Thomas Eschenhagen, Roberto Bolli, Thomas Braun, Loren J. Field, Bernd K. Fleischmann, Jonas Frisén, Mauro Giacca, Joshua M. Hare, Steven R. Houser, Richard T. Lee, Eduardo Marbán, James F. Martin, Jeffery D. Molkentin, Charles E. Murry, Paul R. Riley, Pilar Ruiz-Lozano, Hesham A. Sadek, Mark A. Sussman and Joseph A. Hill
Circulation. 2017;CIRCULATIONAHA.117.029343, originally published July 6, 2017
Kiave-Yune Howangyin, Ivana Zlatanova, Cristina Pinto, Anta Ngkelo, Clément Cochain, Marie Rouanet, José Vilar, Mathilde Lemitre, Christian Stockmann, Bernd K. Fleischmann, Ziad Mallat and Jean-Sébastien Silvestre
Circulation. 2016;133:826-839, originally published January 27, 2016
Kiave-Yune Howangyin, Ivana Zlatanova, Cristina Pinto, Anta Ngkelo, Clément Cochain, Marie Rouanet, José Vilar, Mathilde Lemitre, Christian Stockmann, Bernd K. Fleischmann, Ziad Mallat, Jean-Sébastien Silvestre
Circulation March 2016, 133 (9) 826-839; DOI: https://doi.org/10.1161/CIRCULATIONAHA.115.020857
Cardiac healing is impaired in Mertk−/−/Mfge8−/− mice. A, Echocardiographic analysis 14 days after myocardial infarction. Ejection fraction (%), left ventricular end-diastolic volume (μL), and left ventricular end-systolic volume (μL) are shown for wild-type (WT), Mertk−/−, Mfge8−/−, and Mertk−/−/Mfge8−/− mice. Results are minimum to maximum values. n=6 mice per group. *P<0.05, **P<0.01 vs WT mice (Kruskal-Wallis 1-way ANOVA). B, Quantitative analysis of infarct size, collagen content, capillary density, and number of apoptotic cells. Results are presented as scatterplots with mean bar. n=6 mice per group. *P<0.05, **P<0.01 vs WT mice (Kruskal-Wallis 1-way ANOVA). Representative photomicrographs are shown. Arrows point to area of interest. Bar, 100 μm. Mertk indicates myeloid-epithelial-reproductive protein tyrosine kinase; Mfge8, milk fat globule epidermal growth factor; Tunel, terminal deoxynucleotidyl transferase dUTP nick-end labeling; and WGA, wheat germ agglutinin.
Kiave-Yune Howangyin, Ivana Zlatanova, Cristina Pinto, Anta Ngkelo, Clément Cochain, Marie Rouanet, José Vilar, Mathilde Lemitre, Christian Stockmann, Bernd K. Fleischmann, Ziad Mallat, Jean-Sébastien Silvestre
Circulation March 2016, 133 (9) 826-839; DOI: https://doi.org/10.1161/CIRCULATIONAHA.115.020857
Bone marrow (BM)–derived myeloid-epithelial-reproductive protein tyrosine kinase (Mertk) and milk fat globule epidermal growth factor (Mfge8) improve cardiac remodeling after myocardial infarction (MI). A, Mfge8 and Mertk mRNA levels in fluorescence-activated cell sorter–sorted Ly6CHigh and Ly6CLow monocytes (MO)/macrophages (Mϕ; CD45+CD11b+ Ly6G− Ly6CHigh/Low), neutrophils (CD45+CD11b+Ly6G+), T lymphocytes (CD45+CD3+), and F4/80-positive macrophages (CD45+/CD11b+/Ly6G−/Ly6C+) isolated from wild-type (WT) mice with MI. Cardiac tissue was harvested at day 1 (neutrophils and T lymphocytes), day 3 (Ly6CHigh and Ly6CLow monocytes/macrophages), and day 7 (F4/80-positive macrophages) after the onset of MI. Results are mean±SEM. n=3 to 4 mice per group. B, Echocardiographic analysis 14 days after MI. Ejection fraction (%), left ventricular end-diastolic volume (μL), and left ventricular end-systolic volume (μL) are shown for lethally irradiated WT mice (Host) transplanted with BM cells isolated from WT, Mertk−/−, Mfge8−/−, and Mertk−/−/Mfge8−/− mice. Results are minimum to maximum values. n=8 to 20 mice per group. *P<0.05, ***P<0.001 vs WT BM chimeras (Kruskal-Wallis 1-way ANOVA). C, Quantitative analysis of infarct size (top left), collagen content (top right), number of apoptotic cells (bottom left), and capillary density (bottom right). Results are presented as scatterplots with mean bar. n=8 mice per group. *P<0.05, **P<0.01, ***P<0.001 vs WT BM chimeras (Kruskal-Wallis 1-way ANOVA).
