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

项目摘要/摘要： 肺动脉高压(PAH)，一种进行性和致命性的疾病，其特征是毛细血管前 伴有血管闭塞损害和肺动脉压严重升高的疾病可归因于这两种疾病 血管松弛丧失和严重的血管重构。严重的细胞凋亡失调，或 细胞程序性死亡，在肺动脉平滑肌细胞(PASMC)中防止异常的周转 PASMC在血管重塑中起重要作用。目前批准的治疗目标 血管扩张和PAH进展缓慢，但靶向逆转血管重塑的药物，尤其是 可以作为逆转疾病的有效疗法的细胞死亡尚未确定。我们最近 在啮齿动物的全身和肺血管中发现了光感受器或视蛋白，并 阐明了它们介导血管松弛对蓝光的反应的途径。在肺动脉中 我们最近发现，蓝光照射也选择性地诱导细胞死亡 在PASMC中来自多环芳烃的模型。相反，在正常的PASMC中，只有蓝光才能诱导细胞凋亡 当视蛋白活性的负调节因子G蛋白偶联受体激酶-2(GRK2)被抑制时。因此， 我建议的重点是探索这一有趣的发现，并阐明蓝光 诱导细胞凋亡。我们将使用严重PAH的SUGEN低氧大鼠模型来分离PASMC 体外实验。在特定的目标1中，我们将确定视蛋白受体的表达是增加还是 在PAH PASMC中，GRK2的表达降低。在特定目标2中，我们将测量细胞内K+([K+]i)以 确定蓝光暴露是否通过外流增加PAH PASMC对细胞凋亡的敏感性 K+通过环核苷酸门控的K+通道，在特定的目标3，我们将测量切割(活性)的caspase-3 水平和活性，以确定蓝光暴露是否增加PAH PASMCs中caspase-3的活性。 随着本申请中提出的研究的完成，我们将描述一部全新的小说， 波长特定的光激活分子开关，可用于治疗PAH 通过选择性地恢复异常的PASMC中的细胞凋亡来预防和潜在地逆转重塑。这些资金 这一奖励不仅将允许申请者接受实现这一目标所需的培训，而且还 提供机会开始开发体内应用结果的方法。

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.