Blocking the NLRP3 inflammasome reduces osteogenic calcification and M1 macrophage polarization in a mouse model of calcified aortic valve stenosis

Background and aims: Activated innate immune cells infiltrating the valve and their secreted cytokines drive the differentiation of valve interstitial cells into myofibroblastic and osteoblastic phenotypes in calcified aortic valve stenosis (CAVS). In this study, we investigated how NLRP3 inhibition with CY-09 reduces aortic valve stenosis and calcification. Methods: ApoE(-/-) mice were fed a high-fat diet for 24 weeks with or without intraperitoneal injection of 2.5 mg/ kg/day NLRP3 inhibitor CY-09 for 42 consecutive days, while the control group mice were fed a normal diet. The valve function was monitored by echocardiography; calcified nodules were assessed by Von Kossa staining; and calcification-related molecules, inflammatory factors, and white leucocyte influx into the valve were assessed by immunohistochemistry, TUNEL assay, and PCR. Results: Mice treated with CY-09 exhibited improved aortic valve function and reduced valve calcification deposition. CY-09 intervention significantly downregulated the elevated expression of the NLRP3 inflammasome pathway molecules NLRP3, caspase-1, and IL-18 and the osteogenic calcification markers RUNX2, SPARC, and BMP2 in stenotic valves, while the number of apoptotic cells and dystrophic calcification markers CDH11 and alpha-SMA did not change significantly. Inhibition of NLRP3 activity also reduced the ratio of M1/M2 macrophages, prevented the shift of macrophages towards the M1 phenotype, and downregulated the levels of the proinflammatory factors IL-6 and TNF-alpha. Conclusions: This study provides a proof-of-concept that pharmacological inhibition of the NLRP3 inflammasome is a feasible strategy for alleviating aortic valve calcification and stenosis.