Maternal vascular adaptation to pregnancy is critically important in order to expand the capacity for blood flow through the uteroplacental unit to meet the needs of the developing fetus. Failure of the maternal vasculature to properly adapt can result in hypertensive disorders of pregnancy such as preeclampsia (PE). Herein, we review the endocrinology of maternal adaptation to pregnancy and contrast this with that of PE. Our focus is specifically on those hormones with directly impact endothelial cell function and dysfunction, as endothelial cell dysfunction is a hallmark of PE. A variety of growth factors and cytokines are present in normal vascular adaptation to pregnancy but have also been shown to be circulating at abnormal levels in PE pregnancies. Many of these factors promote endothelial dysfunction when present at abnormal levels by acutely inhibiting key Ca2+ signaling events and chronically promoting the breakdown of endothelial cell-cell contacts. Increasingly, our understanding of how the contributions of the placenta, immune cells, and the endothelium itself promote the endocrine milieu of PE is becoming more clear. We then describe in detail how the complex endocrine environment of PE effects endothelial cell function, why this has contributed to the difficulty in fully understanding and treating this disorder, and how a focus on signaling convergence points of many hormones may be a more successful treatment strategy.

Endothelium-mediated vasodilation is specifically enhanced in uterine circulation during pregnancy, and production of nitric oxide (NO) is increased in response to a wide array of agonists. Uterine artery endothelial cells from nonpregnant (NP-UAECs) or pregnant (P-UAECs) ewes maintained in culture still show a pregnancy-enhanced difference in ATP-stimulated endothelial NO synthase (eNOS; official symbol NOS3) activation, even though NOS3 protein, purinergic receptors, and associated cell signaling proteins are expressed at equal levels. We have also shown that the pregnancy-enhanced endothelial cell NO response to ATP requires an enhanced and sustained capacitative entry phase that is likely mediated via canonical transient receptor potential protein/inositol 1,4,5-trisphosphate receptor type 2 interaction. In this study, we now show by simultaneous video imaging of individual Fura-2-loaded cells that the pregnancy-enhanced capacitative entry phase is not continuous and equal in all cells, but is in fact mediated as a series of periodic [Ca(2+)](i) bursts within individual cells. Not only does pregnancy increase the number of bursts over a longer time period in individual cells, but also a greater proportion of cells exhibit this burst activity, and at high cell density this occurs in a synchronous manner. The mediator of cell synchronization is connexin 43 (Cx43) gap junctions because 1) Cx43 is readily detectable by Western blot analysis in UAECs, whereas Cx40 and Cx37 are weakly detected or absent, and 2) pregnancy-specific enhancement of [Ca(2+)](i) bursts by ATP is blocked by inhibitory loop peptides selective to Cx43 ((43,37)GAP27) but not by a scrambled control peptide or (40)GAP27 or (40,37)GAP26 peptides, which are specific to Cx40 or Cx37. The relationship between Ca(2+) bursts and NOS3 activation is further established by the finding that (43,37)GAP27 inhibits ATP-stimulated NOS3 activation but has no effect on cell mitogenesis. We conclude that it is pregnancy-enhanced gap junction communication between cells that underlies pregnancy enhancement of capacitative entry via TRPC3 and, in turn, NOS3 activation. Such improved gap junction function allows greater and more sustained [Ca(2+)](i) responses to agents such as ATP within a single cell, as well as the additional recruitment of greater numbers of cells to the response in a coordinated and synchronous manner to support enhanced NO production.

Pregnancy is a time of greatly increased uterine blood flow to meet the needs of the growing fetus. Increased uterine blood flow is also observed in the follicular phase of the ovarian cycle. Simultaneous fura-2 and 4,5-diaminofluoresceine (DAF-2) imaging reveals that cells of the uterine artery endothelium (UA Endo) from follicular phase ewes produce marginally more nitric oxide (NO) in response to ATP than those from luteal phase. However, this is paralleled by changes in NO in response to ionomycin, suggesting this is solely due to higher levels of endothelial nitric oxide synthase (eNOS) protein in the follicular phase. In contrast, UA Endo from pregnant ewes (P-UA Endo) produces substantially more NO (4.62-fold initial maximum rate, 2.56-fold overall NO production) in response to ATP, beyond that attributed to eNOS levels alone (2.07-fold initial maximum rate, 1.93-fold overall with ionomycin). The ATP-stimulated intracellular free calcium concentration ([Ca2+]i) response in individual cells of P-UA Endo comprises an initial peak followed by transient [Ca2+]i bursts that are limited in the luteal phase, not altered in the follicular phase, but are sustained in pregnancy and observed in more cells. Thus pregnancy adaptation of UA Endo NO output occurs beyond the level of eNOS expression and likely through associated [Ca2+]i cell signaling changes. Preeclampsia is a condition of a lack of UA Endo adaptation and poor NO production/vasodilation and is associated with elevated placental VEGF165. While treatment of luteal NP-UA Endo and P-UA Endo with VEGF165 acutely stimulates a very modest [Ca2+]i and NO response, subsequent stimulation of the same vessel with ATP results in a blunted [Ca2+]i and an associated NO response, with P-UA Endo reverting to the response of luteal NP-UA Endo. This demonstrates the importance of adaptation of cell signaling over eNOS expression in pregnancy adaptation of uterine endothelial function and further implicates VEGF in the pathophysiology of preeclampsia.

In pregnancy, vascular nitric oxide (NO) production is increased in the systemic and more so in the uterine vasculature, so supporting maximal perfusion of the uterus. This high level of functionality is matched in the umbilical vein, and in corresponding disease states such as preeclampsia, reduced vascular responses are seen in both uterine artery and umbilical vein. In any endothelial cell, NO actually produced by endothelial nitric oxide synthase (eNOS) is determined by the maximum capacity of the cell (eNOS expression levels), eNOS phosphorylation state, and the intracellular [Ca2+]i concentration in response to circulating hormones or physical forces. Herein we discuss how pregnancy specific reprogramming of NO output is determined as much by pregnancy adaptation of [Ca2+]i signaling responses as it is by eNOS expression and phosphorylation. By examining changes in [Ca2+]i signaling responses from HHVE, UAEC, and HUVEC in (where appropriate) nonpregnant, normal pregnant, and pathological pregnant (preeclamptic) state, it is clear that pregnancy adaptation of NO output occurs at the level of sustained phase ‘capacitative entry’ [Ca2+]i response, and the adapted response is lacking in preeclamptic pregnancies. Further, gap junction function is an essential permissive regulator of the capacitative response and impairment of NO output results from any inhibitor of gap junction function, or capacitative entry using TRPC channels. Identifying these [Ca2+]i signaling mechanisms underlying normal pregnancy adaptation of NO output not only provides novel targets for future treatment of diseases of pregnancy, but may also apply to other common forms of hypertension.