Abstract

INTRODUCTIONGestational diabetes mellitus (GDM) is associated with fetoplacental endothelial dysfunction, which may be induced by hyperglycemia. We hypothesized that endothelial exosomes, which are extracellular nanovesicles affecting endothelial function, play a role in the high glucose (HG)-induced endothelial dysfunction. METHODSExosomes were isolated from HUVECs incubated with basal glucose (5.5 mmol/L; HUVEC- BG; exo-BG) and from HUVECs incubated with HG for 24 h (25 mmol/L; HUVEC-HG; exo-HG) in exosome-free medium. Exosomes were isolated and characterized by ultracentrifugation, sucrose gradient, electron microscopy, nanotracking analysis and Western blotting. HUVEC-BG and HUVEC-HG were exposed to exo-BG and exo-HG in two different concentrations: 5 μg and 1 μg exosome protein/mL. The exosomal effect on endothelial cell function was determined by wound healing assay, expression of endothelial nitric oxide synthase (eNOS), human cationic amino acid transporter type 1 (hCAT-1), vascular endothelial growth factor (VEGF) and intracellular adhesion molecule type 1 (ICAM-1) by Western blotting, qPCR or flow cytometry. RESULTSHG increased the exosomal release from HUVECs, endothelial wound healing and expression of phosphorylated (P∼Ser1177)-eNOS, hCAT-1, VEGF and ICAM-1. Exo-HG also increased endothelial cell wound healing, P∼Ser1177-eNOS, hCAT-1 and ICAM-1 expression in HUVEC-BG. Exo-BG reverted the effect of HG on endothelial cell wound healing and hCAT-1 mRNA expression to normal values. DISCUSSIONOur results show that HG may induce endothelial dysfunction in HUVECs and that exosomes from HUVEC-HG mimicked some of the effects of HG. This study contributes to the unraveling of the mechanism by which hyperglycemia affects the fetoplacental vasculature in GDM.

Highlights

  • Gestational diabetes mellitus (GDM) is associated with fetoplacental endothelial dysfunction, which may be induced by hyperglycemia

  • In human umbilical vein endothelial cells (HUVECs) from women diagnosed with GDM, an impaired endothelial function has been demonstrated

  • This effect could be partly mimicked by exposing HUVEC-BG to exosomes from HUVEC-high glucose (HG)

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Summary

Introduction

Gestational diabetes mellitus (GDM) is associated with fetoplacental endothelial dysfunction, which may be induced by hyperglycemia. The exosomal effect on endothelial cell function was determined by wound healing assay, expression of endothelial nitric oxide synthase (eNOS), human cationic amino acid transporter type 1 (hCAT-1), vascular endothelial growth factor (VEGF) and intracellular adhesion molecule type 1 (ICAM-1) by Western blotting, qPCR or flow cytometry. RESULTS: HG increased the exosomal release from HUVECs, endothelial wound healing and expression of phosphorylated (P∼Ser1177)-eNOS, hCAT-1, VEGF and ICAM-1. Various changes associated with low NO bioactivity have been found in the L-Arginine/nitric oxide (NO) signaling pathway in HUVECs derived from GDM pregnancies, such as increased expression of human cationic amino acid transporter type 1 (hCAT-1), increased NO production and increased phosphorylated Ser1177 endothelial nitric oxide synthase (P∼Ser1177-eNOS) [9,11], as compared to HUVECs from healthy pregnancies

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