Estrogen enhancement of endothelial vasodilatory factors

描述（由申请人提供）：绝经后的女性心肌梗死和中风的风险急剧增加。内源性雌激素的丧失与更年期相关，导致心血管风险增加。一个重要的，最近建立的雌激素的血管保护功能是增强内皮一氧化氮（NO）的分泌，促进血管舒张和对抗血栓形成。内皮细胞分泌NO是通过Ca2+激活内皮NO合成酶（eNOS）来调控的。我的研究目的是阐明雌激素和雷洛昔芬（一种选择性雌激素受体调节剂）促进内皮血管扩张因子（EDRF）释放的细胞和分子机制，包括NO、前列环素（PGI2）和内皮依赖性超极化因子（EDHF）。我提出，雌激素和雷洛昔芬结合受体后，它们改变基因表达和/或离子运输机制，通过增加Ca2+内流和/或减少内皮细胞中的Ca2+挤出来提高[Ca2+] I。目的：我将测试雌激素和雷洛昔芬治疗对哪些可能的靶分子（如eNOS， EDHF）的组合有影响。此外，我将评估非基因组受体的激活是否通过生理相关浓度的雌激素调节内皮细胞中钙依赖性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。实验将在取自不同组大鼠的完整瓣膜内皮和主动脉环以及取自人脐静脉的内皮细胞（HUVECs）中进行。EDRF释放将使用生物测定法测量。生物测定使我们能够确定制剂中EDRF的性质及其细胞作用。细胞内Ca2+在刺激EDRF分泌中的作用将使用比例脲- 2荧光法确定。基因表达改变的程度将通过RT-PCR、Western blots和免疫组织化学进行评估。了解雌激素心脏保护作用的机制有望为开发新的治疗策略做出重大贡献，从而将合成雌激素类似物的有益心脏保护作用与雌激素活性的不良成分分离开来。对这些保护机制的深入了解预计最终也会对男性人群有益。

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.