Derek Boeldt

Pregnancy Enhances Sustained Ca2+ Bursts and Endothelial Nitric Oxide Synthase Activation in Ovine Uterine Artery Endothelial Cells Through Increased Connexin 43 Function

Abstract 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 [Ca2+]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 [Ca2+]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 Ca2+ 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 [Ca2+]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.

Vascular adaptation in pregnancy and endothelial dysfunction in preeclampsia

Maternal vascular adaptation to pregnancy is critically important 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 that directly influence 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. However, they 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 clearer. We then describe in detail how the complex endocrine environment of PE affects 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.

[Ca2+]i signaling vs. eNOS expression as determinants of NO output in uterine artery endothelium: relative roles in pregnancy adaptation and reversal by VEGF165

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 ([Ca(2+)](i)) response in individual cells of P-UA Endo comprises an initial peak followed by transient [Ca(2+)](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 [Ca(2+)](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 VEGF(165). While treatment of luteal NP-UA Endo and P-UA Endo with VEGF(165) acutely stimulates a very modest [Ca(2+)](i) and NO response, subsequent stimulation of the same vessel with ATP results in a blunted [Ca(2+)](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.

The loss of sustained Ca2+ signaling underlies suppressed endothelial nitric oxide production in preeclamptic pregnancies: implications for new therapy

Approximately 8% of pregnancies are complicated by preeclampsia (PE), a hypertensive condition characterized by widespread endothelial dysfunction. Reduced nitric oxide (NO) output in PE subjects has been inferred but not directly measured, and there is little understanding of why this occurs. To address this we have used direct imaging of changes in intracellular Ca(2+) concentration ([Ca(2+)]i) and NO in umbilical vein endothelium of normal and PE subjects that is still intact and on the vessel luminal surface. This was achieved by dissection and preloading with fura 2 and DAF-2 imaging dyes, respectively, before subsequent challenge with ATP (100 μM, 30 min). As a control to reveal the content of active endothelial nitric oxide synthase (eNOS) per vessel segment, results were compared with a maximal stimulus with ionomycin (5 μM, 30 min). We show for the first time that normal umbilical vein endothelial cells respond to ATP with sustained bursting that parallels sustained NO output. Furthermore, in subjects with PE, a failure of sustained [Ca(2+)]i bursting occurs in response to ATP and is associated with blunted NO output. In contrast, NO responses to maximal [Ca(2+)]i elevation using ionomycin and the levels of eNOS protein are more similar between groups than the responses to ATP. When the endothelial cells from PE subjects are isolated and allowed to recover in culture, they regain the ability under fura 2 imaging to show multiple [Ca(2+)]i bursts otherwise seen in the cells from normal subjects. Thus novel clinical therapy aimed at restoring function in vivo may be possible.

Altered VEGF-stimulated Ca2+ signaling in part underlies pregnancy-adapted eNOS activity in UAEC.

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 Ca(2+) imaging and arginine-to-citrulline conversion eNOS activity assays to assess the importance of VEGF-stimulated Ca(2+) responses in pregnancy-related changes in NO production in UAEC. In this study, we show that pregnancy-induced changes in VEGF-stimulated Ca(2+) responses could account in part for the greater capacity of VEGF to stimulate eNOS in UAECs from pregnant versus non-pregnant animals. VEGF-stimulated Ca(2+) 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 Ca(2+) entry, and were more sensitive to a submaximal dose of VEGF. In UAECs from pregnant and non-pregnant animals Ca(2+) 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 [Ca(2+)]i are necessary for eNOS activation in UAECs, and pregnancy-induced changes in Ca(2+) responses could also in part explain the pregnancy-specific adaptive increase in eNOS activity in UAECs.

Pregnancy, Programming and Preeclampsia: Gap Junctions at the Nexus of Pregnancy-induced Adaptation of Endothelial Function and Endothelial Adaptive Failure in PE

The challenge of pregnancy to the mother requires that her own metabolic and endocrine needs be met while also taking on the literally growing demands of the unborn child. While all of the mothers organs require continued support, the uterus and now added placenta must also develop substantially. One critical area of adaptation is thus the ability to provide added blood flow over and above that already serving the preexisting maternal organs. Previous reviews have covered in detail how this is achieved from an endocrine or indeed vascular physiology standpoint and we will not repeat that here. Suffice it to say in addition to new vessel growth, there is also the need to achieve reduced vascular resistance through maintenance of endothelial vasodilation, particularly through NO and PGI2 production in response to multiple agonists and their associated cell signaling systems. In this review, we continue our focus on pregnancy adaptive changes at the level of cell signaling, with a particular emphasis now on the developing story of the critical role of gap junctions. Remapping of cell signaling itself beyond changes in individual hormones and respective receptors brings about global changes in cell function, and recent studies have revealed that such post-receptor changes in cell signaling are equally if not more important in the process of pregnancy adaptation of endothelial function than the upregulated expression of vasodilator synthetic pathways themselves. The principle significance, however, of reviewing this aspect of pregnancy adaptation of endothelial cell function is that these same gap junction proteins that mediate pregnancy-adapted changes in vasodilatory signaling function may also be the focal point of failure in diseased pregnancy, and clues as to how and why are given by comparing studies of Cx43 functional suppression at wound sites with studies of preeclamptic pregnancy. If preeclamptic pregnancy is indeed a pregnancy misconstrued by the body in endocrine terms to be a wound, then the kinases so activated that correspondingly suppress Cx43 function in the vascular endothelium may also be valid pharmacologic targets for novel therapies in the near future.

