Vitamin K alleviates ferroptosis and calcification in aortic valve disease

Introduction  

Calcific aortic valve disease (CAVD) is the most common valve disease and is associated with high morbidity and mortality. Despite its prevalence, there is no medical treatment to slow CAVD progression, which is characterized by chronic inflammatory and calcifying remodeling processes lasting for decades. 

Epidemiological data suggest that dietary intake of vitamin K may reduce CAVD incidence, while vitamin K antagonists appear to promote disease. However, randomized trials have not yet confirmed the efficacy of vitamin K supplementation in modulating CAVD progression, and the precise mechanisms by which vitamin K affects CAVD remain to be resolved. 

This study investigates the mechanisms of action of vitamin K in modulating valvular calcification.

Methods and Results  

To examine the effects of vitamin K on CAVD, C57BL/6J mice with wire injury-induced aortic valve stenosis were fed either a standard chow diet or a vitamin K2-enriched diet for six weeks (Fig 1A). Echocardiography revealed that vitamin K2-fed mice had significantly lower peak velocity and mean pressure gradient, indicating protection against aortic valve stenosis (Fig 1B). Vitamin K2 supplementation did not alter ejection fraction but was associated with reduced left ventricular mass, highlighting beneficial remodeling of the left ventricle (Fig 1C). Multi-color flow cytometry demonstrated a decrease in circulating monocytes, T cells, and B cells in the vitamin K2 group, suggesting anti-inflammatory effects (Fig 1D). Importantly, vitamin K2 did not affect hemostasis or promote pro-coagulability.

Human aortic valve samples were collected from patients undergoing valve replacement surgery for CAVD, and valvular interstitial cells (VICs) were isolated (Fig 2A). VICs were cultured in either a control medium (CM) or a pro-calcifying medium (PCM) with sodium dihydrogen phosphate and ascorbic acid. Both vitamin K1 and K2 inhibited VIC calcification, as indicated by alizarin red and hydroxyapatite crystal staining, and downregulated key calcification markers such as ALPL and RUNX2 (Fig 2B). Vitamin K also upregulated matrix Gla protein (MGP), a vitamin K-dependent inhibitor of calcification. 

Since vitamin K has recently been identified as a potent inhibitor of ferroptosis — a novel cell death pathway characterized by excessive lipid peroxidation — we investigated the role of ferroptosis in calcification. We found that VIC calcification was associated with lipid peroxidation and ferroptosis. Vitamin K protected VICs against ferroptosis by reversing lipid peroxidation induced by the GPX-4 inhibitor RSL3 (Fig 2C).

Further investigation into the vitamin K cycle showed that the canonical vitamin K-cycling enzyme VKORC1 was downregulated in VICs exposed to PCM, whereas VKORC1L1 expression remained stable (Fig 2D). VKORC1L1 is an enzyme known for its anti-oxidative properties. siRNA-mediated knockdown of VKORC1 and VKORC1L1 in VICs revealed that only VKORC1L1 silencing promoted lipid peroxidation and significantly increased expression of ALPL, a key calcification marker (Fig 2E). 

Conclusion

Vitamin K alleviates murine aortic valve stenosis and valvular cell calcification by suppressing ferroptosis and enhancing anti-oxidative mechanisms mediated by VKORC1L1. These findings suggest that vitamin K, via VKORC1L1, may offer a therapeutic approach for modulating CAVD progression.