Colchicine Blocks Abdominal Aortic Aneurysm Development by Maintaining Vascular Smooth Muscle Cell Homeostasis

Abstract

Development of non-surgical treatment of human abdominal aortic aneurysm (AAA) has clinical significance. Colchicine emerges as an effective therapeutic regimen in cardiovascular diseases. Yet, whether colchicine slows AAA growth remain controversy. Here, we demonstrated that daily intragastric administration of low-dose colchicine blocked AAA formation, prevented vascular smooth muscle cell (SMC) phenotype switching and apoptosis, and vascular inflammation in both peri-aortic CaPO₄ injury and subcutaneous angiotensin-II infusion induced experimental AAA mice models. Mechanistically, colchicine increased global mRNA stability by inhibiting the METTL14/YTHDC1-mediated m6A modification, resulting in increased sclerostin (SOST) expression and consequent inactivation of the WNT/β-catenin signaling pathway in vascular SMCs from mouse AAA lesions and in cultured human aortic SMCs. Moreover, human and mouse AAA lesions all showed increased m6A methylation, decreased SOST expression, and skewed synthetic SMC de-differentiation phenotype, compared to those without AAA. This study uncovers a novel mechanism of colchicine in slowing AAA development by using the METTL14/SOST/WNT/β-catenin axis to control vascular SMC homeostasis in mouse aortic vessels and in human aortic SMCs. Therefore, use of colchicine may benefit AAA patients in clinical practice.

Keywords: N6-methyladenosine; abdominal aortic aneurysm; colchicine; sclerostin; vascular smooth muscle cell.

Figure 1

Colchicine prevents peri-aortic CaPO₄ injury-induced AAA. A) Abdominal aortic diameters of saline and colchicine-treated mice. B) Elastin fragmentation grade. C) Lesion α-SMA⁺ SMCs positive area. D) Lesion Mac2⁺ macrophage-positive area. E) Lesion CD31⁺ microvessel numbers. F) Immunofluorescent staining of media and adventitia α-SMA (green) and TUNEL (red) double positive SMCs. Arrows indicate TUNEL-positive cells. G) Immunofluorescent staining of media and adventitia α-SMA (green) and Ki67 (red) double positive SMCs. Arrows indicate Ki67-positive cells. Representative images are shown to the left (B-G). Scale: 100 µm (B-E) and 200 µm (F/G). Data are mean ± SEM, n=12 per group. *P<0.05, ****P<0.0001, Student's t test.

Figure 2

Colchicine does not influence AAA lesion neutrophil activation, but increases lesion SMC tubulin depolymerization in CaPO₄ injury-induced AAA. A) ELISA analysis of plasma NET levels from saline and colchicine-treated mice. B) Immunofluorescent staining of Ly6G (green) and α-tubulin (red) to detect lesion neutrophil accumulation and tubulin depolymerization. Arrows indicate Ly6G-positive neutrophils. C) ELISA analysis of plasma GDF 15 levels from saline and colchicine-treated mice. D) Immunoblot analysis of JNK phosphorylation in AAA lesions, livers, and kidneys from saline and colchicine-treated mice. E) Immunofluorescent staining of α-SMA (green) and α-tubulin (red) to detect SMC tubulin depolymerization. Arrows indicate α-SMA-positive SMCs. F) Immunofluorescent staining of CD68 (green) and α-tubulin (red) to detect macrophage tubulin depolymerization. Arrows indicate CD68-positive macrophages. Representative images of B, D, E, and F are shown to the left. Scale: 100 μm, inset: 25 μm. G) Immunofluorescent staining of α-tubulin (red) to detect tubulin depolymerization in cultured human aortic SMCs stimulated with 20 ng/ml PDGF-BB and treatment with or without 1 nM colchicine (n=4). Scale: 50 µm. Data are mean ± SEM, n=12 per group. *P<0.05, ***P<0.001. Student's t test (A, B, and D-F) or nonparametric Mann-Whitney U test (C).

