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PI3K p110γ deletion attenuates murine atherosclerosis by reducing macrophage proliferation but not polarization or apoptosis in lesions.

Zotes TM, Arias CF, Fuster JJ, Spada R, Pérez-Yagüe S, Hirsch E, Wymann M, Carrera AC, Andrés V, Barber DF - PLoS ONE (2013)

Bottom Line: Atherosclerosis is an inflammatory disease regulated by infiltrating monocytes and T cells, among other cell types.We analyzed atherosclerosis development in LDLR(-/-)p110γ(+/-) and LDLR(-/-)p110γ(-/-) mice, and performed expression and functional assays in tissues and primary cells from these and from p110γ(+/-) and p110γ(-/-) mice.Lack of p110γ in LDLR(-/-) mice reduces the atherosclerosis burden.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Oncology, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid, Spain.

ABSTRACT
Atherosclerosis is an inflammatory disease regulated by infiltrating monocytes and T cells, among other cell types. Macrophage recruitment to atherosclerotic lesions is controlled by monocyte infiltration into plaques. Once in the lesion, macrophage proliferation in situ, apoptosis, and differentiation to an inflammatory (M1) or anti-inflammatory phenotype (M2) are involved in progression to advanced atherosclerotic lesions. We studied the role of phosphoinositol-3-kinase (PI3K) p110γ in the regulation of in situ apoptosis, macrophage proliferation and polarization towards M1 or M2 phenotypes in atherosclerotic lesions. We analyzed atherosclerosis development in LDLR(-/-)p110γ(+/-) and LDLR(-/-)p110γ(-/-) mice, and performed expression and functional assays in tissues and primary cells from these and from p110γ(+/-) and p110γ(-/-) mice. Lack of p110γ in LDLR(-/-) mice reduces the atherosclerosis burden. Atherosclerotic lesions in fat-fed LDLR(-/-)p110γ(-/-) mice were smaller than in LDLR(-/-)p110γ(+/-) controls, which coincided with decreased macrophage proliferation in LDLR(-/-)p110γ(-/-) mouse lesions. This proliferation defect was also observed in p110γ(-/-) bone marrow-derived macrophages (BMM) stimulated with macrophage colony-stimulating factor (M-CSF), and was associated with higher intracellular cyclic adenosine monophosphate (cAMP) levels. In contrast, T cell proliferation was unaffected in LDLR(-/-)p110γ(-/-) mice. Moreover, p110γ deficiency did not affect macrophage polarization towards the M1 or M2 phenotypes or apoptosis in atherosclerotic plaques, or polarization in cultured BMM. Our results suggest that higher cAMP levels and the ensuing inhibition of macrophage proliferation contribute to atheroprotection in LDLR(-/-) mice lacking p110γ. Nonetheless, p110γ deletion does not appear to be involved in apoptosis, in macrophage polarization or in T cell proliferation.

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Intracellular cAMP levels are higher in LDLR-/−p110γ−/− than in LDLR−/−p110γ+/−macrophages.LDLR−/−p110γ+/− and LDLR−/−p110γ−/− BMM were allowed to differentiate and were lysed. (A) ELISA was used to determine intracellular cAMP levels in unstimulated BMM lysates (t = 0). The graph shows basal intracellular cAMP levels in LDLR−/−p110γ−/− vs. LDLR−/−p110γ+/− BMM (n = 4 experiments). Mean ± SD; Student’s t-test, p<0.01. (B) Western blot of BMM extracts to detect protein-bound cAMP after M-CSF stimulation (0, 24 and 48 h). (C) Bands in (B) were quantified with ImageJ and measured as arbitrary units (AU). Mean ± SD of four independent experiments, each with one mouse/genotype. Student’s t-test, p<0.05 and p<0.01. (D) Western blot of BMM extracts to detect p-CREB and total CREB (t = 0).
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pone-0072674-g004: Intracellular cAMP levels are higher in LDLR-/−p110γ−/− than in LDLR−/−p110γ+/−macrophages.LDLR−/−p110γ+/− and LDLR−/−p110γ−/− BMM were allowed to differentiate and were lysed. (A) ELISA was used to determine intracellular cAMP levels in unstimulated BMM lysates (t = 0). The graph shows basal intracellular cAMP levels in LDLR−/−p110γ−/− vs. LDLR−/−p110γ+/− BMM (n = 4 experiments). Mean ± SD; Student’s t-test, p<0.01. (B) Western blot of BMM extracts to detect protein-bound cAMP after M-CSF stimulation (0, 24 and 48 h). (C) Bands in (B) were quantified with ImageJ and measured as arbitrary units (AU). Mean ± SD of four independent experiments, each with one mouse/genotype. Student’s t-test, p<0.05 and p<0.01. (D) Western blot of BMM extracts to detect p-CREB and total CREB (t = 0).

