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Pregnancy-Dependent Changes in Cell Signaling Underlie Changes in Differential Control of Vasodilator Production in Uterine Artery Endothelial Cells¹

Perinatal Research Laboratories, Departments of Obstetrics/Gynecology (I.M.B., J.A.S., T.D., J.M.C., J.Z., R.R.M.) and Meat/Animal Science (R.R.M.), University of Wisconsin, Madison, Wisconsin 53715; and the Center for Perinatal Biology, Loma Linda University School of Medicine (L.Z.), Loma Linda, California 92350

Address all correspondence and requests for reprints to: Ian M Bird, Ph.D., Department Obstetrics and Gynecology, Perinatal Research Laboratories, University of Wisconsin, 7E Meriter Hospital/Park, 202 South Park Street, Madison, Wisconsin 53715. E-mail: imbird{at}facstaff.wisc.edu

During pregnancy, the uterine vasculature shows a marked increase in vasodilator production [prostacyclin (PGI₂) and nitric oxide (NO)] in response to a number of agonists including angiotensin II (AII) and ATP. As a consequence vascular resistance is kept low, and uterine blood flow is maximized to meet the needs of the growing fetus. Studies of the molecular basis underlying this change in control of endothelial NO and PGI₂ production have been hampered by the lack of availability of a suitable cell model. To that end we have developed and characterized a new ovine uterine artery endothelial cell (UAEC) culture model derived from nonpregnant (NP) or pregnant (P) ewes. Endothelial cells were isolated from pregnant (120–130 days; n = 6) and nonpregnant (n = 4) ewes and maintained in primary culture. Endothelial cells at passage 4 showed uniform expression of endothelial nitric oxide synthase (eNOS; an endothelial marker) as well as AII type 1 receptor and growth factor receptors and uniform uptake of acetylated low density lipoprotein (a property of endothelial cells not shared by fibroblasts or vascular smooth muscle cells), thus demonstrating cell purity. Expressions of eNOS, cyclooxygenase-1, PGI₂ synthase, cytosolic phospholipase A₂, AII type 1 receptor, and growth factor receptors are also maintained at passage 4. Mitogenesis is maintained in response to basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), and vascular endothelial growth factor (VEGF) in both NP-UAEC and P-UAEC. The differential production of vasodilators by NP-UAEC and P-UAEC is maintained in a manner similar to that previously reported in vivo. Thus, P-UAEC make NO in response to AII, ATP, bFGF, EGF, and VEGF, whereas NP-UAEC make NO in response to bFGF, EGF, and VEGF only. Similarly, P-UAEC make PGI₂ in response to AII, ATP, bFGF, and VEGF, whereas NP-UAEC make PGI₂ only in response to ATP and VEGF. As both cytosolic phospholipase A₂ and eNOS may be regulated by both Ca²⁺ and protein kinases, we investigated the effects of these agonists on Ca²⁺ mobilization and ERK-1/2 phosphorylation. ATP consistently elevates Ca²⁺ levels in both P-UAEC and NP-UAEC. All other agonists were without acute (0–4 min) effect on Ca²⁺ in P-UAEC or NP-UAEC. In contrast, all agonists stimulated an acute (10 min) phosphorylation of ERK-1/2 in P-UAEC, whereas only EGF stimulated activation in NP-UAEC. P-UAEC production of PGI₂ by agonists of both heptahelical receptors and growth factor receptors correlates closely with ERK-2 phosphorylation alone. For NO, this correlation holds for heptahelical receptor agonists, but additional signaling pathways are also implicated for bFGF and VEGF. In contrast, in NP-UAEC the lack of ERK-2 phosphorylation in response to all agonists other than EGF, and the dissociation between NO or PGI₂ production and ERK-2 phosphorylation suggest that alternate pathways play a predominant role.

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