Stimulation of Collateral Vessel Growth by Inhibition of Galectin 2 in Mice Using a Single-Domain Llama-Derived Antibody

Abstract

Background In the presence of arterial stenosis, collateral artery growth (arteriogenesis) can alleviate ischemia and preserve tissue function. In patients with poorly developed collateral arteries, Gal-2 (galectin 2) expression is increased. In vivo administration of Gal-2 inhibits arteriogenesis. Blocking of Gal-2 potentially stimulates arteriogenesis. This study aims to investigate the effect of Gal-2 inhibition on arteriogenesis and macrophage polarization using specific single-domain antibodies. Methods and Results Llamas were immunized with Gal-2 to develop anti-Gal-2 antibodies. Binding of Gal-2 to monocytes and binding inhibition of antibodies were quantified. To test arteriogenesis in vivo, Western diet-fed LDLR.(low-density lipoprotein receptor)-null Leiden mice underwent femoral artery ligation and received treatment with llama antibodies 2H8 or 2C10 or with vehicle. Perfusion restoration was measured with laser Doppler imaging. In the hind limb, arterioles and macrophage subtypes were characterized by histology, together with aortic atherosclerosis. Llama-derived antibodies 2H8 and 2C10 strongly inhibited the binding of Gal-2 to monocytes (93% and 99%, respectively). Treatment with these antibodies significantly increased perfusion restoration at 14 days (relative to sham, vehicle: 41.3±2.7%; 2H8: 53.1±3.4%, P=0.016; 2C10: 52.0±3.8%, P=0.049). In mice treated with 2H8 or 2C10, the mean arteriolar diameter was larger compared with control (vehicle: 17.25±4.97 μm; 2H8: 17.71±5.01 μm; 2C10: 17.84±4.98 μm; P<0.001). Perivascular macrophages showed a higher fraction of the M2 phenotype in both antibody-treated animals (vehicle: 0.49±0.24; 2H8: 0.73±0.15, P=0.007; 2C10: 0.75±0.18, P=0.006). In vitro antibody treatment decreased the expression of M1-associated cytokines compared with control (P<0.05 for each). Atherosclerotic lesion size was comparable between groups (overall P=0.59). Conclusions Inhibition of Gal-2 induces a proarteriogenic M2 phenotype in macrophages, improves collateral artery growth, and increases perfusion restoration in a murine hind limb model.

Keywords: antibody; collateral circulation; macrophage; murine model; perfusion defect.

Figure 1

Llama immune response. Antibody titers of both immunized llamas to recombinant mGal‐2 (A) and recombinant hGal‐2 (B), measured in the sera collected on days 0, 28, and 43 of the immunization period. Serum is diluted 1000× before measurement. hGAL‐2 indicates human galectin 2; mGal‐2, murine galectin 2.

Figure 2

Inhibition of binding of Gal‐2 (galectin 2) to murine monocytes by single‐domain antibodies. Binding of labeled Gal‐2 to murine monocytes, analyzed by flow cytometry, and the influence of single‐domain antibodies (VHHs). A, Cells (10⁵ cells/well) were incubated with either Gal‐2 alone (no VHH), Gal‐2 with 1 of the 41 selected periplasmic fraction VHHs, or without any addition (−). B, Cells were incubated with no addition, Gal‐2 alone (gray bars), or Gal‐2 with 2H8, 1C11, 2C10, or 2D8. VHH indicates variable domain of heavy chains.

Figure 3

Perfusion restoration. Restoration of hind limb perfusion after femoral artery ligation. Perfusion was quantified noninvasively by laser Doppler perfusion imaging (A). Measurements were taken postoperatively (post‐op) and 2, 7, and 14 days after femoral artery ligation. Restoration of hind limb perfusion was significantly increased in animals treated with either 2H8 (n=14) or 2C10 (n=14) compared with control (n=25) (B). Repeated‐measures ANOVA (RM‐ANOVA) was used for overall analysis; a mixed model using time and treatment as input was used for time point analysis.

Figure 4

Perivascular macrophage characteristics. Analysis of perivascular macrophage characteristics by quadruple fluorescent immunohistochemistry. A–E, A typical example of an a arteriole with perivascular macrophages, stained for the pan‐macrophage marker F4/80 (A), cell nuclei (B), SMA (smooth muscle actin; C), and M2 marker mannose receptor (D). E, Merged image. Total number of macrophages (F) and fraction of M2 macrophages (G) were quantified. Animals used: control, n=10; 2C10, n=11; 2H8, n=14. ANOVA plus Dunnett test was used for analysis.

Figure 5

Atherosclerosis. Anti–Gal‐2 treatment does not affect atherosclerosis. A, Representative microscopic images of hematoxylin and eosin–stained aortic root sections of Western diet–fed LDLR−/−.Leiden mice: control, n=10; 2C10, n=13; 2H8, n=11. Magnification ×9. B, Plaque area in the aortic root of animals treated with control, 2H8, or 2C10 was comparable between groups using ANOVA. Gal‐2 indicates galectin 2; LDLR−/−, low‐density lipoprotein receptor–null.

Figure 6

Gal‐2–induced RNA‐expression. RNA expression levels of murine isolated bone marrow effector cells (A–C) and human isolated monocytes (D–F). Cells (100 000 cells/well) were stimulated for 3 hours with medium only (calibrator sample, not shown), Gal‐2, Gal‐2 with 2C10 antibody, Gal‐2 with 2H8 antibody, Gal‐2 with an irrelevant control single‐domain antibody (control VHH), 2C10 antibody alone, 2H8 antibody alone, or control VHH antibody alone. After stimulation, RNA expression of M1‐associated genes IFN‐β (interferon β; A and D), IL6 (interleukin 6; B and E), and TNF‐α (tumor necrosis factor α; C and F) were quantified. Expression is displayed as fold increase to the calibrator sample (medium only) and relative to reference gene GAPDH. All experiments were executed in triplicate. Trend analysis was performed separately for samples stimulated with and without Gal‐2. Post hoc analysis was performed with a Dunnett test using the control VHH samples as the reference group. Gal‐2 indicates galectin 2; hGAL‐2, human galectin 2; IFN‐β, interferon β; IL6, interleukin 6; mGal‐2, murine galectin 2; TNF‐α, tumor necrosis factor‐α; VHH, variable domain of heavy chains.