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

项目总结/摘要： 肺动脉高压（PAH）是一种进行性和致命的疾病，其特征是毛细血管前病变， 伴有血管闭塞性病变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塑。细胞凋亡的显著失调，或 肺动脉平滑肌细胞（PASMCs）中的程序性细胞死亡阻止了肺动脉平滑肌细胞（PASMCs）的异常代谢。 PASMCs和显著有助于血管重塑。目前批准的治疗目标 血管舒张和PAH进展缓慢，但靶向逆转血管重塑的药物，特别是 细胞死亡，可以作为有效的治疗方法来扭转疾病还有待确定。我们最近 发现啮齿动物全身和肺血管中存在光感受器或视蛋白， 阐明了它们介导血管舒张反应蓝光的途径。肺动脉 平滑肌细胞（PASMCs），我们最近发现蓝光照射也选择性地诱导细胞死亡 在PASMCs中，来自PAH模型。与此相反，在正常PASMCs中，仅蓝光可诱导凋亡 当G蛋白偶联受体激酶-2（GRK 2），视蛋白活性的负调节剂，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。我们将采用Sugen-低氧大鼠重度PAH模型，分离PASMC用于 体外实验在具体目标1中，我们将确定视蛋白受体的表达是否增加， 在PAH PASMCs中GRK 2的表达降低。在具体目标2中，我们将测量细胞内K+（[K+]i）， 确定蓝光暴露是否增加PAH PASMC对凋亡的易感性， 在特异性目标3中，我们将测量切割的（活性）caspase-3 水平和活性，以确定蓝光暴露是否增加PAH PASMCs中的半胱天冬酶-3活性。 随着本申请中提出的研究的完成，我们将表征一种完全新颖的， 波长特异性，光激活分子开关，可用于治疗PAH， 通过选择性恢复异常PASMC中的凋亡来预防并潜在地逆转重塑。资金 从这个奖项将不仅允许必要的培训申请人实现这一目标，而且 为开始开发用于结果的体内应用的方法提供了机会。

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.