Differential control of uterine artery endothelial monolayer integrity by TNF and VEGF is achieved through multiple mechanisms operating inside and outside the cell - Relevance to preeclampsia.

Uterine artery endothelium undergoes a form of functional adaptation during pregnancy because of an increase in Cx43 communication, resulting in increased Ca2+/IP3 exchange and more synchronous and sustained vasodilator production. We have shown previously that acute exposure to growth factors and TNF can block this adaptation through ERK and/or Src-mediated Cx43 phosphorylation. In preeclampsia such adapted function is already missing, but while elevated TNF is associated with this condition, particularly after 28 weeks (late PE), elevated circulating VEGF165 is not. Given PE is a long term condition emerging in the second half of pregnancy, and is often associated with added edema, we now compare the chronic effects of these two factors on the cell monolayer in order to establish if the breakdown of junctional adherens and tight junctional assemblies in which Cx43 resides could also explain loss of vasodilatory function. We report that while TNF can degrade monolayer integrity even in the 0.1-1 ng/ml physiologic range, VEGF up to 10 ng/ml does not. In addition, the progressive action of TNF is mediated through Src and ERK signaling to promote internalization and destruction of VE-Cadherin (VE-Cad) and ZO-1, as well as the expression and secretion of a variety of proteases. At least one protein degraded from the extracellular space is VE-Cad, resulting in release of a shed VE-Cad protein product, and consistent with monolayer breakdown being sensitive to both Src and MEK/ERK kinase inhibitors and the general protease inhibitor GM6001. We conclude that the greater association of TNF with 'late' PE is as much due to its longer term destabilizing effects on junctional assemblies as it is to acute closure of Cx43 channels themselves. New therapies aimed at stabilizing these junctional assemblies may help treat this hypertensive condition.

Cytokine concentrations direct endothelial function in pregnancy and preeclampsia.

Endothelial dysfunction is a prominent feature of preeclampsia, a hypertensive disorder of pregnancy, and contributes to multiple symptoms characteristic of the syndrome. A myriad of growth factors and cytokines are dysregulated in preeclampsia as compared to normal pregnancy, however, a complete appreciation of the effect of changing concentrations of these factors on endothelial function is lacking. In this study, we evaluate the effect of a variety of growth factors and cytokines on Ca2+ signaling and monolayer integrity. We report that VEGF165, TNFα, EGF, and IL-1ß either improve or inhibit Ca2+ signaling depending on dose, whereas TNFα and IL-1ß reduce monolayer integrity and bFGF increases monolayer integrity. Additionally, to model the effects of combinations of growth factors and cytokines, we screened for Ca2+ signaling changes in response to 16 dose combinations of VEGF165 and TNFα together. This revealed an optimal combination capable of supporting pregnancy-adapted Ca2+ signaling, and that changes in either VEGF165 or TNFα dose would result in a shift toward suppressed function. This study shows in detail how growth factor or cytokine concentration effects endothelial cell function. Such data can be used to model how changing growth factor and cytokine levels in normal pregnancy may contribute to healthy endothelial function and in preeclampsia may promote endothelial dysfunction. The results of VEGF165 and TNFα combination treatments suggest that more complex growth factor and cytokine combination modeling may be important in order to more accurately understand the effects of circulating factors on the endothelial function.

Conjugated linoleic acid improves endothelial Ca2+ signaling by blocking growth factor and cytokine-mediated Cx43 phosphorylation.

Sustained Ca2+ burst signaling is crucial for endothelial vasodilator production and is disrupted by growth factors and cytokines. Conjugated linoleic acid (CLA), a Src inhibitor in certain preparations, is generally regarded as safe during pregnancy by the FDA. Multiple CLA preparations; t10, c12 or c9, t11 CLA, or a 1:1 mixture of the two were administered before growth factor or cytokine treatment. Growth factors and cytokines caused a significant decrease in Ca2+ burst numbers in response to ATP stimulation. Both t10, c12 CLA and the 1:1 mixture rescued VEGF165 or TNFα inhibited Ca2+ bursts and correlated with Src-specific phosphorylation of connexin 43. VEGF165, TNFα, and IL-6 in combination at physiologic concentrations revealed IL-6 amplified the inhibitory effects of lower dose of VEGF165 and TNFα. Again, the 1:1 CLA mixture was most effective at rescue of function. Therefore, CLA formulations may be a promising treatment for endothelial dysfunction in diseases such as preeclampsia.

