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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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Lesion apoptosis is unaffected by p110γ deletion.Atherosclerotic plaques were analyzed in LDLR−/−p110γ+/− and LDLR−/−p110γ−/− mice after a two-month high-fat diet. (A) Representative photomicrographs of TUNEL immunofluorescent staining for lesion apoptosis in aortic sections from LDLR−/−p110γ+/− (n = 8) and LDLR−/−p110γ−/− mice (n = 8) (top); percentage of TUNEL+ relative to total cells in the delimited lesion area (bottom). Bar = 50 μm. (B) Representative photomicrographs of cleaved caspase-3 immunofluorescent staining for lesion apoptosis in aortic sections from LDLR−/−p110γ+/− (n = 5) and LDLR−/−p110γ−/− mice (n = 5) (top); percentage of cleaved caspase-3+ relative to total cells in the delimited lesion area (bottom). Bar = 50 μm. Mean ± SD; Student’s t-test.
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pone-0072674-g003: Lesion apoptosis is unaffected by p110γ deletion.Atherosclerotic plaques were analyzed in LDLR−/−p110γ+/− and LDLR−/−p110γ−/− mice after a two-month high-fat diet. (A) Representative photomicrographs of TUNEL immunofluorescent staining for lesion apoptosis in aortic sections from LDLR−/−p110γ+/− (n = 8) and LDLR−/−p110γ−/− mice (n = 8) (top); percentage of TUNEL+ relative to total cells in the delimited lesion area (bottom). Bar = 50 μm. (B) Representative photomicrographs of cleaved caspase-3 immunofluorescent staining for lesion apoptosis in aortic sections from LDLR−/−p110γ+/− (n = 5) and LDLR−/−p110γ−/− mice (n = 5) (top); percentage of cleaved caspase-3+ relative to total cells in the delimited lesion area (bottom). Bar = 50 μm. Mean ± SD; Student’s t-test.

Mentions: Macrophage apoptosis has been implicated in plaque progression [16]. We measured total apoptosis in lesions by TUNEL (Figure 3A) and cleaved caspase-3 (Figure 3B) immunofluorescent staining of aortic sinus sections from LDLR−/−p110γ+/− and LDLR−/−p110γ−/− mice. Lesion area was delimited for TUNEL staining with the help of smooth muscle cells (SMC), which limit lesion area and are autofluorescent, and for cleaved caspase-3 staining by adding Mac-3 staining to the SMC guide; some lesion apoptotic cells are not Mac-3+. We detected a tendency toward lower apoptotic rates in LDLR−/−p110γ−/− compared to LDLR−/−p110γ+/− mice (Figure 3A, 3B), although the differences were not significant.


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)

Lesion apoptosis is unaffected by p110γ deletion.Atherosclerotic plaques were analyzed in LDLR−/−p110γ+/− and LDLR−/−p110γ−/− mice after a two-month high-fat diet. (A) Representative photomicrographs of TUNEL immunofluorescent staining for lesion apoptosis in aortic sections from LDLR−/−p110γ+/− (n = 8) and LDLR−/−p110γ−/− mice (n = 8) (top); percentage of TUNEL+ relative to total cells in the delimited lesion area (bottom). Bar = 50 μm. (B) Representative photomicrographs of cleaved caspase-3 immunofluorescent staining for lesion apoptosis in aortic sections from LDLR−/−p110γ+/− (n = 5) and LDLR−/−p110γ−/− mice (n = 5) (top); percentage of cleaved caspase-3+ relative to total cells in the delimited lesion area (bottom). Bar = 50 μm. Mean ± SD; Student’s t-test.
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Related In: Results  -  Collection

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pone-0072674-g003: Lesion apoptosis is unaffected by p110γ deletion.Atherosclerotic plaques were analyzed in LDLR−/−p110γ+/− and LDLR−/−p110γ−/− mice after a two-month high-fat diet. (A) Representative photomicrographs of TUNEL immunofluorescent staining for lesion apoptosis in aortic sections from LDLR−/−p110γ+/− (n = 8) and LDLR−/−p110γ−/− mice (n = 8) (top); percentage of TUNEL+ relative to total cells in the delimited lesion area (bottom). Bar = 50 μm. (B) Representative photomicrographs of cleaved caspase-3 immunofluorescent staining for lesion apoptosis in aortic sections from LDLR−/−p110γ+/− (n = 5) and LDLR−/−p110γ−/− mice (n = 5) (top); percentage of cleaved caspase-3+ relative to total cells in the delimited lesion area (bottom). Bar = 50 μm. Mean ± SD; Student’s t-test.
Mentions: Macrophage apoptosis has been implicated in plaque progression [16]. We measured total apoptosis in lesions by TUNEL (Figure 3A) and cleaved caspase-3 (Figure 3B) immunofluorescent staining of aortic sinus sections from LDLR−/−p110γ+/− and LDLR−/−p110γ−/− mice. Lesion area was delimited for TUNEL staining with the help of smooth muscle cells (SMC), which limit lesion area and are autofluorescent, and for cleaved caspase-3 staining by adding Mac-3 staining to the SMC guide; some lesion apoptotic cells are not Mac-3+. We detected a tendency toward lower apoptotic rates in LDLR−/−p110γ−/− compared to LDLR−/−p110γ+/− mice (Figure 3A, 3B), although the differences were not significant.

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