Figure 3. Effect of GPx-1 deficiency and MAPK signaling pathways inhibitors on macrophage proliferation.

Macrophages were collected after differentiation of thioglycollate-elicited mouse peritoneal macrophages for 3 days in 10 ng/ml MCSF. A left panel, macrophages (2,5×10⁴ cells) were incubated with MCSF or oxLDL, respectively, and BrdU for another 48 hours. Subsequently, the proliferative activity was investigated with a BrdU-based chemiluminescence assay and expressed as relative proliferation rate relating to control cells from ApoE^−/− mice without stimulus. Data represent means ± SD of 4 to 5 separate experiments. *p<0.05 or **p<0,01 above the histogram indicate statistically significant differences between the different genotypes and below the histogram compared with cells without treatment of MCSF or oxLDL. A right panel, macrophages from GPx-1^−/−ApoE^−/− mice were pretreated with 10 µM ebselen for 1 h and incubated with 10 µg/ml oxLDL and BrdU for another 48 h. The proliferation rate was expressed as the relative proliferation rate relating to the proliferative activity of untreated cells. Data represent means ± SD of 3 independent experiments. B, macrophages of GPx-1^−/−ApoE^−/− (upper panel) and ApoE^−/− (lower panel) mice were pre-incubated with 75 µM PD98059 or 15 µM U0126, respectively, for 1 h and then incubated with 10 ng/ml MCSF (left) or 10 µg/ml oxLDL (right) and BrdU for another 48 h. The proliferation rate was expressed as the relative proliferation rate relating to the proliferative activity of untreated cells. Data represent means ± SD of 3 to 5 independent experiments. C, Representative immunohistochemical staining for the proliferation marker PCNA in atherosclerotic lesions of the aortic sinus demonstrating more pronounced positive nuclear staining in GPx-1^−/−ApoE^−/− mice (left panel) than ApoE^−/− mice (right panel). The vessel lumen is to the upper left-hand corner. The demarcation between intima and media is indicated by arrowheads.