Anaplerotic reprogramming of endothelial cells in pulmonary hypertension

肺动脉高压（PAH）是一种危及生命的疾病，医疗需求未得到满足。目前， 现有的治疗未能显著降低PAH进展和死亡率，5年内仍接近50% 诊断后。远端肺动脉的癌样增生是肺动脉高压的主要原因。 肺血管阻力导致右心衰竭最近的研究强调了代谢的关键作用， 重编程触发肺血管重塑。然而，这种特殊的机械联系， 将代谢重编程与肺血管细胞不受控制的增殖联系起来， 确立了习然而，缺乏这方面的知识会产生一个严重的障碍， 针对血管重塑的治疗方法。在上一个赠款周期，我们发现， 患者和动物模型中PAH肺中的氧化应激导致硝化介导的Akt活化。 通过酪氨酸Y350的硝化激活Akt诱导丙酮酸羧化酶（PC）的过表达，导致 到回补刺激重塑。我们报道了抑制Akt硝化或PC介导的 回补导致临床前模型中PAH的显著衰减。在细胞培养实验中， Akt硝化改变肺动脉内皮细胞（PAEC）的形态、增殖率和基因表达， 表情微阵列分析显示内皮细胞到间质细胞的多种标志物上调， 图2示出了响应于Akt硝化（PDGFRa、TGFbR、SMAD 3、RUNX 2）的过渡（EndMT）。为了确定可能的 为了研究EndMT的作用机制，我们对PC相互作用组进行了质谱分析。我们找到了一个 通过Akt硝化抑制减弱PC与细胞周期蛋白依赖性激酶5（CDK 5）的结合。我们的数据表明 该PC可以激活细胞质中的CDK 5。CDK 5反过来磷酸化RUNX 2-一种公认的介导剂 间充质转化事实上，最近的两篇出版物显示，CDK和RUNX 2信号转导在细胞内的激活， 肺动脉高压患者。然而，这些报告并没有提供机械的见解。在目前的提案中，我们 假设Akt硝化触发PC在胞质溶胶中的表达和积累，导致 PAH中CDK 5/RUNX 2轴介导的EndMT和血管重塑我们将测试这个假设与 以下目的：1）阐明硝化（Y350）介导的Akt激活在EndMT事件中的作用; 2） 确定胞质PC是否在CDK 5/RUNX 2轴激活中起关键作用; 3）检查 靶向蛋白降解剂（PROTAC）对EndMT的体内作用。

Pulmonary arterial hypertension (PAH) is a life-threatening disease with unmet medical needs. Currently, available therapies fail to substantially reduce PAH progression and mortality, which remains near 50% five years after diagnosis. The cancer-like proliferation of the distal pulmonary arteries is the primary cause of increased pulmonary vascular resistance, leading to right heart failure. Recent studies highlighted a critical role of metabolic reprogramming in triggering pulmonary vascular remodeling. However, the particular mechanistic link that connects the metabolic reprogramming with the uncontrolled proliferation of pulmonary vascular cells has not been established. Nevertheless, the lack of this knowledge generates a critical barrier that prevents effective therapeutics that target vascular remodeling. During the previous grant cycle, we showed that increased oxidative stress in the PAH lungs in patients and animal models results in the nitration mediated Akt activation. The activation of Akt via nitration of tyrosine Y350 induces overexpression of Pyruvate Carboxylase (PC), leading to anaplerotic stimulation of remodeling. We reported that inhibition of both Akt nitration or PC-mediated anaplerosis resulted in marked attenuation of PAH in preclinical models. In cell culture experiments, we observed that Akt nitration changes the pulmonary artery endothelial cells (PAEC) morphology, proliferation rate, and gene expressions. The microarray profiling showed upregulation of multiple markers of Endothelial to Mesenchymal Transition (EndMT) in response to Akt nitration (PDGFRa, TGFbR, SMAD3, RUNX2). To identify the possible mechanisms of EndMT, we performed a mass spectrometry analysis of PC interactome. We found a direct binding of PC to the Cyclin-Dependent Kinase 5 (CDK5) attenuated by Akt nitration inhibition. Our data indicate that PC could activate CDK5 in the cytosol. CDK5, in turn, phosphorylates RUNX2 – a well-established mediator of mesenchymal transition. Indeed, two recent publications showed activation of CDKs and RUNX2 signaling in PAH patients. However, these reports did not provide mechanistic insights. In the current proposal, we hypothesize that Akt nitration triggers PC expression and accumulation in the cytosol leading to activation of the CDK5/RUNX2 axis-mediated EndMT and vascular remodeling in PAH. We will test this hypothesis with the following aims: 1) To elucidate the role of nitration (Y350) mediated Akt activation in EndMT events; 2) To determine whether cytosolic PC plays a key role in CDK5/RUNX2 axis activation; 3) To examine the effect of targeted protein degraders (PROTACs) on EndMT in vivo.