ORIGINAL ARTICLE Human supraphysiological gestational weight gain and fetoplacental vascular dysfunction F Pardo 1 , L Silva 1 , T Sáez 1 , R Salsoso 1 , J Gutiérrez 1,2 , C Sanhueza 1 , A Leiva 1 and L Sobrevia 1,3,4 OBJECTIVE: Human foetal development and growth in an environment of maternal obesity associates with high risk of cardiovascular disease and adverse neonatal outcome. We studied whether supraphysiological gestational weight gain results in human fetoplacental endothelial dysfunction and altered fetoplacental vascular reactivity. METHODS: Primary cultures of human umbilical vein endothelial cells (HUVECs) and umbilical vein rings were obtained from pregnant women (112 total of patients recruited, 7 patients dropped out) exhibiting prepregnancy normal weight that ended with a physiological (pGWG (n = 67), total weight gain 11.5–16 kg, rates of weight gain ⩽ 0.42 kg per week) or supraphysiological (spGWG (n = 38), total weight gain 416 kg, rates of weight gain 40.42 kg per week) gestational weight gain (reference values from US Institute of Medicine guidelines). Vascular reactivity to insulin (0.1–1000 nmol l - 1 , 5 min) in KCl-preconstricted vein rings was measured using a wire myograph. Protein levels of human equilibrative nucleoside transporter 1 (hENT1), total and Ser 1177 - or Thr 495 -phosphorylated endothelial nitric oxide synthase (eNOS) were detected by western blot or immunofluorescence, and adenosine transport (0–250 μmol l - 1 adenosine, 2 μCi ml - 1 [ 3 H]adenosine, 20 s, 25 °C) was measured in the presence or absence of 1 μmol l - 1 nitrobenzylthioinosine (hENT1 inhibitor) or 10 μmol l - 1 chlorpromazine (CPZ, endocytosis inhibitor) in HUVECs. RESULTS: spGWG associates with reduced NOS activity-dependent dilation of vein rings (P = 0.001), lower eNOS expression and higher Thr 495 (P = 0.044), but unaltered Ser 1177 eNOS phosphorylation. hENT1-adenosine maximal transport activity was reduced (P = 0.041), but the expression was increased (P = 0.001) in HUVECs from this group. CPZ increased hENT1-adenosine transport (P = 0.040) and hENT1 plasma membrane accumulation only in cells from pGWG. CONCLUSION: spGWG in women with a normal prepregnancy weight causes lower fetoplacental vascular reactivity owing to the downregulation of eNOS activity and adenosine transport in HUVECs. Maternal spGWG is a detrimental condition for human fetoplacental endothelial function and reducing these alterations could result in a better neonate outcome. International Journal of Obesity (2015) 39, 1264–1273; doi:10.1038/ijo.2015.57 INTRODUCTION Obesity courses with multiple systemic complications including hypertension, dyslipidaemia, diabetes mellitus and insulin resistance. 1,2 Pregnancy is a natural phenomenon where the mother courses with physiological gestational weight gain (pGWG). However, a group of women showing normal body mass index (BMI) before they get pregnant (that is, prepregnancy) or at early stages of pregnancy courses with a supraphysiological gestational weight gain (spGWG) ending pregnancy with obesity (BMI ⩾ 30 kg m -2 ). 3–5 According to the 2009 US Institute of Medicine (IOM) guidelines for GWG in women with normal prepregnancy BMI, 6 pregnant women showing a total weight gain (tWG) 11.5–16 kg and rates of weight gain (rWG) for the 2nd and 3rd trimester of pregnancy ⩽ 0.42 kg per week correspond to pGWG, and those with tWG 416 kg and rWG 40.42 kg per week correspond to spGWG. During pregnancy, it is crucial to maintain a pGWG to ensure appropriate foetus development and growth. 6 However, intrauterine early-life development in an environment of maternal obesity or spGWG increases the risk of cardiovascular disease 7 and adverse neonatal outcome (for example, preterm delivery and umbilical cord arterial blood pH o7.1). 5,8 Even when women who were obese before pregnancy show endothelial dysfunction, 9 and spGWG associates with increased adiposity at birth 10 and with long-term implications on offspring cardiometa- bolic risk factors at young adult age, 5 it is unknown whether spGWG affect the human fetoplacental vasculature. 5,6,11–13 The human placenta lacks innervation; 14 thus, the local release of vasoactive molecules from the endothelium such as nitric oxide (NO) or adenosine, or systemic circulating factors such as insulin and adenosine, have key roles to maintain fetoplacental vascular function. 15 Adenosine and insulin cause vasodilation in most vascular beds, 16,17 including the human umbilical vein, 15,18,19 a phenomenon primarily dependent on adenosine uptake via the human equilibrative nucleoside transporters 1 (hENT1), an Na + -independent membrane trans- porter with apparent K m ~ 100–200 μmol l - 1 , sensitive to inhibition by o 1 μmol l - 1 nitrobenzylthioinosine (NBTI), with a minor contribution of hENT2 (apparent K m ~ 50–250 μmol l - 1 , inhibited by 41 μmol l - 1 NBTI), 13 and endothelial NO synthase (eNOS) activity. These mechanisms are crosslinked and associate with fetoplacental endothelial dysfunction in gestational diabetes mellitus (GDM); 20 however, nothing is known regarding these mechanisms in the human fetoplacental vasculature in spGWG. 13 1 Cellular and Molecular Physiology Laboratory (CMPL), Division of Obstetrics and Gynaecology, School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, Santiago, Chile; 2 Cellular Signalling and Differentiation Laboratory (CSDL), Faculty of Health Sciences, Universidad San Sebastián, Santiago, Chile; 3 Department of Physiology, Faculty of Pharmacy, Universidad de Sevilla, Seville, Spain and 4 University of Queensland Centre for Clinical Research (UQCCR), Faculty of Medicine and Biomedical Sciences, University of Queensland, Herston, QLD, Australia. Correspondence: Dr F Pardo or Dr A Leiva or Professor L Sobrevia, Cellular and Molecular Physiology Laboratory (CMPL), Division of Obstetrics and Gynaecology, School of Medicine, Faculty of Medicine, Pontificia Universidad Católica de Chile, PO Box 114-D, Santiago, Chile. E-mail: fpardo@med.puc.cl or aalevia@uc.cl or sobrevia@med.puc.cl Received 14 January 2015; revised 11 March 2015; accepted 29 March 2015; accepted article preview online 14 April 2015; advance online publication, 19 May 2015 International Journal of Obesity (2015) 39, 1264 – 1273 © 2015 Macmillan Publishers Limited All rights reserved 0307-0565/15 www.nature.com/ijo