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

Abstract

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.

Keywords: Calcific aortic valve disease; Cholesterol transport; Forkhead Box S1; Inflammasome; Valvular interstitial cells.

Graphical Abstract

In the early stages of calcific aortic valve disease, lipid deposition occurs in the aortic valve and oxidized LDL activates FOXS1 transcription. As a transcription factor, FOXS1 inhibits BSCL2 transcription by binding to the promoter region, thereby suppressing the PPARγ–LXRα–ABC transporter axis, leading to obstructed cholesterol efflux, cholesterol accumulation in valvular interstitial cells (VICs), activation of the NLRP3 inflammasome, and promotion of osteogenic differentiation of VICs, which ultimately exacerbates valvular calcification.

Figure 1

Identification of oxLDL-induced FOXS1 in VICs of patients with CAVD. (A) Venn diagram comparing the up-regulated DEGs in GEO datasets (GSE148219, GSE76717, and GSE153555) with human TF list. (B) qRT-PCR analysis of the genes in VICs treated with oxLDL. Unpaired two-tailed Student’s t-test (n = 6). (C) Correlation analysis between FOXS1 and RUNX2 in human aortic valves. Two-tailed Pearson correlation analysis. (D) Western blot of ALP, RUNX2, and FOXS1 expression in aortic valves. Unpaired two-tailed Student’s t-test (n = 20). (E) Uniform manifold approximation and projection (UMAP) visualization of cells in mice aortic valves and violin plot of FOXS1 expression levels in different cell types. (F) Alizarin Red staining and immunohistochemical staining of FOXS1 in aortic valves from CAVD patients or controls. Scale bar = 100 μm. (G) Immunofluorescence staining of FOXS1 and vimentin in aortic valves from CAVD patients or controls. 4′,6-diamidino-2-phenylindole (DAPI) was used for nuclear counterstaining. Scale bar = 50 μm. (H) Immunofluorescence staining of FOXS1 in mice aortic valves with or without HFD-fed. DAPI was used for nuclear counterstaining. Scale bar = 100 μm. (I and J) Western blot analysis of ALP, RUNX2, and FOXS1 expression in VICs treated with oxLDL at different concentrations and different time points. One-way ANOVA followed by Bonferroni multiple comparisons test (n = 6). Values are the mean ± SD. **P < 0.01, ***P < 0.001.

Figure 2

FOXS1 promotes oxLDL-induced osteogenic differentiation of VICs. (A) Western blot analysis of ALP, RUNX2, and FOXS1 in VICs with si-FOXS1 and oxLDL (n = 6). (B) Alizarin Red staining, calcium content, and ALP staining of VICs treated with si-FOXS1 and oxLDL (n = 6). Scale bar = 50 μm. (C) Western blot analysis of ALP, RUNX2, and Flag-FOXS1 in VICs with Ad-FOXS1 and oxLDL (n = 6). (D) Alizarin Red staining, calcium content, and ALP staining of VICs treated with Ad-FOXS1 and oxLDL (n = 6). Scale bar = 50 μm. Values are the mean ± SD. P values were calculated using one-way ANOVA followed by Bonferroni multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

FOXS1 deficiency attenuates aortic valve calcification in vivo. (A and B) The severity of aortic valve stenosis in Apoe^−/− and Apoe^−/−Foxs1^−/− mice fed with HFD was evaluated using echocardiography. Pulsed-wave Doppler examination was conducted across the aortic valve to measure peak transvalvular jet velocity (A) and the mean transvalvular pressure gradient (B). (C) HE, Von Kossa, Alizarin Red, and Masson’s staining of aortic valves from Apoe^−/− and Apoe^−/−Foxs1^−/− mice fed with HFD. Values are the mean ± SD. Data were analysed by unpaired two-tailed Student’s t-test (n = 10). **P < 0.01, ***P < 0.001.

