The retinal microenvironment in diabetic retinopathy

描述（由申请人提供）：糖尿病视网膜病变是成人视网膜疾病最常见的原因之一。许多分子和组织学变化已被证实，现在认识到神经和血管问题存在。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VEGF的增加。缺氧和酸中毒都是已知的VEGF调节因子，但糖尿病患者视网膜氧和pH变化的幅度和时间过程尚不清楚。有关这些参数的更多信息将有助于更好地了解疾病进展，并最终可能导致在VEGF引起损伤之前了解可靶向的途径。我们将对链脲佐菌素（STZ）诱导的糖尿病大鼠和无糖尿病的酸中毒大鼠进行肠内PO2和pH的记录。为了将这些变化与其他事件联系起来，我们还将测量重要的血管参数（血流量、渗漏、分子信号）。特定目的1：糖尿病视网膜病变中的氧气。视网膜组织缺氧与VEGF的增加有关，VEGF在视网膜病变的背景期和增生期都有发现。有相当多的间接证据支持糖尿病视网膜病变中缺氧的存在，但直接证据非常有限。我们将对糖尿病1年以上大鼠的视网膜内氧合进行研究。我们的假设是，在这段时间内，视网膜内PO2会减少。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一个更敏感的指标，并且比基线PO2的变化发生得更早。我们在糖尿病患者12周的早期研究中发现视网膜PO2增加。我们的假设是，这是由于血流量增加，我们现在将对此进行调查。特定目的2：糖尿病视网膜病变的酸中毒。在某些组织中，VEGF受酸中毒的调节，但在成人视网膜中pH值在控制VEGF中的作用尚不清楚。我们将描述糖尿病大鼠视网膜pH值长达1年。我们的假设是，内视网膜的酸性会比正常情况下至少持续几个月。我们还假设分子机制在减少酸中毒中起作用，如碳酸酐酶的增加和酸的去除机制。这些将通过mRNA和蛋白质测量进行评估。特异性目的3：视网膜酸中毒是否足以上调VEGF？发现糖尿病视网膜是酸性的，将开启它是调节VEGF的因素之一的可能性，但不会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产生酸中毒。我们将首先建立一种治疗方案，产生与糖尿病相似的视网膜内pH值，然后评估VEGF和渗漏的变化。我们的假设是，酸中毒本身就足以增加VEGF，产生导致渗漏的分子变化，并导致渗漏本身。

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.