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Endothelial and macrophage-specific deficiency of P38α MAPK does not affect the pathogenesis of atherosclerosis in ApoE-/- mice.

Kardakaris R, Gareus R, Xanthoulea S, Pasparakis M - PLoS ONE (2011)

Bottom Line: The p38α Mitogen-Activated Protein Kinase (MAPK) regulates stress- and inflammation-induced cellular responses.Surprisingly, although p38α-deficiency strongly attenuated oxidized LDL-induced expression of molecules responsible for monocyte recruitment in endothelial cell cultures in vitro, endothelial-specific p38α ablation in vivo did not affect atherosclerosis development.Similarly, macrophage specific deletion of p38α did not affect atherosclerotic plaque development in ApoE(-/-) mice.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular Medicine, Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany.

ABSTRACT

Background: The p38α Mitogen-Activated Protein Kinase (MAPK) regulates stress- and inflammation-induced cellular responses. Factors implicated in the development of atherosclerosis including modified low-density lipoprotein (LDL), cytokines and even shear stress induce p38 activation in endothelial cells and macrophages, which may be important for plaque formation. This study investigates the effects of endothelial- and macrophage-specific deficiency of p38α in atherosclerosis development, in Apolipoprotein E deficient (ApoE(-/-)) mice.

Methodology/principal findings: ApoE(-/-) mice with macrophage or endothelial cell-specific p38α deficiency were fed a high cholesterol diet (HCD) for 10 weeks and atherosclerosis development was assessed by histological and molecular methods. Surprisingly, although p38α-deficiency strongly attenuated oxidized LDL-induced expression of molecules responsible for monocyte recruitment in endothelial cell cultures in vitro, endothelial-specific p38α ablation in vivo did not affect atherosclerosis development. Similarly, macrophage specific deletion of p38α did not affect atherosclerotic plaque development in ApoE(-/-) mice.

Conclusions: Although previous studies implicated p38α signaling in atherosclerosis, our in vivo experiments suggest that p38α function in endothelial cells and macrophages does not play an important role in atherosclerotic plaque formation in ApoE deficient mice.

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Similar plaque characteristics, cytokine, chemokine and adhesion molecule expression in p38αEC-KO/ApoE−/− and ApoE−/− mice.Staining and quantification at the aortic sinus of p38αEC–KO/ApoE−/− mice for: (A) Collagen content, Masson Trichrome staining (blue fibres, indicated by arrows) (B) Foam cell content, MOMA2 staining (red, indicated by arrows). (C) Necrotic core area, clear areas in lesions that did not stain for toluidine blue (nec = necrosis). (D) Relative mRNA levels of adhesion molecules, chemokines and pro-inflammatory cytokines (from left to right) of aortal arches from p38αEC-KO/ApoE−/− and ApoE−/− mice after 10 weeks on HCD. P38αEC-KO/ApoE−/− females, n = 9; ApoE−/− females, n = 6. Scale bar 50 µm. Error bars represent SD.
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pone-0021055-g003: Similar plaque characteristics, cytokine, chemokine and adhesion molecule expression in p38αEC-KO/ApoE−/− and ApoE−/− mice.Staining and quantification at the aortic sinus of p38αEC–KO/ApoE−/− mice for: (A) Collagen content, Masson Trichrome staining (blue fibres, indicated by arrows) (B) Foam cell content, MOMA2 staining (red, indicated by arrows). (C) Necrotic core area, clear areas in lesions that did not stain for toluidine blue (nec = necrosis). (D) Relative mRNA levels of adhesion molecules, chemokines and pro-inflammatory cytokines (from left to right) of aortal arches from p38αEC-KO/ApoE−/− and ApoE−/− mice after 10 weeks on HCD. P38αEC-KO/ApoE−/− females, n = 9; ApoE−/− females, n = 6. Scale bar 50 µm. Error bars represent SD.

