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: 8327780
• 负责人: William J. Pearce
• 金额: $ 21.47万
• 依托单位: LOMA LINDA UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8327780
• 关键词: 1-Phosphatidylinositol 3-Kinase; 8-((4-chlorophenyl)thio)cyclic-3' ,5' -GMP; Acclimatization; Actins; Address; Adult; Arteries; Automobile Driving; Biology; Blood Vessels; Cells; 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; 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; Pulmonary Circulation; 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，并且会产生许多代谢，内分泌和 发育中的胎儿的功能变化。其中，血管重塑是最广泛的 正如我们已经证明的那样，研究了胎儿肺和脑循环。然而 已经详细研究过低氧血管重塑的功能后果 驱动低氧血管重塑的机制尚不清楚且在很大程度上未被研究。基于证据 缺氧增加了血管内皮生长因子（VEGF），并且可以对 非内皮细胞，该提案探讨了慢性缺氧对动脉的影响的假设 结构和功能是通过VEGF对血管的直接和间接营养作用介导的 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 使用内皮脊椎脑大脑动脉的常氧和低氧器官培养 VEGF对平滑肌的直接营养作用，由FLK-1或FLT-1受体介导的PI3-激酶介导 或地图激酶途径。第二个推论预测慢性缺氧增强了VEGF的能力 通过直接对动脉内皮作用，对血管平滑肌发挥间接营养作用。 具体目标2将使用内皮胎儿胎儿脑动脉的常氧和低氧器官培养 定义NO和内皮素-1内皮释放在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活化剂8-PCPTCGMP）的影响所定义 NO合成酶抑制剂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 在血管平滑肌上，通过直接影响血管周围交感神经神经。具体目标3 将使用内皮INTACT和内皮细胞脑化的常氧和低氧器官培养 通过上宫颈神经节切除术在128 d妊娠的胎儿中取消的胎儿动脉并收获14 几天后，以确定去甲肾上腺素和神经肽-Y的直接作用。所有实验将分析 通过以下更改对VEGF的响应：1）通过主动和被动应力应变测量的收缩力； 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）的丰度 通过蛋白质印迹测量的链，SMI肌球蛋白，SM2肌球蛋白和非肌肉肌球蛋白）； 3） 使用校准荧光的6收缩蛋白的透射分布的形态计量学 免疫组织化学。这些实验将对非血管生成进行前所未有的评估 VEGF及其在对慢性缺氧的胎儿脑血管重塑反应中的作用