The retinal microenvironment in diabetic retinopathy

描述(申请人提供)：糖尿病视网膜病变是成人视网膜疾病最常见的原因之一。许多分子和组织学改变已经被证实，现在人们认识到神经和血管问题的存在。血管功能障碍最重要的因素是血管内皮生长因子(VEGF)的升高，导致血管渗漏和血管生成。不幸的是，我们仍然不知道是什么导致了血管内皮生长因子的增加。缺氧和酸中毒都是已知的血管内皮生长因子调节因子，但糖尿病患者视网膜氧和pH变化的幅度和时间进程尚不清楚。更多关于这些参数的信息将有助于更好地了解疾病的进展，并最终可能导致了解在血管内皮生长因子造成损害之前可以靶向的途径。我们将在链脲佐菌素(STZ)诱导的糖尿病大鼠以及没有糖尿病的酸中毒大鼠的视网膜内记录PO2和pH。为了将这些变化放在其他事件的背景下，我们还将测量重要的血管参数(血流量、渗漏、分子信号)。具体目的1：糖尿病视网膜病变中的氧气。视网膜组织缺氧与视网膜病变的背景和增殖期的血管内皮生长因子的增加有关。有相当多的间接支持糖尿病视网膜病变中存在低氧，但直接证据非常有限。我们将描述糖尿病大鼠视网膜内氧合的特征。我们的假设是，随着时间的推移，视网膜内的PO2将会下降。我们还将研究糖尿病患者视网膜氧调节的变化。我们的假设是，PO2调节的变化将是微环境变化的更敏感的指标，并且比基线PO2的变化发生得更早。我们在早期的一项研究中发现，糖尿病12周时视网膜中的PO2升高。我们的假设是，这是由于血流增加，我们现在将进行调查。特定目的2：糖尿病视网膜病变中的酸中毒。在一些组织中，血管内皮生长因子受到酸中毒的调节，但在成人视网膜中，pH在控制血管内皮生长因子中的作用尚不清楚。我们将对糖尿病长达一年的大鼠的视网膜pH值进行表征。我们的假设是，至少在未来几个月内，视网膜内的酸性程度将高于正常水平。我们还假设，减少酸中毒的分子机制发挥作用，例如增加碳酸氢酶和酸去除机制。这些都将通过mRNA和蛋白质的测量进行评估。具体目标3：视网膜酸中毒是否足以上调血管内皮生长因子？发现糖尿病视网膜是酸性的，这将开启一种可能性，即它是调节血管内皮生长因子的因素之一，但不能告诉我们它是否重要。因此，在没有糖尿病的情况下，我们会通过碳酸酐酶抑制而产生酸中毒。我们将首先建立一种治疗方案，产生类似于糖尿病的视网膜内pH值，然后评估血管内皮生长因子的变化和渗漏。我们的假设是，仅酸中毒就足以增加血管内皮生长因子，产生作为渗漏基础的分子变化，并导致渗漏本身。 公共卫生相关性：该项目将提供有关糖尿病视网膜病变中受损的视网膜代谢过程的信息，糖尿病视网膜病变是糖尿病的一种主要致盲并发症。这项工作将通过测量患有或不患有实验性糖尿病的大鼠视网膜局部的氧气和pH值来完成。我们希望阐明疾病是如何发展的，并测试有关氧气和pH调节的假说，这可能为早期和较小侵入性的治疗提供洞察力。

DESCRIPTION (provided by applicant): Diabetic retinopathy is one of the most common causes of retinal disease in adults. Many molecular and histological changes have been demonstrated, and it is now recognized that neural as well as vascular problems exist. The most important contributor to vascular dysfunction is the elevation of vascular endothelial growth factor (VEGF), which causes vascular leakage and angiogenesis. Unfortunately, we still do not know what underlies the increase in VEGF. Hypoxia and acidosis are both known regulators of VEGF, but the magnitude and time course of changes in retinal oxygen and pH are not known in diabetes. More information about these parameters will allow a better understanding of disease progression, and ultimately may lead to an understanding of pathways that can be targeted before VEGF causes damage. We will make intraretinal recordings of PO2 and pH in rats with streptozotocin (STZ)-induced diabetes, and in rats with acidosis in the absence of diabetes. To place these changes in the context of other events, we will also measure important vascular parameters (blood flow, leakage, molecular signals). SPECIFIC AIM 1: Oxygen in diabetic retinopathy. Retinal tissue hypoxia has been implicated in the increased VEGF that is found in both the background and proliferative phases of retinopathy. There is considerable indirect support for the existence of hypoxia in diabetic retinopathy, but very limited direct evidence. We will characterize intraretinal oxygenation in rats with up to 1 year of diabetes. Our hypothesis is that inner retinal PO2 will decrease over this time. We will also investigate changes in the regulation of retinal oxygen in diabetics. Our hypothesis is that changes in regulation of PO2 will be a more sensitive indicator of changes in the microenvironment and occur earlier than changes in baseline PO2. We found increased PO2 in the retina in an earlier study at 12 weeks of diabetes. Our hypothesis is that this was due to increased blood flow, which we will now investigate. SPECIFIC AIM 2: Acidosis in diabetic retinopathy. VEGF is regulated by acidosis in some tissues but the role of pH in controlling VEGF in the adult retina is unknown. We will characterize retinal pH in rats with up to 1 year of diabetes. Our hypothesis is that the inner retina will be more acidic than normal for at least a few months. We also hypothesize that molecular mechanisms come into play to reduce acidosis, such as increased carbonic anhydrase and acid removal mechanisms. These will be evaluated with mRNA and protein measurements. SPECIFIC AIM 3: Is retinal acidosis sufficient to upregulate VEGF? Finding that the diabetic retina is acidotic will open the possibility that it is one of the factors that regulates VEGF, but will not tell us whether it is important. Consequently, we will produce acidosis in the absence of diabetes, via carbonic anhydrase inhibition. We will first establish a treatment protocol that yields intraretinal pH values similar to diabetes, and then evaluate changes in VEGF and leakage. Our hypothesis is that acidosis alone will be sufficient to increase VEGF, produce molecular changes that underlie leakage, and cause leakage itself. PUBLIC HEALTH RELEVANCE: This project will provide information about retinal metabolic processes that are impaired in diabetic retinopathy, a major blinding complication of diabetes. This work will be done by measuring oxygen and pH locally in the retina of rats with or without experimental diabetes. We hope to clarify how the disease progresses, and test hypotheses about oxygen and pH regulation, which may offer insight into earlier and less invasive treatments.