Actions of Estrogen in Uterine Artery Endothelium

本研究的总体假设是，雌激素在UA内皮细胞（UAEC）与膜雌激素受体（ER）结合后的急性（小于120分钟）作用导致eNOS磷酸化并与caveolin-1分离，并与热休克蛋白90 （HSP90）相关，从而直接和/或间接通过丝裂原活化蛋白激酶（MAPK）途径增加eNOS活性。然而，慢性（超过2小时）雌激素对UAEC的作用导致UA内皮小洞蛋白-1表达下调，这是er介导的，至少部分是通过膜er介导的MAPK激活，MAPK易位到细胞核中刺激AP-1转录因子，从而抑制小洞蛋白-1的表达。此外，在发情周期、ERT和妊娠期间，UA内皮中与eNOS和caveolin-1 HSP90相关的eNOS和caveolin-1水平的变化以及UBF的升高之间存在直接关系。为了解决这一假设，我们将以绵羊UA内皮为实验靶点，研究以下具体目标。特异性目的1：进一步表征雌激素对细胞外信号调节激酶（ERK2/1）和其他MAPK家族成员（JNK、p38mapk和ERK5）的激活，并确定雌激素和膜不透性E2b-BSA在UAEC中导致ERK激活的膜ER启动信号。具体目的2：建立雌激素在UAEC中刺激eNOS活性、NO生成、eNOS磷酸化和caveolin-1解离以及与HSP90的相关性的时间和剂量依赖性。具体目的3：确定雌激素是否通过MAPK磷酸化eNOS、eNOS与caveolin-1分离并通过MAPK途径与HSP90关联来刺激UAEC中NO的产生。具体目的4：在UAEC中，确定雌激素激活的MAPK是否易位到细胞核内刺激AP-1 （Fos/Jun二聚体）转录因子从而下调caveolin-1的表达，以及caveolin-1的下调是否与小窝减少或缺失有关。具体目的5：确定在短期（0-120分钟）ERT期间，体内UA内皮小巢蛋白-1/HSP90相关eNOS的量是否发生变化，以及在长期（天）ERT、卵巢周期和妊娠期间，体内UA内皮小巢蛋白-1的表达是否发生改变，以及这些变化是否与UBF升高有关。在怀孕期间，UBF大幅增加，以便为胎儿的发育提供足够的氧气和营养供应。妊娠期血液供应不足可导致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，这项拨款申请将为围产期保健提供重要的临床意义。由于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，它也可能对系统性心血管系统产生相关影响。

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.