ABSTRACT
BACKGROUND: The angiotensin II type 2 receptor (AT2R) has been suggested to have an athero-protective role, however no studies have investigated the effect of direct stimulation of this receptor in atherosclerosis. Thus this study aimed to determine the effect of direct AT2R stimulation in setting of atherosclerosis, using the known AT2R agonist, CGP42112. METHODS AND RESULTS: Apolipoprotein E-deficient (ApoE(-/-)) mice were fed a high fat (21%) diet for 16 weeks, with subcutaneous infusions of CGP42112 (1, 5 or 10 µg/kg/min) administered via osmotic mini-pumps in the final 4 weeks. CGP42112 treatment at all doses significantly improved endothelial function (p<0.001) when compared to acetylcholine mediated-vasorelaxation in aorta taken from vehicle-treated ApoE(-/-) mice. In aortic segments adjacent to those used for vascular reactivity studies, CGP42112 treatment at all doses concomitantly increased eNOS immunoreactivity and protein levels whilst superoxide (O2(-)) production was significantly (p<0.01) decreased compared to levels measured in aorta from vehicle-treated ApoE(-/-) mice. Moreover, CGP42112 (1 µg/kg/min) treatment significantly attenuated (p<0.05) atherosclerotic lesion progression (assessed as both lipid deposits and luminal encroachment in thoracic aorta and aortic arch) and significantly increased plaque stability in the brachiocephalic artery, a region normally prone to rupture. Both the vaso- and athero-protective effects of CGP42112 (1 µg/kg/min) were reversed with co-infusion of the AT2R antagonist, PD123319, but not the MasR antagonist, A779. CONCLUSION: For the first time we have shown that direct stimulation of the AT2R improves endothelial function, reduces atherosclerotic lesion progression and mediates plaque stability with these effects at least partly due to restoration of nitric oxide bioavailability.
Subject(s)
Apolipoproteins E , Atherosclerosis/drug therapy , Cardiotonic Agents/therapeutic use , Plaque, Atherosclerotic/drug therapy , Receptor, Angiotensin, Type 2/agonists , Animals , Apolipoproteins E/deficiency , Atherosclerosis/metabolism , Cardiotonic Agents/pharmacology , Cohort Studies , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Oligopeptides/pharmacology , Oligopeptides/therapeutic use , Plaque, Atherosclerotic/metabolism , Receptor, Angiotensin, Type 2/metabolismABSTRACT
Liraglutide, a once-daily glucagon-like peptide-1 receptor (GLP-1R) agonist, has been approved as a new treatment for type 2 diabetes and is the subject of a clinical trial programme to evaluate the effects on cardiovascular disease and safety. The current study aimed to determine the in vivo effect of liraglutide on progression of atherosclerotic vascular disease in the apolipoprotein E-deficient (ApoE(-/-)) mouse model and identify underlying mechanisms responsible. Liraglutide treatment inhibited progression of early onset, low-burden atherosclerotic disease in a partially GLP-1R-dependent manner in the ApoE(-/-) mouse model. In addition, liraglutide treatment inhibited progression of atherosclerotic plaque formation and enhanced plaque stability, again in a partially GLP-1R-dependent manner. No significant effect of liraglutide on progression of late onset, high-burden atherosclerotic disease was observed. In addition, no significant endothelial cell dysfunction was identified in ApoE(-/-) mice with early onset, low-burden atherosclerotic disease, although significant prevention of weight gain was observed in liraglutide-treated mice using this dietary protocol. Taken together, these results suggest a potential role for liraglutide in the prevention and stabilisation of atherosclerotic vascular disease together with possible protection against major cardiovascular events.
