Vascular AT2R expression and function during pregnancy

摘要 在怀孕期间，内皮血管舒张增强以降低血压和增加子宫血流量。 妊娠增强的血管舒张功能是通过血管紧张素2型表达的增加实现的 受体（AT 2 R）在子宫和全身血管内皮。在先兆子痫中， 由于AT 2 R表达和活性下调，内皮反应失败， 功能障碍、血管阻力增加和高血压。先兆子痫影响所有怀孕的5%-8%， 治疗仅限于放松血管平滑肌的抗高血压药。没有治疗方法 直接解决内皮衰竭。先兆子痫的病因尚不清楚，但妊娠特异性 血管系统中AT 2 R的上调可能是理解疾病起源的重要关键 以及母体器官功能的根本原因。有机会研究新的口腔 非肽化合物21是重要的和有价值的。根据可靠的初步数据，我们建议， 妊娠通过与配体激活的雌激素的差异结合选择性上调内皮AT 2 R 受体（即ERβ vs ERα）与AT 2 R启动子中的功能性雌激素反应元件（ERE）的结合。失败 妊娠期间血管AT 2 R上调在内皮细胞功能障碍中发挥作用 先兆子痫的血管收缩，因此，该系统的激活逆转了先兆子痫的血管收缩， 功能障碍目的1将首先建立内皮中AT 2 R的妊娠特异性上调，然后 通过使用ER特异性激动剂、拮抗剂，确定ER α或β在AT 2 R上调中的机制作用， 啮齿动物模型中的siRNA。AT 2 R转录机制将决定配体的差异结合- 原代人子宫动脉AT 2 R启动子中激活的ER和转录因子对推定的ERE的作用 内皮细胞，使用ChiP和EMSA/ supershift测定，然后使用报告基因测定来确定它们的 功能.目标2将转向临床翻译相关性。我们将确定血管AT 2 R是否 参与先兆子痫内皮功能障碍，以及AT 2 R激活是否挽救血管功能障碍。测试 AT 2 R介导的机制，血管舒张途径的EDHF、NO和PGI 2途径， 测定此外，eNOS的表达及其活性状态--EDHF和PGI 2的信号成分， 途径以及硝酸盐/亚硝酸盐和PGI 2的生产和膜电位的变化-将是 测定了目的3验证AT 2 R在体内的作用。我们将使用2个妊娠期高血压模型来测试 2种选择性AT 2 R激动剂（CGP-42112肽和化合物21）对母体、胎盘和胎儿的作用 与先兆子痫有关的胎儿异常这些研究将提供新的信息， 增加内皮AT 2 R，该过程的失败导致先兆子痫内皮功能障碍。的 这些目标的积极转化意义在于它们评估了内皮细胞的治疗效用， 在先兆子痫受试者中潜在安全的靶向治疗。

ABSTRACT In pregnancy, endothelial vasodilation is enhanced to decrease blood pressure and increase uterine blood flow. Pregnancy-enhanced vasodilatory function is achieved through an increase in expression of angiotensin type 2 receptor (AT2R) in the endothelium of uterine and systemic vasculature. In preeclampsia, this adaptive endothelial response fails due to downregulation of AT2R expression and activity, with associated endothelial dysfunction, increased vascular resistance, and hypertension. Preeclampsia affects 5%–8% of all pregnancies, and treatment is limited to antihypertensives that relax vascular smooth muscle. There is no treatment to directly address endothelial failure. The cause of preeclampsia is not known, but the pregnancy-specific upregulation of AT2R in the vasculature may hold important keys to understanding the origins of the disease and the underlying causes of maternal organ function. The opportunity to study the effects of novel oral nonpeptide Compund21 is significant and valuable. Based on solid preliminary data, we propose that pregnancy selectively upregulates endothelial AT2R via differential binding of ligand-activated estrogen receptor (ie, ERβ vs ERα) to a functional estrogen response element (ERE) in the AT2R promoter. Failure of vascular AT2R upregulation during pregnancy plays a role in endothelial cell dysfunction and vasoconstriction in preeclampsia and, consequently, activation of this system reverses preeclamptic vascular dysfunction. Aim 1 will first establish pregnancy-specific upregulation of AT2R in the endothelium and then define the mechanistic role of ER α or β in upregulation of AT2R by using ER-specific agonists, antagonists, and siRNA in rodent models. AT2R transcription mechanisms will determine differential binding of ligand- activated ER and transcription factors to putative ERE in AT2R promoter in primary human uterine artery endothelial cells, using ChiP and EMSA/ supershift assays, and then use reporter assays to determine their functionality. Aim 2 will move towards clinical translational relevance. We will determine if vascular AT2R is involved in preeclamptic endothelial dysfunction and if AT2R activation rescues vascular dysfunction. To test the AT2R-mediated mechanisms, EDHF, NO, and PGI2 pathways of vascular relaxation pathways will be determined. Also, the expression of eNOS and its activity state—signaling components of EDHF and PGI2 pathways as well as nitrate/ nitrite and PGI2 production and changes in membrane potential—will be measured. Aim 3 will verify the AT2R effects in vivo. We will use 2 models of gestational hypertension to test the effect of 2 selective AT2R agonists (CGP-42112 peptide and Compound 21) on the maternal, placental, and fetal abnormalities associated with preeclampsia. These studies will provide new information of how pregnancy increases endothelial AT2R and that failure of this process leads to preeclamptic endothelial dysfunction. The positive translational significance of these aims is that they evaluate the therapeutic utility of an endothelium- targeted therapy which is potentially safe in preeclampsia subjects.