TNF-alpha inhibits pregnancy-adapted Ca2+ signaling in uterine artery endothelial cells.

Enhancement of vasodilation of uterine arteries during pregnancy occurs through increased connexin (Cx)43 gap junction (GJ) communication supporting more frequent and sustained Ca2+ 'bursts'. Such adaptation is lacking in subjects with preeclampsia (PE). Here we show TNF-alpha, commonly increased in PE subjects, inhibits Cx43 function and Ca2+ bursts in pregnancy-derived ovine uterine artery endothelial cells (P-UAEC) via Src and MEK/ERK phosphorylation of Cx43, and this can be reversed by PP2 or U0126. Of relevance to humans: (1) the nutraceutical Src antagonist t10, c12 CLA also recovers Ca2+ bursting in P-UAEC. (2) TNF-alpha can reduce and PP2 rescue Ca2+ bursting and NO output in human umbilical vein endothelium (HUV Endo) preparations. (3) Treatment of HUV Endo from PE subjects with PP2 alone can rescue bursting and NO output. We conclude TNF-alpha acts via Src more than MEK/ERK to inhibit GJ Cx43 function in PE subjects, and CLA may offer a potential therapy.

Cyclic Nucleotides Differentially Regulate Cx43 Gap Junction Function in Uterine Artery Endothelial Cells From Pregnant Ewes.

Cell-cell communication is dependent on GJ (gap junction) proteins such as Cx43 (connexin 43). We previously demonstrated the importance of Cx43 function in establishing the enhanced pregnancy vasodilatory phenotype during pregnancy in uterine artery endothelial cells from pregnant (P-UAEC) ewes. Cx43 is regulated by elevating cAMP and PKA (protein kinase A)-dependent Cx43 S365 phosphorylation-associated trafficking and GJ open gating, which is opposed by PKC (protein kinase C)-dependent S368 phosphorylation-mediated GJ turnover and closed gating. However, the role of cyclic nucleotide-mediated signaling mechanisms that control Cx43 and GJ function in P-UAECs is unknown. We hypothesize that cAMP will mediate increases in S365 phosphorylation, thereby, enhancing GJ trafficking and open gating, while cGMP will stimulate S368, but not S365, phosphorylation to enhance GJ turnover and closed gating in P-UAECs. Treatment with 8-Bromo (8-Br)-cAMP signal significantly (P<0.05) increased nonphosphorylated S365 signal and total Cx43 phosphorylation, but not S368 phosphorylation, while 8-Br-cGMP significantly (P<0.05) increased Cx43 C-terminus-S365 signal, S368, and total Cx43 phosphorylation. Inhibition of PKA, but not PKG (protein kinase G), abrogated the 8-Br-cAMP-stimulated increase in nonphosphorylated S365 and total Cx43 phosphorylation and inhibited S368 below basal levels, whereas inhibition of PKG blocked (P<0.05) the 8-bromo-cGMP-stimulated rises in nonphosphorylated S365, total Cx43, and S368 phosphorylation levels in P-UAECs. Functional studies showed that 8-Br-cAMP increased dye transfer and sustained calcium bursts, while 8-Br-cGMP decreased both. Thus, in P-UAECs, only 8-Br-cAMP and not 8-Br-cGMP effectively enhances nonphosphorylated S365 and total Cx43 expression that correspondingly reduces S368 phosphorylation, allowing increased GJ communication. This provides new insights into the regulatory mechanisms behind Cx43 function and GJ communication.