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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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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.
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pone-0072063-g003: 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.

Mentions: We investigated whether proliferative acitivity of monocyte-derived macrophages might account for the increased cellularity of early atherosclerotic lesions in GPx-1−/−ApoE−/− mice [16]. The proliferation rate of mouse peritoneal macrophages was assessed with a BrdU-based chemiluminescence assay (Figure 3 A, left panel). MCSF (10 ng/ml) significantly induced the proliferation of macrophage from GPx-1−/−ApoE−/− and ApoE−/− control mice, respectively (p<0,01). Moreover, macrophages from GPx-1−/−ApoE−/− mice showed significantly more BrdU incorporation than macrophages from ApoE−/− control mice (p<0.05). Likewise, oxLDL had significant effects on macrophage proliferation at 10 and 20 µg/ml in GPx-1−/−ApoE−/− mice and again, macrophages from GPx-1−/−ApoE−/− mice showed significantly more BrdU incorporation than macrophages from ApoE−/− control mice at any concentration of oxLDL (p<0,05). Interestingly, the GPx mimic ebselen led to a significant decrease of GPx-1−/−ApoE−/− macrophage proliferation (p<0,05, Figure 3 A, right 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)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750037&req=5

pone-0072063-g003: 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.
Mentions: We investigated whether proliferative acitivity of monocyte-derived macrophages might account for the increased cellularity of early atherosclerotic lesions in GPx-1−/−ApoE−/− mice [16]. The proliferation rate of mouse peritoneal macrophages was assessed with a BrdU-based chemiluminescence assay (Figure 3 A, left panel). MCSF (10 ng/ml) significantly induced the proliferation of macrophage from GPx-1−/−ApoE−/− and ApoE−/− control mice, respectively (p<0,01). Moreover, macrophages from GPx-1−/−ApoE−/− mice showed significantly more BrdU incorporation than macrophages from ApoE−/− control mice (p<0.05). Likewise, oxLDL had significant effects on macrophage proliferation at 10 and 20 µg/ml in GPx-1−/−ApoE−/− mice and again, macrophages from GPx-1−/−ApoE−/− mice showed significantly more BrdU incorporation than macrophages from ApoE−/− control mice at any concentration of oxLDL (p<0,05). Interestingly, the GPx mimic ebselen led to a significant decrease of GPx-1−/−ApoE−/− macrophage proliferation (p<0,05, Figure 3 A, right 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