Anti-Galectin-2 Antibody Treatment Reduces Atherosclerotic Plaque Size and Alters Macrophage Polarity

Abstract

Background: Galectins have numerous cellular functions in immunity and inflammation. Short-term galectin-2 (Gal-2) blockade in ischemia-induced arteriogenesis shifts macrophages to an anti-inflammatory phenotype and improves perfusion. Gal-2 may also affect other macrophage-related cardiovascular diseases.

Objectives: This study aims to elucidate the effects of Gal-2 inhibition in atherosclerosis.

Methods: ApoE -/- mice were given a high-cholesterol diet (HCD) for 12 weeks. After 6 weeks of HCD, intermediate atherosclerotic plaques were present. To study the effects of anti-Gal-2 nanobody treatment on the progression of existing atherosclerosis, treatment with two llama-derived anti-Gal-2 nanobodies (clones 2H8 and 2C10), or vehicle was given for the remaining 6 weeks.

Results: Gal-2 inhibition reduced the progression of existing atherosclerosis. Atherosclerotic plaque area in the aortic root was decreased, especially so in mice treated with 2C10 nanobodies. This clone showed reduced atherosclerosis severity as reflected by a decrease in fibrous cap atheromas in addition to decreases in plaque size.The number of plaque resident macrophages was unchanged; however, there was a significant increase in the fraction of CD206⁺ macrophages. 2C10 treatment also increased plaque α-smooth muscle content, and Gal-2 may have a role in modulating the inflammatory status of smooth muscle cells. Remarkably, both treatments reduced serum cholesterol concentrations including reductions in very low-density lipoprotein, low-density lipoprotein, and high-density lipoprotein while triglyceride concentrations were unchanged.

Conclusion: Prolonged and frequent treatment with anti-Gal-2 nanobodies reduced plaque size, slowed plaque progression, and modified the phenotype of plaque macrophages toward an anti-inflammatory profile. These results hold promise for future macrophage modulating therapeutic interventions that promote arteriogenesis and reduce atherosclerosis.

Fig. 1

Treatment with two anti-galectin-2 (Gal-2) antibodies, 2H8 and 2C10, reduces plaque area and changes the plaque phenotype. ( A ) Plaque area in the aortic roots of ApoE ^−/− mice under control, 2H8, or 2C10 antibody treatments. Mean value with standard deviation, n  = 19. * p ≤ 0.05. ** p ≤ 0.01. *** p ≤ 0.001. **** p ≤ 0.0001. ( B ) Qualitative categorization of plaque phenotype following hematoxylin and eosin staining. FCA, fibrous cap atheroma; PIT, pathological intimal thickening; IT, intimal thickening. Fisher's exact test, p  = 0.0129. Representative images of aortic roots stained with hematoxylin and eosin under control ( C ), 2H8 ( D ), and 2C10 ( E ) conditions are shown. Scale bar is 150 µm.

Fig. 2

An increased ratio of macrophages in the plaque is anti-inflammatory with modified cholesterol efflux regulatory protein levels. Percentage of the plaques staining positive for the macrophage marker, Mac3 ( A ), the anti-inflammatory macrophage marker CD206 ( E ), ABCA-1 ( I ), and ABCG-1 ( M ) are shown. Representative pictures of each stain from control, 2H8, and 2C10 treatment groups, respectively, are shown ( B – D , F – H , N – P ). * p ≤ 0.05. ** p ≤ 0.01. *** p ≤ 0.001. **** p ≤ 0.0001. Scale bars represent 150 µm.

Fig. 3

There is more smooth muscle actin present in the plaques of anti-galectin-2 (Gal-2) treated mice, yet galectin-2 has no effect on smooth muscle cell proliferation and a mild inflammatory effect. Representative images of control ( A ), 2H8 ( B ), and 2C10 ( C ) conditions. Scale bars represent 150 µm. Percentage of plaque area positive for α-smooth muscle actin (αSMA) ( D ) under control, 2H8, and 2C10 conditions. Mean values with standard deviation. n  = 19. 5-bromo-2-deoxyuridine (BrdU) incorporation assay ( E ) to measure vascular smooth muscle cell (VSMC) proliferation under different concentrations of galectin-2. n  = 5. Relative quantitative polymerase chain reaction (qPCR) gene expression vs. control of three markers of inflammation, interleukin (IL)-1β ( F ), tumor necrosis factor-α (TNF-α) ( G ), and CCL2 ( H ) stimulated with galectin-2 for 4 and 24 hours. * p ≤ 0.05. ** p ≤ 0.01. *** p ≤ 0.001. **** p ≤ 0.0001. Standard deviation shown, n  = 3.

Fig. 4

Serum cholesterol concentrations are reduced in anti-galectin-2 (Gal-2) treated mice while the main cholesterol genesis pathways in the liver are unaltered. Total serum cholesterol concentrations in anti-Gal-2 treated mice are shown ( A ) under control, 2H8, and 2C10 treatments with fractions therein of high-density lipoprotein (HDL) ( B ), low-density lipoprotein (LDL) ( C ), and very low-density lipoprotein (VLDL) ( D ) concentrations also shown. The fold change on four key SREBP1 ( E ) and SREBP2 ( F ) targets is shown under various conditions including stimulation with galectin is also shown. Mean values with standard deviation ( A – F ). n  = 17 ( A – D ), n  = 4 ( E , F ). * p ≤ 0.05. ** p ≤ 0.01. *** p ≤ 0.001. **** p ≤ 0.0001.