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

VEGF 对子宫动脉内皮功能的积极作用与消极作用......

• 批准号: 7189522
• 负责人: IAN M. BIRD
• 金额: $ 15.2万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2012-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7189522
• 关键词: Agonist; Antibodies; Arteries; Biological Assay; Birds; Cell Communication; Cell physiology; Cells; Closure; Collaborations; Condition; Connexin 43; Coupled; Development; Disease Progression; Dose; Endothelial Cells; Endothelium; Excision; Exposure to; Family; Functional disorder; G-Protein-Coupled Receptors; Gap Junctions; Growth Factor; Heterodimerization; Heterotrimeric GTP-Binding Proteins; Image; Knock-out; Location; MEKs; Measures; Mediating; Mediator of activation protein; Methods; Monitor; Outcome; Pathway interactions; Phosphorylation; Phosphotransferases; Physiological; Pre-Eclampsia; Pregnancy; Production; Protein Isoforms; Receptor Protein-Tyrosine Kinases; Relative (related person); Research Personnel; Role; Series; Signal Pathway; Small Interfering RNA; System; U-0126; VEGF121 gene; VEGF165; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-2; Vascular Endothelial Growth Factors; base; density; human PLCB3 protein; member; phospholipase C beta; phospholipase C gamma; programs; receptor; response; therapeutic target

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.

我们在了解激酶和 Ca2+ 在激活 子宫动脉内皮细胞 (UAEC) 中的 eNOS 及其在妊娠期间如何改变。特别增强 妊娠期间UAEC 中NO 的产生是通过激酶对eNOS 的敏化来实现的。第二 独立地，适应包括开发一系列持续的 Ca2+ 爆发，以响应 激动剂，例如 ATP，通过异三聚体 G 蛋白刺激 PLC-β3。这是循环电容输入的一种形式 由 TRPC3 与 IP3R2 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 本身水平上进行调节，而是在通过 CX43 间隙连接增强细胞间通讯的水平上进行调节。 我们还表明 VEGF 通过酪氨酸激酶受体 VEGFR2 激活 eNOS，因此提出了可能性 PLC gamma 可能被激活，而不是 PLC-beta。此外，VEGF 只能刺激 Ca2+ 反应 约 25% 的细胞，而且 Ca2+ 反应不包括 P- 中 ATP 所见的重复 Ca2+ 爆发 阿拉伯联合酋长国。此外，细胞的VEGF预处理可以抑制UAEC随后对ATP的反应， 显然是通过 ERK-1/2 途径磷酸化并关闭 CX43 间隙连接。这样的效果 之前过度暴露于 VEGF 会消除一种对妊娠有益的适应，即持续 通过增强间隙连接功能进行 Ca2+ 反应。问题是我们能否识别出两者之间的机制差异？ 受体或信号通路介导 VEGF 的有益作用与有害作用，以干预 “病态妊娠”？现在很明显，在许多其他细胞系统中，VEGFR2 功能可以通过以下方式调节： 与 VEGFR1 或 NP-1 受体异二聚化。因此，第一个问题是是否存在 VEGFR1 或 NP-1 对 VEGFR2 在介导 VEGF 反应中的生理作用有任何影响，特别是对于 怀孕。第二个是建立 VEGF 动员 Ca2+ 的时间差异的基础，而不是 通过特别关注 PLC gamma 与 beta 3 在每种情况下的作用和位置，以及 ATP 的影响 去除每个响应上的 PLC gamma。第三是更全面地了解其机制（即激酶介导 磷酸化），VEGF 预处理可能会改变间隙连接功能，从而改变对经典疗法的反应 异三聚体 G 蛋白偶联受体，例如受 ATP 作用的受体。这引导我们实现 -Sp 目标 1)：建立 VEGFR1 和 NP-1 在调节 VEGFR2 Ca2+ 动员、ERK-1/2 激活和激活中的相对作用 NP 与 P-UAEC 中 eNOS 的比较。 Sp 目标 2)：建立 PLC gamma 在介导 VEGFR1 与 VEGFR2/NP-1 中的作用 NP 和 P-UAEC 中 Ca2+ 的耦合动员。 Sp 目标 3)：建立 VEGFR1 与 VEGFR2 的相对作用以及 NP-1 通过 MEK/ERK 介导的 CX43 磷酸化阻断细胞间通讯。 Sp 目标 4)：在 与项目 II、III 和核心 C 合作，检查项目 1 中确定的机制路径是否存在 和/或在项目 II 和 III 中调节或衍生的内皮细胞中发生改变。我们希望通过追求这些 研究，我们将能够更好地理解生长因子调节UAEC功能的机制 和功能障碍，并确定对抗功能障碍的潜在治疗靶点。