Positive vs. Negative Effects of VEGF on Uterine ARtery Endothelial Function...

我们在理解激酶和Ca 2+在激活细胞中的作用方面取得了实质性进展。 子宫动脉内皮细胞（UAEC）中的eNOS及其在妊娠中的变化。特别加强 妊娠期间UAEC中NO的产生是通过激酶使eNOS敏感化来实现的。其次 并且独立地，适应包括响应于以下的持续系列的Ca 2+爆发的发展： 激动剂如ATP，其通过异源三聚体G蛋白刺激PLC-β 3。这是一种循环式容性进入 通过TRPC 3与IP 3R 2的相互作用介导。尽管如此，TRPC通道开放的妊娠特异性增强 不是在TRPC本身的水平上调节，而是在通过CX43间隙连接增强的细胞间通讯的水平上调节。 我们还发现VEGF通过酪氨酸激酶受体VEGFR 2激活eNOS，因此增加了这种可能性。 PLC γ可能被激活而不是PLC β。此外，VEGF只能刺激细胞中的Ca 2+反应。 约25%的细胞，并进一步表明，Ca 2+反应不包括在P- UAEC。此外，VEGF预处理细胞可抑制UAEC随后对ATP的反应性， 显然是通过磷酸化并通过ERK-1/2途径关闭CX43缝隙连接。因此， 在过度表达VEGF之前， 通过增强的间隙连接功能的Ca 2+响应。问题是，我们能不能找出 受体或信号通路介导VEGF的有益作用与有害作用，以便干预 “病态妊娠”？在许多其他细胞系统中，现在很明显，VEGFR 2功能可以通过以下方式调节： 与VEGFR 1或NP-1受体异二聚化。因此，第一个问题是， VEGFR 1或NP-1对VEGFR 2在介导对VEGF的应答中的生理学作用有任何影响，特别是在 怀孕第二个是建立VEGF对Ca 2+动员的时间差异的基础， 通过特别关注PLC γ与β 3在每种情况下的作用和位置，以及 去除每个响应上的PLC γ。第三是更全面地了解机制（即激酶介导 VEGF预处理可通过其改变间隙连接功能，从而改变对经典的免疫反应性。 异源三聚体G蛋白偶联受体，例如由ATP作用的那些。这使我们-SP目标1）：建立 VEGFR 1和NP-1在调节VEGFR 2的Ca 2+动员、ERK-1/2的活化和活化中的相对作用 eNOS在NP与P-UAEC中的表达。目的2）：确定PLC γ在介导VEGFR 1与VEGFR 2/NP-1中的作用 NP和P-UAEC中Ca 2+的偶联动员。Sp目的3）：建立VEGFR 1与VEGFR 2的相对作用， NP-1通过MEK/ERK介导的CX43磷酸化阻断细胞间通讯。目标4）：在 与项目II、III和核心C合作，检查项目1中确定的机制途径是否存在 和/或在项目II和III中调节或衍生的内皮细胞中改变。我们希望，通过追求这些目标， 研究，我们将能够更好地了解生长因子调节UAEC功能的机制 和功能障碍，并确定潜在的治疗目标，以打击功能障碍。

We have made substantial progress in our understanding of the roles of both kinases and Ca2+ in the activation of eNOS in uterine artery endothelial cells (UAEC) and how this may be altered in pregnancy. Specifically enhanced production of NO in UAEC during pregnancy is achieved through the sensitivitization of eNOS via kinases. Secondly and independently, the adaptation includes the development of a sustained series of Ca2+ bursts in response to agonists such as ATP that stimulate PLC-beta 3 via heterotrimeric G-proteins. This is a form of cyclic capacitative entry mediated by TRPC3 interaction with IP3R2. Nonetheless, pregnancy specific enhancement of TRPC channel opening is not regulated at the level of TRPC itself but at the level of enhanced cell-cell communication via CX43 Gap junctions. We have also shown VEGF activates eNOS through the tyrosine kinase receptor VEGFR2 and so raises the possibility that PLC gamma may be activated rather than PLC-beta. In addition VEGF can only stimulate a Ca2+ response in about 25% of cells and further that the Ca2+ response does not include the repeated Ca2+ bursts seen for ATP in P- UAEC. Furthermore, VEGF pretreatment of cells can inhibit the subsequent responsiveness of UAEC to ATP, apparently by phosphorylating and so closing the CX43 Gap junctions via the ERK-1/2 pathway. As such the effect of prior overexposure to VEGF is to remove one of the otherwise beneficial adaptations to pregnancy, namely sustained Ca2+ responses via enhanced Gap junction function. The question is can we identify a mechanistic difference in the receptor or signaling pathway mediating the beneficial actions vs deleterious actions of VEGF in order to intervene in 'diseased pregnancy'? In many other cell systems it is now apparent that VEGFR2 function can be modulated by heterodimerization with VEGFR1 or NP-1 receptor. The first question therefore is if the presence or absence of VEGFR1 or NP-1 has any effect on the physiologic role of VEGFR2 in mediating responses to VEGF, particularly with pregnancy. The second is to establish the basis of the temporal differences in Ca2+ mobilization by VEGF as opposed to ATP by focusing specifically on the roles and locations of PLC gamma vs beta 3 in each case, and the effect of removal of PLC gamma on each response. The third is to understand more fully the mechanism (ie kinase mediate phosphorylation) by which VEGF pretreatment may alter Gap junction function and so responsiveness to classical heterotrimeric G protein coupled receptors such as those acted on by ATP. This leads us to -Sp Aim 1): Establish the relative roles of VEGFR1 and NP-1 in modulating VEGFR2 mobilization of Ca2+, activation of ERK-1/2, and activation of eNOS in NP vs P-UAEC. Sp Aim 2): Establish the roles of PLC gamma in mediating VEGFR1 vs VEGFR2/NP-1 coupled mobilization of Ca2+ in NP and P-UAEC. Sp Aim 3): Establish the relative roles of VEGFR1 vs VEGFR2 and NP-1 in blocking cell-cell communication through MEK/ERK mediated CX43 phosphorylation. Sp Aim 4): In collaboration with Projects II, III and Core C, Examine if the mechanistic pathways identified in Project 1 are present and or altered in endothelial cells conditioned or derived in Projects II and III. It is our hope that by pursuing these studies, we will be better able to understand the mechanisms by which growth factors act to regulate UAEC function and dysfunction and identify potential therapeutic targets to combat dysfunction.