Sex-specific fetal programming of adult vascular dysfunction and hypertension

描述（由申请人提供）：流行病学研究显示，妊娠受损妇女所生儿童的心血管（CV）疾病风险增加，如先兆子痫、PCOS、蛋白质或能量限制、肥胖、压力和吸烟，但其发病机制仍不完全清楚。作为在这些妊娠病理学中观察到的常见因素之一，母体睾酮（T）升高可能导致CV疾病的胎儿编程。事实上，我们最近的研究表明，母体T升高导致大鼠后代高血压表型的发展。为了理解这些机制，本项目提出了两个中心假设。首先，产前T诱导性别特异性高血压的发病和严重程度，这些高血压反应是由出生后T水平的增加介导的。第二，出生后T的增加通过血管平滑肌（VSM）蛋白激酶C（PKC）和内皮细胞EDHF/NO表达/功能的性别特异性功能障碍诱导高血压反应。为了验证这些假设，我们提出了一系列的实验，在我们建立的怀孕大鼠模型，并检查他们的后代。提出了三个具体的目标：1）确定母亲T升高是否程序后代的高血压，男性比女性更明显的影响，以及出生后T增加是否先于高血压发作。我们将遥测血压（BP）的渐进性变化，并测量T水平，以建立高血压的发病和严重程度与出生后T水平变化之间的关系，机械地确定出生后T增加是否是BP增加的关键因素。2)评估VSM中性别特异性高血压机制。我们将研究PKC同工酶的表达谱在亚细胞组分，其磷酸化状态和功能活性，并研究雄激素调节PKC表达的机制，通过评估结合T推定ARE在PKC启动子芯片和报告基因测定。3)剖析内皮功能受损的性别特异性机制。我们将研究EDHF和NO介导的途径，并通过确定EDHF组分SK 3和IK 1通道和连接蛋白（CX 37，CX 40和CX 47）的mRNA和蛋白水平，它们的亚细胞定位以及使用血管反应性和膜电位研究的功能活性来评估受损EDHF介导的血管舒张的机制。我们将通过评估eNOS的表达、其活性、NO的产生和信号事件来研究NO介导的血管舒张功能受损的作用。我们预计，在子宫内T暴露将导致性别特异性高血压的影响，通过上调不同的血管PKC同工酶和差异内皮功能障碍的男性和女性的血管，这可能是通过调节出生后的T水平的变化。这些结果将为理解成人CV功能障碍的胎儿编程提供新的分子基础，并提高我们对血管功能障碍性别差异的认识，为制定预防和治疗高血压的性别特异性策略提供令人兴奋的机会。

DESCRIPTION (provided by applicant): Epidemiological studies show increased risk of cardiovascular (CV) diseases in children born to women with compromised pregnancies, such as in preeclampsia, PCOS, protein or energy restriction, obesity, stress, and smoking, but its pathogenesis remains incompletely understood. As one of the common factors observed in these pregnancy pathologies, elevated maternal testosterone (T) is likely to contribute to the fetal programming of CV disorders. Indeed, our recent studies demonstrate that elevated maternal T causes development of hypertensive phenotypes in rat offspring. To understand the mechanisms, 2 central hypotheses are proposed in this project. First, prenatal T induces sex-specific onset and severity of hypertension, and these hypertensive responses are mediated by postnatal increases in T levels. Second, increase in postnatal T induces hypertensive responses through sex-specific dysfunctions in vascular smooth muscle (VSM) protein kinase C (PKC) and endothelial EDHF/NO expression/function. To test these hypotheses, we propose a series of experiments in our established pregnant rat model and examine their offspring. Three specific aims are proposed: 1) Determine whether elevated maternal T programs offspring's hypertension, with more pronounced effect in males than females, and if postnatal T increase precedes hypertension onset. We will telemetrically monitor progressive changes in blood pressure (BP) and measure T levels to establish a relationship between onset and severity of hypertension and changes in postnatal T levels, mechanistically determining if postnatal T increase is the key contributing factor for BP increase. 2) Evaluate the sex-specific hypertensive mechanisms in VSM. We will examine the PKC isoenzyme expression profile in subcellular fractions, its phosphorylation status, and functional activity and examine mechanisms by which androgens regulate PKC expression by assessing binding of T to putative ARE in PKC promoter by ChiP and reporter assays. 3) Dissect the sex-specific mechanisms of impaired endothelial functions. We will examine the EDHF- and NO-mediated pathways and evaluate the mechanisms for impaired EDHF-mediated vasodilation by determining mRNA and protein levels of EDHF components SK3 and IK1 channels and connexins (CX37, CX40, and CX47), their subcellular localization, and functional activity using vascular reactivity and membrane potential studies. We will investigate the role of impaired NO-mediated vasodilator function by assessing the expression of eNOS, its activity, NO production, and signaling events. We expect that in utero T exposure will cause gender-specific hypertensive effects through upregulation of distinct vascular PKC isoenzymes and differential endothelial dysfunctions in the male and female vasculature, which may be regulated through postnatal changes in T levels. The results will provide a novel molecular basis to the understanding of fetal programming of adult CV dysfunction and improve our knowledge of sex differences in vascular dysfunction, providing an exciting opportunity to devise sex-specific strategies for prevention and treatment of hypertension.