VEGF gene expression in CNS microvascular pericyte

描述（由申请人提供）：为了应对环境氧在血脑屏障（BBB）细胞中的波动，经历许多复杂的适应性措施，以维持组织稳态和止血。当由于中枢神经系统疾病的病理生理学改变了氧的可用性和利用平衡时，这些适应性反应尤为重要。在微血管水平上，涉及内皮细胞和周细胞的氧响应信号机制调节血管生成、血管通透性和代谢。我们建议使用GaSPak 100缺氧系统（Becton Dickinson and Company Sparks， MD）研究体外缺氧暴露后中枢神经系统周细胞的早期反应。暴露于低氧（1%）15分钟内，周细胞合成并释放环戊酮前列腺素。HPLC和免疫技术检测PGD2和脱水产物delta12pGJ2的升高。没有合成Delta12pGD2。周细胞组成性地表达血管内皮细胞生长因子（VEGF） mRNA的五种交替剪接变体中的三种，但几乎不表达蛋白质。低氧暴露增加mRNA水平以及VEGF蛋白的合成和释放。在常压条件下，将Delta12pGJ2或15-deoxyDelta12/14PGJ2添加到周细胞中，以剂量依赖性的方式增加VEGF蛋白的合成和释放。结果表明，CNS周细胞产生的PGD是调控缺氧血管生成反应的早期信号分子。我们将进一步研究周细胞对VEGF的反应：目的1：确定缺氧诱导VEGF基因表达上调的机制。我们还将初步评估翻译和抑制因子elF-4E和4eb的作用。目的2：确定COX-1在周细胞VEGF对缺氧反应中的作用。目的3：确定COX-1介导的周细胞PGD/PGJ释放在缺氧中的作用。我们认为中枢微血管周细胞在血管缺氧应激反应中起着重要的调节作用，环戊酮前列腺素参与了周细胞的应激反应。

DESCRIPTION (provided by applicant): In response to fluctuations in environmental oxygen in the cells of the blood brain barrier (BBB) undergo a number of complex adaptive measures in order to maintain tissue homeostasis and hemostasis. These adaptive responses are particularly important when the balance of oxygen availability and utilization is altered as a result of the pathophysiology of CNS disease. At the microvascular level oxygen responsive signaling mechanisms involving both the endothelial cell and the pericyte regulate angiogenesis, vascular permeability and metabolism. We propose to investigate early responses of the CNS pericyte following in vitro exposure to hypoxia using the GaSPak 100 hypoxia system (Becton Dickinson and Company Sparks, MD.) Within 15 minutes of exposure to low oxygen (1%) pericytes synthesize and release cyclopentenone prostaglandins. Increased PGD2 and the dehydration product delta12pGJ2 were detected by HPLC and by immune techniques. Delta12pGD2 was not synthesized. Pericytes constitutively express three of the five alternate splice variants of vascular endothelial cell growth factor (VEGF) mRNA, but little to no protein. Exposure to low oxygen increased mRNA levels as well as the synthesis and release of VEGF protein. Addition of either Delta12pGJ2 or 15-deoxyDelta12/14PGJ2 to pericyte under normoxic conditions increased the synthesis and release of VEGF protein in a dose dependent manner. Results suggest that PGD produced by the CNS pericyte is an early signaling molecule in regulation of the angiogenic response to hypoxia. We will further investigate the pericyte VEGF response: Aim #l: Determine the mechanism of hypoxia induced upregulation of VEGF gene expression. We will also evaluate the role of translation initially and inhibiting factor elF-4E and 4EBprincipal investigator. Aim#2: To determine the role of COX-1 in pericyte VEGF response to hypoxia. Aim#3: To determine the role of the COX-1 mediated release of pericyte PGD/PGJ in hypoxia. We propose that the CNS microvascular pericyte is a regulatory cell important in the vascular response to hypoxic stress and that cyclopentenone prostagladins are involved in the pericyte stress response.