Anaplerotic reprogramming of endothelial cells in pulmonary hypertension

肺动脉高压中内皮细胞的回补重编程

• 批准号: 10441800
• 负责人: Ruslan Rafikov
• 金额: $ 64.88万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2026-05-01
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10441800
• 关键词: Affinity; Animal Model; Antioxidants; Attenuated; Binding; Cell Culture Techniques; Cell Proliferation; Cells; Cellular Morphology; Cessation of life; Complex; Cyclin-Dependent Kinase 5; Cytosol; Data; Development; Diagnosis; Disease; Disease Progression; Distal; Endothelial Cells; Endothelium; Event; Gene Activation; Gene Expression; Genetic; Goals; Grant; Knowledge; Lead; Life; Link; Lung; MADH3 gene; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mediator of activation protein; Medical; Mesenchymal; Metabolic; Mitochondria; Mus; Oxidative Stress; Pathologic; Pathway interactions; Patients; Peptides; Play; Point Mutation; Pre-Clinical Model; Proteins; Publications; Pulmonary Hypertension; Pulmonary Vascular Resistance; Pulmonary artery structure; Pyruvate Carboxylase; Reporting; Resistance; Role; Signal Transduction; Stress; Testing; Therapeutic Intervention; Tyrosine; Up-Regulation; Vascular Proliferation; Vascular remodeling; Work; attenuation; experimental study; in vivo; insight; mortality; mouse model; new therapeutic target; nitration; novel; overexpression; pre-clinical; prevent; pulmonary arterial hypertension; pulmonary artery endothelial cell; pulmonary vascular cell proliferation; pulmonary vascular remodeling; response; right ventricular failure; therapeutic target; therapeutically effective; vector

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.

肺动脉高压（PAH）是一种威胁生命的疾病，具有未满足的医疗需求。现在， 可用的疗法无法大大降低PAH的进展和死亡率，五年保持接近50％ 诊断后。圆盘肺动脉的癌症样增殖是增加的主要原因 肺血管抗性，导致右心力衰竭。最近的研究强调了代谢的关键作用 在触发肺血管重塑时重新编程。但是，特定的机械链接 将代谢重编程与肺血管细胞的不受控制的增殖连接连接 建立了。然而，缺乏这种知识会产生一个关键的障碍，以防止有效 靶向血管重塑的治疗。在上一个赠款周期中，我们表明增加了 患者和动物模型中PAH肺中的氧化应激导致硝化介导的Akt激活。 通过酪氨酸Y350硝化而激活AKT会诱导丙酮酸羧化酶（PC）的过表达，前导 进行复杂化重塑的刺激。我们报道了抑制Akt硝化或PC介导的 在临床前模型中，旋律严重导致PAH明显衰减。在细胞培养实验中，我们观察到 Akt硝化改变了肺动脉内皮细胞（PAEC）形态，增殖率和基因 表达。微阵列谱图显示内皮多个标记到间充质的上调 响应Akt硝化（PDGFRA，TGFBR，SMAD3，RUNX2），过渡（EndMT）。确定可能 EndMT的机制，我们对PC相互作用组进行了质谱分析。我们找到了一个直接的 PC与Cyclin依赖性激酶5（CDK5）的结合通过Akt硝酸抑制减弱。我们的数据指示 该PC可以激活细胞质中的CDK5。 CDK5，反过来磷酸化Runx2 - 一个建立的介体 间充质转变。确实，最近的两个出版物显示了CDK和Runx2信号的激活 PAH患者。但是，这些报告没有提供机械见解。在当前的提议中，我们 假设AKT硝化触发PC的表达并在细胞质中积累，导致激活 CDK5/RUNX2轴介导的EndMT和PAH中的血管重塑。我们将用 以下目的：1）阐明硝酸（Y350）在EndMT事件中介导的Akt激活的作用； 2）到 确定胞质PC是否在CDK5/RUNX2轴激活中起关键作用； 3）检查 在体内EndMT上的靶向蛋白质降解器（Protac）。