Figure 3

Colchicine inhibits SMC phenotypic switching in per-aortic CaPO₄ injury-induced AAA in mice. A) Immunofluorescent staining of α-SMA (green) and TAGLN (red) double positive SMCs in AAA lesions. Arrows indicate α-SMA and TAGLN double positive SMCs. B) Immunofluorescent staining of α-SMA (green) and KLF4 (red) double positive SMCs in AAA lesions. Arrows indicate α-SMA and KLF4 double positive SMCs. C) Immunofluorescent staining of α-SMA (green) and CD68 (red) double positive SMCs in AAA lesions. Arrows indicate α-SMA and CD68 double positive SMCs. Scale in A-C: 100 μm. D) Human aortic SMCs were treated with PDGF-BB (20 ng/ml) with or without colchicine (1 nM) for 24 hours and harvested for immunoblot analysis of TAGLN, α-SMA, KLF4, CD68 and GAPDH (n=4). E) Gelatin gel zymography analysis of AAA lesions. F) ELISA analysis of plasma TNF-α, IL-1β and IL-6 levels. G) RT-PCR analysis of lesion ACTA2, TAGLN, MYOCD, MYH11, CNN1, KLF4, MMP2, MMP9, IL-6 TNF-α and IL-1β. Data are mean ± SEM, n=12 per group. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, Student's t test (A, B, C, E, F and G) or two-way ANOVA followed by Bonferroni post hoc test (D).

Figure 4

AAA lesion gene expression and SMC activation in Ang II-infused Apoe^-/- mice and peri-aortic CaPO₄-injured mice received saline or colchicine treatment. A) Volcano plot depicted the differentially expressed genes in AAA lesions from Ang II-infused Apoe^-/- mice (n=4/each). B) Top enriched Gene Ontology (GO) biological process terms in Ang II-infused AAA lesions. Individual GO terms were sorted by adjusted P values. C) Gene Set Enrichment Analysis (GSEA) showed WNT signaling pathway in Ang II-infused AAA lesions. D) Immunofluorescent staining of media α-SMA (green) and β-catenin (red) double positive SMCs in CaPO₄ injury-induced AAA lesions. Scale: 100 μm. Arrows indicate β-catenin accumulation in media α-SMA-positive SMC nuclear, n=12 per group. E) Immunoblot analysis of p-GSK3β and β-catenin (active) expression in CaPO₄ injury-induced AAA lesions, n=8 per group. F) Immunoblot detection of nuclear β-catenin in CaPO₄ injury-induced AAA lesions, n=8 per group. G) Human aortic SMCs were treated with PDGF-BB (20 ng/ml) with or without colchicine (1 nM) for 24 hours and harvested for immunoblot analysis of p-GSK3β and β-catenin, n=4. H) Human aortic SMCs were treated with PDGF-BB (20 ng/ml) with or without colchicine (1 nM) or WNT agonist (10 μM) for 24 hours and harvested for immunoblot analysis of SOST, β-catenin, p-GSK3β, GSK3β, TAGLN, α-SMA, KLF4 and CD68, n=4. Data are mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, Student's t test (D-F) or two-way ANOVA followed by Bonferroni post hoc test (G and H).

Figure 5

SOST Silencing abrogates the protective effects of colchicine on peri-aortic CaPO₄ injury-induced AAA. A) Abdominal aortic diameters. B) Immunofluorescent staining of α-SMA (green) and SOST (red) to detect lesion SOST expression. C) Elastin fragmentation grade. D) Lesion α-SMA⁺ SMCs positive area. E) Lesion CD31⁺ microvessel numbers. F) Lesion Mac2⁺ macrophages-positive area. G-J) Immunofluorescent staining detected media α-SMA (green) and TAGLN (red) double positive (G), α-SMA (green) and KLF4 (red) double positive (H), α-SMA (green) and CD68 (red) double positive (I), and α-SMA (green) and β-catenin (red) double positive (J) SMCs. Arrows indicate media double positive SMCs. Representative images are shown to the left. Scale: 100 µm. Data are mean ± SEM, n=8 per group. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, Student's t test.