Mentions: Measurement of bodyweight and cholesterol levels before and after the HCD treatment revealed no differences between p38αEC-KO/ApoE−/− and their ApoE−/− littermate controls, which showed similarly increased cholesterol levels and body weight after HCD feeding (Figure S2A). After 10 weeks on HCD, mice were sacrificed, and atherosclerotic lesion development was assessed in the whole aorta by en face staining with Sudan IV (Figure 2B), but also at the aortic sinus by histological analysis of consecutive sections followed by cross-sectional plaque area quantification (Figure 2C). This analysis did not reveal differences in lesion size between p38αEC-KO/ApoE−/− and their ApoE−/− littermates, either in the whole aorta or at the aortic sinuses. To further characterize the lesions, we assessed collagen and foam cell content and necrotic core formation. Quantification of collagen on sections stained with Masson Trichrome did not reveal differences in collagen content in plaques from p38αEC-KO/ApoE−/− mice compared to ApoE−/− controls (Figure 3A). In addition, staining with a MOMA2 antibody to detect macrophages showed a similar foam cell content in plaques from p38αEC-KO/ApoE−/− mice compared to ApoE−/− controls (Figure 3B). Finally, quantification of necrotic core area in sections from the aortic sinus did not reveal differences between p38αEC-KO/ApoE−/− and ApoE−/− controls (Figure 3C). Taken together, our experiments showed that endothelial p38α deficiency does not affect atherosclerotic plaque size or composition in ApoE−/− mice. These results were surprising in light of our in vitro findings, showing that p38α was essential for oxLDL-induced expression of adhesion molecules and chemokines that are important for monocyte recruitment in atherosclerosis. We therefore assessed the expression of a number of cytokines, chemokines and adhesion molecules in aortic roots from p38αEC-KO/ApoE−/− and ApoE−/− control mice at the end of the HCD treatment. These experiments did not show any significant differences in cytokine, chemokine or adhesion molecule expression between p38αEC-KO/ApoE−/− and ApoE−/− control mice (Figure 3D). Thus, our results show that endothelial cell-specific ablation of p38α does not affect the development of atherosclerosis in the ApoE−/− model of the disease.


Endothelial and macrophage-specific deficiency of P38α MAPK does not affect the pathogenesis of atherosclerosis in ApoE-/- mice.

Kardakaris R, Gareus R, Xanthoulea S, Pasparakis M - PLoS ONE (2011)

Similar plaque characteristics, cytokine, chemokine and adhesion molecule expression in p38αEC-KO/ApoE−/− and ApoE−/− mice.Staining and quantification at the aortic sinus of p38αEC–KO/ApoE−/− mice for: (A) Collagen content, Masson Trichrome staining (blue fibres, indicated by arrows) (B) Foam cell content, MOMA2 staining (red, indicated by arrows). (C) Necrotic core area, clear areas in lesions that did not stain for toluidine blue (nec = necrosis). (D) Relative mRNA levels of adhesion molecules, chemokines and pro-inflammatory cytokines (from left to right) of aortal arches from p38αEC-KO/ApoE−/− and ApoE−/− mice after 10 weeks on HCD. P38αEC-KO/ApoE−/− females, n = 9; ApoE−/− females, n = 6. Scale bar 50 µm. Error bars represent SD.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111465&req=5

