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The epidermal growth factor receptor (EGFR) is expressed robustly in the placenta, and critical processes of pregnancy such as placental growth and trophoblast fusion are dependent on EGFR function. However, the role that aberrant EGFR signaling might play in the etiology and/or maintenance of preeclampsia (PE) remains largely unexplored. Recently, we have shown that overexpression of EGFR in cultured uterine artery endothelial cells (UAEC), which express little endogenous EGFR, remaps responsiveness away from vascular endothelial growth factor receptor (VEGFR) signaling and toward EGFR, suggesting that endothelial EGFR expression may be kept low to preserve VEGFR control of angiogenesis. Here we will consider the evidence for the possibility that the endothelial dysfunction observed in PE might in some cases result from elevation of endothelial EGFR. During pregnancy, trophoblasts are known to synthesize large amounts of EGFR protein, and the placenta regularly releases syncytiotrophoblast-derived exosomes and microparticles into the maternal circulation. Although there are no reports of elevated EGFR gene expression in preeclamptic endothelial cells, the ongoing shedding of placental vesicles into the vascular system raises the possibility that EGFR-rich vesicles might fuse with endothelium, thereby contributing to the symptoms of PE by interrupting angiogenesis and blocking pregnancy-adapted vasodilatory function.

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+]iare 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.

In pregnancy, vascular nitric oxide (NO) production is increased in the systemic and more so in the uterine vasculature, thereby supporting maximal perfusion of the uterus. This high level of functionality is matched in the umbilical vein, and in corresponding disease states such as pre-eclampsia, reduced vascular responses are seen in both uterine artery and umbilical vein. In any endothelial cell, NO actually produced by endothelial NO synthase (eNOS) is determined by the maximum capacity of the cell (eNOS expression levels), eNOS phosphorylation state, and the intracellular [Ca2+]iconcentration 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+]isignaling responses as it is by eNOS expression and phosphorylation. By examining the changes in [Ca2+]isignaling responses from human hand vein endothelial cells, uterine artery endothelial cells, and human umbilical vein endothelial cells in (where appropriate) nonpregnant, normal pregnant, and pathological pregnant (pre-eclamptic) state, it is clear that pregnancy adaptation of NO output occurs at the level of sustained phase ‘capacitative entry’ [Ca2+]iresponse, and the adapted response is lacking in pre-eclamptic pregnancies. Moreover, 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+]isignaling 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.

We have previously shown that endothelial cells (EC) derived from the uterine artery (UA) of both pregnant (P-UAEC) and nonpregnant (NP-UAEC) ewes show a biphasic intracellular free Ca2+ ([Ca2+]i) response after ATP stimulation. In each case, the initial transient peak, caused by the release of Ca2+ from the intracellular Ca2+ stores, is mediated by purinergic receptor-Y2 and is very similar in both cell types. However, the sustained phase in particular, caused by the influx of extracellular Ca2+, is heightened in the P-UAEC, and associates with an increased ability of the cells to demonstrate enhanced capacitative Ca2+ entry (CCE) via store-operated channels (SOCs). Herein we demonstrated that the difference in the sustained [Ca2+]i response is maintained for at least 30 min. When 2-aminoethoxydiphenyl borate (2APB) (an inhibitor of the inosital 1,4,5-trisphosphate receptor (IP3R) and possibly SOC) was used in conjunction with ATP, it was capable of completely inhibiting CCE. Since 2APB can inhibit SOC in some cell types and 2APB was capable of inhibiting CCE in the UAEC model, the role of SOC in CCE was first evaluated using the classical inhibitor La3+. The ATP-induced sustained phase was inhibited by 10 μM La3+, implying a role for SOC in the [Ca2+]i response. Since canonical transient receptor potential channels (TRPCs) have recently been identified as putative SOCs in many cell types, including EC, the expression levels of several isoforms were evaluated in UAEC. Expression of TRPC3 and TRPC6 channels in particular was detected, but no significant difference in expression level was found between NP- and P-UAEC. Nonetheless, we were able to show that IP3R2 interacts with TRPC3 in UAEC, forming a protein complex, and that this interaction is considerably enhanced in an agonist sensitive manner by pregnancy. Thus, while IP3R and TRPC isoforms are not altered in their expression by pregnancy, enhanced functional interaction of TRPC3 with IP3R2 may underlie pregnancy-enhanced CCE in the UAEC model and so explain the prolonged [Ca2+]i sustained phase seen in response to ATP.

