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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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Related in: MedlinePlus

Comparable atherosclerotic plaque formation in p38αEC-KO/ApoE−/− and ApoE−/− mice after 10 weeks of HCD.(A) Analysis of deletion efficiency by immunoblotting in p38αEC-KO/ApoE−/− and their ApoE−/− littermates after 5 weeks of tamoxifen diet and 10 weeks of HCD, by MACS sorting of MLECs with CD146. CD146+ = MLECs, CD146− = other cells. P38EC-KO/Apo−/− females, n = 9; ApoE−/− females, n = 6. (B) Quantification of lesion area on whole aorta from female p38αEC-KO/ApoE−/− and ApoE−/− mice. En face Sudan IV staining of plaques. P38αEC-KO/ApoE−/− females, n = 14; ApoE−/− females, n = 13. Scale bar, 2 mm. (C) Quantification of lesion area on atherosclerotic plaques at the aortic sinus of p38αEC-KO/ApoE−/− and ApoE−/− mice. Plaques are marked by arrows on aortal cross sections at the height of the aortic sinus. P38αEC-KO/ApoE−/− females, n = 14; ApoE−/− females, n = 13; p38αEC-KO/ApoE−/− males, n = 14; ApoE−/− males, n = 15. Scale bar, 0.2 mm.
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pone-0021055-g002: Comparable atherosclerotic plaque formation in p38αEC-KO/ApoE−/− and ApoE−/− mice after 10 weeks of HCD.(A) Analysis of deletion efficiency by immunoblotting in p38αEC-KO/ApoE−/− and their ApoE−/− littermates after 5 weeks of tamoxifen diet and 10 weeks of HCD, by MACS sorting of MLECs with CD146. CD146+ = MLECs, CD146− = other cells. P38EC-KO/Apo−/− females, n = 9; ApoE−/− females, n = 6. (B) Quantification of lesion area on whole aorta from female p38αEC-KO/ApoE−/− and ApoE−/− mice. En face Sudan IV staining of plaques. P38αEC-KO/ApoE−/− females, n = 14; ApoE−/− females, n = 13. Scale bar, 2 mm. (C) Quantification of lesion area on atherosclerotic plaques at the aortic sinus of p38αEC-KO/ApoE−/− and ApoE−/− mice. Plaques are marked by arrows on aortal cross sections at the height of the aortic sinus. P38αEC-KO/ApoE−/− females, n = 14; ApoE−/− females, n = 13; p38αEC-KO/ApoE−/− males, n = 14; ApoE−/− males, n = 15. Scale bar, 0.2 mm.

Mentions: The results of the above described experiments suggested that p38α activity in endothelial cells might play an important role for the development of atherosclerotic plaques and encouraged us to study in vivo the effect of endothelial cell specific p38α ablation, in the development of atherosclerosis. For this purpose we used Cre-loxP-mediated conditional targeting of p38α in ApoE-deficient mice, which spontaneously develop atherosclerotic plaques due to elevated blood cholesterol levels, a pathology that is further aggravated upon feeding with a HCD. Thus, we crossed mice carrying loxP-flanked p38α alleles with Tie2ERT2Cre transgenics, which mediate tamoxifen-inducible Cre recombination specifically in endothelial cells [23] and subsequently with ApoE−/− mice. To induce Cre-mediated excision of the loxP-flanked p38α allele in endothelial cells, we fed groups of 6–8 week old p38αEC-KO/ApoE−/− and their p38αFL/FL/ApoE−/− littermates that did not carry the Tie2ERT2Cre transgene with a diet containing tamoxifen (400 mg/kg tamoxifen citrate, 5% sucrose in phytoestrogen-free chow) for 5 consecutive weeks [24]. The mice were subsequently placed on HCD for 10 weeks to facilitate the development of atherosclerotic plaques. To assess the efficiency of p38α ablation, we measured p38α expression in primary endothelial cells isolated by CD146-mediated magnetic cell sorting (MACS) from the lungs of mice, at the end of the HCD feeding. Flow cytometric analysis of cell fractions collected during MACS sorting showed efficient separation of CD146+ and CD146− fractions (Figure S1). Immunoblot analysis of protein lysates from whole lung, CD146+ and CD146− cell fractions showed efficient ablation of p38α in endothelial cell isolates from p38αEC-KO/ApoE−/− mice, indicating that tamoxifen-treatment induced efficient ablation of p38α in the vascular endothelium that persisted during the period of HCD feeding (Figure 2A).


