Journal: eLife

Article Title: Cell-autonomous and non-cell-autonomous effects of Arginase 2 on cardiac aging

doi: 10.7554/eLife.94794

Figure Lengend Snippet: ( A ) Arg2 mRNA levels of male and female young (3–4 months) and old (20–22 months) wild type (wt) heart tissues analyzed by qRT-PCR. Rps12 served as the reference (n=8–9 animals per group); ( B ) Representative histological images of heart interstitial and perivascular fibrosis in young and old wt and Arg2 -/- female mice. Fibrosis is shown by the blue-colored Trichrome Masson’s staining. Scale bar = 50 µm. (n=5–7 mice per group); ( C ) Quantification of total fibrotic area in cardiac tissue (% of total area); ( D ) Hydroxyproline content of mouse heart from young and old wt and Arg2 -/- female mice. (n=4 mice in each group); ( E ) Representative confocal images showing immunofluorescence staining of PDGF-Rα (green, fibroblasts marker) in young and old wt and Arg2 -/- heart tissue. DAPI (blue) is used to stain nuclei. Scale bar = 50 µm; ( F ) Relative PDGF-Rα signal quantification of confocal images (n=4 per each group). ( G ) Representative confocal images showing immunofluorescence staining of p16 (green, senescent marker) in young and old wt and Arg2 -/- heart tissue. DAPI (blue) is used to stain nuclei. Scale bar = 50 µm; ( H ) Percentage of p16 + nuclei in the four groups (n=4). The values shown are mean ± SD. Data are presented as the fold change to the young- wt group, except for panel D . *p≤0.05, **p≤0.01, ***p≤0.005, ****p≤0.001 between the indicated groups. wt, wild-type mice; Arg2 -/- , Arg2 gene knockout mice.

Article Snippet: Human cardiac fibroblasts (HCF) from adult human heart tissue were purchased from Innoprot ( P10452 ) and cultured in the proprietary fibroblast culture medium (P60108-2, Innoprot) composed as follows: 500 ml of fibroblast basal medium, 25 ml of FBS, 5 ml of fibroblast growth supplement-2, and 5 ml of penicillin/streptomycin in the Poly-L-Lysine (PLL) coated flasks and dishes.

Techniques: Quantitative RT-PCR, Staining, Immunofluorescence, Marker, Gene Knockout

Journal: eLife

Article Title: Cell-autonomous and non-cell-autonomous effects of Arginase 2 on cardiac aging

doi: 10.7554/eLife.94794

Figure Lengend Snippet: ( A ) Confocal microscopy illustration of immunofluorescence double staining of ARG2 (red) and TNNT (green; cardiomyocytes marker). Scale bar = 25 µm; ( B ) Bright-field microscopy images of isolated wt cardiomyocytes and primary cardiac fibroblasts. The immunoblot shows the level of ARG2 in both cardiomyocytes and fibroblasts upon exposure to hypoxia (1% O 2 ) for 24 hr. C indicates freshly isolated cardiomyocytes used as control, N and H indicate normoxia and hypoxia conditions, and K indicates kidney tissue extract used as positive control. Tubulin served as protein loading control; ( C ) mRNA expression levels of Arg2 in cardiomyocytes (card) and non-cardiomyocytes (non-c) cells isolated from old wt and Arg2 -/- female mouse hearts. Gapdh served as the reference. (n=3–5 mice per group); ( D to G ) Representative confocal images of old wt mouse heart showing co-localization of ( D ) ARG2 (red) and MAC-2 (green, mouse macrophage marker), ( E ) ARG2 (red) and CD31 (green, endothelial marker), ( F ) ARG2 (red) and PDGF-Rα (green, fibroblasts marker), and ( G ) ARG2 and α-smooth muscle actin (α-SMA; green, smooth muscle cell/myofibroblasts marker); ( H to M ) Representative confocal images of human heart tissue showing co-localization of ( H ) ARG2 (green) and TNNT (red, cardiomyocytes marker), ( I ) ARG2 (red) and CD31 (green, endothelial marker), ( J ) ARG2 (red) and CD-68 (green, macrophage marker), ( K ) ARG2 (red) and vimentin (green, fibroblast marker), and ( L–M ) ARG2 (red) and α-smooth muscle actin (α-SMA; green), ( L ) myofibroblasts and ( M ) smooth muscle cell marker. DAPI (blue) stains cell nuclei. Scale bar = 20 µm. Each experiment was repeated with 3–5 animals.

Article Snippet: Human cardiac fibroblasts (HCF) from adult human heart tissue were purchased from Innoprot ( P10452 ) and cultured in the proprietary fibroblast culture medium (P60108-2, Innoprot) composed as follows: 500 ml of fibroblast basal medium, 25 ml of FBS, 5 ml of fibroblast growth supplement-2, and 5 ml of penicillin/streptomycin in the Poly-L-Lysine (PLL) coated flasks and dishes.

