Role of Endothelin System and NAD(P)H Oxidase in Retinal Arteriolar Dysfunction

描述(申请人提供)：急性视网膜缺血期损害随后的视网膜血流供应，并与几种导致视力损害和失明的眼部疾病有关。初始视网膜缺血后视网膜血流量减少的实验证据表明，内皮功能障碍可能导致持续性视网膜损伤。参与调节视网膜血流的两个重要的内皮衍生因子是血管扩张剂一氧化氮(NO)和血管收缩因子内皮素-1(ET-1)。我们的初步研究表明，通过升高眼压(IOP)导致的视网膜缺血可削弱缓激肽诱导的猪视网膜小动脉NO介导的扩张和增强ET-1介导的收缩。缺血前玻璃体内注射超氧阴离子清除剂坦普尔或内皮素转换酶抑制剂磷酰胺可维持血管扩张为缓激肽。尽管这些初步研究表明ET-1和氧化应激参与了血管功能障碍，但它们之间的相互关系以及导致所观察到的损伤的信号事件仍有待阐明。在此，我们假设缺血性损伤激活了依赖蛋白激酶C(PKC)的血管内皮细胞系统，导致通过NAD(P)H氧化酶产生超氧化物，继而增加Rho/Rho激酶的激活，从而增加血管张力，减少NO介导的血管扩张。由于我们的长期目标是了解导致未来血管治疗的视网膜血管运动功能的生理和病理生理调节的信号机制，目前的应用将通过识别导致急性视网膜缺血后血管功能损害的原因因素和细胞机制而朝着这一目标迈出第一步。我们将通过追求三个具体目标来验证上述假设：(1)确定增强的ECA和PKC活性是否有助于缺血诱导的视网膜小动脉功能障碍。(2)探讨内皮素A/B受体和血管p38丝裂原活化蛋白激酶/NAD(P)H氧化酶信号通路的激活与视网膜小动脉功能障碍的关系。(3)确定血管Rho/Rho激酶信号增强是否参与缺血诱导的视网膜小动脉功能障碍。我们将使用体内和体外方法结合各种细胞/分子技术来整合这三个目的，以阐明导致视网膜小动脉功能障碍的潜在机制和信号通路。这些研究的结果对于促进我们对视网膜缺血相关视网膜血管疾病的发病机制的理解是至关重要的，并可能为未来的治疗干预提供新的靶点。眼内正常血流量的减少或视网膜的缺血与几种导致视力损害和失明的眼病有关。最初的视网膜缺血后视网膜血流量减少的实验证据表明，小血管，即小动脉，通过产生或对化学物质一氧化氮做出反应而变宽或扩张的能力降低，可能导致视网膜的持续性损害。导致血管功能损害的一个潜在机制是视网膜内内皮素水平升高。内皮素是一种蛋白质，可以通过导致血管崩溃或收缩，或者通过增加氧自由基的产生来影响血管，而氧自由基可以破坏一氧化氮。然而，这两个事件在促进视网膜缺血后视网膜血管功能异常方面的确切作用及其之间的潜在联系仍不清楚。这一建议的目的是为了更好地了解导致视网膜小血管功能损害的机制，这将有助于开发视网膜缺血性疾病的新疗法。

DESCRIPTION (provided by applicant): Acute periods of retinal ischemia impair subsequent supply of retinal blood flow and have been associated with several ocular diseases leading to visual impairment and blindness. Experimental evidence of diminished retinal blood flow after the initial retinal ischemia suggests that endothelial dysfunction may contribute to persistent retinal damage. Two important endothelium-derived factors involved in regulating retinal blood flow are vasodilator nitric oxide (NO) and vasoconstrictor endothelin-1 (ET-1). Our preliminary studies showed that retinal ischemia via elevated intraocular pressure (IOP) impaired bradykinin-induced NO-mediated dilation and enhanced ET-1-mediated constriction in pig retinal arterioles. Intravitreal administration of superoxide scavenger TEMPOL or endothelin-converting enzyme (ECE) inhibitor phosphoramidon before ischemia preserved vasodilation to bradykinin. Although these pilot studies suggest the involvement of ET-1 and oxidative stress in vascular dysfunction, their interrelationship and the signaling events contributing to the observed impairment remain to be elucidated. Herein, we hypothesize that ischemic insult activates the protein kinase C (PKC)-dependent vascular endothelin system, which leads to superoxide production via NAD(P)H oxidase and a subsequent increase in Rho/Rho kinase activation for the increased vascular tone and a reduced NO-mediated vasodilation. Since our long-term goal is to understand the signaling mechanisms responsible for physiological and pathophysiological regulation of retinal vasomotor function leading to future vascular therapy, the present application will serve the initial step toward this goal by identifying the causal factor and cellular mechanisms contributing to the impairment of vascular function following acute retinal ischemia. We will test the aforementioned hypothesis by pursuing three specific aims: (1) Determine whether enhanced ECE and PKC activities contribute to ischemia-induced dysfunction of retinal arterioles. (2) Determine whether activation of endothelin A/B receptors and vascular p38 mitogen-activated protein kinase/NAD(P)H oxidase signaling contributes to ischemia-induced dysfunction of retinal arterioles. (3) Determine whether enhanced vascular Rho/Rho kinase signaling contributes to ischemia-induced dysfunction of retinal arterioles. We will use both in-vivo and in-vitro approaches with various cellular/molecular techniques to integrate these three aims for elucidating the underlying mechanisms and signaling pathways responsible for the ischemia-induced arteriolar dysfunction in the retina. The results derived from these studies are essential to advance our understanding in the pathogenesis of retinal vascular disease associated with retinal ischemia and may suggest novel targets for future therapeutic interventions. Reduction in normal blood flow or ischemia to the retina in the eye has been associated with several ocular diseases leading to visual impairment and blindness. Experimental evidence of diminished retinal blood flow after the initial retinal ischemia suggests that reduction in the ability of small blood vessels, the arterioles, to widen or dilate by producing or responding to the chemical nitric oxide may contribute to persistent damage of the retina. A potential mechanism leading to the impairment of blood vessel function is elevated levels of endothelin in the retina. Endothelin is a protein that can impact blood vessels by causing them to collapse or constrict, or by increasing the production of oxygen-derived free radicals, which can damage nitric oxide. However, the precise roles of and potential link between these two events in contributing to this abnormal function of retinal vessels following retinal ischemia remain unknown. The goal of this proposal is to gain a better understanding into the mechanisms leading to the functional damage of the small blood vessels in the retina, which will be helpful for development of new therapies for retinal ischemic disease.