Impact of glutathione peroxidase-1 deficiency on macrophage foam cell formation and proliferation: implications for atherogenesis

Abstract

Clinical and experimental evidence suggests a protective role for the antioxidant enzyme glutathione peroxidase-1 (GPx-1) in the atherogenic process. GPx-1 deficiency accelerates atherosclerosis and increases lesion cellularity in ApoE(-/-) mice. However, the distribution of GPx-1 within the atherosclerotic lesion as well as the mechanisms leading to increased macrophage numbers in lesions is still unknown. Accordingly, the aims of the present study were (1) to analyze which cells express GPx-1 within atherosclerotic lesions and (2) to determine whether a lack of GPx-1 affects macrophage foam cell formation and cellular proliferation. Both in situ-hybridization and immunohistochemistry of lesions of the aortic sinus of ApoE(-/-) mice after 12 weeks on a Western type diet revealed that both macrophages and - even though to a less extent - smooth muscle cells contribute to GPx-1 expression within atherosclerotic lesions. In isolated mouse peritoneal macrophages differentiated for 3 days with macrophage-colony-stimulating factor (MCSF), GPx-1 deficiency increased oxidized low density-lipoprotein (oxLDL) induced foam cell formation and led to increased proliferative activity of peritoneal macrophages. The MCSF- and oxLDL-induced proliferation of peritoneal macrophages from GPx-1(-/-)ApoE(-/-) mice was mediated by the p44/42 MAPK (p44/42 mitogen-activated protein kinase), namely ERK1/2 (extracellular-signal regulated kinase 1/2), signaling pathway as demonstrated by ERK1/2 signaling pathways inhibitors, Western blots on cell lysates with primary antibodies against total and phosphorylated ERK1/2, MEK1/2 (mitogen-activated protein kinase kinase 1/2), p90RSK (p90 ribosomal s6 kinase), p38 MAPK and SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase), and immunohistochemistry of mice atherosclerotic lesions with antibodies against phosphorylated ERK1/2, MEK1/2 and p90RSK. Representative effects of GPx-1 deficiency on both macrophage proliferation and MAPK phosphorylation could be abolished by the GPx mimic ebselen. The present study demonstrates that GPx-1 deficiency has a significant impact on macrophage foam cell formation and proliferation via the p44/42 MAPK (ERK1/2) pathway encouraging further studies on new therapeutic strategies against atherosclerosis.

Figure 1. Localization of GPx-1 in mice atherosclerotic lesions.

GPx-1 mRNA and protein expression, macrophages and SMCs in sequential sections of the aortic sinus of ApoE^−/− (A, C) and GPx-1^−/−ApoE^−/− (B) mice. A, GPx-1 mRNA expression was detected by in situ-hybridization (upper panels) and both macrophages and SMCs were detected by immunohistochemistry (middle panels, see Methods). Upper left panel: anti-sense probe; upper right panel: corresponding section hybridized with the sense probe for GPx-1 (no signal). B, Control sections of GPx-1^−/−ApoE^−/− mice showed no expression of GPx-1 mRNA, neither with the anti-sense nor with the sense probe (upper panels). The representative atherosclerotic lesion containes both macrophages (lower left panel) and SMCs (lower right panel). C, Representative double immunohistochemical staining for GPx-1 (brown) and macrophages (red; left panel) and GPx-1 (brown) and SMCs (red; right panel) in ApoE^−/− mice. Note the close intermingling and overlapping of the different antigens predominantly within the inner parts of the intima. In A to C, the vessel lumen is to the upper left-hand corner. The demarcation between intima and media is indicated by arrowheads.

Figure 2. Lipoprotein staining and effect of GPx-1 deficiency on oxLDL induced foam cell formation.