Techniques: Confocal Microscopy, Immunofluorescence, Double Staining, Marker, Microscopy, Isolation, Western Blot, Control, Positive Control, Expressing

Journal: eLife

Article Title: Cell-autonomous and non-cell-autonomous effects of Arginase 2 on cardiac aging

doi: 10.7554/eLife.94794

Figure Lengend Snippet: ( A ) Collagen production measured as hydroxyproline content in mouse wt fibroblasts treated with conditioned media (CM) from young and old wt splenic cells (96 hr of incubation) (n=3 mice in each group). mRNA expression levels of ( B ) Mmp2 , ( C ) Mmp9, and ( D ) Col1a1 in wt fibroblasts treated with conditioned media (CM) from old wt and Arg2 -/- splenic cells (96 hr of incubation) were analyzed by qRT-PCR. Gapdh served as the reference. (n=4 mice per group). Data are expressed as fold change to respective control groups. *p≤0.05 between the indicated groups. MØ, splenic macrophage; CM, conditioned media; wt, wild-type mice; Arg2 -/- , Arg2 gene knockout mice.

Article Snippet: Human cardiac fibroblasts (HCF) from adult human heart tissue were purchased from Innoprot ( P10452 ) and cultured in the proprietary fibroblast culture medium (P60108-2, Innoprot) composed as follows: 500 ml of fibroblast basal medium, 25 ml of FBS, 5 ml of fibroblast growth supplement-2, and 5 ml of penicillin/streptomycin in the Poly-L-Lysine (PLL) coated flasks and dishes.

Techniques: Incubation, Expressing, Quantitative RT-PCR, Control, Gene Knockout

Journal: eLife

Article Title: Cell-autonomous and non-cell-autonomous effects of Arginase 2 on cardiac aging

doi: 10.7554/eLife.94794

Figure Lengend Snippet: ( A ) Collagen production measured as hydroxyproline content in mouse wt fibroblasts treated with conditioned media (CM) from old, wt, and Arg2 -/- splenic cells (96 hr incubation) (n=3 mice in each group). The values shown are mean ± SD. mRNA expression levels of ( B ) Fib , ( C ) Col3a1, and ( D ) Tgfb1 in wt fibroblasts treated with conditioned media (CM) from old wt and Arg2 -/- splenic cells (96 hr incubation) were analyzed by qRT-PCR. Gapdh served as the reference. (n=4 mice per group); ( E ) Hydroxyproline content in fibroblasts treated with CM from old wt splenic cells (96 hr incubation). ILRa (50 ng/ml) is used to prevent IL-1β binding to its receptor (n=4 independent experiments). ( F ) Immunoblotting analysis of ARG2 and vimentin in human cardiac fibroblasts (HCFs) upon Arg2 gene overexpression. GAPDH served as protein loading control; ( G ) qRT-PCR analysis of mRNA expression levels Arg2 in HCF cells; ( H ) The plot graph shows the quantification of the vimentin signals on immunoblots shown in panel F. (n=3 independent experiments); ( I ) qRT-PCR analysis of mRNA expression levels of Col3a1 in HCF cells. Gapdh served as the reference. (n=6 independent experiments); ( J ) Representative confocal images of human cardiac fibroblasts (HCF) upon transfection with rAd-CMV-Con/ Arg2 for 48 hr. MitoSOX (Red) is used to stain mitochondrial ROS (mtROS). TEMPO (10 μmol/l) is used to prevent mtROS generation. DAPI (blue) stains cell nuclei. Scale bar = 50 µm. ( K ) Mitosox signal quantification (n=3 independent experiments); ( L ) qRT-PCR analysis of mRNA expression levels of Col3a1 in HCF cells treated as indicated. Gapdh served as the reference. (n=5 independent experiments). ( M ) Immunoblotting analysis of ARG2 and HIF-1α in human cardiac fibroblasts (HCFs) upon 1% hypoxia incubation for 48 h. Tubulin served as a protein loading control; ( N ) Representative confocal images of HCFs under normoxia (21% O 2 ) or hypoxia (1% O 2 ) for 48 hr. MitoSOX (Red) is used to stain mitochondrial ROS (mtROS). TEMPO (10 μmol/l) is used to prevent mtROS generation. DAPI (blue) stains cell nuclei. Scale bar = 50 µm. ( O ) Representative confocal images of HCFs upon transfection with shRNA for Arg2 gene silencing under normoxia or hypoxia. MitoSOX (Red) is used to stain mitochondrial ROS (mtROS). DAPI (blue) stains cell nuclei. Scale bar = 25 µm. ( P ) MitoSOX signal quantification of images shown in panel O (n=3 independent experiments). Data are expressed as fold change to respective control group. *p≤0.05, **p≤0.01, ***p≤0.005 and ****p≤0.001 between the indicated groups. MØ, splenic macrophage; Con, control.

Article Snippet: Human cardiac fibroblasts (HCF) from adult human heart tissue were purchased from Innoprot ( P10452 ) and cultured in the proprietary fibroblast culture medium (P60108-2, Innoprot) composed as follows: 500 ml of fibroblast basal medium, 25 ml of FBS, 5 ml of fibroblast growth supplement-2, and 5 ml of penicillin/streptomycin in the Poly-L-Lysine (PLL) coated flasks and dishes.

Techniques: Incubation, Expressing, Quantitative RT-PCR, Binding Assay, Western Blot, Over Expression, Control, Transfection, Staining, shRNA