Fetal Hypoxemia and Endothelium Derived Nitric Oxide

描述(申请人提供)：胎儿缺氧是导致胎儿发病率和死亡率的主要原因。胎儿对低氧应激的适应能力对其生存至关重要。一氧化氮(NO)作为冠脉流量和心肌收缩能力的重要调节因子，在心脏保护中发挥着重要作用，而一氧化氮合酶(NOS)的基因表达对缺氧敏感。我们推测，慢性缺氧通过增加冠脉循环中eNOS来源的NO和心肌细胞iNOS来源的NO而导致心脏损伤，从而上调胎儿心脏中的一氧化氮合酶途径，从而起到心脏保护作用。为了验证这一点，将妊娠豚鼠暴露在慢性低氧(10.5%O2持续14天)中，并在5个特定目的下对胎儿心脏进行检查：目的1)验证慢性低氧增加胚胎豚鼠心脏冠脉和心脏一氧化氮合酶基因表达和血管生成的假说。利用免疫荧光技术对常氧(NMX)和低氧(HPX)胎儿心脏中NOS/cGMP/PKG通路的基因/蛋白表达进行定量和蛋白质定位。目的2)验证慢性低氧致胎鼠心脏损伤的假说。在分离的胎儿心脏标本中，将定量检测细胞凋亡(Bax/Bcl2表达，TUNEL)和冠脉血管生成(VEGF，VEGFR1，VEGFR2，Ang1，Ang2表达)，并测量冠脉流量和收缩力的功能反应。目的3)验证宫内抑制诱导型一氧化氮合酶(INOS)衍生的NO和ROS的生成对胎儿心脏缺氧损伤的保护作用。将NO和ROS抑制剂应用于怀孕母亲，并测量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)验证诱导型一氧化氮合酶(INOS)来源的一氧化氮(NO)刺激分离的胎儿心肌细胞(FCM)产生ROS的假说。INOS来源的NO刺激ROS的机制将在培养的NMX和HPX胎儿心脏FCM中进行研究。目的5)验证出生前低氧通过诱导型一氧化氮合酶途径增加子代豚鼠动脉血压的假说。将测量年龄匹配的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，缺氧改变了胎儿心脏中一氧化氮合酶的表达，导致了出生前和出生后的适应性和适应性不良反应。这将确认iNOS来源的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.