DOI: 10.5061/dryad.hx3ffbgrt
title: Cell-autonomous and non-cell-autonomous effects of Arginase 2 on cardiac aging

description: Aging is a predominant risk factor for heart disease. Aging heart reveals
 low-grade chronic inflammation, cell apoptosis, cardiac fibrosis, and
 increased vulnerability to ischemic injury. The underlying molecular
 mechanisms responsible for the cardiac aging and its susceptibility to
 injury are not fully understood. Although literature reports a role for
 mitochondrial Arginase 2 (ARG2) in heart failure, contradictory results
 are reported. How ARG2 participates in cardiac aging is still unknown. In
 this study, we demonstrate that Arg2 is not expressed in cardiomyocytes
 from aged mice and humans, but upregulated in non-myocyte cells, including
 macrophages, fibroblasts, endothelial cells. Mice with genetic deficiency
 of Arg2 (Arg2^-/-^) are protected from age-associated cardiac
 inflammation, myocyte apoptosis, interstitial and perivascular fibrosis,
 endothelial-mesenchymal transition (EndMT), and susceptibility to ischemic
 injury. Further experiments show that ARG2 mediates IL-1b release from
 macrophages of old mice, contributing to the cardiac aging phenotype. In
 addition, ARG2 enhances mitochondrial reactive oxygen species (mtROS) and
 activates cardiac fibroblasts that is inhibited by inhibition of mtROS.
 Thus, our study demonstrates a non-cell-autonomous effect of ARG2 on
 cardiomyocytes, fibroblasts, and endothelial cells mediated by IL-1b from
 aging macrophages as well as a cell-autonomous effect of ARG2 through
 mtROS in fibroblasts contributing to cardiac aging phenotype.
description: # Cell-autonomous and non-cell-autonomous effects of Arginase 2 on cardiac
 aging Dataset DOI: [10.5061/dryad.hx3ffbgrt](10.5061/dryad.hx3ffbgrt) ##
 Description of the data and file structure **Dataset Overview** This
 dataset contains the processed data used to test the hypothesis that the
 mitochondrial enzyme Arginase 2 (ARG2) contributes to cardiac aging.
 Specifically, the study demonstrates that ARG2 is not expressed in
 cardiomyocytes from aged mice and human cardiac tissue samples, but is
 upregulated in non-myocyte cell populations of the aging heart, including
 macrophages, fibroblasts, and endothelial cells. The dataset covers a
 range of measurements performed in both young and old wild-type (wt) mice
 and *Arg2* knockout (*Arg2^-/-^*) mice. The assessments include markers of
 age-associated cardiac inflammation, cardiomyocyte apoptosis, interstitial
 and perivascular fibrosis, endothelial-to-mesenchymal transition (EndMT),
 and susceptibility to ischemic injury. Data were generated using
 conventional and standardized molecular biology techniques such as
 real-time quantitative PCR (RT-qPCR), Western blotting, and
 immunofluorescence microscopy followed by quantitative image analysis. All
 individual data points underlying each graph or figure panel are reported
 in the associated data files. **Data Organization** The data are organized
 in files named according to the figure they correspond to in the
 associated manuscript. For example: Figure_1-source_data_1.zip contains
 data relevant to Figure 1. ### Files and variables
 **Figure1-source_data_1** (GraphPad Prism file) This file contains
 experimental data underlying the analyses related to cardiac fibrosis and
 aging in wild type (Wt) and *Arg2* knockout (KO; Arg2^-/-^) mice. The file
 includes multiple data tables, each corresponding to a different
 experimental measurement. For each dataset, experimental groups are
 organized in separate columns. Experimental groups: -          Wt = wild
 type animals -          KO = Arg2^-/-^ animals -          Y = young
 animals -          O = old animals Both male and female animals are
 included where indicated. Contents of the data tables: -          Figure
 1A: Expression levels of ARG2 in young and old male and female Wt mice
 -          Figure 1C: Percentage of fibrotic area in Wt vs. *Arg2^-/-^*
 (young and old) -          Figure 1D: Hydroxyproline content of left
