VEGF gene expression in CNS microvascular pericyte

描述(由申请人提供)：为了响应环境的波动，血脑屏障(BBB)细胞中的氧经历了一系列复杂的适应性措施，以维持组织的动态平衡和止血。当由于中枢神经系统疾病的病理生理学改变了氧的利用和利用的平衡时，这些适应性反应尤其重要。在微血管水平上，涉及内皮细胞和周细胞的氧反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 Spark，MD)研究体外缺氧后CNS周细胞的早期反应。在暴露于低氧(1%)的15分钟内，周细胞合成并释放环戊酮前列腺素。用高效液相色谱法和免疫技术检测到PGD2和脱水产物delta12pGJ2的增加。Delta12pGD2未合成。周细胞结构性地表达血管内皮细胞生长因子(VEGF)的五种交替剪接变体中的三种，但几乎不表达蛋白质。低氧暴露可增加血管内皮细胞生长因子蛋白的合成和释放，并增加其基因表达水平。在常氧条件下向周细胞中加入Delta12pGJ2或15-脱氧Delta12/14PGJ2均可促进VEGF蛋白的合成和释放，并呈剂量依赖性。结果提示，中枢神经系统周细胞产生的PGD是调控血管新生反应的早期信号分子。我们将进一步研究周细胞血管内皮生长因子的反应：目的#L：确定缺氧诱导血管内皮生长因子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：探讨环氧合酶-1在周细胞血管内皮生长因子低氧反应中的作用。目的#3：探讨环氧合酶-1(COX-1)介导的周细胞PGD/PGJ释放在缺氧中的作用。我们认为，中枢神经系统微血管周细胞是血管对低氧应激反应的重要调节细胞，环戊烯酮前列腺素参与了周细胞应激反应。

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.