Impairment of circulating metabolites induces endothelial dysfunction in adolescents exposed to gestational diabetes in utero

Background Offspring born to women with gestational diabetes (GD) are at increased risk of developing atherosclerotic cardiovascular disease (ASCVD) later in life. The underlying molecular mechanisms remain poorly understood. Purpose To determine whether the circulating milieu of GD adolescents affects vascular homeostasis and identify underlying metabolic mediators. Methods Plasma samples were collected from adolescents (aged 9-15 years) born to normoglycemic (n=8) or GD (n=12) mothers. Aortas from C57BL/6 mice were incubated for 24 hours with 10% plasma from both groups. Endothelium-dependent (EDR) and -independent (EIR) relaxations to acetylcholine (10-9 to 10-4,5 M) and sodium nitroprusside (10-10 to 10-5,5 M) with and without antioxidants Tempol (10-4 M) or NAC (10-5 M) were assessed by wire myography. Furthermore, the expression of oxidative/inflammatory genes (RT-PCR) and endothelial activation (monocyte adhesion assay) were evaluated in human aortic endothelial cells (HAEC) after exposure to 10% plasma from control and GD adolescents. Nitric oxide (NO) production was measured with diaminofluorescein (DAF) fluorescence. Metabolomic analysis of plasma samples was carried out with LC-MS. Results Aortas exposed to GD plasma exhibited reduced EDR compared to those incubated with control plasma, whereas EIR were unaffected. EDR impairment was rescued by tempol and NAC, confirming oxidative stress as a key mediator. HAEC exposed to GD plasma showed mRNA overexpression of pro-oxidant genes and increased ROS production. Expression of inflammatory genes and monocyte adhesion were also enhanced, suggesting an inflammatory endothelial phenotype. Metabolomic profiling of plasma samples revealed 19 significantly dysregulated metabolites in GD-adolescents. Levels of N-monomethylarginine (NMMA), a NO synthase inhibitor, were increased, whereas 3-hydroxyanthranilic acid (3-HAA), a metabolite with anti-inflammatory properties, was decreased. Supplementation of GD plasma with L-arginine reversed the NMMA-driven effects, restoring NO production both in vitro and ex vivo, while the addition of 3-HAA decreased inflammatory mRNA/protein expression and monocyte adhesion. 3-HAA supplementation of plasma caused downregulation of NOX4 mRNA/protein and ROS production. Conclusion Our results demonstrate that adolescents exposed to GD in utero exhibit an altered circulating metabolome that impairs endothelial function, potentially contributing to their elevated ASCVD risk. This metabolic imbalance may represent a putative target to mitigate ASCVD risk in this population.