Mentions: Since p110γ regulates cAMP levels in cardiomyocytes and microglia [32], [33], [34] we tested whether this is the case in macrophages, using ELISA to measure intracellular cAMP levels in LDLR−/−p110γ+/− and LDLR−/−p110γ−/− mouse BMM. Basal cAMP levels were higher in LDLR−/−p110γ−/− compared to LDLR−/−p110γ+/− BMM (Figure 4A). In an alternative approach, we measured differences in protein-bound cAMP in Western blot, using an antibody that recognizes protein-bound cAMP [39]. In macrophages, this antibody recognizes major three bands of 24, 33 and 35 kDa, as determined when BMM from p110γ+/−mice were differentiated in vitro and FSK-stimulated, and cAMP detected by Western blot (Figure S3). Again, basal cAMP levels were significantly higher in BMM lysates from LDLR−/−p110γ−/− than from LDLR−/−p110γ+/− mice and this difference was sustained after M-CSF stimulation (Figure 4B, 4C).


PI3K p110γ deletion attenuates murine atherosclerosis by reducing macrophage proliferation but not polarization or apoptosis in lesions.

Zotes TM, Arias CF, Fuster JJ, Spada R, Pérez-Yagüe S, Hirsch E, Wymann M, Carrera AC, Andrés V, Barber DF - PLoS ONE (2013)

Intracellular cAMP levels are higher in LDLR-/−p110γ−/− than in LDLR−/−p110γ+/−macrophages.LDLR−/−p110γ+/− and LDLR−/−p110γ−/− BMM were allowed to differentiate and were lysed. (A) ELISA was used to determine intracellular cAMP levels in unstimulated BMM lysates (t = 0). The graph shows basal intracellular cAMP levels in LDLR−/−p110γ−/− vs. LDLR−/−p110γ+/− BMM (n = 4 experiments). Mean ± SD; Student’s t-test, p<0.01. (B) Western blot of BMM extracts to detect protein-bound cAMP after M-CSF stimulation (0, 24 and 48 h). (C) Bands in (B) were quantified with ImageJ and measured as arbitrary units (AU). Mean ± SD of four independent experiments, each with one mouse/genotype. Student’s t-test, p<0.05 and p<0.01. (D) Western blot of BMM extracts to detect p-CREB and total CREB (t = 0).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3750002&req=5

