In pregnancy, the uterine vasculature undergoes dramatic vasodilatory adaptations. Previously, vascular endothelial growth factor (VEGF) has been shown to stimulate endothelial nitric oxide synthase (eNOS) in uterine artery endothelial cells (UAECs) derived from pregnant ewes to a greater extent than those from non-pregnant ewes in a manner not fully explained by changes in the phosphorylation of eNOS. In this study, we used Fura-2 Ca²⁺ imaging and arginine-to-citrulline conversion eNOS activity assays to assess the importance of VEGF-stimulated Ca²⁺ responses in pregnancy-related changes in NO production in UAEC. In this study, we show that pregnancy-induced changes in VEGF-stimulated Ca²⁺ responses could account in part for the greater capacity of VEGF to stimulate eNOS in UAECs from pregnant versus non-pregnant animals. VEGF-stimulated Ca²⁺ responses in UAECs from pregnant and non-pregnant animals were mediated through VEGF receptor 2 and were detected in roughly 15% of all cells. There were no pregnancy-specific differences in area under the curve or peak height. UAECs from pregnant animals were more consistent in the time to response initiation, had a larger component of extracellular Ca²⁺ entry, and were more sensitive to a submaximal dose of VEGF. In UAECs from pregnant and non-pregnant animals Ca²⁺ responses and eNOS activation were sensitive to the phospholipase C/inositol 1,4,5-trisphosphate pathway inhibitors 2-aminoethoxydiphenylborane and U73122. Thus, changes in VEGF-stimulated [Ca²⁺]_i are necessary for eNOS activation in UAECs, and pregnancy-induced changes in Ca²⁺ responses could also in part explain the pregnancy-specific adaptive increase in eNOS activity in UAECs.

Abstract

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-1β either improve or inhibit Ca²⁺ signaling depending on dose, whereas TNFα and IL-1β 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.