Role of Vascular Endothelial Growth Factor in Hypoxic Remodeling of Ovine Cer

慢性胎儿缺氧在产科病理中很常见，并产生大量的代谢、内分泌和 胎儿发育过程中的功能变化。其中，血管重塑是应用最广泛的 研究，特别是胎儿的肺循环和脑循环，正如我们已经证明的那样。鉴于 低氧血管重塑的功能后果已被详细研究，主要 推动缺氧性血管重塑的机制仍不清楚，而且在很大程度上还没有研究。基于证据 血管内皮生长因子(VEGF)可因缺氧而增加，并可对血管产生营养作用 非内皮细胞，这一建议探索了慢性低氧对动脉的影响的假设 血管内皮生长因子对血管的直接和间接营养作用 平滑的肌肉。这一主要假设得出了三个推论。第一个推论预测 慢性缺氧可增强血管内皮细胞生长因子对血管平滑肌的直接营养作用。具体目标1将 用常氧和低氧培养的胎儿脑血管内皮细胞测定 Flk-1或Flt-1受体PI3-Kinase介导的血管内皮生长因子对血管的直接营养作用 或MAP-Kinase途径。第二个推论预测，慢性缺氧增强了血管内皮生长因子的能力 通过直接作用于动脉内皮细胞，对血管平滑肌产生间接的营养作用。 特定目标2将使用内皮完整的胎儿大脑动脉的常氧和低氧器官培养来 明确血管内皮细胞释放NO和内皮素-1在血管内皮生长因子对脑血管的影响中的作用 由NO供体SNAP，蛋白激酶G激活剂8-pCPTcGMP， 一氧化氮合酶抑制剂L-NAME、内皮素-1和选择性内皮素拮抗剂BQ-123。 第三个推论预测，慢性缺氧增强了血管内皮生长因子发挥间接营养作用的能力 通过直接作用于血管周围交感神经而对血管平滑肌产生影响。具体目标3 将使用常氧和低氧培养的内皮完整和去内皮的脑组织 妊娠128d经颈上神经节切除术失神经的胎儿动脉 几天后确定去甲肾上腺素和神经肽-Y的直接作用。所有的实验都会分析 对血管内皮生长因子的反应通过以下方面的变化：1)通过主动和被动应力-应变测量的收缩；2) 6种不同的收缩蛋白(a-肌动蛋白。肌球蛋白轻链激酶，20 kDa肌球蛋白光 链、SMI肌球蛋白、SM2肌球蛋白和非肌肉肌球蛋白)通过蛋白质印迹检测；以及3) 6种收缩蛋白跨壁分布的校准荧光形态计量学研究 免疫组织化学。这些实验将使对非血管生成的 血管内皮生长因子在慢性缺氧胎儿脑血管重塑中的作用

Chronic fetal hypoxia is common in obstetric pathologies, and produces numerous metabolic, endocrine and functional changes in the developing fetus. Among these, vascular remodeling is one ofthe most widely studied, particulariy in the fetal pulmonary and cerebral circulations, as we have demonstrated. Whereas functional consequences of hypoxic vascular remodeling have been examined in detail, the primary mechanisms driving hypoxic vascular remodeling remain unclear and largely unstudied. Based on evidence that Vascular Endothelial Growth Factor (VEGF) is increased by hypoxia, and can exert trophic effects on non-endothelial cells, this proposal explores the hypothesis that the effects of chronic hypoxia on arterial structure and function are mediated via both direct and indirect trophic effects of VEGF on vascular smooth muscle. This main hypothesis gives rise to three corollaries. The first corollary predicts that chronic hypoxia enhances the direct trophic effects of VEGF on vascular smooth muscle. Specific Aim 1 will use normoxic and hypoxic organ cultures of endothelium-denuded fetal cerebral arteries to determine the direct trophic effects of VEGF on smooth muscle as mediated by either Flk-1 or Flt-1 receptors, PI3-Kinase or MAP-Kinase pathways. The second corollary predicts that chronic hypoxia enhances the ability of VEGF to exert indirect trophic effects on vascular smooth muscle through direct effects on arterial endothelium. Specific Aim 2 will use normoxic and hypoxic organ cultures of endothelium-intact fetal cerebral arteries to define the role of endothelial release of NO and endothelin-1 in the effects of VEGF on cerebrovascular smooth muscle as defined by the effects ofthe NO donor SNAP, the Protein Kinase G activator 8-pCPTcGMP, the NO synthase inhibitor L-NAME, endothelin-1 and the selective endothelin antagonist BQ-123. The third corollary predicts that chronic hypoxia enhances the ability of VEGF to exert indirect trophic effects on vascular smooth muscle through direct effects on the perivascular sympathetic innervation. Specific Aim 3 will use normoxic and hypoxic organ cultures of endothelium-intact and endothelium-denuded cerebral arteries from fetuses denervated via superior cervical ganglionectomy at 128 d gestation and harvested 14 days later to determine the direct effects of norepinephrine and neuropeptide-Y. All experiments will analyze responses to VEGF via changes in: 1) contractility via active and passive stress-strain measurements; 2) abundances of 6 different contractile proteins (a-actin. Myosin Light Chain Kinase, 20 kDa Myosin Light Chain, SMI myosin, SM2 myosin, and non-muscle myosin) measured via Western blots; and 3) morphometry of the transmural distribution of the 6 contractile proteins using calibrated fluorescent immunohistochemistry. These experiments will enable an unprecedented evaluation ofthe non-angiogenic effects of VEGF and their roles in fetal cerebrovascular remodeling responses to chronic hypoxia