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)

Comparable atherosclerotic plaque formation in p38αEC-KO/ApoE−/− and ApoE−/− mice after 10 weeks of HCD.(A) Analysis of deletion efficiency by immunoblotting in p38αEC-KO/ApoE−/− and their ApoE−/− littermates after 5 weeks of tamoxifen diet and 10 weeks of HCD, by MACS sorting of MLECs with CD146. CD146+ = MLECs, CD146− = other cells. P38EC-KO/Apo−/− females, n = 9; ApoE−/− females, n = 6. (B) Quantification of lesion area on whole aorta from female p38αEC-KO/ApoE−/− and ApoE−/− mice. En face Sudan IV staining of plaques. P38αEC-KO/ApoE−/− females, n = 14; ApoE−/− females, n = 13. Scale bar, 2 mm. (C) Quantification of lesion area on atherosclerotic plaques at the aortic sinus of p38αEC-KO/ApoE−/− and ApoE−/− mice. Plaques are marked by arrows on aortal cross sections at the height of the aortic sinus. P38αEC-KO/ApoE−/− females, n = 14; ApoE−/− females, n = 13; p38αEC-KO/ApoE−/− males, n = 14; ApoE−/− males, n = 15. Scale bar, 0.2 mm.
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pone-0021055-g002: Comparable atherosclerotic plaque formation in p38αEC-KO/ApoE−/− and ApoE−/− mice after 10 weeks of HCD.(A) Analysis of deletion efficiency by immunoblotting in p38αEC-KO/ApoE−/− and their ApoE−/− littermates after 5 weeks of tamoxifen diet and 10 weeks of HCD, by MACS sorting of MLECs with CD146. CD146+ = MLECs, CD146− = other cells. P38EC-KO/Apo−/− females, n = 9; ApoE−/− females, n = 6. (B) Quantification of lesion area on whole aorta from female p38αEC-KO/ApoE−/− and ApoE−/− mice. En face Sudan IV staining of plaques. P38αEC-KO/ApoE−/− females, n = 14; ApoE−/− females, n = 13. Scale bar, 2 mm. (C) Quantification of lesion area on atherosclerotic plaques at the aortic sinus of p38αEC-KO/ApoE−/− and ApoE−/− mice. Plaques are marked by arrows on aortal cross sections at the height of the aortic sinus. P38αEC-KO/ApoE−/− females, n = 14; ApoE−/− females, n = 13; p38αEC-KO/ApoE−/− males, n = 14; ApoE−/− males, n = 15. Scale bar, 0.2 mm.
Mentions: The results of the above described experiments suggested that p38α activity in endothelial cells might play an important role for the development of atherosclerotic plaques and encouraged us to study in vivo the effect of endothelial cell specific p38α ablation, in the development of atherosclerosis. For this purpose we used Cre-loxP-mediated conditional targeting of p38α in ApoE-deficient mice, which spontaneously develop atherosclerotic plaques due to elevated blood cholesterol levels, a pathology that is further aggravated upon feeding with a HCD. Thus, we crossed mice carrying loxP-flanked p38α alleles with Tie2ERT2Cre transgenics, which mediate tamoxifen-inducible Cre recombination specifically in endothelial cells [23] and subsequently with ApoE−/− mice. To induce Cre-mediated excision of the loxP-flanked p38α allele in endothelial cells, we fed groups of 6–8 week old p38αEC-KO/ApoE−/− and their p38αFL/FL/ApoE−/− littermates that did not carry the Tie2ERT2Cre transgene with a diet containing tamoxifen (400 mg/kg tamoxifen citrate, 5% sucrose in phytoestrogen-free chow) for 5 consecutive weeks [24]. The mice were subsequently placed on HCD for 10 weeks to facilitate the development of atherosclerotic plaques. To assess the efficiency of p38α ablation, we measured p38α expression in primary endothelial cells isolated by CD146-mediated magnetic cell sorting (MACS) from the lungs of mice, at the end of the HCD feeding. Flow cytometric analysis of cell fractions collected during MACS sorting showed efficient separation of CD146+ and CD146− fractions (Figure S1). Immunoblot analysis of protein lysates from whole lung, CD146+ and CD146− cell fractions showed efficient ablation of p38α in endothelial cell isolates from p38αEC-KO/ApoE−/− mice, indicating that tamoxifen-treatment induced efficient ablation of p38α in the vascular endothelium that persisted during the period of HCD feeding (Figure 2A).

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