pone-0072674-g004: Intracellular cAMP levels are higher in LDLR-/−p110γ−/− than in LDLR−/−p110γ+/−macrophages.LDLR−/−p110γ+/− and LDLR−/−p110γ−/− BMM were allowed to differentiate and were lysed. (A) ELISA was used to determine intracellular cAMP levels in unstimulated BMM lysates (t = 0). The graph shows basal intracellular cAMP levels in LDLR−/−p110γ−/− vs. LDLR−/−p110γ+/− BMM (n = 4 experiments). Mean ± SD; Student’s t-test, p<0.01. (B) Western blot of BMM extracts to detect protein-bound cAMP after M-CSF stimulation (0, 24 and 48 h). (C) Bands in (B) were quantified with ImageJ and measured as arbitrary units (AU). Mean ± SD of four independent experiments, each with one mouse/genotype. Student’s t-test, p<0.05 and p<0.01. (D) Western blot of BMM extracts to detect p-CREB and total CREB (t = 0).
Mentions: Since p110γ regulates cAMP levels in cardiomyocytes and microglia [32], [33], [34] we tested whether this is the case in macrophages, using ELISA to measure intracellular cAMP levels in LDLR−/−p110γ+/− and LDLR−/−p110γ−/− mouse BMM. Basal cAMP levels were higher in LDLR−/−p110γ−/− compared to LDLR−/−p110γ+/− BMM (Figure 4A). In an alternative approach, we measured differences in protein-bound cAMP in Western blot, using an antibody that recognizes protein-bound cAMP [39]. In macrophages, this antibody recognizes major three bands of 24, 33 and 35 kDa, as determined when BMM from p110γ+/−mice were differentiated in vitro and FSK-stimulated, and cAMP detected by Western blot (Figure S3). Again, basal cAMP levels were significantly higher in BMM lysates from LDLR−/−p110γ−/− than from LDLR−/−p110γ+/− mice and this difference was sustained after M-CSF stimulation (Figure 4B, 4C).

Bottom Line: Atherosclerosis is an inflammatory disease regulated by infiltrating monocytes and T cells, among other cell types.We analyzed atherosclerosis development in LDLR(-/-)p110γ(+/-) and LDLR(-/-)p110γ(-/-) mice, and performed expression and functional assays in tissues and primary cells from these and from p110γ(+/-) and p110γ(-/-) mice.Lack of p110γ in LDLR(-/-) mice reduces the atherosclerosis burden.

View Article: PubMed Central - PubMed

Affiliation: Department of Immunology and Oncology, Centro Nacional de Biotecnología-Consejo Superior de Investigaciones Científicas (CNB-CSIC), Madrid, Spain.

ABSTRACT
Atherosclerosis is an inflammatory disease regulated by infiltrating monocytes and T cells, among other cell types. Macrophage recruitment to atherosclerotic lesions is controlled by monocyte infiltration into plaques. Once in the lesion, macrophage proliferation in situ, apoptosis, and differentiation to an inflammatory (M1) or anti-inflammatory phenotype (M2) are involved in progression to advanced atherosclerotic lesions. We studied the role of phosphoinositol-3-kinase (PI3K) p110γ in the regulation of in situ apoptosis, macrophage proliferation and polarization towards M1 or M2 phenotypes in atherosclerotic lesions. We analyzed atherosclerosis development in LDLR(-/-)p110γ(+/-) and LDLR(-/-)p110γ(-/-) mice, and performed expression and functional assays in tissues and primary cells from these and from p110γ(+/-) and p110γ(-/-) mice. Lack of p110γ in LDLR(-/-) mice reduces the atherosclerosis burden. Atherosclerotic lesions in fat-fed LDLR(-/-)p110γ(-/-) mice were smaller than in LDLR(-/-)p110γ(+/-) controls, which coincided with decreased macrophage proliferation in LDLR(-/-)p110γ(-/-) mouse lesions. This proliferation defect was also observed in p110γ(-/-) bone marrow-derived macrophages (BMM) stimulated with macrophage colony-stimulating factor (M-CSF), and was associated with higher intracellular cyclic adenosine monophosphate (cAMP) levels. In contrast, T cell proliferation was unaffected in LDLR(-/-)p110γ(-/-) mice. Moreover, p110γ deficiency did not affect macrophage polarization towards the M1 or M2 phenotypes or apoptosis in atherosclerotic plaques, or polarization in cultured BMM. Our results suggest that higher cAMP levels and the ensuing inhibition of macrophage proliferation contribute to atheroprotection in LDLR(-/-) mice lacking p110γ. Nonetheless, p110γ deletion does not appear to be involved in apoptosis, in macrophage polarization or in T cell proliferation.

Show MeSH
Related in: MedlinePlus