Regulating Specific Growth Factor Signaling Using Immobilized Branched Ligands

VEGF-binding peptide ligands are incorporated into hydrogel microspheres and reduce the amount of growth factor in solution. VEGF binding affinity is enhanced by creating ligands with a dimer structure. The spheres are able to knock down VEGF-mediated HUVEC growth and reduce calcium signaling. The binding interaction is reversible, allowing the spheres to be used as a VEGF delivery vehicle.

Phosphorylation of Ser-279/282 and Tyr-265 positions on Cx43 as possible mediators of VEGF-165 inhibition of pregnancy-adapted Ca2+ burst function in ovine uterine artery endothelial cells.

Normal pregnancy requires increased uterine endothelial cell driven vasodilation that is related to increases in sustained Ca2+ signaling via increased connexin 43 (Cx43) gap junction function. Preeclampsia, a hypertensive disorder of pregnancy associated with endothelial dysfunction, is also linked with down regulation of Ca2+ driven vasodilator production and increased levels of vascular endothelial growth factor (VEGF). Cx43 function can be acutely down-regulated by phosphorylation of multiple inhibitory residues and VEGF is known to promote phosphorylation of Cx43. Herein, we show that VEGF-165 promotes Cx43 phosphorylation at Ser-279/282 and Tyr-265 residues and blocks pregnancy-adapted Ca2+ signaling in ovine uterine artery endothelial cells (UAEC). Pharmacological Src and ERK kinase pathway inhibitors (PP2 and U0126) reverse these phosphorylations and rescue Ca2+ signaling. We also report a nutraceutical Src inhibitor, t10,c12 conjugated linoleic acid (10,12 CLA), rescues Ca2+ signaling in UAEC and therefore may have therapeutic potential for preeclampsia.

Positive versus negative effects of VEGF165 on Ca2+ signaling and NO production in human endothelial cells

The role increased vascular endothelial growth factor (VEGF) plays in vascular function during normal vs. preeclamptic pregnancy has been a source of some controversy of late. In this study, we seek to understand how VEGF165 influences vasodilator production via Ca2+ signaling mechanisms in human endothelial cells. We utilize human umbilical vein endothelial cells (HUVEC) as well as intact ex vivo human umbilical vein (HUV Endo) to address direct stimulation of Ca2+ and NO by VEGF165 alone, as well as the effect of VEGF165 on subsequent ATP-stimulated Ca2+ signaling and NO production. We show that VEGF165 stimulates Ca2+ responses in both HUVEC and HUV Endo, which results in a corresponding increase in NO production in HUV Endo. Longer-term VEGF165 pretreatment then inhibits sustained Ca2+ burst responses to ATP in HUVEC and HUV Endo. This is paralleled by a corresponding drop in ATP-stimulated NO production in HUV Endo, likely through inhibition of Cx43 gap-junction function. Thus, although VEGF165 makes a small initial positive impact on vasodilator production via direct stimulation of Ca2+ responses, this is outweighed by the greater subsequent negative impact on Ca2+ bursts and vasodilator production promoted by more potent agonists such as ATP. Overall, elevated levels of VEGF165 associated with preeclampsia could contribute to the endothelial dysfunction by preventing Ca2+ bursts to other agonists including but not limited to ATP. NEW & NOTEWORTHY In this manuscript, we show that VEGF levels associated with preeclampsia are a net negative contributor to potential vasodilator production in both a human ex vivo and in vitro endothelial cell model. Therefore, pharmacological targeting of VEGF-stimulated signaling pathways could be a novel treatment modality for preeclampsia-related hypertension.

Changes in Ca2+ Signaling and Nitric Oxide Output by Human Umbilical Vein Endothelium in Diabetic and Gestational Diabetic Pregnancies

ABSTRACT Diabetes (DM) complicates 3%–10% of pregnancies, resulting in significant maternal and neonatal morbidity and mortality. DM pregnancies are also associated with vascular dysfunction, including blunted nitric oxide (NO) output, but it remains unclear why. Herein we examine changes in endothelial NO production and its relationship to Ca2+ signaling in endothelial cells of intact umbilical veins from control versus gestational diabetic (GDM) or preexisting diabetic subjects. We have previously reported that endothelial cells of intact vessels show sustained Ca2+ bursting in response to ATP, and these bursts drive prolonged NO production. Herein we show that in both GDM and DM pregnancies, the incidence of Ca2+ bursts remains similar, but there is a reduction in overall sustained phase Ca2+ mobilization and a reduction in NO output. Further studies show damage has occurred at the level of NOS3 protein itself. Since exposure to DM serum is known to impair normal human umbilical vein endothelial cell (HUVEC) function, we further studied the ability of HUVEC to signal through Ca2+ after they were isolated from DM and GDM subjects and maintained in culture for several days. These HUVEC showed differences in the rate of Ca2+ bursting, with DM > GDM = control HUVEC. Both GDM- and DM-derived HUVEC showed smaller Ca2+ bursts that were less capable of NOS3 activation compared to control HUVEC. We conclude that HUVEC from DM and GDM subjects are reprogrammed such that the Ca2+ bursting peak shape and duration are permanently impaired. This may explain why ROS therapy alone is not effective in DM and GDM subjects.