Kiave-Yune Howangyin, Ivana Zlatanova, Cristina Pinto, Anta Ngkelo, Clément Cochain, Marie Rouanet, José Vilar, Mathilde Lemitre, Christian Stockmann, Bernd K. Fleischmann, Ziad Mallat, Jean-Sébastien Silvestre
Circulation March 2016, 133 (9) 826-839; DOI: https://doi.org/10.1161/CIRCULATIONAHA.115.020857
Bone marrow (BM)–derived myeloid-epithelial-reproductive protein tyrosine kinase (Mertk) and milk fat globule epidermal growth factor (Mfge8) do not affect the number of cardiac monocytes/macrophages in the infarcted heart. A, Quantification of the number of green fluorescent protein (GFP)–positive Ly6C monocytes/macrophages (CD45+CD11b+Ly6G−Ly6CHigh/Low) by ImageStream analysis in the ischemic heart of α-actin–GFP+ mice transplanted with wild-type (WT) or Mertk−/−/Mfge8−/− BM cells. Representative images of ImageStream assay are shown. Results are mean±SEM. n=4 per group **P<0.01 vs WT chimeras (Mann–Whitney test). B, Cells harvested from WT mice transplanted with BM cells isolated from WT (BM WT) or Mertk−/−/Mfge8−/− (BM Mertk−/−/Mfge8−/−) mice were analyzed by flow cytometry at days 1, 3, 5, 7, and 10 after myocardial infarction. Results are expressed as the number of cells per 1 mg tissue. Representative examples of neutrophil (CD11b+ Ly6G+), Ly6CHigh or Ly6CLow monocyte (CD11b+Ly6G−F4/80−Ly6CHigh or Low), and Ly6CHigh or Ly6CLow macrophage (CD11b+Ly6G−F4/80+Ly6CHigh or Low) staining are shown. Results are mean±SEM. n=4 to 5 mice per group (Mann–Whitney test).
Kiave-Yune Howangyin, Ivana Zlatanova, Cristina Pinto, Anta Ngkelo, Clément Cochain, Marie Rouanet, José Vilar, Mathilde Lemitre, Christian Stockmann, Bernd K. Fleischmann, Ziad Mallat, Jean-Sébastien Silvestre
Circulation March 2016, 133 (9) 826-839; DOI: https://doi.org/10.1161/CIRCULATIONAHA.115.020857
Bone marrow (BM)–derived myeloid-epithelial-reproductive protein tyrosine kinase (Mertk) and milk fat globule epidermal growth factor (Mfge8) govern vascular endothelial growth factor A (VEGFA) release. A, Quantitative analysis of cytokines and VEGFA protein levels in the cardiac tissue of lethally irradiated wild-type (WT) mice transplanted with BM-derived cells isolated from WT (BM WT) or Mertk−/−/Mfge8−/− (BM Mertk−/−/Mfge8−/−) animals at days 1, 3, 5, 7, and 10 after myocardial infarction. Results are mean±SEM. n=4 to 5 mice per group. *P<0.05 vs WT BM chimeras at day 3 (Mann–Whitney test). B, VEGFA and interleukin (IL)-10 mRNA levels in Ly6CHigh and Ly6CLow monocytes/macrophages (CD11b+Ly6G−Ly6CHigh/Low) sorted by fluorescence-activated cell sorter from the cardiac tissue of WT mice transplanted with BM from WT or Mertk−/−/Mfge8−/− mice at day 3 after myocardial infarction. Results are mean±SEM. n=3 to 4 mice per group. *P<0.05, **P<0.01 vs WT chimeras (Mann–Whitney test). nd indicates not detected; TGFβ, transforming growth factor-β; and TNFα, tumor necrosis factor-α.
Kiave-Yune Howangyin, Ivana Zlatanova, Cristina Pinto, Anta Ngkelo, Clément Cochain, Marie Rouanet, José Vilar, Mathilde Lemitre, Christian Stockmann, Bernd K. Fleischmann, Ziad Mallat, Jean-Sébastien Silvestre
Circulation March 2016, 133 (9) 826-839; DOI: https://doi.org/10.1161/CIRCULATIONAHA.115.020857
Milk fat globule epidermal growth factor (Mfge8) and myeloid-epithelial-reproductive protein tyrosine kinase (Mertk) control an M2-like activation mode and macrophage-derived vascular endothelial growth factor A (VEGFA) release. cDNA of bone marrow (BM)–differentiated macrophages from wild-type (WT) or Mertk−/−/Mfge8−/− mice was analyzed by reverse transcription–quantitative polymerase chain reaction. A, Mertk and Mfge8 mRNA levels. Results are mean±SEM. n=4 to 5 mice per group. #P<0.5, ##P<0.001 vs interleukin (IL)-4–treated macrophages (Kruskal-Wallis 1-way ANOVA). B, mRNA levels for the markers of the M1-like mode: NOS2 and cox2. Results are mean±SEM. n=4 to 5 mice per group. C, mRNA levels for the markers of the M2-like mode: Ym1, Arg1, VCAM-1, and RELMα. Results are mean±SEM. n=4 to 5 mice per group. D, VEGFA protein levels were quantified in the supernatants of differentiated macrophages by ELISA. Results are mean±SEM. n=4 to 5 mice per group. IFNγ indicates interferon-γ; and LPS, lipopolysaccharide. B through D: *P<0.05, **P<0.01 vs WT of the same stimulated group of macrophages (Mann–Whitney test).