Subject(s)
Atherosclerosis/drug therapy , Diabetes Mellitus, Type 2/drug therapy , Glucagon-Like Peptide 1/analogs & derivatives , Hypoglycemic Agents/therapeutic use , Plaque, Atherosclerotic/drug therapy , Receptors, Glucagon/agonists , Animals , Apolipoproteins E/deficiency , Apolipoproteins E/metabolism , Atherosclerosis/diagnosis , Disease Models, Animal , Disease Progression , Endothelial Cells/drug effects , Glucagon-Like Peptide 1/therapeutic use , Glucagon-Like Peptide-1 Receptor , Liraglutide , Mice , Mice, Inbred C57BL , Mice, Knockout , Plaque, Atherosclerotic/diagnosisABSTRACT
The glucagon like peptide-1 receptor (GLP-1R) agonist liraglutide attenuates induction of plasminogen activator inhibitor type-1 (PAI-1) and vascular adhesion molecule (VAM) expression in human vascular endothelial cells (hVECs) in vitro and may afford protection against endothelial cell dysfunction (ECD), an early abnormality in diabetic vascular disease. Our study aimed to establish the dependence of the in vitro effects of liraglutide on the GLP-1R and characterise its in vivo effects in a mouse model of ECD. In vitro studies utilised the human vascular endothelial cell line C11-STH and enzyme-linked immunosorbent assays (ELISA) for determination of PAI-1 and VAM expression. In vivo studies of vascular reactivity and immunohistochemical analysis were performed in the ApoE(-/-) mouse model. In vitro studies demonstrated GLP-1R-dependent liraglutide-mediated inhibition of stimulated PAI-1 and VAM expression. In vivo studies demonstrated significant improvement in endothelial function in liraglutide treated mice, a GLP-1R dependent effect. Liraglutide treatment also increased endothelial nitric oxide synthase (eNOS) and reduced intercellular adhesion molecule-1 (ICAM-1) expression in aortic endothelium, an effect again dependent on the GLP-1R. Together these studies identify in vivo protection, by the GLP-1R agonist liraglutide, against ECD and provide a potential molecular mechanism responsible for these effects.
Subject(s)
Apolipoproteins E/deficiency , Atherosclerosis/prevention & control , Endothelial Cells/drug effects , Endothelium, Vascular/drug effects , Glucagon-Like Peptide 1/analogs & derivatives , Hypoglycemic Agents/pharmacology , Receptors, Glucagon/agonists , Vascular Cell Adhesion Molecule-1/metabolism , Animals , Apolipoproteins E/genetics , Atherosclerosis/genetics , Atherosclerosis/metabolism , Atherosclerosis/physiopathology , Cell Line, Transformed , Cyclic AMP-Dependent Protein Kinases/metabolism , Disease Models, Animal , Dose-Response Relationship, Drug , Endothelial Cells/metabolism , Endothelium, Vascular/metabolism , Endothelium, Vascular/physiopathology , Enzyme-Linked Immunosorbent Assay , Glucagon-Like Peptide 1/pharmacology , Glucagon-Like Peptide-1 Receptor , Humans , Immunohistochemistry , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/metabolism , Liraglutide , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , NF-kappa B/genetics , NF-kappa B/metabolism , Nitric Oxide Synthase Type III/metabolism , Plasminogen Activator Inhibitor 1/metabolism , RNA, Messenger/metabolism , Receptors, Glucagon/metabolism , Tumor Necrosis Factor-alpha/metabolism , Vascular Cell Adhesion Molecule-1/genetics , Vasodilation/drug effects , Vasodilator Agents/pharmacologyABSTRACT
Novel AT(2)R ligands were designed by substituting individual ß-amino acid in the sequence of the native ligand angiotensin II (Ang II). Relative ATR selectivity and functional vascular assays (in vitro AT(2)R-mediated vasorelaxation and in vivo vasodepressor action) were determined. In competition binding experiments using either AT(1)R- or AT(2)R- transfected HEK-293 cells, only ß-Asp(1)-Ang II and Ang II fully displaced [(125)I]-Ang II from AT(1)R. In contrast, ß-substitutions at each position of Ang II exhibited AT(2)R affinity, with ß-Tyr(4)-Ang II and ß-Ile(5)-Ang II exhibiting ≈ 1000-fold AT(2)R selectivity. In mouse aortic rings, ß-Tyr(4)-Ang II and ß-Ile(5)-Ang II evoked vasorelaxation that was sensitive to blockade by the AT(2)R antagonist PD123319 and the nitric oxide synthase inhibitor L-NAME. When tested with a low level of AT(1)R blockade, ß-Ile(5)-Ang II (15 pmol/kg per minute IV for 4 hours) reduced blood pressure (BP) in conscious spontaneously hypertensive rats (ß-Ile(5)-Ang II plus candesartan, -24 ± 4 mm Hg) to a greater extent than candesartan alone (-11 ± 3 mm Hg, n=7, P<0.05), an effect that was abolished by concomitant PD123319 infusion. However, in an identical experimental protocol, ß-Tyr(4)-Ang II had no influence on BP (n=10), and it was less stable than ß-Ile(5)-Ang II in plasma stability assays. Thus, this study demonstrated that a single ß-amino acid substitution resulted in a compound that demonstrated both in vitro vasorelaxation and in vivo depressor activity via AT(2)R. This approach to the design and synthesis of novel AT(2)R-selective peptidomimetics shows great potential to provide insight into AT(2)R function.
