Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA336

Poster Communications

Pregestational maternal obesity-associated human umbilical vein endothelial dysfunction results from endoplasmic reticulum stress

R. Villalobos-Labra1, M. Subiabre1, L. Silva1,2, M. Farías-Jofré1, L. Sobrevia1,3,4

1. Pontificia Universidad Católica de Chile, Santiago, Chile. 2. University Medical Centre Groningen (UMCG), University of Groningen, Groningen, Netherlands. 3. Universidad de Sevilla, Seville, Spain. 4. UQ Centre for Clinical Research (UQCCR), University of Queensland, Brisbane, Queensland, Australia.

Pregestational maternal obesity (PGMO) is associated with an adverse cardio-metabolic outcome in the offspring (1). A key step in the development of cardio-metabolic alterations is the endothelial dysfunction (2). Endothelial dysfunction associates with endoplasmic reticulum (ER) stress (3), a condition seen in human umbilical vein endothelial cells (HUVECs) from PGMO (4). However, whether HUVECs from PGMO show ER stress-dependent endothelial dysfunction is unknown. The aim of this study was to determine whether HUVECs from women with PGMO show endothelial dysfunction due to ER stress. HUVECs were isolated (collagenase digestion) from full term normal (n = 10) or PGMO (n = 12) pregnancies collected at the Clinical Hospital UC-CHRISTUS (Chile) after informed consent approved by the UC-CHRISTUS ethics committee. The investigation conforms to the principles outlined in the Declaration of Helsinki. Cells (passage 3) were incubated (8 h) without or with tunicamycin (5 µmol/L, ER stress inducer), tauroursodeoxycholic acid (TUDCA, 100 µmol/L, ER stress reducer), or both. L-Arginine transport kinetics (0-1000 mmol/L L-arginine, 3 μCi/mL L-[3H]arginine, 1 min incubation, 37°C), the protein abundance of the human cationic amino acids transporter-1 (hCAT-1), total endothelial nitric oxide synthase (eNOS), eNOS phosphorylated in serine1177 (pSer1177-eNOS) and threonine495 (pThr495-eNOS) protein abundance was determined by western blot. NOS activity was estimated in cells preloaded with 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) in the absence or presence of NG-nitro-L-arginine methyl ester (L-NAME, 100 µmol/L, NOS inhibitor). Values are mean ± S.E.M., compared by unpaired ANOVA, P<0.05. Cells from PGMO showed higher maximal L-arginine transport capacity (Vmax/Km) (1.5 ± 0.3 fold), hCAT-1 protein abundance (1.6 ± 0.3 fold), and pThr495-eNOS (1.7 ± 0.3 fold) compared with cells from normal pregnancies. However, pSer1177-eNOS and NO generation were reduced (42 ± 12 and 42 ± 15%, respectively) in HUVECs from PGMO compared with normal pregnancies. TUDCA blocked the effects of PGMO. Incubation of cells from normal pregnancies with tunicamycin increased the Vmax/Km (2.2 ± 0.4 fold) and the hCAT-1 protein abundance (1.5 ± 0.2 fold) but reduced the NO generation (37 ± 20%). In conclusion, HUVECs from women with PGMO show endothelial dysfunction due to PGMO-induced ER stress.

Where applicable, experiments conform with Society ethical requirements