 ventricular (LV) tissue in Wt vs. *Arg2^-/-^*^ ^(young and old) -         
 Figure 1F: PDGFRα positive signal across groups -          Figure 1H:
 Number of P16-positive nuclei across groups Each table is accompanied by a
 simple graph generated in GraphPad Prism to visualize the data
 distribution. **Figure_1_–_figure_supplement_1-source_data1.zip**
 (GraphPad Prism file) This file contains experimental data underlying the
 analyses related to *Arg2* and *Arg1* expression in Wt and *Arg2* knockout
 (KO; *Arg2^-/-^*) mice. The file includes multiple data tables, each
 corresponding to a different experimental measurement. For each dataset,
 experimental groups are organized in separate columns. Experimental
 groups: -          Wt = wild type animals -          KO = *Arg2^-/-^*
 animals -          Y = young animals -          O = old animals -         
 6mo, 18mo and 27mo = 6-, 18- and 27-months old animals Both male and
 female animals are included where indicated. Contents of the data tables:
 -          Figure 1 – figure supplement 1-source data1_A: Expression
 levels of Arg1 in young and old female Wt and Arg2-/- mice -         
 Figure 1 – figure supplement 1-source data1_B: Expression levels of Arg1
 in young and old male Wt mice -          Figure 1 – figure supplement
 1-source data1_C: Expression levels of Arg2 in 6-, 18- and 27-months old
 Wt mice -          Figure 1 – figure supplement 1-source data1_D:
 Expression levels of Arg1 in 6-, 18- and 27-months old Wt mice Each table
 is accompanied by a simple graph generated in GraphPad Prism to visualize
 the data distribution. **Figure_1_–_figure_supplement_2-source_data1.zip**
 (GraphPad Prism file) This file contains experimental data underlying the
 analyses related to collagen genes (*Col1a1* and *Col3a1*) expression in
 Wt and *Arg2* knockout (KO; Arg2^-/-^) mice. The file includes multiple
 data tables, each corresponding to a different experimental measurement.
 For each dataset, experimental groups are organized in separate columns.
 Experimental groups: -          Wt = wild type animals -          KO =
 *Arg2^-/-^* animals -          Y = young animals -          O = old
 animals Contents of the data tables: -          Figure 1 – figure
 supplement 1-source data2_A: Expression levels of *Col1a1* in young and
 old female Wt and *Arg2^-/-^* mice -          Figure 1 – figure supplement
 1-source data2_B: Expression levels of *Col3a1* in young and old female Wt
 and *Arg2^-/-^* mice Each table is accompanied by a simple graph generated
 in GraphPad Prism to visualize the data distribution.
 **Figure_2-source_data_1.zip** (GraphPad Prism file) This file contains
 experimental data underlying the analyses related to cardiac inflammation
 and macrophage characterization (infiltrating Vs resident cells) in Wt and
 *Arg2* knockout (KO; *Arg2^-/-^*) young and old mice. The file includes
 multiple data tables, each corresponding to a different experimental
 measurement. For each dataset, experimental groups are organized in
 separate columns. Experimental groups: -          Wt = wild type animals
 -          KO = *Arg2^-/-^* animals -          Y = young animals
 -          O = old animals Contents of the data tables: -          Figure
 2A: Expression levels of *Adgre1* (gene name of the macrophage specific
 marker F4-80) in young and old female Wt and *Arg2^-/-^* mice -         
 Figure 2B: Expression levels of *Mcp1* in Wt vs. *Arg2^-/-^* (young and
 old females) -          Figure 2C: Expression levels of *Il1b* in Wt vs.
 *Arg2^-/-^* (young and old females) -          Figure 2D: Expression
 levels of *Tnfa* in Wt vs. *Arg2^-/-^* (young and old females) -         
 Figure 2G: F4-80 positive cells across groups -          Figure 2H: LYVE1
 (resident marker) and F4-80 double positive cells across groups -         
 Figure 2I: CCR2 (infiltrating marker) and F4-80 double positive cells
 across groups Each table is accompanied by a simple graph generated in
 GraphPad Prism to visualize the data distribution.
