Metformin protects against vascular calcification through the selective degradation of Runx2 by the p62 autophagy receptor

Vascular calcification is associated with aging, type 2 diabetes and atherosclerosis, and increases the risk of cardiovascular morbidity and mortality. It is an active, highly regulated process that resembles physiological bone formation. It has previously been established that pharmacological doses of metformin alleviate arterial calcification through AMPK-activated autophagy, however the specific pathway remains elusive. In the present study we hypothesized that metformin protects against arterial calcification through the direct autophagic degradation of Runx2.
Calcification was blunted in VSMCs by metformin in a dose-dependent manner (0.5mM - 1.5mM) compared to control cells (P<0.01). VSMCs cultured under high
-phosphate (Pi) conditions in the presence of metformin (1mM) showed
asignificant increase in LC3 puncta following bafilomycin-A1 (Baf-A; 5n M) treatment compared to control cells (P<0.001). Furthermore, reduced expression of Runx2 was observed in the nuclei of metformin-treated calcifying VSMCs (P<0.0001).
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Evaluation of the functional role of autophagy through Atg3 knockdown in VSMCsshowed aggravated Pi-induced calcification (P<0.0001), failure to induce autophagy (punctate LC3) (P<0.001) and increased nuclear Runx2 expression (p<0.0001) in VSMCs cultured under high Pi conditions in the presence of metformin (1mM). Mechanistic studies employing three-way co-immunoprecipitation (co-IP) with Runx2, p62 and LC3 revealed that p62 binds to both LC3 and Runx2 upon metformin treatment in VSMCs. Furthermore, immunoblotting with LC3 revealed that Runx2 specifically binds with p62 and LC3-II in metformin-treated calcified VSMCs.
Lastly, we investigated the importance of the autophagy pathway in vascular calcification in a clinical setting. Ex vivo clinical analyses of calcified diabetic lower limb artery tissues highlighted a negative association between Runx2 and LC3 in the vascular calcification process. These studies suggest that exploitation of metformin and its analogues may represent a novel therapeutic strategy for clinical intervention through the induction of AMPK/Autophagy Related 3 (Atg3)-dependent autophagy and the subsequent p62-mediated autophagic degradation of Runx2.