Actions of Estrogen in Uterine Artery Endothelium

这项研究的总体假设是，雌激素对尿路内皮细胞(UAEC)的急性作用(不到120分钟)与膜上雌激素受体(ER)结合导致eNOS磷酸化和与小窝蛋白-1解离，并与热休克蛋白90(HSP90)结合，从而直接或/或通过丝裂原活化蛋白激酶(MAPK)途径间接增加eNOS活性。然而，长期(超过2小时)雌激素对UAEC的作用导致UA内皮细胞Caveoln-1表达下调，这是由ER介导的，至少部分是通过膜ER介导的MAPK激活，MAPK转位到细胞核内刺激AP-1转录因子，从而抑制Caveolin-1的表达。此外，在动情周期、ERT和妊娠期间，UA内皮细胞小窝蛋白-1、HSP90相关eNOS和小窝蛋白-1水平的变化与UBF的升高有直接关系。为了解决这一假设，将以绵羊尿酸内皮细胞为实验对象，研究以下具体目标。具体目的1：进一步研究雌激素对细胞外信号调节蛋白激酶(ERK2/1)和其他MAPK家族成员(JNK、p38MAPK和ERK5)的激活作用，并确定雌激素激活ERK的膜信号通路和膜上不通透性的E2b-BSA。具体目的2：建立雌激素刺激UAEC内eNOS活性、NO产生、eNOS磷酸化和小窝蛋白-1解离以及与HSP90之间的关系的时间和剂量依赖关系。目的3：确定雌激素是否通过MAPK磷酸化eNOS，eNOS与小窝蛋白-1解离，并通过MAPK途径与HSP90结合，从而刺激UAEC产生NO。具体目的4：确定在UAEC中，雌激素激活的MAPK是否移位到细胞核内，从而刺激AP-1(Fos/Jun二聚体)转录因子，从而下调小凹-1的表达，以及小凹-1的下调是否与小窝的减少或丢失有关。具体目的5：探讨短期(0-120min)ERT期间体内UA内皮小窝蛋白-1/HSP90相关eNOS表达的变化，以及长期(天数)ERT、卵巢周期和妊娠期间UA内皮小窝蛋白-1表达的变化，以及这些变化是否与UBF升高有关。UBF在怀孕期间显著增加，以便为发育中的胎儿提供足够的氧气和营养供应。妊娠期血液供应不足可能导致IUGR、先兆子痫和新生儿出生体重下降，这反过来又与新生儿发病率呈负相关。因此，这项赠款申请将在围产期保健方面提供重要的临床意义。它也可能在全身心血管系统中有相关的影响，因为雌激素被认为是年轻女性患心血管疾病风险低于男性的主要原因。

The overall hypothesis of this grant is that acute (less than 120 min) actions of estrogen on UA endothelial cells (UAEC) upon binding to membrane estrogen receptor (ER) lead to eNOS phosphorylation and dissociation from caveolin-1 as well as association with heat shock protein 90 (HSP90) thereby increasing eNOS activity directly and/or indirectly via the mitogen- activated protein kinase (MAPK) pathway. Whereas, chronic (greater than 2hr) estrogen actions on UAEC result in down- regulation of UA endothelial caveoln-1 expression, and this is ER-mediated and at least in part through membrane ER-mediated activation of MAPK which translocates into the nucleus to stimulate the AP-1 transcription factors thereby inhibiting caveolin-1 expression. Furthermore, there are direct relationships between changes in caveolin-1 HSP90 associated eNOS and caveolin-1 levels in UA endothelium and rises in UBF during the estrous cycle and ERT and pregnancy. To address this hypothesis, the following specific aims will be studied using sheep UA endothelium as the experimental target. Specific aim 1: to further characterize activation of extracellular signal- regulated kinases (ERK2/1) and other MAPK family members (JNK, p38mapk, and ERK5) by estrogen and to determine the membrane ER- initiated signaling that results in ERK activation in response to estrogen and the membrane impermeable E2b-BSA in UAEC. Specific aim 2: to establish the time- and dose-dependency of estrogen stimulated eNOS activity, NO production, eNOS phosphorylation and dissociation from caveolin-1 as well as association with HSP90 in UAEC. Specific aim 3: to establish if estrogen stimulation of NO production in UAEC is through MAPK phosphorylation of eNOS and eNOS dissociation from caveolin-1 and association with HSP90 via the MAPK pathway. Specific aim 4: to establish in UAEC if estrogen activated MAPK translocates into the nucleus to stimulate the AP-1 (Fos/Jun dimers) transcription factors thereby down-regulating caveolin-1 expression, and if down-regulation of caveolin-1 is associated with reduced or lost of caveolae. Specific aim 5: to establish if the amounts of UA endothelial caveolin-1/HSP90 associated eNOS changes in vivo during short- term (0-120 min) ERT, and UA endothelial caveolin-1 expression is altered in vivo during long-term (days) ERT, ovarian cycle, and pregnancy, and if these changes are associated with rises in UBF. UBF increases substantially during pregnancy in order to provide sufficient oxygen and nutrient supply for the development of the growing fetus. Insufficient blood supply during pregnancy can result in IUGR, preeclampsia, and decreased neonatal birthweight, which in turn inversely correlates to neonatal morbidity. Thus, this grant application will provide important clinical implications in perinatal healthcare. It may also have relevant implications in the systemic cardiovascular system since estrogen is believed to be the major reason that young women have lower risk in cardiovascular diseases than men.