Estrogen enhancement of endothelial vasodilatory factors

描述（由申请人提供）：绝经后的妇女发生心肌梗死和中风的风险显著增加。与绝经相关的内源性雌激素的丧失导致心血管风险增加。雌激素的一个重要的、最近建立的血管保护功能是增强内皮一氧化氮（NO）分泌，其促进血管舒张并拮抗血栓形成。内皮细胞分泌的NO通过Ca 2+激活内皮细胞NO合成酶（eNOS）来调节。本研究的目的是阐明雌激素和选择性雌激素受体调节剂雷洛昔芬增强内皮血管舒张因子（EDRF）包括NO、前列环素（PGI 2）和内皮依赖性超极化因子（EDHF）释放的细胞和分子机制。我建议雌激素和雷洛昔芬结合到它们的受体后，它们改变基因表达和/或离子转运机制，通过增强内皮细胞中的Ca 2+内流和/或减少Ca 2+外排来提高[Ca 2 +]i。目的：我将测试哪种可能的靶分子组合（例如，eNOS、EDHF）受雌激素和雷洛昔芬治疗的影响。此外，我将评估是否激活非基因组受体的生理相关浓度的雌激素调节内皮细胞中的钙依赖性eNOS。所获得的知识有望为雌激素和雷洛昔芬治疗在心血管保护中的作用提供新的见解。将在取自不同组大鼠的完整瓣膜内皮和主动脉环以及人脐静脉（HUVEC）内皮细胞中进行实验。将使用生物测定法测量EDRF释放。生物测定使我们能够鉴定EDRF的性质及其在我们的制剂中的细胞作用。细胞内Ca 2+在刺激EDRF分泌中的作用将使用比率fura- 2荧光法测定。将通过RT-PCR、蛋白质印迹和免疫组织化学评估基因表达改变的程度。了解雌激素的心脏保护作用的机制，预计将大大有助于新的治疗策略的发展，从而合成雌激素类似物的有益的心脏保护作用可能会从雌激素活性的不良成分分离。对这些保护机制的深入了解预计最终也将对男性人群有益。

DESCRIPTION (provided by applicant): The risk of myocardial infarction and stroke increases dramatically in women after menopause. Loss of endogenous estrogen associated with menopause contributes to this increased cardiovascular risk. An important, recently established vasoprotective function of estrogen is enhancement of endothelial nitric oxide (NO) secretion which promotes vasodilation and antagonizes thrombosis. NO secreted by the endothelium is regulated through Ca2+ activation of endothelial NO synthase (eNOS). The objective of my research is to elucidate the cellular and molecular mechanisms whereby estrogen and raloxifene, a selective estrogen receptor modulator, enhance release of endothelial vasodilatory factors (EDRF) including NO, prostacyclin (PGI2), and endothelium-dependent hyperpolarizing factors (EDHF). I propose that after estrogen and raloxifene bind to their receptors, they alter gene expression and/or ion transport mechanisms raising [Ca2+]i through enhanced Ca2+ influx and/or decreased Ca2+ extrusion in endothelial cells. Aims: I will test which combination of the plausible target molecules (e.g., eNOS, EDHF) are affected by estrogen and raloxifene treatment. In addition, I will evaluate whether activation of non-genomic receptors by physiologically relevant concentrations of estrogen modulates calcium-dependent eNOS in endothelial cells. The knowledge gained is expected to provide new insight into the role of estrogen and raloxifene therapy in cardiovascular protection. Experiments will be performed in intact valvular endothelium and aortic rings taken from different groups of rats as well as in endothelial cells derived from human umbilical veins (HUVECs). EDRF release will be measured using Bioassay. Bioassay allows us to identify the nature of EDRF and its cellular action(s) in our preparation. The role of intracellular Ca2+ in stimulation of EDRF secretion will be determined using ratiometric fura- 2 fluorimetry. The extent of altered gene expression will be assessed by RT-PCR, Western blots, and immunohistochemistry. Understanding the mechanisms underlying the cardioprotective action of estrogen is expected to contribute significantly to the development of new therapeutic strategies, whereby the beneficial cardioprotective effects of synthetic estrogen analogues might be separated from the undesirable components of estrogen activity. The enhanced insight into these protective mechanisms is expected to eventually also be beneficial for the male population.