Gli1 promotes the phenotypic transformation of valve interstitial cells through Hedgehog pathway activation exacerbating calcific aortic valve disease

Abstract

Calcific aortic valve disease (CAVD) is the most prevalent human valve disease worldwide. Multiple factors induce "irreversible" pathological changes in the aortic valve leaflets, resulting in changes in cardiac hemodynamics, eventually leading to heart failure. However, no effective pharmaceutical interventions have been found and prosthetic valve replacement is the only curative approach. Glioma-associated oncogene 1 (Gli1) exerts a regulatory role on cardiovascular diseases, and it is already a therapeutic target to combat tumors. Our research aimed to explore the role and basic mechanism of Gli1 in CAVD, to pave the way for the discovery of effective drugs in the treatment of CAVD. Human aortic valve tissues were obtained to evaluate Gli1 expression and primary valve interstitial cells (VICs) were used to perform related experiments. The results showed that Gli1 promoted cell proliferation and significantly accelerated cell osteogenic transformation through the up-regulation of the osteogenic factors Runx2 and Alp, in turn through the AKT signaling pathway by targeting P130cas expression. Furthermore, Gli1 was activated by TGF-β and sonic hedgehog through the canonical and non-canonical Hedgehog signaling pathways in VICs. Our results indicated that Gli1 promoted cell proliferation and accelerated cell osteogenic transformation in VICs, providing a new strategy for the therapy of CAVD by targeting Gli1.

Keywords: AKT signaling pathway; Calcific aortic valve disease (CAVD); Hedgehog signaling pathway; glioma-associated oncogene 1 (Gli1); osteogenic transformation.

Figure 1

Analysis of Gli1 expression in aortic valve tissues from CAVD and healthy samples. (A) and (B) western blotting analysis showed that Gli1, Runx2, ALP proteins increased significantly in CAVD samples compared with normal ones. (C) RT-PCR was applied to detect the mRNA expression level of Gli1 and Runx2 in 10 pairs of aortic valve samples from CAVD and normal patients. Data are expressed as the mean ± SD, n=10. Data were analyzed using one-way ANOVA, (*) p < 0.05 indicates a significant difference. (D) HE, Von Kossa staining along with Gli1 Immunohistochemical staining were performed in calcified aortic valves and normal ones. (E) Dual immunofluorescence staining of Gli1, α-SMA (α-smooth muscle actin), Vimentin, Runx2, and DAPI were performed in calcified aortic valves and normal ones.

Figure 2

Total RNA-seq analysis after regulating the expression of Gli1 in VICs. (A) and (B) The box plot and correlation heat map the in the control group, Gli1 group, and Gant61 group. (C) volcano map of differentially expressed genes (DEGs) in these three different groups. Gli1 group versus control group: up-regulation 1224 genes and down-regulation 1264 genes; Gant61 group versus control group: up-regulation 1148 genes and down-regulation 1354 genes. FC (fold change) > 1 was accepted as positive DEGs. (D) Venn interaction of DEGs of the three groups compared with each other and 308 common DEGs were found. (E) KEGG pathway enrichment of common DEGs, bubble colors (deep) indicate the degree of enrichment (-Log10(P-value)), bubble size indicates gene counts matched the pathway enrichment, and rich factor indicates the matched gene counts in the integrated pathway background genes. (F) GO biological function enrichment of common DEGs, blue bars represent the degree of enrichment (-Log10(P-value)), red polyline showed the gene numbers involved.

Figure 3

Gli1 promotes the proliferation of VICs and regulates the cell cycle. (A) Molecular structure of Gant61. (B) IC50 of Gant61 on VICs, concentrations were transferred to Log(c). (C) CCK-8 assay was performed in the control group and Gant61 group. (D) Ki-67 and Edu staining in VICs were performed in both control and Gant61 groups. (E) Semi-quantitative statistics on the percentage of Ki-67 positive cells and Edu positive cells. (F) Ki-67 and Edu staining in VICs under different treatments: control, Gli1, and Gli1+Gant61. (G) Semi-quantitative statistics on the percentage of Ki-67 positive cells and Edu positive cells. (H) and (I) Flow cytometry cell cycle of VICs in a different group and semi-quantitative statistics of each phase ratio. (J) and (K) RT-PCR and western blotting analysis of cell cycle-related factors in different groups. (L) Semi-quantitative statistics on the cyclin B protein. Data are expressed as the mean ± SD. Data were analyzed using one-way ANOVA, (*) p < 0.05 indicates a significant difference.

