Spatial and temporal expression, and statin responsiveness of galectin-1 and galectin-3 in murine atherosclerosis

Abstract

Background and objectives: Existing data on the spatiotemporal expression patterns of a variety of galectins in murine atherosclerosis are limited. We investigated the expression levels of galectins, and their in vivo spatiotemporal expression patterns and statin responsiveness in the inflamed atherosclerotic plaques of apolipoprotein E (apoE)(-/-) mice.

Materials and methods: Galectins expression patterns in aortic atherosclerotic plaques and serum galectin-3 levels were investigated in 26-week-old apoE(-/-) (n=6) and C57BL/6 mice (n=9). To investigate the spatial and temporal patterns of galectin-1 and galectin-3 in plaques, high-cholesterol diet-fed 26-week-old (n=12) and 36-week-old apoE(-/-) mice (n=6) were sacrificed and their aortas were examined for galectins' expression using immunoblot analysis and immunohistochemical stain. 36-week-old apoE(-/-) mice were treated with atorvastatin (n=3, 0.57 mg/kg/day) for the evaluation of its effect on aortic galectins' expression.

Results: Immunoblot analyses showed that galectin-1 and galectin-3 were the predominant galectins expressed in murine atherosclerosis. The serum galectin-3 level was significantly higher in apoE(-/-) mice (p<0.001). While galectin-1 was weakly expressed in both intimal plaques and the media of atherosclerotic aortas, galectin-3 was heavily and exclusively accumulated in intimal plaques. Galectin-3 distribution was colocalized with plaque macrophages' distribution (r=0.66). As the degree of plaque extent and inflammation increased, the intraplaque galectin-3 expression levels proportionally elevated (p<0.01 vs. baseline), whereas galectin-1 expression had not elevated (p=0.14 vs. baseline). Atorvastatin treatment markedly reduced intraplaque galectin-3 and macrophage signals (p<0.001 vs. baseline), whereas it failed to reduce galectin-1 expression in the aortas.

Conclusion: Galectin-3 is the predominant gal and is colocalized with macrophages within atherosclerotic plaques. Intraplaque galectin-3 expression reflects the degree of plaque inflammation.

Keywords: Atherosclerosis; Galectin 1; Galectin 3; Macrophages.

Fig. 1

A: immunoblots of different galectin proteins such as gal-1, gal-2, gal-3 and gal-8 in the aortas of C57BL/6 and apoE^-/- mice. B: densitometry analyses of aortic gal proteins. C: serum gal-3 levels of 26-week-old chow diet-fed C57BL/6 (n=6) and high cholesterol diet-fed apoE^-/- mice (n=6). apoE: apolipoprotein E.

Fig. 2

Differential spatial expression pattern of macrophage, gal-1, and gal-3 proteins in atherosclerotic plaques of 26-week-old apoE^-/- mice aortic roots (n=3). A: hematoxylin and eosin (H&E) staining. B, C and D: immunohistochemical (IHC) staining for macrophage, gal-1, and gal-3 proteins. apoE: apolipoprotein E.

Fig. 3

Differential temporal expression pattern of gal-1 and gal-3 proteins in atherosclerotic plaques of 26-week-old apoE^-/- mice versus 36-week-old apoE^-/- mice (n=3). A: IHC staining for gal-1 protein in aortic roots of 26-week-old apoE^-/- mice (left panel) versus 36-week-old apoE^-/- mice (right panel). B: IHC staining for gal-3 protein in aortic roots of 26-week-old apoE^-/- mice (left panel) vs. 36-week-old apoE^-/- mice (right panel). C: quantitative histologic analyses of intraplaque gal-1 and gal-3 protein contents between 26-week-old apoE^-/- mice and 36-week-old apoE^-/- mice. IHC: immunohistochemical, apoE: apolipoprotein E.

Fig. 4

Correlation analysis of in vivo plaque gal-3 protein and plaque macrophage content in the entire apoE^-/-, mice cohort including 26-week-old, 36-week-old apoE^-/- (n=3), treated with or without atorvastatin. apoE: apolipoprotein E.

Fig. 5

Statin responsiveness of intraplaque gal-1 and gal-3 proteins in 36-week-old apoE^-/- mice (n=3). A: representative aortic root sections of salinetreated apoE^-/- mice for control and atorvastatin-treated apoE^-/- mice. B: images (original magnification ×200) from left to right demonstrate adjacent sections of immunoreactive macrophage, immunoreactive gal-1, immunoreactive gal-3, and multicolor immunofluorescent macrophage (red) with gal-3 (green) proteins. C: quantitative histologic analyses of intraplaque gal-1 and gal-3 protein contents between saline-treated and atorvastatin-treated 36-week-old apoE^-/- mice. D: immunoblots of different galectin proteins such as gal-1 and gal-3 in the aortas of saline-treated apoE^-/- (n=3) mice and atorvastatin treated apoE^-/- (n=3) mice. E: densitometry analyses of aortic gal-1 and gal-3 proteins. GAPDH: glyceraldehyde phosphate dehydrogenase, apoE: apolipoprotein E.