Metformin Protects against the Degeneration of Aortic Valves

Objectives: The molecular actions of metformin, including its hypoglycemic effect are mediated via inhibition of mitochondrial ATP production and activation of AMP-activated protein kinases (AMPK), an enzyme that coordinates cell growth and autophagy. However, the list of AMPK-independent actions of metformin is rapidly increasing. In this study, we aimed at investigating 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 were cultured in vitro under pro-degenerative (pd, β-glycerophosphate and CaCl2) or pro-calcifying (pc, NaH2PO4) conditions and treated with metformin. Calcium deposition was evaluated by alizarin red staining. Alkaline phosphatase (AP) was analyzed colorimetrically in supernatants and histologically in AV leaflets. Gene and protein expression were analyzed by quantitative RT-PCR and by Western blot, respectively.

Result: Metformin efficiently increases AMPK phosphorylation of VICs (p < 0.001) and results in distinct anti-degenerative ramifications of VIC cultures (p < 0.001, n = 16) and AV leaflets (up to 8 weeks, n = 10, p < 0.01) under both conditions. The protective effect of metformin is not mediated via autophagic responses. While activation of AMPK (AICAR) prevents the degeneration of VICs to a certain degree (p < 0.01), inhibition of AMPK (compound C, p < 0.01)-, nitrite oxide synthase (L-Name, p < 0.01), as well as sirtuin-1 (EX527, p < 0.05) - and peroxisome proliferator-activated receptor-gamma coactivator 1 α (PGC-1α, SR18292, p < 0.001) signaling partially reverses the protective effect of metformin. Expression of osteopontin (2.3 fold) and osteocalcin (1.7 fold) increases (p < 0.01), transforming growth factor β (0.4 fold, p < 0.01) and bone morphogenetic protein 2 (0.2 fold, p < 0.01) as well as collagens (1A1, 0.4 fold; 3A1, 0.4 fold; 5A1, 0.3 fold; p < 0.05) are downregulated by metformin. AP activity is inhibited in supernatants of VICs (p < 0.001) and AV leaflets (p < 0.001) as well as in AV tissue.

Conclusion: Metformin prevents degeneration of VICs and AV leaflets. The protective effects are not dependent on autophagic mechanisms and partially mediated via AMPK signaling, 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.

Publication History

Article published online:
19 February 2021