Effects of the angiotensin II type-1 receptor antagonist telmisartan on endothelial activation induced by advanced glycation endproducts Serena Del Turco, Teresa Navarra, Giuseppina Basta, Raffaele De Caterina # CNR Institute of Clinical Physiology, Pisa, Italy,# G. d Annunzio University, Chieti, Italy
Background I Angiotensin and cardiovascular disease Vasoconstriction Aldosterone synthesis VSMC tone Oxidative stress NADPH oxidase ROS LOX-1 Angiotensin II Inflammation Leukocyte infiltration Permeability Activation of signaling (NF-κB ) Endothelial dysfunction NO production Vasoconstriction Platelet aggregation Adhesion molecule expression, Growth factor and cytokine release 1 Tissue remodeling Proliferation of VSMC MMP activation
Background II Ang II, AT 1 R and endothelial dysfunction Ang II AT 1 R Endothelial cell NADPH Oxidase Gq enos CAM ROS NO ERK ET-1 expression VSMC growth contraction NF-κB VCAM-1 ICAM-1 E-Selectin ONOO - DNA damage Vasorelaxation Vascular protection Endothelial dysfunction
Background III ARBs and ACE-inhibitors Angiotensinogen Renin Ang I (1-10) Bradykinin ACE ACE inhibitors LOSARTAN TELMISARTAN OLMESARTAN ARBs Ang II (1-8) Kinin fragment AT 1 -receptor (detrimental effects) AT 2 -receptor (beneficial effects)
Background IV Pleiotropic effects of telmisartan agonist of PPAR-γ amelioration of Insulin resistence improvement of lipid profile favorable fat redistribution beneficial effects on: - vascular function - cardiac remodeling - renal function
Aim of the study We hypothesize that part of the anti-atherogenic properties of ARBs are mediated by a reversal of specific (Ang-II-mediated) and nonspecific (TNF-α- or AGEs-mediated) induction of endothelial dysfunction. We studied the anti-inflammatory and anti-oxidant effects of telmisartan on adhesion molecule expression in an in vitro system mimicking the vascular milieu in inflammatory and diabetic conditions.
Methods Cell culture and experimental design Cultured human umbilical vein endothelial cells (HUVEC) were pretreated with or without telmisartan (TEL) (10, 100 μmol/l) for 30 minutes, and then stimulated with AGEs (200 μmol/l) or TNF-α (20 ng/ml) for 18h to induce vascular adhesion molecule-1 (VCAM-1) and intercellular molecule-1 (ICAM-1), and for 10h to induce E-Selectin expression. Losartan and its active metabolite EXP-3174 (both at 10, 100 μmol/l), were used as control. Detection of adhesion molecule expression Assay of cell surface adhesion molecules was carried out by cell surface enzyme immunoassay (EIA) using mouse anti-human antibody against VCAM-1, ICAM-1 and E- Selectin. Detection of intracellular ROS generation Generation of intracellular ROS (mostly hydrogen peroxide) was assessed by carboxydi-chloro-fluoresceinediacetate (DCHF-DA) fluorescence.
VCAM-1 expression (OD mu, mean SD) VCAM-1 expression (OD mu, mean SD) Results I Telmisartan reduced VCAM-1 expression induced by TNF-α..and by AGEs 700 600 500 400 300 200 100 0 (TEL 0 μmol/l) (TEL 10 μmol/l) (TEL 100 μmol/l) 1 2 + TNF-α (10 ng/ml) P<0.001 vs stimulation with TNF-α 200 150 100 50 (TEL 0 μmol/l) (TEL 10 μmol/l) (TEL 100 μmol/l) P<0.001 vs stimulation with AGEs 0 1 2 + AGEs (200 μg/ml)
E-Selectin expression (OD mu, mean SD)v E-Selectin expression (OD mu, mean SD) Results II Telmisartan increased E-Selectin expression after TNF-α stimulation 1000 800 600 400 200 but prevented its induction by AGEs 0 (TEL 0 μmol/l) (TEL 10 μmol/l) (TEL 100 μmol/l) + TNF-α (10 ng/ml) 1 2 P<0.001 vs stimulation with TNF-α 50 40 30 20 10 0 (TEL 0 μmol/l) (TEL 10 μmol/l) (TEL 100 μmol/l) + AGEs (200 μg/ml) 1 2 P<0.001 vs stimulation with AGEs
Results III 1. Telmisartan did not affect ICAM-1 expression induced by AGEs or TNF-α; 2. Loosartan, a hydrophilic ARB, and its active metabolite, EXP3174, a compound with anti-inflammatory properties, did not influence adhesion molecule expression induced by TNF-a or AGEs.
Intracellular ROS production (Fluorescence intensity a.u., mean±sd) Results IV Telmisartan (10 μmol/l) prevented the increase of intracellular ROS after AGEs stimulation but not after TNF-α stimulation. (A) control + TEL (10 µmol/l) (B) 100 TEL (0 µmol/l) TEL (10 µmol/l) P<0.05 80 AGEs (200 µg/ml) 60 40 20 TNF-a (10 ng/ml) 0 1 2 3 + AGEs (200 µg/ml) + TNF-a (10 ng/ml)
Conclusions Our results demonstrate that telmisartan: - has peculiar regulatory mechanisms on adhesion molecule expression induced by either AGEs or TNF-α; - AT1R antagonism was not involved in the inhibitory effect madiated by TEL and highlighting the peculiar nature of TEL among ARBs; - such anti-inflammatory properties may disclose a role for this drug in the pro-atherogenic vascular milieu occurring in diabetes