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Preeclampsia is associated with altered Ca²⁺ regulation and NO production in human fetal venous endothelial cells¹

Centre for Cardiovascular Biology & Medicine, Guy’s, King’s & St. Thomas’ Schools of Biomedical Sciences and Medicine, King’s College London, Guy’s Campus, London SE1 1UL, UK

²Correspondence: Center for Cardiovascular Biology & Medicine, New Hunt’s House (Rm. 2.34B), GKT School of Biomedical Sciences, King’s College London, Guy’s Campus, London SE1 1UL, UK. E-mail: giovanni.mann{at}kcl.ac.uk

SPECIFIC AIMS

Preeclampsia (PE) is a leading cause of fetal and maternal mortality and is associated with abnormalities in maternal and fetal circulation. Our aim was to investigate whether fetal endothelial cells have abnormal free cytosolic calcium ([Ca²⁺]_i) regulation that could be related to fetal vascular dysfunction.

PRINCIPAL FINDINGS

1. Ca²⁺ influx after histamine stimulation is reduced in PE endothelial cells
Fura-2 ratio fluorescence was used as a measure of [Ca²⁺]_i in human umbilical vein endothelial cells from normal, PE, and age-matched preterm (PT) deliveries. The basal [Ca²⁺]_i was slightly but significantly elevated in PE (compared with normal or PT); peak [Ca²⁺]_i response to histamine stimulation was normal in PE. However, the plateau phase of the response to histamine (which reflects Ca²⁺ influx) was significantly depressed (Fig. 1 ), with the median value being reduced by 43% (P<0.002). We also treated endothelial cells with histamine in Ca²⁺-free solution to Ca²⁺-deplete internal stores, with cyclopiazonic acid present to inhibit the store Ca²⁺ATPase and prevent any resequestration of Ca²⁺. We then measured the rise and fall of [Ca²⁺]_i when extracellular Ca²⁺ was added to or removed from cells and found that the rate of rise of [Ca²⁺]_i in PE cells was reduced by 65% (P<0.01) with no significant difference in the subsequent fall of [Ca²⁺]_i. This confirmed that Ca²⁺ influx was inhibited in PE and not that plasma membrane Ca²⁺ATPase was stimulated.

View larger version (22K): [in this window] [in a new window]   Figure 1. Basal and histamine-stimulated [Ca2+] i levels in endothelial cells from normal and PE pregnancies. A) Endothelial cells were maintained in Ca2+ containing solution and basal and histamine-stimulated peak and plateau [Ca2+]i levels were monitored in single cells using the 340/380 nm fluorescence ratio. Basal, peak, and plateau [Ca2+]i levels measured in cells from normal term () and PE (•) pregnancies are summarized in scatter plots shown in panel B. Ratio denotes the peak [Ca2+]i or plateau [Ca2+]i minus the basal [Ca2+]i level. Bars denote the mean ± SE of measurements in 22 normal and 17 PE endothelial cell cultures. Significant differences between groups were assessed using Mann-Whitney and Kruskal-Wallis nonparametric ANOVA tests, **P < 0.002.

2. Ba²⁺, Mn²⁺, and Gd³⁺ permeabilities are altered in PE endothelial cells
We used Fura-2 to investigate whether PE affected permeability to Mn²⁺ and Ba²⁺, both of which can be detected by Fura-2. Basal and histamine stimulated Mn²⁺ influx were substantially increased in PE (by 71%, P<0.05, and 232%, P<0.005, respectively), which for the stimulated influx was contrary to expectations based on the Ca²⁺ data. Ba²⁺, however, showed the same trend as Ca²⁺, with a 64% (P<0.03) reduction of influx in histamine-stimulated PE cells compared with normal. Suspecting modified divalent cation entry in PE, we tested to see whether Gd³⁺ differentially inhibited Ca²⁺ or Mn²⁺ entry in PE cells. We found that permeability to Gd³⁺ was elevated in PE (Fura-2 fluorescence is also sensitive to Gd³⁺) but undetectable in normal cells. The inhibitory action of Gd³⁺ on Ca²⁺ and Mn²⁺ entry was not significantly altered in PE.

3. Basal and histamine-stimulated eNOS activity is elevated in PE
We next examined whether the reduced Ca²⁺ influx under histamine-stimulated conditions correlated with reduced eNOS activity by measuring intracellular cGMP (an index of NO) in the presence of phosphodiesterase inhibition. We found no difference in [cGMP] between normal and PT cells, but NO production in PE cells was altered in a number of ways (Fig. 2 ). Basal and histamine-stimulated [cGMP] were significantly elevated. We tested sensitivity to the eNOS inhibitor L-NAME and found that the L-NAME-resistant part of the elevation of cGMP was higher in PE for basal and stimulated levels. Immunoblots for eNOS and soluble guanylyl cyclase (sGC) proteins relative to -tubulin revealed no difference for sGC between normal, PT, and PE cells. However, eNOS expression in PT and PE cells was reduced compared with normal cells.

