Role of Non-visual Opsins in Light-dependent Apoptosis of Pulmonary Arterial Smooth Muscle Cells in Pulmonary Arterial Hypertension

非视觉视蛋白在肺动脉高压肺动脉平滑肌细胞光依赖性凋亡中的作用

• 批准号: 10025576
• 负责人: Gautam Sikka
• 金额: $ 6.13万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-13 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10025576
• 关键词: Affect; Age; Apoptosis; Apoptotic; Award; Biological Assay; Biology; Blood Vessels; Blood capillaries; CASP3 gene; Caspase; Cell Death; Cells; Characteristics; Cleaved cell; Control Animal; Coupled; Cyclic Nucleotides; DNA Nucleotidylexotransferase; Disease; Disease Progression; Drug Targeting; Enzymes; Exposure to; Funding; G protein coupled receptor kinase; G-Protein-Coupled Receptors; Gender; Genetic; Goals; Hypoxia; Immunoblot Analysis; In Vitro; Label; Lesion; Light; Literature; Lung; Measures; Mediating; Messenger RNA; Methods; Modeling; Molecular; Morbidity - disease rate; Morphology; Nuclear; Opsin; Pathway interactions; Patients; Pharmaceutical Preparations; Pharmacology; Phosphotransferases; Photoreceptors; Phototherapy; Potassium Channel; Predisposition; Process; Proteins; Pulmonary artery structure; Pulmonary vessels; Race; Rattus; Regulation; Relaxation; Research; Research Personnel; Resistance; Rodent; Role; Signal Transduction; Small Interfering RNA; Smooth Muscle Myocytes; Specificity; Stains; Stimulus; Testing; Time; Training; Vascular Diseases; Vascular remodeling; Vasodilation; Viral; Work; beta-arrestin; differential expression; effective therapy; experimental study; in vivo; insight; lung hypoxia; mortality; nexin; novel; overexpression; pressure; prevent; programs; protein expression; pulmonary arterial hypertension; receptor; response; side effect; skills; therapeutic target; voltage

PROJECT SUMMARY/ ABSTRACT: Pulmonary arterial hypertension (PAH), a progressive and deadly condition characterized by pre-capillary disease with vaso-occlusive lesions and severe elevations in pulmonary arterial pressure, is attributed to both loss of vascular relaxation and severe vascular remodeling. Significant dysregulation of apoptosis, or programmed cell death, in pulmonary arterial smooth muscle cells (PASMCs) prevents turnover of abnormal PASMCs and contributes significantly to vascular remodeling. Currently approved treatments target vasodilation and slow progression of PAH, but drugs targeting reversal of vascular remodeling, and particularly cell death, that can serve as effective therapies to reverse disease have yet to be identified. We recently discovered the presence photoreceptors, or opsins, in rodent systemic and pulmonary blood vessels and elucidated the pathway by which they mediate vasorelaxation in response to blue light. In pulmonary arterial smooth muscle cells (PASMCs), we recently found that blue light exposure also selectively induces cell death in PASMCs from a model of PAH. In contrast, in normal PASMCs, apoptosis can be induced by blue light only when G protein-coupled receptor kinase-2 (GRK2), a negative regulator of opsin activity, is inhibited. Thus, the focus of my proposal is to explore this intriguing finding and elucidate the mechanism by which blue light induces apoptosis. We will employ the Sugen-hypoxia rat model of severe PAH to isolate PASMCs used in in vitro experiments. In Specific Aim 1, we will determine whether expression of opsin receptors is increased or expression of GRK2 is decreased, in PAH PASMCs. In Specific Aim 2, we will measure intracellular K+ ([K+]i) to determine if blue light exposure increases the susceptibility to apoptosis, in of PAH PASMC through efflux of K+ via cyclic nucleotide-gated K+ channels, and in Specific Aim 3, we will measure cleaved (active) caspase-3 levels and activity to determine whether blue light exposure increases caspase-3 activity in PAH PASMCs. With the completion of the research proposed in this application, we will characterize a completely novel, wavelength-specific, light-activated molecular switch, which could be harnessed for treatment of PAH to prevent and potentially reverse remodeling by selectively restoring apoptosis in abnormal PASMCs. The funds from this award will not only allow for the training necessary for the applicant to realize this goal, but also provide the opportunity to begin developing methods for in vivo application of the results.

项目摘要/摘要： 肺动脉高压（PAH），一种进行性和致命状态，其特征是前毛细血管前的 具有血管熟悉病变和肺动脉压的严重升高的疾病归因于 血管松弛和严重的血管重塑的丧失。细胞凋亡的明显失调或 肺动脉平滑肌细胞（PASMC）中的程序性细胞死亡可防止异常营业额 PASMC并为血管重塑做出重大贡献。目前批准的治疗目标 PAH的血管舒张和缓慢的进展，但针对血管重塑的逆转药物，尤其是 可以用作逆转疾病的有效疗法的细胞死亡尚未确定。我们最近 在啮齿动物系统和肺血管中发现了存在感受器或Opsin的存在，以及 阐明了它们响应蓝光而介导血管延缓的途径。在肺动脉中 平滑肌细胞（PASMC），我们最近发现蓝光暴露还有选择地诱导细胞死亡 在PASH的PASMC中。相反，在正常的PASMC中，仅蓝光可以诱导凋亡 当G蛋白偶联受体激酶-2（GRK2）（opsin活性的负调节剂）被抑制时。因此， 我的提议的重点是探索这个有趣的发现并阐明蓝光的机制 诱导凋亡。我们将采用严重PAH的Sugen-Hypoxia大鼠模型来分离使用的PASMC 体外实验。在特定目标1中，我们将确定Opsin受体的表达是否增加或 在PAH PASMC中，GRK2的表达降低。在特定的目标2中，我们将测量细胞内K+（[K+] i） 确定蓝光暴露是否会增加对PASMC的凋亡的敏感性 K+通过循环核苷酸门控的K+通道，在特定的目标3中，我们将测量裂解（活动）caspase-3 确定蓝光暴露是否会增加caspase-3活性的水平和活性。 随着本应用程序提出的研究的完成，我们将表征一个完全新颖的， 波长特异性的，光激活的分子开关，可以利用用于治疗PAH 通过选择性恢复异常PASMC的凋亡，预防并可能反向重塑。资金 来自该奖项不仅允许申请人实现此目标所需的培训，还可以 提供机会开始开发用于体内应用结果的方法。