Association of serum irisin levels with endothelial dysfunction markers in an in vitro model of HUVEC cells challenged with serum from pediatric patients with Diabetes Mellitus and Metabolic Syndrome

Abstract

Background and aims: Endothelial dysfunction is a crucial pathophysiological mechanism for cardiovascular diseases and a potential consequence of metabolic disorders. Preclinical studies have revealed that irisin, an adipomyokine, can exert beneficial effects in preventing endothelial dysfunction by decreasing the production of oxidative stress, promoting endothelial production of nitric oxide (NO), and increasing the endothelial cell viability. However, these studies have the limitation of studying single-damage inductors for endothelium, evading the representativeness of the combined adverse metabolic extracellular milieu in humans. We first aimed to characterize the role of irisin on dual damage in an in vitro model of hyperglycemia and hyperlipidemia. We further investigated the associations between irisin serum levels and endothelial dysfunction markers in an in vitro model of human umbilical vein endothelial cells (HUVEC) challenged with the serum from pediatric patients diagnosed with Type 2 Diabetes Mellitus (T2DM) and Metabolic Syndrome (MetS).

Methodology: We characterized the effects of recombinant irisin (0-20 nM) after 24 hours of treatment with high glucose and high palmitic acid on cell viability, cell migration and adhesion, and endothelial NO production. In a second stage, we obtained serum samples from a cohort of 55 pediatric patients aged 6 to 16 years old. These patients were grouped depending on their metabolic status into T2DM patients (n=21), MetS (n=18) patients, and healthy controls (n=16). We performed cell viability assays and measured endothelial NO production in individual HUVEC cell cultures challenged for 24 hours with the serum of these patients.

Results: Treatment with recombinant irisin (10 nM) demonstrated a significant maintenance of endothelial cell viability in culture with hyperlipidemic and hyperglycemic media. Endothelial cell viability was supported mainly by significantly reducing late-apoptotic cell populations. Functionally, irisin remarkably maintained the endothelial NO production in these conditions while improving endothelial cell adhesion to a 5% gelatin extracellular matrix substrate. Assays involving patients revealed significantly lower serum irisin levels in the MetS and T2DM groups than in controls. HUVEC cell cultures challenged with the serum of patients with T2DM and MetS showed substantially lower endothelial NO production, lower cell viability, and higher rates of late apoptotic and necrotic cells. We observed significant positive associations between irisin serum levels and NO production (R= 0.27, p<0.05) and cell viability (R=0.37, p<0.05). Negative correlations between serum irisin levels and rates of late apoptotic cells (R=-0.41) and necrotic cells (R=-0.32, p<0.05) were identified.

Conclusions: Overall, these results suggest that irisin exerts beneficial effects on endothelial integrity in the presence of adverse metabolic scenarios by maintaining endothelial cell functionality through nitric oxide production, sustaining cell viability, downregulating their late apoptosis. This is the first study to show that serum irisin levels are positively correlated with endothelial cell viability and nitric oxide production, while inversely correlated with late-apoptotic and necrotic endothelial cell rate. The loss of these protection mechanisms in patients with MetS and T2DM, which show low circulant irisin levels, translates into the detrimental functional effects on endothelial cells and their association with endothelial dysfunction markers.