PREGNANCY CHANGES IN UTERINE ARTERY SIGNALING PATHWAYS

在怀孕期间，子宫动脉激动剂诱导的NO和PGI2的产生显著增强，从而导致血管阻力的降低和支持胎儿生长所必需的血流量的增加。比较七螺旋受体激动剂AII和ATP对生长因子bFGF和VEGF在P-UAEC中的作用的研究进一步表明，虽然不是所有的激动剂都能动员这些细胞中的Ca2+，但七螺旋受体和生长因子受体的激活都会导致P-UAEC中细胞外信号调节蛋白激酶1和2 （ERK-1/2）的激活。对于每一类激动剂，这与它们刺激NO产生的能力直接相关（见下文）。此外，NP- UAEC的平行研究表明，包括ATP、AII和TPA在内的多种激动剂不能刺激NO的产生，也不能有效地与ERK-1/2激活结合，而在P-UAEC中，ERK-1/2激活可以有效地结合。进一步识别和理解MAPK细胞信号通路的偶联和功能，以及这种变化在妊娠期是如何变化的是本提案的目标。此外，尚未确定的信号通路也可能介导生长因子而非七螺旋受体在NP-UAEC中产生NO，因此，妊娠引起的UAEC功能变化可能涉及与未确定的ERK-1/2独立通路的协同作用，因为与ERK-1/2激活的耦合增加。具体目标1：确定bFGF或VEGF（而非AII或ATP）对MEK介导的ERK-1/2激活的作用是否通过生长因子受体自磷酸化和随后的适配蛋白（Shc/Grb2/SOS）的磷酸酪氨酸募集而发生，从而导致P-UAEC中MEK的Ras/Raf激活，以及在NP-UAEC中这种激活在什么水平上解耦。特异性目标2：确定AII和ATP介导的MEK激活是否通过独立于生长因子受体磷酸化和适配器蛋白募集的替代途径发生，即在P-UAEC中通过Src/Raf/MEK级联发生，以及在NP-UAEC中这种反应在什么水平上解耦。具体目的3：进一步描述细胞信号在从非妊娠到妊娠过程中的变化，从而进一步阐明这些变化的机制基础。我们将通过：（3A）进一步比较P-UAEC和NP-UAEC中Shc/Grb2/SOS/Ras/Raf通路和Src/Raf/MEK通路响应bFGF、VEGF、All、ATP和phorbol酯（TPA -control）的激活时间过程，以及（3B）进一步研究NP-UAEC和P-UAEC中可能通过P13-激酶/AKT介导的MEK独立信号传导。具体目标4：通过观察选择性抑制剂对信号中间体活性的影响，并将任何这种影响分别与NO产生和eNOS磷酸化的变化联系起来，确定目标1-3中确定的这些信号中间体在NP-UAEC和P-UAEC中激动剂刺激NO产生水平上的重要性。特异性目的5:（5A）将这些观察到的变化与体内妊娠引起的变化联系起来。因此,信号通路中间体是丢失或获得的功能在以前的具体目标1 - 4,或者信使rna编码信号中间是5 b改变,我们将执行西方分析UA内皮和UA部分免疫组织化学杀螨酯,卵泡期,黄体期和怀孕的母羊,rt - pcr定量UA-endothelial细胞信使rna从同一动物确定表达发生在体内的变化。（5B）利用最近发展的分子技术进一步研究P-UAEC与NP-UAEC在mRNA种类上可能存在的差异，从而确定额外的信号分子以及妊娠诱导和/或丢失的其他因素，但目前可能尚未确定。我们相信这些研究将在理解UA内皮功能的正常控制和妊娠诱导对各种血管收缩剂的难治性增加的机制方面取得重大进展，并可能提供内皮细胞功能的新模型，为未来寻找导致子痫前期和IUGR的功能障碍的研究提供基础。

During pregnancy, there is a dramatic enhancement of agonist induced NO as well as PGI2 production by the uterine artery, so leading to a reduction in vascular resistance and increase in blood flow necessary to support the growing fetus. Studies comparing the effects of heptahelical receptor agonists AII and ATP vs growth factors bFGF and VEGF in P- UAEC have further revealed that while not all these agonists can mobilize Ca2+ in these cells, both heptahelical receptors and growth factor receptor activation results in activation of Extracellular signal Regulated Protein Kinase 1 and 2 (ERK-1/2) in P-UAEC, and for each class of agonist this correlates directly to their ability to stimulate NO production in particular (see below). In addition, parallel studies in NP- UAEC have shown a broad range of agonists, including ATP, AII and TPA, fail to stimulate NO production or couple efficiently to ERK-1/2 activation that otherwise couple efficiently in P-UAEC. Further identification and understanding of coupling to and function of the MAPK cell signaling pathways, and how this changes in pregnancy is the goal of this proposal. Furthermore, as yet unidentified signaling pathways may also be mediating the growth factor but not heptahelical receptor effects in NP-UAEC to produce NO, and therefore in turn that pregnancy induced changes in UAEC function may involve a possible synergy with unidentified ERK-1/2 independent pathways due to increased coupling to ERK-1/2 activation. To this end we propose: Specific Aim 1: to establish if the action of bFGF or VEGF, but not AII or ATP on MEK mediated activation of ERK-1/2 occurs via growth factor receptor autophosphorylation and subsequent phosphotyrosine recruitment of adapter proteins (Shc/Grb2/SOS) leading to Ras/Raf activation of MEK in P-UAEC and at what level this is uncoupled in NP-UAEC. Specific Aim 2: to establish if AII and ATP mediated activation of MEK occurs through alternate pathways independent of growth factor receptor phosphorylation and adapter protein recruitment, namely via the Src/Raf/MEK cascade in P-UAEC and at what level this response is uncoupled in NP-UAEC. Specific Aim 3: to further delineate the changes which occur in cell signaling in progression from the nonpregnant to pregnant state and so further clarify the mechanistic basis for these changes. This will be investigated by: (3A) further comparing the time course of activation of the Shc/Grb2/SOS/Ras/Raf pathway and the Src/Raf/MEK pathway in response to bFGF, VEGF, All, ATP and phorbol ester (TPA -control) in P-UAEC vs NP-UAEC, and (3B) to further investigate possible MEK independent signaling via P13- kinase/AKT in NP-UAEC and P-UAEC. Specific Aim 4: To establish the importance of these signaling intermediates so identified in Aims 1-3 at the level of agonist-stimulated NO production in NP-UAEC and P-UAEC by observing the effects of selective inhibitors on activity of signaling intermediates and relating any such effects to changes in NO production and eNOS phosphorylation respectively. Specific Aim 5:(5A) to relate these observed changes back to the pregnancy induced changes in vivo. Thus where signaling pathway intermediates have been shown to be lost or gained at the level of function in previous specific aims 1-4, or where mRNA encoding a signaling intermediate is altered in 5B, we will perform western analysis on UA endothelium and immunohistochemistry on UA sections from ovex, follicular phase, luteal phase and pregnant ewes, or RT-PCR quantification of UA-endothelial cell mRNA from the same animals to determine if a change in expression occurs in vivo. (5B) to use recently developed molecular techniques to investigate further possible differences in mRNA species from P-UAEC vs NP-UAEC and so identify additional signaling molecules as well as other factors which are induced and/or lost by pregnancy but may not yet be identified as such at this time. We believe these studies will provide a significant advance in both the understanding of normal control of UA endothelial function and the mechanism underlying pregnancy-induction of increased refractoriness to a variety of vasoconstrictors in pregnancy, and may also provide a new model of endothelial cell function on which future studies searching for the dysfunction leading to preeclampsia and IUGR may be based.