Introduction: Hyperhomocysteinemia (HHcy) is an independent risk factor of various CVD including hypertension and atherosclerosis. HHcy induces excessive oxidative stress to impair endothelial function by lowering bioavailability of the major endothelium-derived vasodilator NO. Glucagon-like peptide-1 (GLP-1) directly binds to its receptor in vasculature to reduce ROS production and increase NO level. Previously we showed that GLP-1R agonist exenatide ameliorates endothelial dysfunction in hypertensive rats.
Hypothesis: We propose to study the vascular benefits of enzymatically stable GLP-1 analogues like exendin-4 against HHcy-induced endothelial dysfunction.
Methods: Functional assays by organ bath and myograph are carried out to evaluate the effect of exendin-4 on vascular tone, in terms of endothelium-dependent relaxations (EDRs), of Hcy-treated (300 μM) rat and mouse aortae, and porcine circumflex arteries. Confocal microscopy is performed to assess oxidative stress, in terms of DHE staining, in endothelial cells and aortic walls of SD rats. Expression levels of AMPK/eNOS pathway-associated proteins in human umbilical vein endothelial cells (HUVECs) and C57 mice aortae are also studied. In in vivo experiments, C57 mice are fed with diet of increased methionine and decreased folate to induce HHcy, followed by continuous venous injection of exendin-4.
Results and Conclusions: Functional assays show that exendin-4 restores Hcy-impaired EDRs in both SD rat aortae and porcine circumflex arteries, where the latter share closer susceptibility to CVD compared to human arteries. Confocal microscopy shows that exendin-4 pre-treatment in endothelial cells and aortic walls of SD rats reduces DHE fluorescence, indicating its oxidative stress-lowering effect. Moreover, Western blots in HUVECs show that exendin-4 up-regulates the levels of phosphorylated AMPK and eNOS which are inhibited by Hcy. Further in vivo experiments in C57 mice, fed with HHcy-inducing diet, are also carried out to confirm the beneficial role of exendin-4. Together, these findings suggest that exendin-4 attenuates endothelial dysfunction by reducing ROS level via AMPK/eNOS pathway, shedding light on its therapeutic potential against HHcy-related CVD.