Colchicine reduces atherosclerotic plaque vulnerability in rabbits

Abstract

Background and aims: The anti-inflammatory agent colchicine is gaining interest as a treatment for coronary artery disease. However, the effects of colchicine in atherosclerotic animal models are mostly unknown. This study aimed to evaluate colchicine in a rabbit model of atherosclerosis.

Methods: Twenty-two rabbits were fed a 0.5% cholesterol-enriched diet for 10 weeks and then randomized to receive either oral saline (n=11) or colchicine (350 μg/kg/day; n=11) for 6 weeks, with 0.2% cholesterol-diet during the treatment period. We performed intravascular ultrasound imaging (at start and end of treatment) and histology analyses of the descending thoracic aorta. Leucocyte activation was assessed in vitro on blood samples obtained during treatment.

Results: Colchicine prevented positive aortic vascular remodelling (p=0.029 vs placebo). This effect was even more marked at high plasma cholesterol level (third quartile of plasma cholesterol, p=0.020). At high cholesterol level, both atherosclerotic plaque and media areas on histomorphology were reduced by colchicine compared to placebo (p=0.031 and p=0.039, respectively). Plaque fibrosis and macrophage area were reduced by colchicine (Masson's trichrome stain: p=0.038; RAM-11: p=0.026). The plaque vulnerability index, assessed by histology, was reduced by colchicine (p=0.040). Elastin/type I collagen ratio in media was significantly higher with colchicine compared to placebo (p=0.013). At a high level of plasma cholesterol, in vitro LPS challenge revealed a decrease in monocyte activation following treatment with colchicine (p<0.001) and no change in the placebo group (p=0.353).

Conclusions: Colchicine decreases plaque vulnerability with reductions in plaque inflammation, medial fibrosis, outward vascular remodelling and ex vivo monocyte activation.

Keywords: Atherosclerosis; Colchicine; Inflammation; Monocyte activation; Plaque vulnerability; Vascular remodelling.

Graphical abstract

Fig. 1

Colchicine reduces aortic outward remodelling as assessed by IVUS and histomorphometric analyses Plots in (A, B, C) represent adjusted mean changes ± SE in total vessel volume, lumen volume and plaque volume of thoracic aorta, respectively, from weeks 10–16 for placebo (circles, n=10) and colchicine-treated (triangles, n=10) groups at specific values of plasma cholesterol at start of treatment (low, medium and high). Pictures in (D) represent descending thoracic aorta cross sections (8 μm) of placebo and colchicine-treated rabbits, stained with haematoxylin-phloxin-safran (HPS) to assess media and plaque areas (magnification x2; scale bars indicate 1 mm). Plots in (E–H) represent adjusted means ± SE of external elastic lamina (EEL), media, plaque and lumen areas in descending thoracic aorta assessed on HPS-stained sections, for placebo (circles, n=11) and colchicine-treated (triangles, n=11) groups at specific values of plasma cholesterol at start of treatment (low, medium and high). ^#P<0.05, for colchicine vs. placebo.

Fig. 2

Colchicine reduces plaque macrophages Plots in (A–E) represent cellular composition of atherosclerotic plaques assessed by presence of vascular smooth muscle cells (vSMCs, α-SM-actin positive staining area), macrophages (RAM-11⁺ cells) and T-cells (CD3⁺ cells). Phenotype of vSMCs was further characterized by SM1 and SM2 markers. Results are expressed as adjusted means ± SE of each staining for placebo (circles, n=11) and colchicine-treated (triangles, n=11) groups at specific values of plasma cholesterol at start of treatment (low, medium and high). Pictures are representative stainings at high plasma cholesterol value at start of treatment. Scale bars represent 100 μm. ^#P<0.05, ^##P<0.01, for colchicine vs. placebo.

Fig. 3

Colchicine reduces plaque vulnerability index Plaque vulnerability index was calculated as (% RAM-11 staining + % ORO staining) / (% α-SM-actin staining + % type I and % type III collagen staining). Results are expressed as adjusted means ± SE for placebo (circles, n=11) and colchicine-treated (triangles, n=11) groups at specific values of plasma cholesterol at start of treatment (low, medium and high).

Fig. 4

Colchicine reduces medial fibrosis Plots in (A–D) represent types I and III collagen, matrix metalloproteinase 9 (MMP9) and elastin expression assessed in aortic media. Results are expressed as adjusted means ± SE of each staining for placebo (circles, n=11) and colchicine-treated (triangles, n=11) groups at specific values of plasma cholesterol at start of treatment (low, medium and high). The elastin/type I collagen ratio was calculated as an index of aortic elasticity (E). Pictures are representative stainings at high plasma cholesterol value at start of treatment. Scale bars represent 100 μm. ^#p<0.05, ^##p < 0.01, for colchicine vs. placebo.

Fig. 5

Colchicine reduces in vitro activation of circulating monocytes CD11b expression levels from rabbit whole blood samples were measured by flow cytometry analysis in monocyte sub-population after in vitro LPS stimulation (100 ng/mL). The expression of CD11b marker is presented as percent of monocyte population (A–C) and as median fluorescence intensity (MFI; D-F). Results are expressed as adjusted mean changes ± SE from start of treatment at each time points (2, 4 and 6 weeks after treatment) for placebo (circles, n=11) and colchicine-treated (triangles, n=11) groups and at specific values of plasma cholesterol at start of treatment (low, medium and high). ^¤p<0.05, ^¤¤p<0.01, ^¤¤¤p<0.001, for change from start of treatment (2, 4 and 6 weeks after treatment vs. start of treatment); ^#p<0.05, for colchicine vs. placebo.