 **Figure_2_-_figure_supplement_1-source_data1.zip** (TIFF image files)
 This folder contains microscopy images used to validate antibodies for
 macrophage population characterization. These data are provided as
 underlying material for the antibody validation experiments and are not
 restricted to the images shown in the manuscript. F4-80 is used as mouse
 pan-macrophage marker. LYVE1 identifies resident macrophages. CCR2
 identifies infiltrating macrophages. -          LYVE1 / F4-80
 colocalization images: Demonstrates LYVE1 as a marker for resident
 macrophages within the total F4-80⁺ macrophage population. -          CCR2
 / F4-80 colocalization images: Demonstrates CCR2 as a marker for
 infiltrating macrophages within the total F4-80⁺ population. Negative
 control images: provided for both LYVE1/F4-80 and CCR2/F4-80 staining to
 confirm antibody specificity. **Figure_3-source_data_1.zip** (GraphPad
 Prism file) This file contains experimental data underlying the analyses
 related to cardiac apoptosis (TUNEL positive cells were quantified) in Wt
 and *Arg2 *knockout (KO; *Arg2^-/-^*) young and old mice. The file
 includes multiple data tables, each corresponding to a different
 experimental measurement. For each dataset, experimental groups are
 organized in separate columns. Experimental groups: -          Wt = wild
 type animals -          KO = *Arg2^-/-^* animals -          Y = young
 animals -          O = old animals -          Non-myoc = non myocytes
 cells -          Myoc = cardiomyocytes Contents of the data tables:
 -          Figure 3B: TUNEL positive cells across groups -          Figure
 3D: TUNEL positive cardiomyocytes and non-myocytes cells in Wt and
 *Arg2^-/-^* animals Each table is accompanied by a simple graph generated
 in GraphPad Prism to visualize the data distribution.
 **Figure4-source_data_1.zip** (GraphPad Prism file) This file contains
 experimental data underlying the analyses related to endothelial to
 mesenchymal transition (EndMT) in cardiac tissue during aging in Wt and
 Arg2 knockout (KO; *Arg2^-/-^*) young and old mice. The file includes
 multiple data tables, each corresponding to a different experimental
 measurement. For each dataset, experimental groups are organized in
 separate columns. Experimental groups: -          Wt = wild type animals
 -          KO = *Arg2^-/- ^*animals -          Y = young animals
 -          O = old animals Contents of the data tables: -          Table
 4B: protein level of SNAIL (master regulator of EndMT) across groups
 -          Table 4C: protein level of Vimentin (mesenchymal marker) across
 groups -          Table 4D: protein level of CD31 (endothelial marker)
 across groups Each table is accompanied by a simple graph generated in
 GraphPad Prism to visualize the data distribution.
 **Figure4-source_data_2.zip** (PDF file) This file contains uncropped
 western blot membranes used for quantification of protein levels. The
 proteins analysed include SNAIL, Vimentin, and CD31, with normalization to
 the respective loading controls (Tubulin or Vinculin). Each image
 includes: -          The relevant experimental group or condition
 -          The molecular weight marker -          The full, uncropped
 membrane image Representative blot regions used for visualization in the
 associated publication are indicated with a red dotted outline.
 **Figure4-source_data_3.zip** (TIFF image files) This folder contains the
 original TIFF image files used for quantification of protein expression
 shown in the related analysis. In the folder the user will find
 sub-folders with following structure: -          CD31 - raw membrane
 images for CD31 staining -          SNAIL - raw membrane images for SNAIL
 staining -          Tubulin - raw membrane images for Tubulin loading
 control -          Vimentin - raw membrane images for Vimentin staining
 -          Vinculin - raw membrane images for Vinculin loading control
 These raw images provide the underlying data for the quantifications
 reported in the manuscript.
 **Figure_4_–*figure_supplement_1*-_source_data1.zip** (GraphPad Prism
 file) This file contains experimental data underlying the analyses related
 to heart weight to body weight of Wt young mice in comparison to old Wt
 and *Arg2^-/-^* old mice. The file includes one data table where
 experimental groups are organized in separate columns. Experimental
 groups: -          WT = wild type animals -          KO = *Arg2^-/-^*
 animals -          Y = young animals -          O = old animals The table
 is accompanied by a simple graph generated in GraphPad Prism to visualize
 the data distribution. **Figure5-source_data_1.zip** (GraphPad Prism file)
 This file contains experimental data underlying the analyses related
 *Arg2* expression levels in myocytes and non-myocytes cells isolated from
 Wt and *Arg2^-/-^* mice. The file includes one data table where
 experimental groups are organized in separate columns. Experimental
 groups: -          Wt = wild type animals -          KO = *Arg2^-/-^*
 animals -          Non-myoc = non myocytes cells -          Myoc =
 cardiomyocytes The table is accompanied by a simple graph generated in
 GraphPad Prism to visualize the data distribution.
