We have made substantial progress in our understanding of the roles of both kinases and Ca2+ in the activation ofeNOS in uterine artery endothelial cells (UAEC) and how this may be altered in pregnancy. Specifically enhancedproduction of NO in UAEC during pregnancy is achieved through the sensitivitization of eNOS via kinases. Secondlyand independently, the adaptation includes the development of a sustained series of Ca2+ bursts in response toagonists such as ATP that stimulate PLC-beta 3 via heterotrimeric G-proteins. This is a form of cyclic capacitative entrymediated by TRPC3 interaction with IP3R2. Nonetheless, pregnancy specific enhancement of TRPC channel openingis 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 possibilitythat PLC gamma may be activated rather than PLC-beta. In addition VEGF can only stimulate a Ca2+ response inabout 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 ofprior overexposure to VEGF is to remove one of the otherwise beneficial adaptations to pregnancy, namely sustainedCa2+ responses via enhanced Gap junction function. The question is can we identify a mechanistic difference in thereceptor 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 byheterodimerization with VEGFR1 or NP-1 receptor. The first question therefore is if the presence or absence ofVEGFR1 or NP-1 has any effect on the physiologic role of VEGFR2 in mediating responses to VEGF, particularly withpregnancy. The second is to establish the basis of the temporal differences in Ca2+ mobilization by VEGF as opposedto ATP by focusing specifically on the roles and locations of PLC gamma vs beta 3 in each case, and the effect ofremoval of PLC gamma on each response. The third is to understand more fully the mechanism (ie kinase mediatephosphorylation) by which VEGF pretreatment may alter Gap junction function and so responsiveness to classicalheterotrimeric G protein coupled receptors such as those acted on by ATP. This leads us to -Sp Aim 1): Establish therelative roles of VEGFR1 and NP-1 in modulating VEGFR2 mobilization of Ca2+, activation of ERK-1/2, and activationof eNOS in NP vs P-UAEC.
Sp Aim 2) : Establish the roles of PLC gamma in mediating VEGFR1 vs VEGFR2/NP-1coupled mobilization of Ca2+ in NP and P-UAEC.
Sp Aim 3) : Establish the relative roles of VEGFR1 vs VEGFR2 andNP-1 in blocking cell-cell communication through MEK/ERK mediated CX43 phosphorylation.
Sp Aim 4) : Incollaboration with Projects II, III and Core C, Examine if the mechanistic pathways identified in Project 1 are presentand or altered in endothelial cells conditioned or derived in Projects II and III. It is our hope that by pursuing thesestudies, we will be better able to understand the mechanisms by which growth factors act to regulate UAEC functionand dysfunction and identify potential therapeutic targets to combat dysfunction.
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