Phospholipases and retinal angiogenesis

描述（由申请人提供）：花生四烯酸（AA）单独或通过其通过环氧合酶（考克斯）、脂氧合酶（LOX）和细胞色素P450单加氧酶（CYP）途径的代谢在调节细胞信号传导事件中起重要作用，这些事件在维持体内平衡或疾病发病机制中至关重要。磷脂酶A2（PLA 2）、磷脂酶D（PLD）和磷脂酶C（PLC）介导AA响应于刺激的释放。在这三种磷脂酶中，PLA 2在刺激诱导的AA释放中起限速作用。在本拨款申请的前一阶段，我们证明了AA的LOX代谢物在使用来自不同血管床（包括视网膜血管床）的微血管内皮细胞调节血管生成中的有效作用。虽然血管生成对于发育和伤口愈合至关重要，但它在包括癌症和糖尿病视网膜病变在内的各种疾病过程中也起着渐进性作用。大量资料表明，血管生成生长因子，特别是血管内皮生长因子（VEGF）在糖尿病视网膜病变的发病机制中起决定性作用。虽然VEGF诱导的血管生成的信号转导机制已经得到了很好的研究，但AA在其血管生成信号转导事件中的参与还没有研究。为了填补这一空白，我们偶然发现VEGF诱导的AA释放依赖于人视网膜微血管内皮细胞（HRMVEC）中PLD的激活。由于PLA 2而不是二酰基甘油（DAG）脂肪酶的抑制抑制VEGF诱导的AA释放，这是可能的，VEGF诱导的AA释放介导的PLD依赖性PLA 2激活。令人惊讶的是，PLD或PLA 2的抑制也减弱了VEGF诱导的HRMVEC DNA合成，迁移和管形成。基于这些新的发现，我们推测PLD依赖的PLA 2激活在VEGF诱导的视网膜血管生成中起决定性作用。为了验证这一假设，我们将提出并解决以下三个具体目标。目标1.验证PLD介导VEGF诱导的血管生成的假设。目标2.验证PLD依赖性PLA 2激活是VEGF诱导血管生成所必需的假设。目标3.为了验证PLD依赖性PLA 2激活在视网膜血管生成中起决定性作用的假设。在上述三个具体目的中提出的实验结果将提供关于PLD-PLA 2轴在视网膜血管生成调节中的作用以及由此在视网膜疾病中的作用的新信息。公共卫生相关性：血管生成在糖尿病视网膜病变等视网膜疾病中起着重要作用。因此，了解视网膜血管生成的机制对于开发针对这些眼部病变的治疗药物至关重要。本研究计划旨在研究PLD-PLA 2轴在VEGF诱导的视网膜血管生成中的作用，从而研究其在视网膜病变中的作用。

DESCRIPTION (provided by applicant): Arachidonic acid (AA) alone or via its metabolism through the cyclooxygenase (COX), lipooxygenase (LOX) and cytochrome P450 monooxygease (CYP) pathways plays an important role in the regulation of cell signaling events that are either crucial in the maintenance of homeostasis or disease pathogenesis. Phospholipase A2 (PLA2), phospholipase D (PLD) and phospholipase C (PLC) mediate AA release in response to stimulus. Among these three phospholipases, PLA2 plays a rate- limiting role in stimulus-induced AA release. During the previous period of the present grant proposal, we demonstrated a potent role for the LOX metabolites of AA in the regulation of angiogenesis using microvascular endothelial cells from different vascular beds, including retinal vascular bed. While angiogenesis is essential for the development and wound healing, it also plays a progressive role in various disease processes including cancer and diabetic retinopathy. A large body of data showed that angiogenic growth factors, particularly vascular endothelial growth factor (VEGF) plays a determinant role in the pathogenesis of diabetic retinopathy. Although the signaling mechanisms underlying VEGF-induced angiogenesis are fairly well studied, the involvement of AA in its angiogenic signaling events is not explored. Towards filling this gap, we accidentally discovered that VEGF- induced AA release depends on PLD activation in human retinal microvascular endothelial cells (HRMVEC). Since inhibition of PLA2 but not diacylglycerol (DAG) lipase suppressed VEGF-induced AA release, it is likely that VEGF-induced AA release is mediated by PLD-dependent PLA2 activation. Surprisingly, inhibition of PLD or PLA2 also attenuated VEGF-induced HRMVEC DNA synthesis, migration and tube formation. Based on these novel findings, we hypothesize that PLD-dependent PLA2 activation plays a determinant role in VEGF-induced retinal angiogenesis. To test this hypothesis, we will propose and address the following three specific aims. Aim 1. To test the hypothesis that PLD mediates VEGF-induced angiogenesis. Aim 2. To test the hypothesis that PLD- dependent PLA2 activation is required for VEGF-induced angiogenesis. Aim 3. To test the hypothesis that PLD-dependent PLA2 activation plays a determinant role in retinal angiogenesis. The results of the experiments proposed in the above-listed three specific aims will provide novel information in regard to the role of PLD-PLA2 axis in the regulation of retinal angiogenesis and thereby in retinal diseases. PUBLIC HEALTH RELEVANCE: Angiogenesis plays a major role in retinal diseases such as diabetic retinopathy. Understanding the mechanisms underlying retinal angiogenesis is, therefore, crucial in the development of therapeutic agents against these ocular lesions. The present research proposal seeks to study the role of PLD- PLA2 axis in VEGF-induced retinal angiogenesis and thereby in retinopathies.