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

Cheng F, Torzewski M, Degreif A, Rossmann H, Canisius A, Lackner KJ - PLoS ONE (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center, Johannes Gutenberg-University, Mainz, Germany.

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.

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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.
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pone-0072063-g005: 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.

Mentions: These significant effects of GPx-1 deficiency were similar upon stimulation with oxLDL either with or without MCSF (Figure 5 A). In case of p90RSK, oxLDL alone even triggered increased phosphorylation in macrophages of GPx-1−/−ApoE−/− compared with ApoE−/− control mice which could not be observed in unstimulated cells (Figure 5 A, lower panel). Notably, the GPx mimic ebselen almost completely abrogated the phosphorylation of ERK in GPx-1−/−ApoE−/− but had no effect on phosphorylation of ERK in ApoE−/− control mice (Figure 5 A, middle panel).


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

Cheng F, Torzewski M, Degreif A, Rossmann H, Canisius A, Lackner KJ - PLoS ONE (2013)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750037&req=5

pone-0072063-g005: 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.
Mentions: These significant effects of GPx-1 deficiency were similar upon stimulation with oxLDL either with or without MCSF (Figure 5 A). In case of p90RSK, oxLDL alone even triggered increased phosphorylation in macrophages of GPx-1−/−ApoE−/− compared with ApoE−/− control mice which could not be observed in unstimulated cells (Figure 5 A, lower panel). Notably, the GPx mimic ebselen almost completely abrogated the phosphorylation of ERK in GPx-1−/−ApoE−/− but had no effect on phosphorylation of ERK in ApoE−/− control mice (Figure 5 A, middle panel).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Chemistry and Laboratory Medicine, University Medical Center, Johannes Gutenberg-University, Mainz, Germany.

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.

Show MeSH
Related in: MedlinePlus