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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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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.
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pone-0072063-g001: 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.

Mentions: To localize the cellular distribution of GPx-1 gene expression in mice atherosclerotic lesions, we performed in situ-hybridization and immunohistochemistry in lesions of the aortic sinus of GPx-1−/−ApoE−/− and ApoE−/− mice after 12 weeks on the WTD. GPx-1 mRNA expression was detected by in situ-hybridization and both macrophages and SMCs as the main cellular components of atherosclerotic lesions were detected by immunohistochemistry (see Materials and Methods). As shown in Figure 1 A and B, GPx-1 mRNA colocalizes with macrophage-rich areas and - even though to a much less extent - with SMCs. Furthermore, GPx-1 protein expression was detected by double staining for both GPx-1/F4/80 and GPx-1/α-Actin using a monoclonal GPx-1 antibody corroborating that both macrophages and SMCs contribute to GPx-1 expression within atherosclerotic lesions in ApoE−/− mice (Fig. 1 C). Immunohistochemical staining of apo B revealed that both macrophages and SMCs - even though to a much less extent - contribute to foam cell formation in atherosclerotic lesions of GPx-1−/−ApoE−/− and ApoE−/− mice (Fig. 2 A).


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)

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.
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Related In: Results  -  Collection

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pone-0072063-g001: 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.
Mentions: To localize the cellular distribution of GPx-1 gene expression in mice atherosclerotic lesions, we performed in situ-hybridization and immunohistochemistry in lesions of the aortic sinus of GPx-1−/−ApoE−/− and ApoE−/− mice after 12 weeks on the WTD. GPx-1 mRNA expression was detected by in situ-hybridization and both macrophages and SMCs as the main cellular components of atherosclerotic lesions were detected by immunohistochemistry (see Materials and Methods). As shown in Figure 1 A and B, GPx-1 mRNA colocalizes with macrophage-rich areas and - even though to a much less extent - with SMCs. Furthermore, GPx-1 protein expression was detected by double staining for both GPx-1/F4/80 and GPx-1/α-Actin using a monoclonal GPx-1 antibody corroborating that both macrophages and SMCs contribute to GPx-1 expression within atherosclerotic lesions in ApoE−/− mice (Fig. 1 C). Immunohistochemical staining of apo B revealed that both macrophages and SMCs - even though to a much less extent - contribute to foam cell formation in atherosclerotic lesions of GPx-1−/−ApoE−/− and ApoE−/− mice (Fig. 2 A).

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