Introduction:
Aortic valve stenosis (AVS) is a highly prevalent valvular heart disease with significant morbidity and mortality. Nevertheless, there are no medical treatment options available. Epidemiological data suggest that vitamin K intake is inversely correlated with AVS incidence, while vitamin K antagonists appear to promote AVS. However, the underlying mechanisms remain poorly understoood.
Methods and Results:
To investigate the role of vitamin K in AVS, C57BL/6J mice underwent wire injury-induced AVS and were fed either a vitamin K2-(MK7)-enriched or control diet for six weeks (100µg/g chow). Bi-weekly transthoracic echocardiography revealed that vitamin K2 supplementation protected against AVS, displayed by significantly reduced peak velocity and mean gradient while left ventricular ejection fraction was unaffected. Multi-color flow cytometry demonstrated a decrease in circulating monocytes, T cells, and B cells in vitamin K2-fed mice. Plasma proteomic further revealed dysregulations of key calcification mediators, including TGFB1, NOTCH3 and LPL. Histological analyses confirmed a reduction in aortic valvular area and monocyte infiltration in vitamin K2-fed mice. Importantly, vitamin K2 did not affect coagulation.
To assess the direct effects of vitamin K on human valvular interstitial cells (VICs), cells were isolated from AVS patients and cultured under pro-calcifying conditions. Both vitamin K1 and K2 inhibited VIC calcification, as evidenced by alizarin red and hydroxyapatite crystal staining, as well as downregulation of calcification markers including BMP2 and RUNX2. Bulk RNA-Sequencing with GO term and KEGG analyses revealed that Vitamin K inhibited adverse ECM remodelling and innate immune signalling during VIC calcification.
Emerging evidence suggest that Vitamin K functions as a ferroptosis inhibitor. Ferroptosis, a regulated cell death driven by lipid peroxidation, was observed in pro-calcifying VIC cultures. Notably lipid peroxidation (4-HNE staining) co-localized with calcified regions in explanted human AVS valves. Vitamin K1 and K2 protected VIC against ferroptosis by mitigating RSL3-induced lipid peroxidation. In vivo, vitamin K2 supplementation was associated with reduced 4-HNE staining in murine valves.
To translate these findings, C57BL/6J with wire injury-induced AVS were treated with the lipid peroxidation inhibitor liproxstatin-1. Inhibition of lipid peroxidation significantly reduced AVS severity in echocardiography and VIC calcification in vitro, further supporting ferroptosis as a driver of disease progression.
Mechanistically, silencing VKORC1L1, a vitamin K reductase with additional anti-oxidative properties, exacerbated lipid peroxidation and significantly increased ALP expression, a key marker of VIC calcification.
Conclusion:
Vitamin K reduces AVS and VIC calcification by inhibiting ferroptosis and enhancing VKORC1L1-mediated anti-oxidative mechanisms. These findings characterize vitamin K as a potential therapeutic target for AVS and highlight ferroptosis as a novel target in AVS.
