Phospholipases and retinal angiogenesis

描述(申请人提供)：花生四烯酸(AA)单独或通过环氧合酶(COX)、脂氧合酶(LOX)和细胞色素P450单加氧酶(CYP)途径代谢，在调节细胞信号事件中发挥重要作用，这些信号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(PLA2)、磷脂酶D(PLD)和磷脂酶C(PLC)介导AA在刺激下的释放。在这三种磷脂酶中，PLA2在刺激诱导的AA释放中起着限速作用。在本拨款提案的前一阶段，我们利用来自不同血管床(包括视网膜血管床)的微血管内皮细胞，展示了AA的LOX代谢物在调节血管生成中的重要作用。虽然血管生成对创伤的发展和愈合是必不可少的，但它也在包括癌症和糖尿病视网膜病变在内的各种疾病过程中发挥着进行性的作用。大量数据表明，血管生成生长因子，尤其是血管内皮生长因子在糖尿病视网膜病变的发病机制中起决定性作用。尽管对血管生成的信号机制已有较好的研究，但AA在其血管生成信号事件中的作用尚不清楚。为了填补这一空白，我们偶然发现，血管内皮生长因子诱导的AA释放依赖于人视网膜微血管内皮细胞(HRMVEC)中PLD的激活。由于抑制PLA2而不是二酰甘油(DAG)脂肪酶抑制了VEGF诱导的AA释放，因此，VEGF诱导的AA释放可能是由PLD依赖的PLA2激活所介导的。令人惊讶的是，PLD或PLA2的抑制也减弱了VEGF诱导的HRMVEC DNA合成、迁移和管状形成。基于这些新的发现，我们假设PLD依赖的PLA2激活在血管内皮生长因子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。目的1.验证PLD介导血管内皮生长因子诱导血管生成的假说。目的2.验证PLD依赖的PLA2激活是血管生成所必需的假说。目的3.验证PLD依赖的PLA2激活在视网膜血管生成中起决定性作用的假说。上述三个特定目的的实验结果将为PLD-PLA2轴在调节视网膜血管生成中的作用从而在视网膜疾病中的作用提供新的信息。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主要作用。因此，了解视网膜血管生成的机制对于开发针对这些眼部损伤的治疗药物至关重要。目前的研究计划旨在研究PLD-PLA2轴在血管内皮生长因子诱导的视网膜血管生成中的作用，从而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.