A, Immunohistochemical staining of lipoprotein apo B in parallel with staining of macrophages and SMCs in sequential sections of the aortic arch of both GPx-1^−/−ApoE^−/− (upper panels) and ApoE^−/− (lower panels) mice. The vessel lumen is to the upper left-hand corner. The demarcation between intima and media is indicated by arrowheads. B, C, Effect of GPx-1 deficiency on oxLDL induced foam cell formation. After differentiation for 3 days with 10 ng/ml MCSF, mouse peritoneal macrophages were incubated with 5 and 10 µg/ml oxLDL, respectively, for 24 hours. B, Representative photomicrographs of peritoneal macrophages stained with oil-red O (magnification ×20, inserts ×100). C, Quantitative analysis of cellular cholesterol content in mouse peritoneal macrophages. After incubation with oxLDL, the cells were lysed and homogenized and total cholesterol content was quantified by fluorescence measurements. The results were expressed as total cholesterol per cellular protein. Each value represents the mean ± SD of four separate measurements.

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.

Figure 4. Effects of MCSF on the phosphorylation of MAPKs.

After pre-incubation for 3 days with 10 ng/ml MCSF, peritoneal macrophages were incubated for 5 and 15 min with 10 ng/ml MCSF. Cellular protein was extracted and protein samples (0.4 mg/ml) were analyzed by Western blot with specific antibodies: anti-phosphorylated MEK1/2 or anti-MEK1/2 (A, right), anti-phosphorylated ERK1/2 or anti-ERK1/2 (B, right), anti-phosphorylated p90RSK or anti-RSK1/2/3 (C, right), anti-phosphorylated p38 MAPK or anti-p38 MAPK (D, right) and anti-phosphorylated SAPK/JNK or anti-SAPK/JNK (E, right) antibodies (representative experiments). ß-Actin or Actin were used as control. Quantitative results were calculated by band densitometry with the intensity of phosphorylated MEK1/2, ERK1/2, p90RSK, p38 MAPK and SAPK/JNK normalized to the total MEK1/2, ERK1/2, RSK1/2/3, p38 MAPK and SAPK/JNK (A–E, left panels). Data represent mean ± SD of 3 separate experiments. *, **indicate statistically significant differences (*p<0.05, **p<0.01) compared with cells without MCSF treatment.

Figure 5. Effects of ebselen or oxLDL on MAPK phosphorylation and expression of MAPK in mice lesions.

A, After pre-incubation for 3 days with 10 ng/ml MCSF, peritoneal macrophages were incubated for 5 min with 10 µg/ml oxLDL with or without MCSF or MCSF with or without ebselen. Cellular protein was extracted and protein samples (0.4 mg/ml) were analyzed by Western blot with specific antibodies: anti-phosphorylated MEK1/2 or anti-MEK1/2 (A, upper panel, right), anti-phosphorylated ERK1/2 or anti-ERK1/2 (A, middle panel, right) or anti-phosphorylated p90RSK or anti-RSK1/2/3 (A, lower panel, right) antibodies (representative experiments). ß-Actin or Actin were used as control. Quantitative results were calculated by band densitometry with the intensity of phosphorylated MEK1/2, ERK1/2, p90RSK normalized to total MEK1/2, ERK1/2, RSK1/2/3 (A, upper, middle and lower panel, left). Data represent mean ± SD of 5–7 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. B, expression of phosphorylated MEK1/2 and ERK1/2 in parallel with staining of macrophages and SMCs in sequential sections of the aortic arch of both GPx-1^−/−ApoE^−/− (upper panels) and ApoE^−/− (lower panels) mice. There is more pronounced expression of phosphorylated ERK1/2 and MEK1/2 both in macrophages and SMCs of GPx-1^−/−ApoE^−/− compared with ApoE^−/− mice. C, representative double immunohistochemical staining for p-p90RSK (nuclei, brown), macrophages or SMCs (red) in ApoE^−/− mice demonstrating expression of p-p90RSK in macrophages rather than in SMCs (arrowheads). The vessel lumen is to the upper left-hand corner. The demarcation between intima and media is indicated by arrowheads.