Figure 6

Colchicine upregulates SOST expression via METTL14/YTHDC1-mediated m6A hypomethylation. A) Colchicine increases SOST mRNA stability, n=6. B) Dot blot analysis of m6A methylation in human aortic SMCs treated with PDGF-BB (20 ng/ml) with or without colchicine (1 nM), n=4. MB: Methylene blue staining. C) m6A methylation levels in CaPO₄ injury-induced AAA in mice treated with saline or colchicine, n=12 per group. D) Dot blot analysis of m6A methylation in AAA lesions from C. MB: Methylene blue staining. E) RNA-Seq detected the expression of m6A writers, erasers and readers in Ang-II infused-induced AAA lesions in mice treated with saline or colchicine, n=4 per group. F) Immunoblots of METTL14, METTL3 and METTL4 in human aortic SMCs stimulated with PDGF-BB (20 ng/ml) with or without colchicine (1 nM), n=4. G) Immunofluorescent staining of METTL14 (red) and α-SMA (green) in CaPO₄ injury-induced AAA lesions from mice treated with saline or colchicine. Scale: 100μm. Arrows indicate α-SMA and METTL14 double positive cells, n=12 per group. H) Human aortic SMCs were transfected with 100 nM METTL14 siRNA (Si-SOST) or control siRNA (Si-NC) for 24 hours then treated with PDGF-BB (20 ng/ml) with or without colchicine (1 nM) for another 24 hours and harvested for immunoblot analysis of METTL14, SOST, β-catenin (active), TAGLN, α-SMA, KLF4 and CD68, n=4. I) Human aortic SMCs were transfected with 100 nM METTL14 siRNA (Si-SOST) or control siRNA (Si-NC) for 24 hours then treated with PDGF-BB (20 ng/ml) with WNT agonist 1 (10 μM) or with colchicine (1 nM) and WNT agonist 1 for another 24 hours and harvested for immunoblot analysis of TAGLN, α-SMA, KLF4 and CD68, n=4. J) RT-PCR analysis of SOST in CaPO₄ injury-induced AAA lesions from saline- and colchicine-treated mice after MeRIP assays, n=6 per group. K) RT-PCR analysis of SOST in human aortic SMCs treated with PDGF-BB (20 ng/ml) with or without colchicine (1 nM) after MeRIP assays, n=6. L) RT-PCR analysis of SOST in CaPO₄ injury-induced AAA lesions from saline- and colchicine-treated mice after RIP assays, n=4 per group. M) Human aortic SMCs were transfected with 100 nM YTHDC1 siRNA (Si-YTHDC1) or control siRNA (Si-NC) for 24 hours then treated with PDGF-BB (20 ng/ml) with or without colchicine (1 nM) for 24 hours and harvested for RT-PCR analysis of YTHDC1 and SOST, n=4. Data are mean ± SEM. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, Student's t test (A, C, D and G), RStudio (E), or two-way ANOVA followed by Bonferroni post hoc test (B, F, H, I, K, L and M), or nonparametric Mann-Whitney U test (J).

Figure 7

Increased m6A methylation and decreased SOST expression in human AAA lesion. A) m6A methylation levels in human AAA lesions and normal aorta, n=3-6 per group. B-D) Immunofluorescent staining detected METTL14 expression (red) in SMCs (α-SMA, green) (B), SOST expression (red) in SMCs (α-SMA, green) (C), and β-catenin expression (red) in SMCs (α-SMA, green) (D) from AAA lesions and normal aorta, n=3-6 per group. Scale: 100 μm, inset: 25 μm. Representative images are shown to the left. E) ELISA analysis of plasma SOST from AAA patients and healthy donors, n=36 per group. Data are mean ± SEM. *P<0.05, ****P<0.0001, Student's t test.