PP131. Regulation of heme oxygenase/carbon monoxide (ho/co) and l-arginine/nitric oxide (no) pathways by human chorionic gonadotropin (hcg) in human fetal endothelium

Abstract

HO/CO and l-arginine/NO pathways are critical regulators of vascular function in early human pregnancy. HO is the rate-limiting enzyme in heme catabolism leading to the formation of biliverdin, which is subsequently reduced to bilirubin by biliverdin reductase, carbon monoxide, and free iron. HO-1, the inducible isoform of the enzyme, is essential to promote placental function and fetal development (1). HO-1 is also required to maintain nitric oxide synthesis (2). Human chorionic gonadotropin (hCG) is the first blastocyst-derived hormonal signal playing a significant role in implantation and placentation (3). We hypothesized that hCG upregulates HO-1 and thus positively regulates early vascular events in human pregnancy. In this study we used human umbilical vein endothelial cells (HUVECs) to demonstrate that hCG up-regulates mRNA and protein abundance of HO-1. The effect of hCG on l-arginine transport and the expression of hCAT1, the main cationic amino acid transporter involved in endothelial nitric oxide synthesis were evaluated. HUVEC were isolated from normal pregnancies (approval from the Institutional Review Board) by collagenase digestion and grown (37°C, 5% CO2) in medium 199 supplemented with 20% newborn and fetal calf sera. Cells were treated with hCG (0.5-100IU/mL, 0-24hours and CoCl2 (150μM, 6hours) as positive control. Total proteins were separated by SDS-PAGE electrophoresis, transferred to nitrocellulose membranes and incubated with monoclonal anti human HO-1 or anti β-actin and respective secondary antibody conjugated with horseradish peroxidase. Protein bands were detected by enhanced chemiluminiscence. Total RNA was isolated and cDNA was obtained by reverse transcription. PCR assays were performed in a reaction mix containing 0.5μM primers, dNTPs, Taq DNA polymerase, 2 mM MgCl2. Relative abundance of HO-1, hCAT-1 and 28S were determined. l-[(3)H]Arginine transport (100μM, 2 μCi ml(-1), 37°C, 1min) were determined in HUVECs exposed (30min) to hCG (0.5-100IU/mL) and CORM-2 (CO donor). The highest level of HO-1 protein were obtained at 5IU/mL of hCG and declined at higher concentrations. hCG increased HO-1 protein abundance and maximal response was observed at 6 hours (4-fold), decreasing at 24hours exposure. These effects of hCG on HO-1 protein abundance are well correlated with changes in HO-1 mRNA regarding time of exposure and concentration of hCG. L-arginine transport and hCAT-1 mRNA were also increased (2.8-fold and 4.5-fold, respectively) by hCG. Upregulation of HO-1 as well as the increase in l-arginine transport and hCAT-1 mRNA in HUVECs are novel effects of hCG, which occur at physiological plasma concentrations of the hormone, as those found in the first weeks of pregnancy. These changes could be relevant for the maintenance of the NO synthesis in fetal and maternal vasculature, ensuring the predominance of the vasodilator tone. These effects could be also critical for vascular remodelling of uterine circulation and placental vascular development in early stages of pregnancy, the lack of which could result in abnormal placentation as described in preeclampsia and fetal growth restriction.