View larger version (32K): [in this window] [in a new window]   Figure 2. Basal and histamine-stimulated levels of cGMP accumulation in endothelial cells from normal, preterm, and PE pregnancies. Endothelial cell monolayers were incubated in Krebs buffer containing 100 µM L-arginine and 500 µM IBMX. Basal and histamine-stimulated cGMP accumulation was measured in the absence or presence of the NOS inhibitor L-NAME (100 µM). Data denote mean ± SE of triplicate measurements in each of 3 normal, 4 preterm, and 3 PE cell cultures, P < 0.02 vs. normal basal, *P < 0.02 vs. normal stimulated, P < 0.02 vs. normal stimulated + L-NAME.

4. Elevated cGMP in PE does not account for the depressed Ca²⁺ influx
As cGMP has been reported to inhibit Ca²⁺ influx, we examined whether the elevated levels of cGMP could account for the depressed Ca²⁺ plateau in PE. A cell-permeant analog of cGMP (100–500 µM 8-bromo-cGMP) dose-dependently inhibited the plateau phase of the response to histamine in normal (21±4%) and PE cells (15±8%) by a similar amount, which was less than the depression of the plateau seen in PE vs. normal cells.

DISCUSSION

Preeclampsia occurs in 4% of pregnancies and is a leading cause of maternal and fetal morbidity and mortality: the disease is characterized by a pregnancy-related increase in blood pressure and significant proteinuria. Impaired endothelial dependent relaxation in the maternal circulation is a hallmark of PE; however, studies of the feto-placental circulation in PE are rare even though the feto-placental vasculature is often abnormal. There is no consensus regarding the functional status of the feto-placental vessels in PE, although some reports conclude that fetal endothelial cell function is compromised. Studies examining NO production in the fetal circulation in normal and PE pregnancies are controversial, with fetal serum concentrations of nitrite and nitrate either unchanged or elevated. Similar discrepancies have been reported for NO production in the maternal circulation, with lowered or unchanged circulating nitrite and nitrate levels in women with PE.

The present study provides important insights into abnormalities of fetal vascular endothelial cells isolated from women with PE. These alterations in endothelial cell function were sustained during culture in vitro and were characterized by 1) slightly elevated basal [Ca²⁺]_i, 2) decreased histamine-stimulated Ca²⁺ influx, 3) altered membrane permeability to other multivalent cations, and 4) enhanced basal and histamine-stimulated rates of NO production.

Fetal endothelial cells obtained from PE pregnancies had a reduced agonist-stimulated [Ca²⁺] influx that resulted in a reduced plateau phase of the [Ca²⁺] response to histamine. Ca²⁺ entry in umbilical vein endothelial cells is known to be capacitative and triggered by depletion of internal Ca²⁺ stores, with the plateau Ca²⁺ level reflecting a balance between Ca²⁺ influx and efflux. Thus, we used Mn²⁺ influx to confirm that divalent cation entry was indeed inhibited in PE cells, but our measurements revealed a paradoxical increase in the histamine-stimulated permeability to Mn²⁺. Like Mn²⁺, Ba²⁺ is not transported by Ca²⁺ATPases, and thus is another good indicator of unidirectional divalent cation movement. Ba²⁺ influx was reduced in PE by 64% (P<0.03). The disparity between Ca²⁺/Ba²⁺ and Mn²⁺ entry in PE endothelial cells suggests either an alteration of the properties of store-operated Ca²⁺ entry or expression of different types of Ca²⁺ influx channels. To investigate this further, we tested whether PE altered the sensitivity of Ca²⁺ or Mn²⁺ entry to inhibition by Gd³⁺. Although our experiments revealed no significant differences in the inhibition of Mn²⁺ entry by Gd³⁺ between normal and PE cells, we identified increased permeability to Gd³⁺ in PE cells. This increase may correspond to a report that expression of stretch-operated channels, but not the channel conductance or sensitivity to stretch, is increased in PE umbilical vein endothelial cells. In summary, we have established that fetal endothelial cells from PE pregnancies exhibit a decreased permeability to Ca²⁺ and Ba²⁺ but an increased permeability to Mn²⁺ and Gd³⁺: this could be due to phenotypic alteration of a single influx pathway or a shift in the balance between different entry pathways with different relative permeabilities to Ca²⁺, Ba²⁺, Mn²⁺, and Gd³⁺. Recent evidence suggests that various modes of Ca²⁺ entry in nonexcitable cells are mediated by a family of transient receptor potential (TRP) proteins. At least three different isoforms of TRP channels (TRP 1,3,4) are expressed in umbilical vein endothelial cells, and regulation of their expression in normal and PE endothelial cells merits investigation. The recent report that incubation of rat aortic vascular smooth muscle cells with PE serum reduces Ca²⁺ transients and inhibits store-operated entry of Ca²⁺ and Ba²⁺ lends additional support to our hypothesis that PE is associated with phenotypic alterations in the entry pathway(s) for multivalent cations.