Figure 4

FOXS1 mediates osteogenic differentiation of VICs by inhibiting ABCA1 and ABCG1. (A) Schematic of RNA-seq for VICs treated with Ad-FOXS1, si-FOXS1, and negative control. (B) Venn diagram interaction of DEGs of the three groups compared with each other and 409 common DEGs were found. (C) Heatmap showing the top 30 DEGs in the 409 common DEGs. (D and E) GO enrichment (D) and KEGG enrichment (E) of common DEGs. The size of the bubbles corresponds to the number of genes that align with the enrichment, and the rich ratio reflects the count of genes that match within the pool of integrated background genes. (F) Heatmap showing the DEGs of ABC transport in KEGG pathway enrichment. (G) qRT-PCR analysis of the genes in VICs treated with Ad-FOXS1, si-FOXS1, and negative control (n = 6). (H–K) Western blot analysis of protein levels in VICs (n = 6) treated with si-FOXS1 (H), Ad-FOXS1 (I), si-ABCA1 (J), and si-ABCG1 (K). (L) Alizarin Red staining, calcium content, and ALP staining of VICs treated with si-FOXS1 and si-ABCA1 or si-ABCG1 (n = 6). Scale bar = 50 μm. Values are the mean ± SD. P values were calculated using unpaired two-tailed Student’s t-test (G–K) or one-way ANOVA followed by Bonferroni multiple comparisons test (L). **P < 0.01, ***P < 0.001.

Figure 5

Cholesterol transport dysfunction drives NLRP3 inflammasome activation and osteogenic differentiation in VICs. (A and B) VICs were incubated with Bodipy-cholesterol (1 μM) under si-FOXS1, si-ABCA1, and si-ABCG1 for 4 h. Representative images of Bodipy-cholesterol-labelled VICs are shown. And cholesterol efflux of VICs was evaluated (B). Scale bar = 20 μm; n = 6. (C) Western blot analysis of protein levels in VICs treated with cholesterol in different concentrations (n = 6). (D) Western blot analysis of protein levels in VICs treated with cholesterol (10 μg/mL) and MCC950 (10 μM) (n = 6). (E) Alizarin Red staining, calcium content, and ALP staining of VICs treated with Ad-FOXS1 and MCC950 (10 μM) (n = 6). Scale bar = 50 μm. Values are the mean ± SD. P values were calculated using one-way ANOVA followed by Bonferroni multiple comparisons test. **P < 0.01, ***P < 0.001.

Figure 6

IMM-H007 inhibits oxLDL-induced osteogenic differentiation of VICs by increasing ABCA1 levels. (A) Western blot analysis of protein levels in VICs treated with IMM-H007 (10 μM) and oxLDL (n = 6). (B and C) Representative images of Bodipy-cholesterol-labelled VICs treated with Ad-FOXS1 and IMM-H007 (10 μM). And cholesterol efflux of VICs was evaluated (C) (n = 6). Scale bar = 20 μm. (D) Alizarin Red staining, calcium content, and ALP staining of VICs treated with oxLDL and IMM-H007 (10 μM) (n = 6). Scale bar = 50 μm. (E) HE, Von Kossa, and Alizarin Red staining of aortic valves treated with IMM-H007 (10 μM) and oxLDL for 21 days in vitro (n = 6). Scale bar = 1 mm. Values are the mean ± SD. P values were calculated using one-way ANOVA followed by Bonferroni multiple comparisons test. **P < 0.01, ***P < 0.001.

Figure 7

FOXS1 regulated ABCA1 and ABCG1 by inhibiting the BSCL2-mediated PPARγ/LXRα axis. (A) Pie chart showing the distribution of FOXS1 peaks in different genomic regions as indicated in ChIP-seq of VICs. (B) GO molecular function enrichment analysis of FOXS1 peaks. (C and D) Venn diagram interaction of promoters in ChIP-seq and DEGs in RNA-seq. The interaction between the 11 common genes and PPARγ, LXRα (NR1H3), ABCA1, and ABCG1 are analysed (D) using the STRING. (E) Correlation analysis between FOXS1 and BSCL2 in human aortic valves. (F) Immunofluorescence staining of Bscl2 and Pparγ in aortic valves from Apoe^−/− and Apoe^−/−Foxs1^−/− mice fed with HFD (n = 10). Scale bar = 100 μm. DAPI was used for nuclear counterstaining. (G) Western blot analysis of protein levels in VICs treated with si-FOXS1 and si-BSCL2 (n = 6). Values are the mean ± SD. P values were calculated using two-tailed Pearson correlation analysis (E), unpaired two-tailed Student’s t-test (F), or one-way ANOVA followed by Bonferroni multiple comparisons test (G). **P < 0.01, ***P < 0.001.