Ambrisentan reverses the prosenescent and anti-autophagic effect of high glucose on human pulmonary artery endothelial cells exposed to hypoxia

Abstract Background Cardiovascular (CV) comorbidities (systemic hypertension, hyperlipidemia, obesity, diabetes mellitus, peripheral arterial disease, coronary artery disease) are associated with reduced treatment response to specific drugs for group 1 pulmonary arterial hypertension (PAH). Often this epidemiological evidence is the result of misdiagnosis of group 2 PH associated with left heart disease, but it can also be explained as the intrinsic loss of the ability of pulmonary endothelial cells to respond to specific antiremodeling drugs. Aims To evaluate the impact of high glucose and hyperosmolar stress on the responsiveness of human pulmonary artery endothelial cells (hPAECs) to the endothelin receptor antagonist ambrisentan in terms of senescence, autophagic activity, viability and expression of some microRNAs involved in pulmonary arterial remodeling. Methods We incubated hPAECs with high glucose (HG, 30.5 mM) or high mannitol (HM, 25 mM), with/without ambrisentan (0.02 nM) in normoxia (N) or hypoxia (H) for 24 h and tested them for cell viability (MTT assay), protein and miRNA levels, autophagy and senescence (β-Gal assay) in vitro. Results H induced cell death as compared to N (752 ± 44 vs 701 ± 45 Abs units, p = 0.03). H reduced autophagy (Figure 1A-C) and induced senescence (Figure 1D), an effect further potentiated by HG and HM. Treatment with A in N and H significantly reversed the effect of HG but not HM on autophagy (Figure 1A-C) and senescence (Figure 1D). H increased the abundance of antiapoptotic miR124-3, compared to N in vehicle (V)-treated hPAEC (p=0.008) (Figure 2A), and induced a similar effect on antiapoptotic and proliferative miR191-3p, although not statistically significant (Figure 2B). In N, A potentiated the expression of both miR124-3p (p=0.007) and miR191-3p (p=0.02) in HG-treated hPAEC, and only miR191-3p in HM-treated hPAEC (p=0.01). A induced a similar effect on miR191-3p in hPAEC exposed to H+HG (p=0.02), with a non-statistically significant trend on miR124-3p. Conclusions In hPAEC exposed to H, A retains its pro-autophagic and antisenescent effects in an in vitro model mimicking diabetes. miR124-3p and miR191-3p may act as biomarkers of disease and treatment response to specific drugs in patients with PAH.Figure 1:autophagy and senescenceFigure 2:miRNA expression