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

血管内皮生长因子在绵羊神经细胞缺氧重塑中的作用

• 批准号: 8015754
• 负责人: William J. Pearce
• 金额: $ 20.91万
• 依托单位: LOMA LINDA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8015754
• 关键词: 1-Phosphatidylinositol 3-Kinase; 8-((4-chlorophenyl)thio)cyclic-3' ,5' -GMP; Acclimatization; Actins; Address; Arteries; Automobile Driving; Biology; Blood Vessels; Cerebrovascular Circulation; Cervical; Chronic; Contractile Proteins; Cyclic GMP-Dependent Protein Kinases; Development; Discipline of obstetrics; Embryo; Endocrine; Endothelial Cells; Endothelin; Endothelin-1; Endothelium; Evaluation; Exhibits; Family; Fetus; Ganglionectomy; Genetic Transcription; Growth; Growth Factor; Harvest; Hormones; Hypoxia; Hypoxia Inducible Factor; Immunohistochemistry; Light; Lung; Measurement; Measures; Mediating; Metabolic; Mitogen-Activated Protein Kinases; Molecular; Myosin ATPase; Myosin Heavy Chains; Myosin Light Chain Kinase; Myosin Light Chains; Myosin Regulatory Light Chains; NG-Nitroarginine Methyl Ester; Neonatal; Neurons; Nitric Oxide Donors; Nitric Oxide Synthase; Norepinephrine; Organ Culture Techniques; Pathology; Pathway interactions; Phenotype; Physiological; Play; Pregnancy; Process; Proteins; Role; S-Nitroso-N-Acetylpenicillamine; S-nitro-N-acetylpenicillamine; Smooth Muscle; Smooth Muscle Myocytes; Source; Stress; Structure; Testing; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factors; Vascular Endothelium; Vascular Smooth Muscle; Vascular remodeling; Western Blotting; age related; base; cell type; cerebral artery; cerebrovascular; design; fetal; fetus hypoxia; inhibitor/antagonist; interest; morphometry; nerve supply; neuropeptide Y; non-muscle myosin; postnatal; receptor; research study; response; transcription factor

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

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 发育中胎儿的功能变化。其中，血管重塑是最广泛的研究之一。 正如我们所证明的那样，我们进行了研究，特别是在胎儿的肺循环和脑循环中。然而 缺氧血管重塑的功能后果已被详细检查，主要 驱动缺氧血管重塑的机制仍不清楚，并且很大程度上未经研究。基于证据 缺氧时血管内皮生长因子（VEGF）增加，对血管产生营养作用 非内皮细胞，该提案探讨了慢性缺氧对动脉的影响这一假设 结构和功能通过 VEGF 对血管的直接和间接营养作用介导 平滑肌。这个主要假设产生了三个推论。第一个推论预测 慢性缺氧增强了VEGF对血管平滑肌的直接营养作用。具体目标 1 将 使用内皮剥脱胎儿脑动脉的常氧和低氧器官培养物来确定 VEGF 对平滑肌的直接营养作用由 Flk-1 或 Flt-1 受体、PI3 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的营养作用。 具体目标 2 将使用内皮完整的胎儿脑动脉的常氧和低氧器官培养物来 定义内皮释放NO和内皮素-1在VEGF对脑血管的影响中的作用 平滑肌由 NO 供体 SNAP、蛋白激酶 G 激活剂 8-pCPTcGMP 的作用定义， 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VEGF发挥间接营养作用的能力 通过直接影响血管周围交感神经支配而对血管平滑肌产生影响。具体目标 3 将使用内皮完整和内皮剥脱的大脑的常氧和低氧器官培养物 妊娠 128 天时通过颈上神经节切除术去除胎儿的动脉并采集 14 几天后确定去甲肾上腺素和神经肽-Y 的直接作用。所有实验都会分析 通过以下方面的变化对 VEGF 做出反应： 1) 通过主动和被动应力应变测量的收缩性； 2） 6 种不同收缩蛋白（a-肌动蛋白、肌球蛋白轻链激酶、20 kDa 肌球蛋白轻链激酶）的丰度 通过蛋白质印迹法测量链、SMI 肌球蛋白、SM2 肌球蛋白和非肌肉肌球蛋白；和 3) 使用校准荧光对 6 种收缩蛋白的跨壁分布进行形态测量 免疫组织化学。这些实验将使对非血管生成的前所未有的评估成为可能。 VEGF的作用及其在胎儿慢性缺氧脑血管重塑反应中的作用