While many endothelial cell lines exist, few are of human origin with characteristics close to the parent endothelial cell. We derived a subline (HUVEC-CS) of immortalized human umbilical vein endothelial cells (HUVEC-C) that proliferate in standard growth media and exhibit positive acetylated low-density lipoprotein (AcLDL) uptake, express eNOS, CD31 and ve-cadherin, and spontaneously form capillary-like structures when grown on Matrigel. HUVEC-CS also maintain endothelial cell characteristics at the level of mitogenesis, kinase activation and vasodilator production. Like primary HUVEC cells, HUVEC-CS express many of the key proteins necessary for vasodilator production, including epithelial nitric oxide synthase (eNOS), HSP 90, cav-1 and -2, cPLA2, and COX-1 and -2. Prostaglandin I synthase (PGIS) was not detectable by Western blot analysis, consistent with primary HUVEC in which PGI2 production is minimal. Receptors were detected for angiotensin II (AII), bradykinin, ATP and growth factors. ATP induced a dose- and time-dependent rise in the intracellular free Ca2+ concentration ([Ca2+]i). Initially, ATP stimulates P2Y receptors rather than P2X receptors, as demonstrated by the inability of ATP to initiate a Ca2+ response subsequent to emptying of the internal Ca2+ stores by thapsigargin. AII, bradykinin, epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) also caused a rise in [Ca2+]i in a subset of the cells. ATP, basic fibroblastic growth factor (bFGF), EGF and VEGF induced mitogenesis and caused a rise in ERK 2 activation within 10 min. L-Arginine to L-citrulline conversion assays showed that ATP, EGF and VEGF induced a significant rise in eNOS activity, and this correlates with an ability to induce Ca2+ mobilization and ERK 2 activation. In conclusion, HUVEC-CS are indeed endothelial cells and appear to be functionally very similar to primary HUVEC. These cells will prove a valuable tool for future studies in both basic and therapeutic sciences.

The principle mechanisms operating at the level of endothelial nitric oxide synthase (eNOS) itself to control its activity are phosphorylation, the auto-regulatory properties of the protein itself, and Ca2+/calmodulin binding. It is now clear that activation of eNOS is greatest when phosphorylation of certain serine and threonine residues is accompanied by elevation of cytosolic [Ca2+]i. While eNOS also contains an autoinhibitory loop, Rafikov et al. (2011) present the evidence for a newly identified ‘flexible arm’ that operates in response to redox state. Boeldt et al. (2011) also review the evidence that changes in the nature of endothelial Ca2+ signaling itself in different physiologic states can extend both the amplitude and duration of NO output, and a failure to change these responses in pregnancy is associated with preeclampsia. The change in Ca2+ signaling is mediated through altering capacitative entry mechanisms inherent in the cell, and so many agonist responses using this mechanism are altered. The term ‘adaptive cell signaling’ is also introduced for the first time to describe this phenomenon. Finally NO is classically regarded as a regulator of vascular function, but NO has other actions. One proposed role is regulation of steroid biosynthesis but the physiologic relevance was unclear. Ducsay & Myers (2011) now present new evidence that NO may provide the adrenal with a mechanism to regulate cortisol output according to exposure to hypoxia. One thing all three of these reviews show is that even after several decades of study into NO biosynthesis and function, there are clearly still many things left to discover.

Activin and inhibin are structurally related dimeric glycoproteins belonging to the transforming growth factor-β superfamily of proteins which are synthesized and secreted by the granulosa cells of the ovary. Although initially characterized by their ability to influence FSH secretion from pituitary cells, paracrine regulatory roles of these factors on neighboring ovarian theca interna have been suggested. While inhibin has been shown to increase and activin to decrease the production of androgens, the mechanisms of action are not well defined, partly due to difficulties in obtaining adequate numbers of thecal cells from individual patients or animal models. Using a unique human ovarian thecal-like tumor (HOTT) cell culture model system we investigated the biochemical and molecular mechanisms controlling C19 steroidogenesis and the effects of activin and inhibin on the activity and expression of key ovarian thecal steroidogenic enzymes, cholesterol side-chain cleavage cytochrome P450 (P450scc), 3β-hydroxysteroid dehydrogenase (3βHSD) and 17α-hydroxylase/17,20 lyase cytochrome P450 (P450c17). Steroid production, level of steroidogenic enzyme mRNA expression, and enzyme activity following treatment with forskolin, inhibin-A and activin-A were examined. Basal steroid production, enzyme activities, and steroidogenic enzyme mRNA levels were not markedly different following treatment with activin (25 ng/ml) or inhibin (25 ng/ml) alone. Forskolin (10 μm) markedly increased production of both androstenedione (fivefold) and progesterone (threefold) as well as the activity of 3βHSD (sevenfold), and P450c17 (sevenfold) over basal. Forskolin stimulated the expression of mRNA for P450scc (fourfold), 3βHSD (threefold), and P450c17 (eightfold) over basal. Androstenedione accumulation was decreased by 60% in the forskolin plus activin group compared with forskolin alone, while progesterone production was maintained. This was attributed to a reduction of P450c17 mRNA (45% of forskolin alone) and activity (45% of forskolin alone). In contrast, co-treatment with forskolin and inhibin increased androstenedione production by 40% while decreasing progesterone by 40% compared with forskolin alone. Concomitantly, this was associated with a higher P450c17 mRNA expression (1·5-fold) and activity (two-fold) but with minimal effects on the mRNA for 3βHSD and P450scc. HOTT cell responses to activin (0·05–50 ng/ml) and inhibin (0·05–50 ng/ml) in the presence of forskolin demonstrated dose-dependent effects on the steroid accumulation, enzymatic activity and mRNA expression of P450c17. Additionally, the differences seen on mRNA expression of steroidogenic enzymes in response to these factors were time-dependent. In summary, forskolin stimulated C19 steroid production from HOTT cells by increasing the expression of all steroidogenic enzymes examined. Inhibin and activin exerted differential effects on the expression of these enzymes which resulted in alterations in the steroid profile toward production of C19 steroids in the case of inhibin and away from C19 steroids in the case of activin. The influence of these important intraovarian factors on the expression of P450c17, a pivotal enzyme in thecal cell production of C19 steroids, could impact greatly on the follicular milieu of a normal developing follicle as well as in pathophysiological disorders such as polycystic ovarian syndrome. Journal of Endocrinology (1996) 148, 213–221

