Core2 1-6-N-glucosaminyltransferase-I deficiency protects injured arteries from neointima formation in ApoE-deficient mice

Abstract

Objective: Core2 1 to 6-N-glucosaminyltransferase-I (C2GlcNAcT-I) plays an important role in optimizing the binding functions of several selectin ligands, including P-selectin glycoprotein ligand. We used apolipoprotein E (ApoE)-deficient atherosclerotic mice to investigate the role of C2GlcNAcT-I in platelet and leukocyte interactions with injured arterial walls, in endothelial regeneration at injured sites, and in the formation of arterial neointima.

Methods and results: Arterial neointima induced by wire injury was smaller in C2GlcNAcT-I-deficient apoE(-/-) mice than in control apoE(-/-) mice (a 79% reduction in size). Compared to controls, apoE(-/-) mice deficient in C2GlcNAcT-I also demonstrated less leukocyte adhesion on activated platelets in microflow chambers (a 75% reduction), and accumulation of leukocytes at injured areas of mouse carotid arteries was eliminated. Additionally, endothelial regeneration in injured lumenal areas was substantially faster in C2GlcNAcT-I-deficient apoE(-/-) mice than in control apoE(-/-) mice. Endothelial regeneration was associated with reduced accumulation of platelet factor 4 (PF4) at injured sites. PF4 deficiency accelerated endothelial regeneration and protected mice from neointima formation after arterial injury.

Conclusions: C2GlcNAcT-I deficiency suppresses injury-induced arterial neointima formation, and this effect is attributable to decreased leukocyte recruitment to injured vascular walls and increased endothelial regeneration. Both C2GlcNAcT-I and PF4 are promising targets for the treatment of arterial restenosis.

Figure 1. C2GlcNAcT-I deficiency suppresses injury-induced arterial neointima formation

a, Cross-sections of arterial neointima stained with Movat pentachrome and quantification of the size of neointima (I), size of media (M), and ratio of intima to media (n = 12 for both groups). b, Anti-Mac-2 staining of infiltrated macrophages in arterial neointima. c, Anti-α-actin staining of vascular smooth muscle cells (SMCs) in the arterial neointima. The average numbers of cells in the neointima were obtained by analyzing 12 cross sections from 12 injured carotid arteries from mice.

Figure 2. C2GlcNAcT-I deficiency suppresses leukocyte rolling and adhesion on activated platelets and in injured arteries ex vivo and in vivo

a, Leukocyte rolling and adhesion on activated platelets at 1.0 ± 0.1 dyn/cm² within 5 min after whole blood of wt and C2GlcNAcT-I−/− mice was perfused through micro-flow chambers. b to c, Leukocyte rolling and adhesion in injured mouse carotid arteries within the first 5 min after injury. Images were obtained from videotape recordings of the epifluorescence intravital microscope with 10X objective, illustrating rolling (←) and adhering (▲) leukocytes in the injured arteries of wt (left) and C2GlcNAcT-I−/− mice (right). The data points in c represent the means of three separate experiments. *P < 0.05, C2GlcNAcT-I−/− vs. wt mice. d to f, Injured carotid arteries of C2GlcNAcT-I+/+/apoE−/− and C2GlcNAcT-I−/−/apoE−/− mice were collected at 1 hour after wire injury (WI) and immunostained with antibodies specific for platelets (d), neutrophils (e), and macrophages (f). Carotid arteries of five mice were analyzed for each group. g and h, Immunostaining for neutrophils (g) and macrophages (h) on cross-sections of carotid arteries of C2GlcNAcT-I+/+/apoE−/− and C2GlcNAcT-I−/−/apoE−/− mice. Arteries were collected at 7 days after wire injury.

Figure 2. C2GlcNAcT-I deficiency suppresses leukocyte rolling and adhesion on activated platelets and in injured arteries ex vivo and in vivo

a, Leukocyte rolling and adhesion on activated platelets at 1.0 ± 0.1 dyn/cm² within 5 min after whole blood of wt and C2GlcNAcT-I−/− mice was perfused through micro-flow chambers. b to c, Leukocyte rolling and adhesion in injured mouse carotid arteries within the first 5 min after injury. Images were obtained from videotape recordings of the epifluorescence intravital microscope with 10X objective, illustrating rolling (←) and adhering (▲) leukocytes in the injured arteries of wt (left) and C2GlcNAcT-I−/− mice (right). The data points in c represent the means of three separate experiments. *P < 0.05, C2GlcNAcT-I−/− vs. wt mice. d to f, Injured carotid arteries of C2GlcNAcT-I+/+/apoE−/− and C2GlcNAcT-I−/−/apoE−/− mice were collected at 1 hour after wire injury (WI) and immunostained with antibodies specific for platelets (d), neutrophils (e), and macrophages (f). Carotid arteries of five mice were analyzed for each group. g and h, Immunostaining for neutrophils (g) and macrophages (h) on cross-sections of carotid arteries of C2GlcNAcT-I+/+/apoE−/− and C2GlcNAcT-I−/−/apoE−/− mice. Arteries were collected at 7 days after wire injury.

