Metformin Exerts Protective Effects against the Degeneration of Aortic Valves

 

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Objectives: Metformin, widely used as antidiabetic agent, is a complex drug with multiple molecular mechanisms and exhibits diverse protective effects also on cardiac and vascular diseases. The main molecular actions, independent of its hypoglycemic effect, are mediated via activation of AMP-activated protein kinases (AMPK), an enzyme that coordinates control of cell growth, and autophagy. However, the list of AMPK-independent actions of metformin is rapidly increasing. Recent findings demonstrated that metformin usage was independently associated with lower coronary artery calcification and also prevents vascular calcification. However, it remains unclear whether metformin has any protective effects on calcific aortic valve disease (CAVD).

Methods: Ovine valvular interstitial cells (VICs), as well as whole aortic valve (AV) leaflets applied as a novel three-dimensional (3D) model for CAVD were cultured in vitro under prodegenerative (pd, β-glycerophosphate [GP], and CaCl2), conditions and treated with metformin, compound C, AICAR, L-Name, and aminoguanidine. Calcium deposition was evaluated by alizarin red staining. Alkaline phosphatase (AP) was analyzed colorimetrically in supernatants over time and histologically in AV leaflets, migratory behavior by wound healing assay and expression of AMPK and phospho-AMPK by Western blot. Morphological changes of the extracellular matrix (ECM) were assessed histologically by hematoxylin/eosin and Movat’s pentachrome.

Results: Metformin efficiently increased AMPK phosphorylation of VICs and resulted in distinct antidegenerative ramifications of 2D cultures (p < 0.001, n = 23) and AV leaflets (p < 0.01, n = 12). AP levels were decreased in supernatants in 2D (p < 0.001) and 3D (p < 0.001), as well as in AV tissue. Activation of AMPK with AICAR, as well as inhibition, with compound C did not result in antidegenerative effects. Migration of VICs was not affected by AMPK activation, but distinctly inhibited after treatment with compound C (p < 0.01). Inhibition of nitric oxide (NO) synthases (endothelial and inducible) partially reversed the antidegenerative effect of metformin (p < 0.01).

Conclusion: Metformin prevents degeneration of VICs and AV leaflets. The protective effects are partially mediated via AMPK-NO pathway, but further AMPK-independent mechanism must be involved. Our data provide the basis for translational approaches for the application of metformin to prevent CAVD progression.