When bovine adrenocortical cells from the zona fasciculata/reticularis (zfr) are maintained in primary culture, cortisol secretion in response to acute stimulation with ACTH and adrenaline (which activate adenylate cyclase) is seen to increase steadily over the first 48 h, while secretion in response to angiotensin II and acetylcholine (which activate phosphoinositidase C) shows an initial decline in the first 24 h and a recovery to maximum after 48 h. We have investigated whether these discrepant changes in cortisol secretory response to the different agonists are due to changes in formation of the associated second messengers (cAMP or inositol phosphates), or altered coupling of these second messenger signals to steroid secretion. Increases in steroid secretion in response to ACTH and adrenaline were paralleled by increased cAMP. Steroid secretion in response to exogenous 8-bromoadenosine 3′:5′-cyclic monophosphate also increased steadily during this 48-h period. Thus increased responsiveness was due to both increased second messenger formation and increased coupling to the steroid secretory response. The decreased steroid secretory response to angiotensin and acetylcholine after 24 h, and subsequent recovery after 48 h in culture, were accompanied by an increased formation of phosphoinositols after 24 h and a further increase by 48 h. However, the steroid secretory response to a combination of calcium ionophore and the protein kinase C activator, phorbol 12-myristate 13-acetate, was reduced after 24 h and recovered by 48 h of culture. Fura-2-loaded cells also showed an increase in intracellular [Ca2+] after 24 h in culture. Thus the impaired steroid secretory response to angiotensin II and acetylcholine after 24 h of culture was not due to reduced formation of second messengers but to a failure of Ca2+ and diacylglycerol so formed to activate the steroid secretory process. Reversible uncoupling of the steroid secretory response from the Ca2+- and diacylglycerol-based but not the cAMP-based second messengers observed in bovine zfr cells suggests that differential control of steroid secretion and other cell functions may be possible in vivo for activators of phosphoinositidase C, and may explain apparently discrepant results from studies on other in-vitro adrenocortical cell preparations. Journal of Endocrinology (1992) 133, 21–28

Analysis by electron microscopy indicated that after 3 days of primary culture, purified bovine adrenal zonal fasciculata/reticularis (ZF/ZR) cells showed improved integrity of their ultrastructure, with an increased density of lipid droplets and smooth endoplasmic reticulum. The basal cortisol output was significantly (P < 0·05) greater on day 3 of culture than for the freshly isolated cells in six out of seven experiments. Similarly, in six experiments with ACTH (1 nmol/l) and five experiments with angiotensin II (10 nmol/l), the stimulated cortisol secretion was significantly (P < 0·01 for all 11 experiments) higher on day 3 of culture than in freshly isolated cells. No significant increase in cortisol secretion above basal was observed with noradrenaline at any concentration in the freshly isolated cells, whereas a dose-dependent increase in cortisol secretion was observed on day 3 of culture in all of four experiments. These findings were supported by cyclic (c) AMP output measured in one such experiment. Thus the basal cAMP output and that stimulated by ACTH (1 nmol/l) were significantly higher after culture (P < 0·001, n = five wells for basal comparison; P < 0·05, n = three wells for ACTH at 1 nmol/l). In agreement with the cortisol results, cAMP production was unaffected by any concentration of noradrenaline in the freshly isolated cells, whereas a dose-dependent rise was found after culture. Angiotensin II at all concentrations had no effect on cAMP production in freshly isolated or cultured cells. These studies demonstrate that the primary culture of purified bovine ZF/ZR cells increases basal steroidogenesis and leads to an enhanced responsiveness to the physiological stimuli, ACTH and angiotensin II. In addition, primary culture of purified bovine ZF/ZR cells revealed a noradrenaline response which was not readily observed in freshly isolated, purified ZF/ZR cells. Journal of Endocrinology (1989) 121, 317–324