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: 10634626
• 负责人: Ruslan Rafikov
• 金额: $ 63.54万
• 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/10634626
• 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; Life; Link; Lung; MADH3 gene; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Mediator; Medical; Mesenchymal; Metabolic; Mitochondria; Mus; Oxidative Stress; Pathologic; Pathway interactions; Patients; Peptides; Phosphorylation; Play; Point Mutation; Pre-Clinical Model; Proliferating; 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; 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)是一种危及生命的疾病，其医疗需求尚未得到满足。目前， 现有的治疗方法未能显著降低PAH的进展和死亡率，五年内死亡率保持在50%左右 在确诊后。远端肺动脉癌样增殖是增加的主要原因。 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 重编程在触发肺血管重塑中的作用。然而，特定的机械联系是 将代谢重编程与肺血管细胞的不受控制的增殖联系起来 已经建立了。然而，缺乏这种知识会产生一个严重的障碍，阻碍有效的 针对血管重塑的治疗学。在前一个赠款周期中，我们发现 在患者和动物模型中，PAH肺中的氧化应激导致硝化介导的Akt激活。 酪氨酸Y350硝化激活Akt诱导丙酮酸羧基酶(PC)过表达 对重塑的抑制刺激。我们报道了对Akt硝化或PC介导的抑制 在临床前模型中，逆转导致多环芳烃显著降低。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的形态、增殖率和基因 表情。微阵列图谱显示多个内皮细胞标志物向间质细胞的上调 Akt硝化反应的过渡(EndMT)(PDGFRA、TGFbR、SMAD3、RUNX2)。找出可能的 EndMT的作用机制，我们对PC相互作用组质谱进行了分析。我们发现了一条直达 PC与细胞周期蛋白依赖性激酶5(CDK5)的结合被Akt硝化抑制减弱。我们的数据显示 该PC可以激活胞浆中的CDK5。CDK5进而使RUNX2磷酸化--RUNX2是一种公认的介体 间充质转化。事实上，最近的两篇文章显示了CDK和RUNX2信号在 PAH患者。然而，这些报告并没有提供机械性的见解。在目前的提案中，我们 假设Akt硝化触发PC在胞浆中的表达和积聚，从而激活 CDK5/RUNX2轴介导的EndMT与PAH血管重构我们将使用 目的如下：1)阐明硝化(Y350)介导的Akt激活在EndMT事件中的作用；2) 确定胞浆PC是否在CDK5/RUNX2轴激活中起关键作用；3)检测 体内靶向蛋白降解物(PROTAC)对EndMT的作用。

项目成果

Pulmonary arterial hypertension: are we close to the success?

肺动脉高压：我们已经接近成功了吗？

• 发表时间: 2017
• 期刊: Journal of clinical respiratory medicine
• 作者: Rafikova,Olga;Rafikov,Ruslan
• 通讯作者: Rafikov,Ruslan

Cytokine profiling in pulmonary arterial hypertension: the role of redox homeostasis and sex.

• DOI: 10.1016/j.trsl.2022.03.013
• 发表时间: 2022-09
• 期刊: TRANSLATIONAL RESEARCH
• 影响因子: 7.8
• 作者: Rafikov, Ruslan;Rischard, Franz;Vasilyev, Mikhail;Varghese, Mathews V.;Yuan, Jason X-J;Desai, Ankit A.;Garcia, Joe G. N.;Rafikova, Olga
• 通讯作者: Rafikova, Olga

Recurrent inhibition of mitochondrial complex III induces chronic pulmonary vasoconstriction and glycolytic switch in the rat lung.

线粒体复合物 III 的反复抑制可诱导大鼠肺部慢性肺血管收缩和糖酵解转换。

• DOI: 10.1186/s12931-018-0776-1
• 发表时间: 2018
• 期刊: Respiratory research
• 影响因子: 5.8
• 作者: Rafikova,Olga;Srivastava,Anup;Desai,AnkitA;Rafikov,Ruslan;Tofovic,StevanP
• 通讯作者: Tofovic,StevanP

Inositol monophosphatase 1 as a novel interacting partner of RAGE in pulmonary hypertension.

• DOI: 10.1152/ajplung.00393.2018
• 发表时间: 2019-03
• 期刊: American journal of physiology. Lung cellular and molecular physiology
• 作者: R. Rafikov;M. McBride;M. Zemskova;S. Kurdyukov;N. McClain;M. Niihori;P. Langlais;O. Rafikova

Necrosis-Released HMGB1 (High Mobility Group Box 1) in the Progressive Pulmonary Arterial Hypertension Associated With Male Sex.

• DOI: 10.1161/hypertensionaha.120.16118
• 发表时间: 2020-12
• 期刊: Hypertension (Dallas, Tex. : 1979)
• 作者: Zemskova M;McClain N;Niihori M;Varghese MV;James J;Rafikov R;Rafikova O
• 通讯作者: Rafikova O