Fetal Hypoxemia and Endothelium Derived Nitric Oxide

描述（由申请人提供）：胎儿缺氧是胎儿发病和死亡的主要原因。胎儿适应低氧应激的能力对其生存至关重要。一氧化氮（NO）作为冠状动脉血流和心脏收缩力的重要调节剂，在心脏保护中起着重要的作用，而一氧化氮合酶（NOS）的基因表达对低氧敏感。我们假设慢性缺氧通过增加冠状动脉循环中enos来源的NO和心室心肌细胞中inos来源的NO对心脏的损伤来上调胎儿心脏中的NOS通路，从而诱导心脏保护。为此，将怀孕的豚鼠暴露于慢性缺氧（10.5%O2持续14d），并对胎儿心脏进行检查，具体目的有5个：目的1)验证慢性缺氧增加豚鼠胎儿心脏中NOS的冠状动脉和心脏基因表达和血管生成的假设。在常氧（NMX）和缺氧（HPX）的胎儿心脏中，利用免疫荧光技术对NOS/cGMP/PKG通路的基因/蛋白表达进行定量和蛋白定位。目的2)验证慢性缺氧诱导豚鼠胎儿心脏损伤的假说。细胞凋亡（Bax/Bcl2表达，TUNEL）和冠状动脉血管生成（VEGF, VEGFR1, VEGFR2, Ang1， Ang2表达）将被量化，并测量离体胎儿心脏制剂中冠状动脉血流和收缩力的功能反应。目的3)验证子宫内抑制inos来源的NO和ROS生成保护胎儿心脏免受缺氧损伤的假设。将NOS和ROS抑制剂给予孕妇，并测量其在子宫内对胎儿心脏基因表达和冠状动脉/收缩功能的影响。目的4)验证inos来源的NO刺激离体胎儿心肌细胞（FCM）中ROS生成的假设。inos来源的NO刺激ROS的机制将在NMX和HPX胎心培养的FCM中进行研究。目的5)验证产前缺氧通过iNOS途径增加豚鼠子代动脉血压的假说。将测量年龄匹配后代的血压和心脏基因表达/功能反应的无线电遥测。我们认为，缺氧会改变胎儿心脏中NOS的表达，从而导致出生前和出生后的适应和不适应反应。这将确定inos衍生的NO合成是胎儿存活的目标途径。

DESCRIPTION (provided by applicant): Fetal hypoxia is a leading cause of fetal morbidity and mortality. The ability of the fetus to adapt to hypoxic stress is critical for its survival. Nitric oxide (NO) plays an important role in cardioprotection as an important modulator of both coronary flow and cardiac contractility, and gene expression of NO synthase (NOS) is hypoxia-sensitive. We hypothesize that chronic hypoxia upregulates the NOS pathway in fetal hearts inducing cardioprotection by increasing eNOS-derived NO in the coronary circulation and cardiac injury by iNOS-derived NO in the cardiomyocytes of the ventricle. To test this, pregnant guinea pigs will be exposed to chronic hypoxia (10.5%O2 for 14d duration) and hearts of fetuses will be examined in 5 specific aims: Aim 1) To test the hypothesis that chronic hypoxia increases coronary and cardiac gene expression of NOS and angiogenesis in the fetal guinea pig heart. Gene/protein expression of the NOS/cGMP/PKG pathway will be quantified and proteins localized using immunofluorescence in normoxic (NMX) and hypoxic (HPX) fetal hearts. Aim 2) To test the hypothesis that chronic hypoxia induces cardiac injury in the fetal guinea pig heart. Apoptosis (Bax/Bcl2 expression, TUNEL) and coronary angiogenesis (VEGF, VEGFR1, VEGFR2, Ang1, Ang2 expression) will be quantified and functional responses of coronary flow and contractile force measured in isolated fetal heart preparations. Aim 3) To test the hypothesis that in utero inhibition of iNOS-derived NO and ROS generation protects the fetal heart from hypoxia-induced injury. NOS and ROS inhibitors will be administered to pregnant mothers and the in utero effect on fetal heart gene expression and coronary/contractile function measured. Aim 4) To test the hypothesis that iNOS-derived NO stimulates ROS generation in isolated fetal cardiomyocytes (FCM). The mechanism of iNOS-derived NO in stimulating ROS will be studied in cultured FCM derived from NMX and HPX fetal hearts. Aim 5) To test the hypothesis that prenatal hypoxia increases arterial blood pressure in the guinea pig offspring via the iNOS pathway. Radiotelemetry of blood pressure and cardiac gene expression/functional responses of hearts of age-matched offspring will be measured. We propose that hypoxia alters NOS expression in the fetal heart contributing to adaptive and maladaptive responses, both pre- and postnatally. This will identify iNOS-derived NO synthesis as a target pathway for fetal survival.