Metformin mitigates aortic valve degeneration in an ex vivo three-dimensional tissue model

Calcific aortic valve disease (CAVD) is a prevalent valvular disorder that lacks effective pharmacological treatments. Metformin, an oral anti-diabetic drug, has been suggested to protect valvular interstitial cells (VICs) from calcification, but its role in preventing organic phosphate-induced degeneration in VICs and aortic valve (AV) leaflets remains unclear. Using an ex vivo three-dimensional tissue model, we induced degeneration with organic phosphate-rich conditions in ovine VICs and AV leaflets. Metformin treatment reduced calcium deposition, as assessed by alizarin red staining (VICs: p < 0.0001; AV leaflets: p < 0.05), and preserved reduced opacity of AV leaflet (p < 0.01). It inhibited early osteogenic differentiation, evidenced by reduced alkaline phosphatase activity (VICs: p < 0.0001; AV leaflets: p < 0.05), and mitigated gene expression of myofibroblastic markers. Furthermore, metformin improved proliferation (VICs: p < 0.0001; AV leaflets: p < 0.05), lowered lactate dehydrogenase levels (VICs: p < 0.01; AV leaflets: p < 0.001), preserved extracellular matrix architecture, and ameliorated extracellular matrix remodeling at gene expression level. AMPK phosphorylation was increased in both VICs (28%, p < 0.001) and AV leaflets (21%, p < 0.05) under pro-degenerative conditions and metformin treatment. In conclusion, metformin protects against organic phosphate-induced degeneration in both VICs and AV leaflets, highlighting its therapeutic potential in CAVD.