑肌过度的压力和血流导致持续的异常， 使细胞谷氨酰胺和胆固醇代谢的变化永久化的机械传导信号， 促进失调的增殖表型。为了验证这一假设，我们使用了一个独特的， CHD的临床相关动物模型，其重现疾病的早期和进展阶段， 在其他疾病模型中表现不佳。我们的具体目标是：1）确定改变谷氨酰胺的作用， 和胆固醇代谢促进分流肺动脉平滑肌异常增殖表型 肌肉细胞（PASMCs）; 2）确定促进雅普增加的启动和维持机制 分流PASMC中的信号传导，并描述雅普在改变的细胞代谢和增殖中的作用;和3） 在CHD的临床前转化模型中评估代谢生物标志物和治疗靶点。我们将 进行谷氨酰胺和葡萄糖的平行C13稳定同位素分辨通量，并进行靶向操作 谷氨酰胺代谢和胆固醇生物合成途径，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启动和维持机械刺激 在分流平滑肌中诱导机械敏感性转录调节因子雅普。我们还将描述 靶向基因敲除雅普在血管平滑肌代谢和增殖中作用 雅普和Chip分析。最后，我们将对确定的代谢生物标志物进行翻译评价 和代谢集中疗法。本提案中概述的结构化经验将 巩固知识和技能，我需要过渡到一个独立的研究生涯，并实现我的长期 科学和职业目标。此外，所获得的科学认识将有助于我们朝着更 明确定义的PAH儿科患者亚组的特异性治疗，并将揭示重要的生物学 这是一种疾病过程的特征，主要是在其最先进和最严重的形式进行研究。

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.