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

慢性胎儿缺氧在产科病理学中是常见的，并且产生许多代谢、内分泌和代谢异常。 发育中胎儿的功能变化。其中，血管重构是最广泛的一种 研究，特别是在胎儿肺和脑循环，正如我们已经证明。而 缺氧血管重塑的功能后果已被详细研究，主要是 驱动缺氧血管重塑的机制仍然不清楚，并且大部分未被研究。基于证据 血管内皮生长因子（VEGF）在缺氧时增加，并可在缺氧时发挥营养作用。 非内皮细胞，这项建议探讨了慢性缺氧对动脉血管的影响的假设， 结构和功能通过VEGF对血管内皮细胞的直接和间接营养作用介导， 平滑肌这一主要假设产生了三个推论。第一个推论预测， 慢性缺氧增强VEGF对血管平滑肌的直接营养作用。具体目标1将 使用常氧和缺氧器官培养内皮剥脱的胎儿脑动脉，以确定 VEGF对平滑肌的直接营养作用，由Flk-1或Flt-1受体、PI 3-激酶介导 或MAP-激酶途径。第二个推论预测，慢性缺氧增强VEGF的能力， 通过对动脉内皮的直接作用对血管平滑肌产生间接营养作用。 具体目标2将使用内皮完整的胎儿脑动脉的常氧和缺氧器官培养物， 明确血管内皮细胞释放NO和内皮素-1在VEGF对脑血管的影响中的作用， 通过NO供体SNAP，蛋白激酶G激活剂8-pCPTcGMP， NO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VEGF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支配而影响血管平滑肌。具体目标3 将使用内皮完整和内皮剥脱的大脑的常氧和缺氧器官培养物， 孕128 d经上级颈神经节切除术切除去神经支配的胎儿动脉， 天后，以确定去甲肾上腺素和神经肽Y的直接作用。所有实验都将分析 通过以下变化对VEGF的反应：1）通过主动和被动应力-应变测量的收缩性; 2） 6种不同的收缩蛋白（α-肌动蛋白，肌球蛋白轻链激酶，20 kDa肌球蛋白轻链 链、SM 1肌球蛋白、SM 2肌球蛋白和非肌肉肌球蛋白）;以及 使用校准的荧光标记的6种收缩蛋白的跨壁分布的形态计量学 免疫组化这些实验将使非血管生成的前所未有的评估成为可能。 血管内皮生长因子在慢性缺氧胎儿脑血管重构中的作用

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