pone-0021055-g003: Similar plaque characteristics, cytokine, chemokine and adhesion molecule expression in p38αEC-KO/ApoE−/− and ApoE−/− mice.Staining and quantification at the aortic sinus of p38αEC–KO/ApoE−/− mice for: (A) Collagen content, Masson Trichrome staining (blue fibres, indicated by arrows) (B) Foam cell content, MOMA2 staining (red, indicated by arrows). (C) Necrotic core area, clear areas in lesions that did not stain for toluidine blue (nec = necrosis). (D) Relative mRNA levels of adhesion molecules, chemokines and pro-inflammatory cytokines (from left to right) of aortal arches from p38αEC-KO/ApoE−/− and ApoE−/− mice after 10 weeks on HCD. P38αEC-KO/ApoE−/− females, n = 9; ApoE−/− females, n = 6. Scale bar 50 µm. Error bars represent SD.
Mentions: Measurement of bodyweight and cholesterol levels before and after the HCD treatment revealed no differences between p38αEC-KO/ApoE−/− and their ApoE−/− littermate controls, which showed similarly increased cholesterol levels and body weight after HCD feeding (Figure S2A). After 10 weeks on HCD, mice were sacrificed, and atherosclerotic lesion development was assessed in the whole aorta by en face staining with Sudan IV (Figure 2B), but also at the aortic sinus by histological analysis of consecutive sections followed by cross-sectional plaque area quantification (Figure 2C). This analysis did not reveal differences in lesion size between p38αEC-KO/ApoE−/− and their ApoE−/− littermates, either in the whole aorta or at the aortic sinuses. To further characterize the lesions, we assessed collagen and foam cell content and necrotic core formation. Quantification of collagen on sections stained with Masson Trichrome did not reveal differences in collagen content in plaques from p38αEC-KO/ApoE−/− mice compared to ApoE−/− controls (Figure 3A). In addition, staining with a MOMA2 antibody to detect macrophages showed a similar foam cell content in plaques from p38αEC-KO/ApoE−/− mice compared to ApoE−/− controls (Figure 3B). Finally, quantification of necrotic core area in sections from the aortic sinus did not reveal differences between p38αEC-KO/ApoE−/− and ApoE−/− controls (Figure 3C). Taken together, our experiments showed that endothelial p38α deficiency does not affect atherosclerotic plaque size or composition in ApoE−/− mice. These results were surprising in light of our in vitro findings, showing that p38α was essential for oxLDL-induced expression of adhesion molecules and chemokines that are important for monocyte recruitment in atherosclerosis. We therefore assessed the expression of a number of cytokines, chemokines and adhesion molecules in aortic roots from p38αEC-KO/ApoE−/− and ApoE−/− control mice at the end of the HCD treatment. These experiments did not show any significant differences in cytokine, chemokine or adhesion molecule expression between p38αEC-KO/ApoE−/− and ApoE−/− control mice (Figure 3D). Thus, our results show that endothelial cell-specific ablation of p38α does not affect the development of atherosclerosis in the ApoE−/− model of the disease.

Bottom Line: The p38α Mitogen-Activated Protein Kinase (MAPK) regulates stress- and inflammation-induced cellular responses.Surprisingly, although p38α-deficiency strongly attenuated oxidized LDL-induced expression of molecules responsible for monocyte recruitment in endothelial cell cultures in vitro, endothelial-specific p38α ablation in vivo did not affect atherosclerosis development.Similarly, macrophage specific deletion of p38α did not affect atherosclerotic plaque development in ApoE(-/-) mice.

View Article: PubMed Central - PubMed

Affiliation: Centre for Molecular Medicine, Institute for Genetics, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany.

ABSTRACT

Background: The p38α Mitogen-Activated Protein Kinase (MAPK) regulates stress- and inflammation-induced cellular responses. Factors implicated in the development of atherosclerosis including modified low-density lipoprotein (LDL), cytokines and even shear stress induce p38 activation in endothelial cells and macrophages, which may be important for plaque formation. This study investigates the effects of endothelial- and macrophage-specific deficiency of p38α in atherosclerosis development, in Apolipoprotein E deficient (ApoE(-/-)) mice.

Methodology/principal findings: ApoE(-/-) mice with macrophage or endothelial cell-specific p38α deficiency were fed a high cholesterol diet (HCD) for 10 weeks and atherosclerosis development was assessed by histological and molecular methods. Surprisingly, although p38α-deficiency strongly attenuated oxidized LDL-induced expression of molecules responsible for monocyte recruitment in endothelial cell cultures in vitro, endothelial-specific p38α ablation in vivo did not affect atherosclerosis development. Similarly, macrophage specific deletion of p38α did not affect atherosclerotic plaque development in ApoE(-/-) mice.

Conclusions: Although previous studies implicated p38α signaling in atherosclerosis, our in vivo experiments suggest that p38α function in endothelial cells and macrophages does not play an important role in atherosclerotic plaque formation in ApoE deficient mice.

Show MeSH
Related in: MedlinePlus