Colchicine Alleviates Non-Atherosclerotic Vascular Aging by Targeting Endothelial Dysfunction and Inflammatory Status

Figures

• 

  Figure 1. Colchicine induces direct vasorelaxation in mouse aorta via NO–cGMP and EDH pathways. (A), Colchicine concentration–response curves (CRCs) in thoracic aortic rings from SMC-KO and SMC-LM mice. (B), Effect of nitric-oxide synthase inhibition (L-NAME, 100 µmol/L) on colchicine-induced relaxation in SMC-KO rings. (C), Effect of combined EDH blockade (apamin + TRAM-34, each 100 nmol/L) on colchicine-induced relaxation in SMC-LM rings. All experiments were performed in wire-mounted thoracic aortic rings (1.5–2 mm) pre-contracted with U46619 to 80–100% of maximal KCl response. Data are expressed as % relaxation of the pre-constriction. Concentration–response curves were analyzed by repeated-measures ANOVA with Greenhouse–Geisser correction for sphericity and Bonferroni post-hoc tests. *P < 0.05 vs vehicle; n = 6 rings per condition.

• 

  Figure 2. Colchicine treatment protects SMC-KO mice against vascular stiffness. (A), Colchicine decreased vascular stiffness in SMC-KO mice (N=6 per group). Data are mean ± SEM. Statistical analysis was by two-way ANOVA followed by Bonferroni post-hoc test, *P<0.05 for SMC-LM vehicle Vs SMC-KO vehicle,and #P<0.05 for SMC-KO vehicle Vs SMC-KO colchicine. (B), M-Mode (Lower wavy image) and B-Mode (top) images of Pulse Wave Velocity (PWV) measured by ultrasound imaging of mouse abdominal aorta. (C), Strain differences plotted against stress differences in mouse carotid artery suggesting potential shift to more elastin content after colchicine treatment (N=6 per group, except for SMC-LM (vehicle); N=5). Statistical analysis was by a general linear model for repeated measurement. #P<0.05 comparing SMC-KO vehicle Vs SMC-KO colchicine. (D), Corresponding image of mouse carotid artery in pressure myograph at the baseline pressure and physiological pressure of 100 mmHg.

• 

  Figure 3. Colchicine treatment protects SMC-KO mice against vascular remodeling. (A), Colchicine treatment restored the increased carotid intima-media thickness (cIMT) in SMC-KO mice (N=3-7 per group as indicated by dots in the graph, scale bar = 100μm, Red arrows shows the area where the intima-media is thickened). (B), Collagen content, measured by Sirius Red staining, was elevated in SMC-KO mice compared to their littermate controls (N=3-5 per group as indicated by dots in the graph, scale bar = 50μm, Black arrows show increased collagen deposition in the intima-media layer). (C), Colchicine treatment reduced elastin fragmentation in SMC-KO mice (N=3-8 per group as indicated by dots in the graph, scale bar = 50μm, Red arrows shows location of breaks). All the measurements were performed manually by an independent observer blinded to genotype and treatment. Data are presented as mean ± SEM. Statistical analysis was performed using two-way ANOVA followed by Bonferroni post-hoc test; *P<0.05 indicates a significant genotype effect, and #P<0.05 indicates a significant colchicine treatment effect.

• 

  Figure 4. Colchicine improves aging-related endothelial dysfunction by upregulating NO. (A and B), Response to sodium nitroprusside (SNP) and acetylcholine (ACh) in aortic rings of SMC-KO and SMC-LM mice treated with vehicle or colchicine (N=10-19 per group as indicated in each graph). (C and D), contribution of the NO-cGMP (blocked by L-NAME) and Endothelium-Dependent Hyperpolarization EDH (blocked by Apamin + TRAM34) pathways to the vasorelaxant effects of ACh in aortas of vehicle- and colchicine-treated SMC-KO mice (N=10-19 per group as indicated in each graph). (E), ACh-mediated relaxation expressed relative to maximal SNP-induced relaxation (10−4 mol/L) to assess potential synergy between endothelial NO signaling and smooth-muscle sensitivity (N = 11–19). Relaxations (mean ± SEM) have been expressed as a percentage of the preconstriction induced by U46619. *:P<0.05 for genotype, #:P<0.05 for effect of colchicine, **:P<0.05 for pathway contribution for genotype, ##:P<0.05 for pathway contribution for colchicine as analyzed by repeated-measures ANOVA with Greenhouse–Geisser correction for sphericity and Bonferroni post-hoc tests.

• 

  Figure 5. Colchicine reduces inducible NOS and COX-2 in the carotid arterial bed of SMC-KO mice. (A and B), Immunofluorescent staining of the carotid arteries in mice shows elevated iNOS and COX-2 expression, indicated by increased fluorescence intensity. Colchicine treatment effectively restored these markers to baseline levels (N=4). (C), MAPK-p38 was hyperactivated in SMC-KO animals, which was partially reversed by colchicine treatment (N=4), Zoom = 20μm. (D), SMC-KO mice show no changes in AKT-p and (E), NFκB expression. Statistical analysis was performed using one-way ANOVA for panels A-C and unpaired t-test for panels D and E. *:P<0.05 for genotype, #:P<0.05 for effect of colchicine. iNOS; inducible Nitric Oxide Synthase, COX-2; Cyclooxygenase-2, MAPK; Mitogen-activated protein kinases, NFκB, nuclear factor κB; AKT, protein kinase B.