Oxysterols, Estrogen, Receptors Anatgonism and Vascular Disease

描述(申请人提供)：最近的随机临床试验表明，雌激素治疗可能不能对绝经和绝经后妇女的心血管疾病提供保护。有强有力的证据表明，如果治疗失败是在动脉粥样硬化出现后开始的，那么治疗失败的可能性更大，这表明拮抗机制可能在这些情况下起作用。我们发现，聚集在动脉粥样硬化病变中的氧固醇27-羟基胆固醇(27HC)是雌激素受体a(ERa)和雌激素受体(ER)功能的强大拮抗剂，并能减弱雌二醇(E2)诱导的内皮型一氧化氮合酶(ENOS)的上调。我们还发现，不能代谢27HC的雌性CYP7B1-/-小鼠已经损害了E2诱导的再内皮化。该研究的目的是确定27HC是否是内皮ER作用的内源性拮抗剂，从而减弱雌激素相关的心血管保护作用。目的1是确定27HC是否减弱了对血管损伤后新生内膜形成和动脉粥样硬化的与E2相关的保护作用。内源性E2(通过阿那曲唑治疗)和外源性E2的保护研究将在接受血管损伤袖带模型的雌性CYP7B1+/+和CYP7B1-/-小鼠身上进行，以及在来自CYP7B1-/-和apoE-/-或低密度脂蛋白受体-/-小鼠杂交的雌性小鼠身上进行。目的2是确定27HC是否改变膜相关的ER功能。在培养的内皮细胞中，将检测27HC对ER亚型选择性激动剂和针对膜ER的新型雌激素树突状分子结合物(EDC)激活eNOS和上游激酶活性的影响。27HC对膜ER决定的内皮细胞表型的影响，包括对迁移和增殖的刺激以及对单核细胞黏附的拮抗作用，也将被评估。目的3确定27HC修饰的内皮细胞ER功能的核靶点。利用RT-PCR和聚焦微阵列技术，检测内源性或外源性E2状态改变后，Tie2-GFP、Cyp7b+/+与Tie2-GFP、Cyp7b-/-雌性小鼠内皮细胞中已知和未知ER靶基因表达的变化。利用EDC作为激动剂，也将在体外鉴定内皮细胞膜ER下游的27HC靶基因。27HC靶基因的作用将在培养内皮细胞表型的获得和功能丧失的研究中进行测试。目的4阐明27HC作为一种独特的SERM的药理作用。27HC拮抗血管和MCF-7细胞的ER功能，增强Hep G2和Caco-2细胞的ER功能。为了确定细胞特异性的基础，将使用肽噬菌体展示技术来鉴定与Era或ER？结合的多肽，后者是由27HC与E2和已知的SERM唯一调节的。已鉴定的多肽对27HC对ERE-荧光素酶的抑制和刺激作用的影响，以及对27HC通过膜ERA和ER的作用的影响将被评估。通过研究内源性雌激素受体拮抗的新机制，这项研究将增加我们对雌激素治疗失败的过程的基本理解。

DESCRIPTION (provided by applicant): Recent randomized clinical trials suggest that estrogen therapy may not provide protection from cardiovascular disease in menopausal and postmenopausal women. There is strong evidence that treatment failure is more likely if it is initiated after atherosclerosis is present, suggesting that antagonistic mechanisms may be operative under those conditions. We have discovered that the oxysterol 27- hydroxycholesterol (27HC), which accumulates in atherosclerotic lesions, is a potent antagonist of estrogen receptor a (ERa) and ER¿ function, and that it attenuates estradiol (E2)-induced upregulation of endothelial NO synthase (eNOS). We have also found that female Cyp7b1-/- mice incapable of metabolizing 27HC have impaired E2-induced reendothelialization. The OBJECTIVE of the proposed research is to determine if 27HC is an endogenous antagonist of ER action in endothelium, thereby diminishing estrogen-related cardiovascular protection. Aim 1 is to determine if 27HC blunts E2-related protection from neointimal formation after vascular injury and from atherosclerosis. Studies of protection by endogenous E2 (manipulated by anastrazole treatment) and exogenous E2 will be done in female Cyp7b1+/+ versus Cyp7b1-/- mice undergoing a cuff model of vascular injury, and in females derived from crosses of Cyp7b1-/- and apoE-/- or LDL receptor-/- mice. Aim 2 is to determine if 27HC modifies membrane-associated ER function. The impact of 27HC on eNOS and upstream kinase activation by ER subtype selective agonists and a new estrogen dendrimer conjugate (EDC) directed at membrane ER will be determined in cultured endothelium. The impact of 27HC on endothelial cell phenotypes dictated by membrane ER, including the stimulation of migration and proliferation and the antagonism of monocyte adhesion, will also be evaluated. Aim 3 is to identify nuclear targets of ER function in endothelium which are modified by 27HC. Using RT-PCR and focused microarrays, changes in expression of known and unknown ER targets will be determined in endothelium sorted from Tie2-GFP;Cyp7b+/+ versus Tie2-GFP;Cyp7b-/- female mice after altering endogenous or exogenous E2 status. 27HC target genes downstream of membrane ER in endothelium will also be identified in vitro using EDC as agonist. The roles of 27HC target genes will be tested in gain- and loss-of-function studies of cultured endothelial cell phenotypes. Aim 4 is to elucidate the pharmacology of 27HC as a unique SERM. Whereas 27HC antagonizes ER function in vascular and MCF-7 cells, it enhances ER function in Hep G2 and Caco-2 cells. To determine the basis for cell specificity, peptide phage display will be employed to identify peptide binding to ERa or ER¿ which is uniquely modulated by 27HC versus E2 and known SERMs. The impact of identified peptides on inhibitory versus stimulatory effects of 27HC on ERE-luciferase, and also on 27HC actions via membrane ERa and ER¿ will be evaluated. By investigating a novel mechanism of endogenous ER antagonism, the proposed research will increase our fundamental understanding of the processes underlying estrogen treatment failure.