Oxidized LDL-induced FOXS1 mediates cholesterol transport dysfunction and inflammasome activation to drive aortic valve calcification.

AIMS Calcific aortic valve disease (CAVD) is becoming more prevalent with the population ageing; however, there is currently no medical therapy available. During early lipid deposition, low-density lipoprotein (LDL) mediates chronic inflammation and accelerates calcification progression. However, the mechanism still needs to be further explored. METHODS AND RESULTS The study identified the transcription factor FOXS in human valvular interstitial cells (VICs) as a pivotal regulator in aortic valve calcification. Bulk RNA-seq and qRT-PCR analysis were conducted to establish that FOXS1 is induced by oxidized LDL (oxLDL) in VICs. To elucidate the role of FOXS1 in osteogenic differentiation, small interfering RNA and recombinant adenovirus were utilized to modulate FOXS1 expression in VICs. High-fat diet (HFD)-fed Apoe-/-Foxs1-/- mice served as an in vivo model to investigate the role of FOXS1 in aortic valve calcification. Analysis from bulk RNA-seq, qRT-PCR, and western blot indicated significant activation of FOXS1 by oxLDL in VICs, with silencing of FOXS1 inhibiting oxLDL-induced osteogenic differentiation. Deletion of FOXS1 markedly reduced aortic valve calcification in HFD-fed Apoe-/- mice, as shown by decreased calcium deposition in the aortic valve leaflets. RNA-seq and chromatin immunoprecipitation sequencing were performed to reveal the regulatory mechanisms of FOXS1, uncovering direct interactions with the promoter of BSCL2, which subsequently inhibits the expression of ABCA1 and ABCG1 via the PPARγ/LXRα axis. The study demonstrated that FOXS1 mediates VICs' cholesterol transport dysfunction through BSCL2, ABCA1, and ABCG1 using Bodipy-cholesterol and showed that intracellular cholesterol accumulation can activate the NLRP3 inflammasome, promoting osteogenic differentiation of VICs. Additionally, it was found that IMM-H007 and recombinant BSCL2 could reduce aortic valve calcification both in vitro and in vivo. CONCLUSION We identified that an oxLDL-induced transcription factor FOXS1 inhibits ABCA1 and ABCG1 expression via the BSCL2/PPARγ/LXRα axis and promotes cholesterol transport dysfunction and the activation of NLRP3 inflammasome in VICs, thereby accelerating the progression of CAVD.