Oxysterols, Estrogen, Receptors Anatgonism and Vascular Disease

描述（由申请人提供）：最近的随机临床试验表明，雌激素治疗可能不能保护绝经期和绝经后妇女免受心血管疾病的侵害。有强有力的证据表明，如果在动脉粥样硬化出现后开始治疗，治疗失败的可能性更大，这表明在这些情况下，拮抗机制可能是有效的。我们发现，在动脉粥样硬化病变中积累的氧甾醇27-羟基胆固醇（27HC）是雌激素受体a （ERa）和ER¿功能的有效拮抗剂，它可以减弱雌二醇（E2）诱导的内皮NO合成酶（eNOS）的上调。我们还发现不能代谢27HC的雌性Cyp7b1-/-小鼠e2诱导的再内皮化受损。本研究的目的是确定27HC是否是内皮内源性内源性内源性内质网拮抗剂，从而降低雌激素相关的心血管保护作用。目的1是确定27HC是否会减弱e2对血管损伤后新生内膜形成和动脉粥样硬化的保护作用。内源性E2（阿纳唑处理）和外源性E2的保护作用研究将在血管损伤袖口模型的雌性Cyp7b1+/+与Cyp7b1-/-小鼠，以及Cyp7b1-/-与apoE-/-或LDL受体-/-小鼠杂交的雌性小鼠中进行。目的2是确定27HC是否改变膜相关内质网功能。27HC对eNOS和上游激酶活化的影响将在培养的内皮细胞中被内质网亚型选择性激动剂和一种新的针对膜内质网的雌激素树状聚合物偶联物（EDC）所确定。27HC对内皮细胞表型的影响，包括对迁移和增殖的刺激以及单核细胞粘附的拮抗，也将被评估。目的3：确定27HC修饰内皮细胞内质网功能的核靶点。利用RT-PCR和聚焦微阵列技术，确定从Tie2-GFP中分离出来的内皮细胞中已知和未知ER靶点的表达变化；Cyp7b+/+对Tie2-GFP的影响改变内源性或外源性E2状态后的Cyp7b-/-雌性小鼠。在体外使用EDC作为激动剂，内皮细胞内质膜下游的27HC靶基因也将被鉴定。27HC靶基因的作用将在培养内皮细胞表型的功能获得和功能丧失研究中进行测试。目的4是阐明27HC作为一种独特的血清素的药理作用。27HC在血管细胞和MCF-7细胞中拮抗内质网功能，而在Hep G2和Caco-2细胞中增强内质网功能。为了确定细胞特异性的基础，将使用肽噬菌体展示来鉴定与ERa或ER结合的肽，这是由27HC与E2和已知serm唯一调节的。将评估鉴定的肽对27HC对ere -荧光素酶的抑制和刺激作用的影响，以及27HC通过膜ERa和ER¿的作用。通过研究内源性内源性内质网拮抗的新机制，本研究将增加我们对雌激素治疗失败的基本理解。

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.