Actions of Estrogen in Uterine Artery Endothelium

该授权的总体假设是，雌激素在与膜雌激素受体（ER）结合后对UA内皮细胞（UAEC）的急性（小于120分钟）作用导致eNOS磷酸化和从小窝蛋白-1解离以及与热休克蛋白90（HSP 90）结合，从而直接和/或通过促分裂原活化蛋白激酶（MAPK）途径间接增加eNOS活性。 然而，雌激素对UAEC的慢性（大于2小时）作用导致UA内皮小窝蛋白-1表达的下调，并且这是ER介导的，并且至少部分地通过膜ER介导的MAPK活化，MAPK易位到细胞核中以刺激AP-1转录因子，从而抑制小窝蛋白-1表达。 此外，有直接关系的变化之间的窖蛋白-1 HSP 90相关的eNOS和窖蛋白-1水平在UA内皮细胞和上升的UBF在发情周期和ERT和妊娠。 为了解决这一假设，将使用绵羊UA内皮作为实验靶点研究以下具体目标。 具体目标1：进一步表征雌激素对细胞外信号调节激酶（ERK 2/1）和其它MAPK家族成员（JNK、p38 mapk和ERK 5）的激活，并确定导致ERK响应雌激素和UAEC中膜不可渗透的E2 b-BSA而激活的膜ER启动的信号传导。 具体目标2：在UAEC中建立雌激素刺激eNOS活性、NO产生、eNOS磷酸化和与小窝蛋白-1的解离以及与HSP 90的关联的时间和剂量依赖性。 具体目标3：确定雌激素刺激UAEC中NO产生是否是通过eNOS的MAPK磷酸化和eNOS从小窝蛋白-1解离并通过MAPK途径与HSP 90结合。 具体目标4：在UAEC中确定雌激素激活的MAPK是否易位到细胞核中以刺激AP-1（Fos/Jun二聚体）转录因子从而下调小窝蛋白-1表达，以及小窝蛋白-1的下调是否与小窝的减少或丧失相关。具体目标5：以确定在短期（0-120分钟）ERT期间UA内皮小窝蛋白-1/HSP 90相关eNOS的量是否在体内发生变化，以及在长期（天）ERT、卵巢周期和妊娠期间UA内皮小窝蛋白-1表达是否在体内发生变化，以及这些变化是否与UBF升高相关。UBF在怀孕期间大幅增加，以提供充足的氧气和营养供应，供胎儿生长发育。 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，这项拨款申请将提供重要的临床意义，在围产期保健。 它也可能对全身心血管系统产生相关影响，因为雌激素被认为是年轻女性患心血管疾病的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.