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

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 Ca(2+) 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 Ca(2+) 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 Ca(2+) bursts remains similar, but there is a reduction in overall sustained phase Ca(2+) 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 Ca(2+) after they were isolated from DM and GDM subjects and maintained in culture for several days. These HUVEC showed differences in the rate of Ca(2+) bursting, with DM > GDM = control HUVEC. Both GDM- and DM-derived HUVEC showed smaller Ca(2+) 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 Ca(2+) bursting peak shape and duration are permanently impaired. This may explain why ROS therapy alone is not effective in DM and GDM subjects.

The loss of sustained Ca(2+) 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.