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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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Attenuated monocyte recruitment molecule expression in p38α deficient endothelial cells, in vitro.(A) Flow cytometric analysis of primary lung endothelial cells to check for purity of population, after treatment with HTNC for 16 hrs. Cells were stained with CD146 FITC, specific for MLECs. Leukocytes isolated from blood were used as negative controls for CD146 staining (data not shown). (B) Immunoblotting for p38α and total p-p38 on MLEC protein extracts. (C) Relative mRNA expression levels of adhesion molecule VCAM1 and chemokines IP-10, MCP-1 and Gro-KC in MLECs. Cells were isolated from the lung of p38αFL/FL/ApoE−/− and ApoE−/− mice, passaged twice and treated with HTNC for 16 hrs to induce cre recombination. Data shown are representative of two separate experiments. Error bars represent SD.
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pone-0021055-g001: Attenuated monocyte recruitment molecule expression in p38α deficient endothelial cells, in vitro.(A) Flow cytometric analysis of primary lung endothelial cells to check for purity of population, after treatment with HTNC for 16 hrs. Cells were stained with CD146 FITC, specific for MLECs. Leukocytes isolated from blood were used as negative controls for CD146 staining (data not shown). (B) Immunoblotting for p38α and total p-p38 on MLEC protein extracts. (C) Relative mRNA expression levels of adhesion molecule VCAM1 and chemokines IP-10, MCP-1 and Gro-KC in MLECs. Cells were isolated from the lung of p38αFL/FL/ApoE−/− and ApoE−/− mice, passaged twice and treated with HTNC for 16 hrs to induce cre recombination. Data shown are representative of two separate experiments. Error bars represent SD.

Mentions: Vascular endothelial cells have an important role in the pathogenesis of atherosclerosis by expressing adhesion molecules and chemokines that facilitate the recruitment of inflammatory cells into the developing plaque. To address the role of p38α in endothelial cells in atherosclerosis, we studied the contribution of p38α activation in the expression of adhesion molecules and chemokines by endothelial cells, upon stimulation with oxLDL. For these experiments, we used primary mouse lung endothelial cells (MLECs) isolated by Dynabead-mediated negative (LEAF) and positive (CD102) selection from dissociated lung tissue from p38αFL/FL/ApoE−/− or ApoE−/− control mice. To delete p38α in these cells, we used His-TAT-NLS-Cre (HTNC), a transducible Cre recombinase that can be used efficiently to mediate recombination of loxP flanked alleles in culture [21]. Purity of endothelial cell cultures after Cre recombination was assessed by flow cytometric analysis after staining with CD146 (Figure 1A). Immunoblot analysis with p38α specific antibodies revealed a strong reduction of p38α expression in HTNC-treated p38αFL/FL/ApoE−/− MLECs, indicating efficient ablation of p38α (Figure 1B, middle panel). Furthermore, immunoblot with antibodies recognizing phosphorylated p38 revealed that stimulation with 100 µg/ml oxLDL induced strong p38 activation in control (ApoE−/−) cells, while p38α-deficient MLECs showed weak p38 activation. As the phospho-p38 antibody used is not isoform-specific, the weak p38 phosphorylation detected most likely corresponds to phosphorylation of the residual p38α and also other p38 isoforms, such as p38β, which is the other major p38 isoform expressed in endothelial cells [22], (Figure 1B, top panel). We then assessed the oxLDL-induced expression of vascular adhesion molecule 1 (VCAM1) and the chemokines IP-10, MCP-1 and Gro-KC, known to be involved in the recruitment of monocytes into the arterial intima. Quantitative real-time PCR (qRT-PCR) analysis on RNA, isolated at different time points after oxLDL stimulation, revealed induction in expression of VCAM1, IP-10, MCP-1 and Gro-KC in control MLECs, which was strongly reduced in MLECs lacking p38α (Figure 1C).


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)

Attenuated monocyte recruitment molecule expression in p38α deficient endothelial cells, in vitro.(A) Flow cytometric analysis of primary lung endothelial cells to check for purity of population, after treatment with HTNC for 16 hrs. Cells were stained with CD146 FITC, specific for MLECs. Leukocytes isolated from blood were used as negative controls for CD146 staining (data not shown). (B) Immunoblotting for p38α and total p-p38 on MLEC protein extracts. (C) Relative mRNA expression levels of adhesion molecule VCAM1 and chemokines IP-10, MCP-1 and Gro-KC in MLECs. Cells were isolated from the lung of p38αFL/FL/ApoE−/− and ApoE−/− mice, passaged twice and treated with HTNC for 16 hrs to induce cre recombination. Data shown are representative of two separate experiments. Error bars represent SD.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3111465&req=5

pone-0021055-g001: Attenuated monocyte recruitment molecule expression in p38α deficient endothelial cells, in vitro.(A) Flow cytometric analysis of primary lung endothelial cells to check for purity of population, after treatment with HTNC for 16 hrs. Cells were stained with CD146 FITC, specific for MLECs. Leukocytes isolated from blood were used as negative controls for CD146 staining (data not shown). (B) Immunoblotting for p38α and total p-p38 on MLEC protein extracts. (C) Relative mRNA expression levels of adhesion molecule VCAM1 and chemokines IP-10, MCP-1 and Gro-KC in MLECs. Cells were isolated from the lung of p38αFL/FL/ApoE−/− and ApoE−/− mice, passaged twice and treated with HTNC for 16 hrs to induce cre recombination. Data shown are representative of two separate experiments. Error bars represent SD.
Mentions: Vascular endothelial cells have an important role in the pathogenesis of atherosclerosis by expressing adhesion molecules and chemokines that facilitate the recruitment of inflammatory cells into the developing plaque. To address the role of p38α in endothelial cells in atherosclerosis, we studied the contribution of p38α activation in the expression of adhesion molecules and chemokines by endothelial cells, upon stimulation with oxLDL. For these experiments, we used primary mouse lung endothelial cells (MLECs) isolated by Dynabead-mediated negative (LEAF) and positive (CD102) selection from dissociated lung tissue from p38αFL/FL/ApoE−/− or ApoE−/− control mice. To delete p38α in these cells, we used His-TAT-NLS-Cre (HTNC), a transducible Cre recombinase that can be used efficiently to mediate recombination of loxP flanked alleles in culture [21]. Purity of endothelial cell cultures after Cre recombination was assessed by flow cytometric analysis after staining with CD146 (Figure 1A). Immunoblot analysis with p38α specific antibodies revealed a strong reduction of p38α expression in HTNC-treated p38αFL/FL/ApoE−/− MLECs, indicating efficient ablation of p38α (Figure 1B, middle panel). Furthermore, immunoblot with antibodies recognizing phosphorylated p38 revealed that stimulation with 100 µg/ml oxLDL induced strong p38 activation in control (ApoE−/−) cells, while p38α-deficient MLECs showed weak p38 activation. As the phospho-p38 antibody used is not isoform-specific, the weak p38 phosphorylation detected most likely corresponds to phosphorylation of the residual p38α and also other p38 isoforms, such as p38β, which is the other major p38 isoform expressed in endothelial cells [22], (Figure 1B, top panel). We then assessed the oxLDL-induced expression of vascular adhesion molecule 1 (VCAM1) and the chemokines IP-10, MCP-1 and Gro-KC, known to be involved in the recruitment of monocytes into the arterial intima. Quantitative real-time PCR (qRT-PCR) analysis on RNA, isolated at different time points after oxLDL stimulation, revealed induction in expression of VCAM1, IP-10, MCP-1 and Gro-KC in control MLECs, which was strongly reduced in MLECs lacking p38α (Figure 1C).

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