The control of eNOS activity by [Ca²⁺] _i has been studied extensively; the consensus is that it is controlled mainly by Ca²⁺ influx rather than by Ca²⁺ released from internal stores. Although there is overwhelming evidence for endothelial cell activation and impaired dilator function in the maternal circulation in PE, there is no agreement concerning the feto-placental circulation, particularly in relation to the role of NO. We found that L-NAME-sensitive, histamine-stimulated cGMP accumulation was significantly increased in PE compared with normal and PT endothelial cells, implying increased generation of NO in PE. This increased production could not be accounted for by increased eNOS or sGC protein expression, and preliminary experiments suggested that the increased NO production is not due to Ca²⁺-independent activation of eNOS via Akt-mediated serine phosphorylation (unpublished data). Inducible NOS is not expressed in PE umbilical vein endothelial cells, and activation of sGC by CO generated via metabolism of heme by heme oxygenase is unlikely in view of the recent report that constitutive expression of HO-2 in placental villous endothelial cells is reduced in PE. The NO pathway can also be affected by reactive oxygen species; we have not investigated this, but its potential importance is indicated by the fact that supplementation with antioxidants markedly reduces the incidence of PE in women at risk.

Increased NO production appears inconsistent with the reduced Ca²⁺ influx detected in PE cells. A putative explanation for the decreased Ca²⁺ influx but increased NO production in PE is that eNOS is associated with plasmalemmal caveolae, but our Ca²⁺ measurements with Fura-2 report bulk cytosolic [Ca²⁺]. Thus, one might postulate distribution of the Ca²⁺ influx pathway, PMCA, and eNOS in PE cells that allows a greater fraction of the influx of Ca²⁺ to activate eNOS and be exported without reaching the cytosol and detection by Fura-2. However, this explanation is unlikely since we also observed a reduced entry with Ba²⁺, an ion not pumped by the PMCA and therefore a more reliable indicator of the unidirectional influx.

Another interpretation is that elevated intracellular cGMP levels may have suppressed plateau [Ca²⁺]_i, since cGMP has been reported to reduce store-operated Ca²⁺ entry. It is unlikely that elevated cGMP levels in PE cells are directly responsible for the reduced plateau Ca²⁺ phase, as the cell-permanent analog 8-bromo-cGMP caused a similar depression of plateau [Ca²⁺]_i in normal and PE cells (normal 21±4% vs. PE 15±8%) and the maximal depression of the plateau in normal and PE cells was insufficient to account for the low PE plateau. Even though we have identified altered Ca²⁺ regulation and NO production in PE endothelial cells, based on the present data we cannot directly link these two processes.

Perhaps one of the most interesting observations is that pregnancy-associated diseases induce phenotypic changes in the fetal vasculature. In the present study, alterations in Ca²⁺ regulation and NO production persisted during cell culture, suggesting that PE is associated with phenotypic alterations in the function of fetal endothelial cells. This may have implications for long-term ‘programming’ of the fetal cardiovascular system, since programming of the fetus in PE was implied from one clinical study in which men whose mothers suffered from PE were found to be at increased risk of developing hypertension in adulthood.

View larger version (23K): [in this window] [in a new window]   Figure 3. Scheme summarizing the effects of PE. Solid lines show ionic movements or biochemical processes. Dashed lines show modulatory influences, those ending in an arrowhead denote stimulation, and those ending in a transverse bar denote inhibition. Two types of cation influx pathway are shown (1 and 2). Pathway 1 is dominant under normal conditions and pathway 2 is dominant in PE. Pathway 1 is more permeable to Ca2+ and Ba2+ and less permeable to Mn2+ and Gd3+ than pathway 2. The two pathways may be two forms of the same channel. eNOS: endothelial nitric oxide synthase. sGC: soluble guanylyl cyclase; PMCA: plasma membrane Ca2+ATPase; SERCA: sarco- or endoplasmic reticulum Ca2+ATPase.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0916fje; to cite this article, use FASEB J. (March 26, 2002) doi/10.1096/fj.01-0916fje;

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