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Pregnancy Induces an Increase in the Expression of Glyceraldehyde-3-Phosphate Dehydrogenase in Uterine Artery Endothelial Cells

Daniel S. Millican, BS

Hiroaki Itoh, MD

Ronald R. Magness, PhD

Department of Obstetrics and Gynecology, Perinatal Research Laboratories, and the Department of Meat and Animal Science, University of Wisconsin-Madison, Madison, Wisconsin

Ian M. Bird, PhD

University of Wisconsin-Madison, Department of Obstetrics and Gynecology, Perinatal Research Laboratories, 7E Meriter Hospital/Park, 202 South Park Street, Madison, WI 53715

Objective: We determined the effects of pregnancy on glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression in ovine uterine and omental (systemic control) artery endothelial cells (UAEC, OAEC). We also determined in primary cultures of UAEC the effects of exposure to either exogenous nitric oxide (NO) or angiogenic growth factors (basic fibroblast growth factor [bFGF], vascular endothelial growth factor [VEGF], and epidermal growth factor [EGF]) on UAEC GAPDH expression.

Methods: We isolated UAEC to high purity from both nonpregnant (NP; n = 4) and pregnant ewes (P; 110-120 days' gestation, n = 4) by limited collagenase dispersion and immediately extracted total RNA. In additional experiements performed in vitro, ovine UAEC isolated from NP ewes and maintained in culture (n = 3) were exposed to 1) 100 µmol/L sodium nitro-prusside for 0, 6, 12, or 24 hours or 2) 10 ng/mL bFGF, VEGF, EGF, or vehicle for 24 hours. Total RNA was then immediately extracted. A partial ovine GAPDH (oGAPDH) cDNA was isolated by reverse transcriptase polymerase chain reaction (RT/PCR) and sequenced. A one-tube semiquantitative RT/PCR amplification assay was established, and GAPDH mRNA was subsequently quantified in all samples of total RNA. The PCR products were separated by size, quantified by Southern hybridization analysis, and normalized to 28S rRNA content. Expression of GAPDH protein was also measured by Western analysis of endothelium-derived protein from omental (n = 14) and uterine (n = 16) arteries from NP and P ewes.

Results: Pregnnacy was associated with a 4.5-fold increase in GAPDH mRNA levels in UAEC, although in vitro exposure of parimary cultures of UAEC from NP ewes to NO or angiogenic growth factors did not significant change GAPDH mRNA expression. A 1.6-fold increase in GAPDH protein was detected in the uterine artery endothelium of P versus NP ewes, but no corresponding increase was found in omental artery endothelium.

Conclusion: Prengnacy increases the expression of both GAPDH mRNA and protein in UAEC. Furthermore, the pregnancy-induced increase in GAPDH protein in the endothelium of the uterine artery appears specific, as it is not observed in the omental (systemic) artery. This induction is not, however, reproduced in vitro with exogenous NO or angiogenic growth factor treatment (up to 24 hours).

Key Words: Glyceraldehyde-3-phosphate dehydrogenase • uterine artery endothelial cells • nitric oxide • angiogenic growth factors

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