Kiave-Yune Howangyin, Ivana Zlatanova, Cristina Pinto, Anta Ngkelo, Clément Cochain, Marie Rouanet, José Vilar, Mathilde Lemitre, Christian Stockmann, Bernd K. Fleischmann, Ziad Mallat, Jean-Sébastien Silvestre
Circulation March 2016, 133 (9) 826-839; DOI: https://doi.org/10.1161/CIRCULATIONAHA.115.020857
Milk fat globule epidermal growth factor (Mfge8)– and myeloid-epithelial-reproductive protein tyrosine kinase (Mertk)–dependent efferocytosis commands vascular endothelial growth factor A (VEGFA) release and endothelial cell proliferation. Apoptotic thymocytes were injected by intraperitoneal administration in wild-type (WT) mice transplanted with WT or Mertk−/−/Mfge8−/− bone marrow (BM). Peritoneal lavage fluid was harvested 3 hours after injection. A, Amount of efferocytosis by monocytes/macrophages (CD45+/CD11b+/Ly6G−). Results are mean±SEM. n=4 to 5 mice per group. *P<0.05 vs control. B, VEGFA protein levels from the supernatant of peritoneal lavage fluid. Results are mean±SEM. n=4 to 5 mice per group. *P<0.05 vs control. C, Supernatants from peritoneal lavage fluid were challenged for endothelial cell proliferation and apoptosis on cultured murine SVEC4-10 endothelial cells. Percentage of sub G0/G1 (apoptotic cells) and S phase (proliferating cells) was assessed with BrdU staining. Representative images of BrdU and 7-Aminoactinomycyn D (7-AAD) staining are shown. Results are mean±SEM. n=4 to 5 mice per group. *P<0.05 vs WT mice transplanted with WT BM (Mann–Whitney test).
Kiave-Yune Howangyin, Ivana Zlatanova, Cristina Pinto, Anta Ngkelo, Clément Cochain, Marie Rouanet, José Vilar, Mathilde Lemitre, Christian Stockmann, Bernd K. Fleischmann, Ziad Mallat, Jean-Sébastien Silvestre
Circulation March 2016, 133 (9) 826-839; DOI: https://doi.org/10.1161/CIRCULATIONAHA.115.020857
Loss of myeloid cell–derived vascular endothelial growth factor A (VEGFA) precipitates adverse left ventricular remodeling after myocardial infarction (MI). A, Echocardiographic analysis 14 days after MI. Ejection fraction (%), left ventricular end-diastolic volume (μL), and left ventricular end-systolic volume (μL) are shown for wild-type (WT) mice (Host) transplanted with bone marrow (BM)–derived cells isolated from LysMCre−VEGFfl/fl or LysMCre+VEGFfl/fl mice. Results are minimum to maximum values. n=7 to 8 mice per group. *P<0.05 vs WT LysMCre−VEGFfl/fl chimeras (Mann–Whitney test). B, Quantitative analysis of infarct size, collagen content, number of apoptotic cells, and capillary density. Results are presented as scatterplots with mean bar. n=7 to 12 mice per group. *P<0.05, ***P<0.001 vs WT LysMCre−VEGFfl/fl chimeras (Mann–Whitney test).
Kiave-Yune Howangyin, Ivana Zlatanova, Cristina Pinto, Anta Ngkelo, Clément Cochain, Marie Rouanet, José Vilar, Mathilde Lemitre, Christian Stockmann, Bernd K. Fleischmann, Ziad Mallat, Jean-Sébastien Silvestre
Circulation March 2016, 133 (9) 826-839; DOI: https://doi.org/10.1161/CIRCULATIONAHA.115.020857
Loss of myeloid cell–derived vascular endothelial growth factor A (VEGFA) activates an M1 phenotype. cDNA from bone marrow (BM)–derived macrophages from LysMCre−VEGFfl/fl and LysMCre+VEGFfl/fl mice were analyzed by reverse transcription–quantitative polymerase chain reaction. A, mRNA levels for the markers of the M1-like mode: NOS2 and cox2. B, mRNA levels for the markers of the M2-like mode: Ym1, Arg1, VCAM-1, and RELMα. Results are mean±SEM. n=4 to 6 mice per group. *P<0.05, **P<0.01 vs LysMCre−VEGFfl/fl of the same stimulated group of macrophages (Mann–Whitney test). C, Apoptotic thymocytes were injected in wild-type (WT) mice transplanted with LysMCre−VEGFfl/fl or LysMCre+VEGFfl/fl BM. Peritoneal lavage fluid was harvested 3 hours after injection. Left, Amount of efferocytosis by monocytes/macrophages (CD45+/CD11b+/Ly6G-). Right, VEGFA protein levels from the supernatant of peritoneal lavage fluid. Results are mean±SEM. n=2 to 3 mice per group. (Mann–Whitney test). IFNγ indicates interferon-γ; IL, interleukin; and LPS, lipopolysaccharide.