Figure 3. C2GlcNAcT-I deficiency accelerates endothelial regeneration on the arterial luminal surface of the injured area

a, Evans blue staining of injured carotid arteries collected at different time points after wire injury shows the areas that were not covered with newly generated endothelial cells. Quantitative data are shown in the right panel. Results are mean values of five separate experiments for each time point. b, Representative micrographs of cross-sections of injured carotid arteries immunostained with anti-CD31 or anti-VE-cadherin. Arrowheads show the areas negative for CD31 or VE-cadherin. Carotid arteries of five mice for each time point were analyzed for each group.

Figure 4. PF4 in endothelial proliferation in the context of platelet-neutrophil interactions

a, Anti-PF4 immunostaining with an antibody specific for PF4 (red) of cross-sections of injured carotid arteries of C2GlcNAcT-I+/+/apoE−/− and C2GlcNAcT-I−/−/apoE−/− mice. Carotid arteries were collected at 1 hour after wire injury. b, western blot for PF4 in injured carotid arteries of C2GlcNAcT-I+/+/apoE−/− and C2GlcNAcT-I−/−/apoE−/− mice. Results are mean values of three separate experiments. c to e, The covering of the wounded area with proliferating endothelial cells at 12 hour after wounding of the growing endothelial layer in the absence of additional leukocytes and platelets (right image in c), in the presence of activated platelets (d), or a mixture of neutrophils and activated platelets (e). Left images are experiments using activated platelets from PF+/+ mice whereas the right images represent experiments using activated platelets from PF−/− mice. f, Quantative analysis of endothelial proliferation under conditions as described in (c) to (e). g, Endothelial cell (EC) proliferation under the above conditions (as described in (c) to (e) was quantified by measuring BrdU incorporation. Results are mean values of five separate experiments.

Figure 5. Regeneration of endothelial cells in the injured area of carotid arteries and the formation of injury-induced arterial neointima in PF4−/−/apoE−/− mice

a, The number of rolling leukocytes and adherent leukocytes in injured mouse carotid arteries at 5 min after injury. Data were obtained from videotape recordings of the epifluoresence intravital microscope with 10X objective. The data points represent the means of three separate experiments. b to e, Injured carotid arteries of PF4+/+/apoE−/− and PF4−/−/apoE−/− mice were collected at 1 h after wire injury (WI) and immunostained with antibodies specific for platelets (b), PF4 (c), neutrophils (d), and macrophages (e). Carotid arteries of five mice were analyzed for each group. f, Evans blue staining of injured carotid arteries collected at 5 days after wire injury. Quantitative data on regeneration of endothelial cells in injured areas are shown in the right panels. Results are mean values of five separate experiments. g, Movat pentachrome staining of cross-sections of injured carotid arteries from PF4+/+/apoE−/− and PF4−/−/apoE−/− mice. h, Anti-Mac-2 staining of infiltrated macrophages in arterial neointima. i, Anti-α-actin staining of vascular smooth muscle cells (SMCs) in the arterial neointima. Carotid arteries were collected at 4 weeks after injury. The size of intima and media, the intima/media ratio, and the number of macrophages and SMCs in injured arteries were quantified by analyzing 12 cross-sections from 10 injured carotid arteries from mice.

Figure 5. Regeneration of endothelial cells in the injured area of carotid arteries and the formation of injury-induced arterial neointima in PF4−/−/apoE−/− mice

a, The number of rolling leukocytes and adherent leukocytes in injured mouse carotid arteries at 5 min after injury. Data were obtained from videotape recordings of the epifluoresence intravital microscope with 10X objective. The data points represent the means of three separate experiments. b to e, Injured carotid arteries of PF4+/+/apoE−/− and PF4−/−/apoE−/− mice were collected at 1 h after wire injury (WI) and immunostained with antibodies specific for platelets (b), PF4 (c), neutrophils (d), and macrophages (e). Carotid arteries of five mice were analyzed for each group. f, Evans blue staining of injured carotid arteries collected at 5 days after wire injury. Quantitative data on regeneration of endothelial cells in injured areas are shown in the right panels. Results are mean values of five separate experiments. g, Movat pentachrome staining of cross-sections of injured carotid arteries from PF4+/+/apoE−/− and PF4−/−/apoE−/− mice. h, Anti-Mac-2 staining of infiltrated macrophages in arterial neointima. i, Anti-α-actin staining of vascular smooth muscle cells (SMCs) in the arterial neointima. Carotid arteries were collected at 4 weeks after injury. The size of intima and media, the intima/media ratio, and the number of macrophages and SMCs in injured arteries were quantified by analyzing 12 cross-sections from 10 injured carotid arteries from mice.