Figure 1.
Expression of CD40 and TRAFs in the carotid artery wall after ligation injury.
(A) Representative images of immunostaining for CD40 in carotid arteries from WT and CD40−/− mice (n = 5 per group). Scale bars: 20 µm or 100 µm. (B) Quantitative RT-PCR analysis of CD40 expression in WT carotid arteries (n = 5 per group). mRNA levels are normalized to GAPDH. Data are expressed as mean ± SEM. * P<0.05 and ** P<0.01 versus uninjured (uninj.)control. (C) Representative images of immunostaining for TRAF6 in carotid arteries from WT and CD40−/− mice (n = 5 per group). Scale bars: 20 µm. Arrows indicate the internal elastic lamina.
Figure 2.
CD40 deficiency inhibits leukocytes recruitment into the carotid artery wall after ligation injury, and thioglycollate-induced peritoneal leukocyte recruitment.
Representative images of immunostaining for neutrophils (anti-PMN mAb) (A) and for Mac-2-positive monocyte/macrophages (B) in carotid arteries from WT and CD40−/− mice (n = 5 per group). Positively stained cells were quantified as described in Method. Arrows indicate the internal elastic lamina. Scale bars: 20 µm. Data are expressed as mean ± SEM. ** P<0.01 versus corresponding WT. (C) Neutrophil recruitment in a model of thioglycollate-induced peritonitis is impaired in CD40-deficient mice. Mice were treated i.p. with 2 ml of 3% thioglycollate or the same volume of isotonic saline. 5 hours later, total number of neutrophils (×106) in the peritoneal cavity was counted. n = 5 mice/group.
Figure 3.
CD40 deficiency inhibits expression of proinflammatory mediators in the carotid artery wall after ligation injury.
Representative cross-sections from carotid arteries immunostained for ICAM-1 (A), VCAM-1 (B), and MCP-1 (C), as well as their quantitative analysis, in WT and CD40−/− mice (n = 5 per group). Arrows indicate the internal elastic lamina. Scale bars: 20 µm. Data are expressed as mean ± SEM. ** P<0.01 versus corresponding WT. (D) Quantitative RT-PCR analysis of mRNA expression of ICAM-1, VCAM-1, MCP-1, TNF-α, IL-6, and IL-1β, in WT and CD40−/− carotid arteries from WT and CD40−/− mice (n = 5 per group). mRNA levels are normalized to GAPDH. Data are expressed as mean ± SEM. * P<0.05 and ** P<0.01 versus corresponding WT.
Figure 4.
CD40 deficiency inhibits NF-κB activation in the carotid artery wall after ligation injury.
Representative cross-sections from carotid artery immunostained for total NF-κB p65 (A) and activated phospho-p65 (Ser536) (B), as well as their quantitative analysis, in WT and CD40−/− mice (n = 5 per group). Arrows indicate the internal elastic lamina. Scale bars: 20 µm. Data are expressed as mean ± SEM. ** P<0.01 versus corresponding WT.
Figure 5.
CD40 is required for CD40L-induced activation of NF-κB pathway in neutrophils.
(A) NF-κB nuclear translocation was assessed by immunofluorescence staining for NF-κB p65 (green) in wildtype and CD40-deficient neutrophils exposed to CD40L (1.0 µg/ml) for 1 h. Cell nuclei were detected by DAPI (blue). Scale bars: 50 µm. (B) NF-κB nuclear translocation was assessed and quantified as the percentage of the p65 nuclei-positively stained cells to the total cells. Similar results are obtained from three independent experiments. Data are expressed as mean ± SEM. ** P<0.01 versus WT.
Figure 6.
Decreased neointima formation and lumen stenosis after femoral artery wire-denudation in CD40−/− mice.
(A) Representative Elastic-stained sections (level 3) of femoral arteries 21d after injury in WT and CD40−/− mice (left panel). Schematic diagram of femoral artery guidewire-induced injury and tissue microtomy (right panel). Arrows indicate the internal elastic lamina. Scale bars: 50 µm. (B) Intima was measured at the 7 cross-section levels (120-µm intervals), and their mean intimal area was calculated. Intima/media ratio (C) and lumen stenosis ratio (D) at each level as well as their mean ratios were determined. n = 10 mice per group. Data are expressed as mean ± SEM. * P<0.05 and ** P<0.01 versus corresponding WT.