Figure 4

Gli1 accelerates the osteogenic differentiation of VICs. (A) Alizarin Red S staining of the cells under different conditioned cultures. (B) Semi-quantitative statistics of Alizarin Red S stain. (C) RT-PCR of Runx2, Gli2, Gli3 for VICs stimulated with Gli1 and then treated with Gant61 or not for 48h. Data were analyzed using one-way ANOVA, (#) versus control, p < 0.05 indicates a significant difference. (D) and (E) Western blotting and semi-quantification of ALP, Runx2 for VICs stimulated with Gli1 and then treated with Gant61 or not for 72h. Data were analyzed using one-way ANOVA, (*) p < 0.05 indicates a significant difference. (F) and (G) Ex-vivo osteogenic differentiation of aortic valve tissues in different conditions. Observed under the fluorescent microscope and Representative HE, Alizarin Red S, Von Kossa staining figures of each tissue. (H) and (I) Co-IP and cellular immunofluorescence staining of Gli1 and Runx2 proteins in VICs.

Figure 5

Identification of Gli1 candidate target genes related to osteogenic differentiation. (A) and (B) The TF binding motifs of Gli1 and the heatmap of the Gli1 binding site within 2kb from the transcriptional starting site. (C) Peak calling analysis of Gli1. (D) GO biological process enrichment and KEGG pathway enrichment analysis for Gli1 binding genes. (E) Principal Component Analysis (PCA) analysis for three pairs of aortic valves. (F) KEGG signaling pathway enrichment analysis of DEGs between samples from the CAVD group and the control group. (G) Venn's interaction of KEGG signaling pathway enrichment analysis between tissue RNA-seq and cellular RNA-seq. (H) Venn's interaction of the two common pathways with ChIP-seq data. (I) Fold enrichment and p-value of the final target genes according to the ChIP-seq data.

Figure 6

Gli1-P130cas-AKT axis promotes the osteogenic differentiation of VICs. (A) Western blotting for p-AKT, AKT, ALP, p-p130cas, p130cas under the Gli1 treatment with or without MK2206. (B) Statistical analysis of Western Blotting gray values of p-AKT/AKT, p-p130cas/p130cas, ALP, and RUNX2. Data were analyzed using one-way ANOVA, (*) p < 0.05 indicates a significant difference. (C) Calcification nodules were tested by Alizarin Red S after being cultured under different treatments. (D) Semi-quantitation of calcification. Data were analyzed using one-way ANOVA, (*) p < 0.05 indicates a significant difference. (E) Western blotting for p-AKT, AKT, ALP, p-p130cas, p130cas under the Gli1 treatment with or without si-BCAR treatment. (F) Statistical analysis of Western Blotting gray values of p-AKT/AKT, p-p130cas/p130cas, ALP, and RUNX2. Data were analyzed using one-way ANOVA, (*) p < 0.05 indicates a significant difference. (G) Calcification nodules were tested by Alizarin Red S after being cultured under different treatments. (H) Semi-quantitation of calcification. Data were analyzed using one-way ANOVA, (*) p < 0.05 indicates a significant difference.

Figure 7

Gli1 activates through canonical and non-canonical Hedgehog signaling pathways in VICs. (A) PCR test of Gli1, Gli2, Gli3, Ptch1 genes in VICs after SHH treatment with or without GDC-0449. Data were analyzed using one-way ANOVA, (#) p < 0.05 indicates a significant difference. (B and C) Western blotting and immunofluorescence staining of Gli1 in VICs were consistent with gene expression. (D) PCR test of Gli1 and Runx2 gene expression in VICs treated by common calcification-inducing stimulators for 48h. Data were analyzed using one-way ANOVA, (* versus control) p < 0.05 indicates a significant difference. (E) Changes in Gli1 and Gli2 gene expression induced by tgf-β1 within 24 hours. (F) PCR test of Gli1, Gli2, Runx2 genes in VICs stimulated by tgf-β1 with or without SB431542. (G) Western blotting of p-SMAD2, p-SMAD3, SMAD2/3, and Gli1 protein in VICs stimulated by tgf-β1 with or without SB431542. (H) Immunofluorescence staining of Gli1 in VICs under different treatments. (I and J) Western blotting of Gli1 in VICs stimulated by tgf-β1 with si-SMAD2/3 or si-SMAD4.