Maintenance of AMPK activation by metformin is beneficial for suppressing the progress of diabetes-accelerated atherosclerosis via inhibition of vascular smooth muscle cells migration

Maintenance of AMPK activation by metformin is beneficial for suppressing the progress of diabetes-accelerated atherosclerosis via inhibition of vascular smooth muscle cells migration

Abstract:

Background: Our previous work revealed that augmented AMPK activation inhibit cell migration by phosphorylating its substrate Pdlim5. As medial VSMCs contribute to the major composition of atherosclerotic plaques, a hypothesis is raised that modulation of AMPK-Pdlim5 signal pathway could retard the development of atherosclerosis through inhibiting migration of VSMCs. Therefore, we initiate the present study to investigate whether AMPK agonist like metformin is beneficial for suppressing diabetes-accelerated atherosclerosis in a diabetic mouse model induced by streptozotocin and high fat diet.

Methods: For cell experiment, vascular smooth muscle cells (VSMCs) were overexpressed flag fused Pdlim5 and Pdlim5 mutant. Then the engineered VSMCs were introduced with metformin or control drug before determination of phosphorylated Pdlim5 with immunoblotting. For animal work, 8-week-old male ApoE−/−mice were induced diabetes with streptozotocin and then were randomly divided into 8 groups: control group, metformin hydrochloride (300 mg/kg/day) group, wildtype-Pdlim5 (Pdlim5 WT) carried adenovirus (Ad) group, Ad Pdlim5 WT and Met group, Ad Pdlim5 S177A group, Ad Pdlim5 S177A and Met group, Ad Pdlim5 S177D group, Ad Pdlim5 S177D and Met group. All mice were fed with high fat diet after virus infection. At the end, mice were sacrificed to observe atherosclerotic plaques and deposition of VSMCs in plaques. Moreover, 12–15-week-old Myh11-cre-EGFP male mice were accepted ligation of the left carotid artery and randomly divided into control and metformin treatment group. Finally, the injured vessel of Myh11-cre-EGFP mice were isolated to analyze the relationship between AMPK activation and neointima formation.

Results: It was found that AMPK directly phosphorylate Pdlim5 at Ser177 in vitro, and metformin, an AMPK agonist, could induce phosphorylation of Pdlim5 indirectly and inhibition of cell migration as a result. Exogenous expression of phosphomimetic S177D-Pdlim5 inhibits lamellipodia formation and migration in VSMCs. It was also demonstrated that VSMCs contribute to the major composition of injury-induced neointimal lesions, while metformin could alleviate the occlusion of carotid artery in a wire-injury mice model. In order to investigate the function of AMPK-Pdlim5 pathway in the context of pathological condition, ApoE−/− male mice were divided randomly into control, streptozocin and high fat diet-induced diabetes mellitus, STZ + HFD together with metformin or Pdlim5 mutant carried adenovirus treatment groups. The results showed increased plasma lipids and aggravated vascular smooth muscle cells infiltration into the atherosclerotic lesion in diabetic mice compared with control mice. However, metformin alleviated diabetes-induced metabolic disorders and atherosclerosis, as well as decreased VSMCs infiltration in atherosclerotic plaques, while Pdlim5 phospho-abolished mutant carried adenovirus S177A-Pdlim5 undermine this protective function.

Conclusions: The activation of AMPK-Pdlim5 pathway by chemicals like Metformin could inhibit formation of migratory machine of VSMCs and alleviate the progress of atherosclerotic plaques in diabetic mice. The maintenance of AMPK activity is beneficial for suppressing diabetes-accelerated atherosclerosis or metabolic syndrome.

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