 **Figure5-source_data_2.zip** (PDF file) This file contains uncropped
 western blot membranes shown in Figure 5C. The blots show the level of
 ARG2 in cardiomyocytes and fibroblasts isolated from mouse heart upon
 culturing at hypoxic conditions (1% Oxygen). The proteins analysed include
 ARG2 and the respective loading control Tubulin. Each image includes:
 -          The relevant experimental group or condition -          The
 molecular weight marker -          The full, uncropped membrane image
 Experimental groups: -          C = control -          N = normoxia
 -          H = hypoxia 1% oxygen -          Wt = wild type animals
 -          KO = *Arg2^-/-^* animals -          K = mouse kidney protein
 extract Representative blot regions used for visualization in the
 associated publication are indicated with a red dotted outline.
 **Figure5-source_data_3.zip** (TIFF image files) This folder contains the
 original TIFF image shown in the related figure. In the folder the user
 will find both raw membrane images for ARG2 and the related loading
 control Tubulin. **Figure6-source_data_1.zip** (GraphPad Prism file) This
 file contains experimental data underlying the analyses related to IL1β
 protein levels in cardiac tissue during aging in Wt and *Arg2^-/-^* young
 and old mice. The file includes multiple data tables, each corresponding
 to a different experimental measurement. For each dataset, experimental
 groups are organized in separate columns. Experimental groups: -         
 Wt = wild type animals -          KO = *Arg2^-/-^* animals -          O =
 old animals Contents of the data tables: -          Figure 6B: protein
 level of IL1β across groups -          Figure 6D: IL1β positive signal
 across groups Each table is accompanied by a simple graph generated in
 GraphPad Prism to visualize the data distribution.
 **Figure6-source_data_2.zip** (PDF file) This file contains uncropped
 western blot membranes used for quantification of protein levels. The
 proteins analysed include IL1β and the respective loading control Tubulin.
 Each image includes: -          The relevant experimental group or
 condition -          The molecular weight marker -          The full,
 uncropped membrane image Experimental groups: -          Wt = wild type
 animals -          Arg2^-/-^ = Arg2^-/-^ animals -          Young = young
 mice -          Old = old mice -          Raw = RAW 264.7 cell line
 Representative blot regions used for visualization in the associated
 publication are indicated with a red dotted outline.
 **Figure6-source_data_3.zip** (TIFF image files) This folder contains the
 original TIFF image files used for quantification of protein expression
 shown in the related analysis. In the folder the user will find sub-folder
 with following structure: -          IL1β - raw membrane images for IL1β
 -          Tubulin - raw membrane images for Tubulin loading control These
 raw images provide the underlying data for the quantifications reported in
 the manuscript. **Figure7-source_data1.zip** (GraphPad Prism file) This
 file contains experimental data underlying analyses of
 macrophage-cardiomyocyte crosstalk in vitro. The datasets include
 measurements of ARG2 and IL1β protein levels in spleen-derived macrophages
 isolated from Wt and *Arg2^-/-^* mice, both young and old. In addition,
 the file includes data on cardiomyocyte responses to
 macrophage-conditioned media, evaluated by the percentage of
 TUNEL-positive cells as an indicator of apoptosis. The file includes
 multiple data tables, each corresponding to a different experimental
 measurement. For each dataset, experimental groups are organized in
 separate columns. Experimental groups: -          WT = wild type animals
 -          KO = *Arg2^-/-^* animals -          Y = young animals
 -          O = old animals -          ILRa = IL receptor antagonist
 Contents of the data tables: -          Figure 7B: protein level of Arg2
 across groups -          Figure 7C: protein level of IL1β across groups
 -          Figure 7D: protein level of IL1β across groups assessed by
 ELISA of conditioned media -          Figure 7G: % of TUNEL positive
 cardiomyocytes cells Each table is accompanied by a simple graph generated
 in GraphPad Prism to visualize the data distribution.
 **Figure7-source_data2.zip** (PDF file) This file contains uncropped
 western blot membranes used for quantification of protein levels. The
 proteins analysed include ARG2, IL1β and the respective loading control
 Tubulin. Each image includes: -          The relevant experimental group
 or condition -          The molecular weight marker -          The full,
 uncropped membrane image Experimental groups: -          MØ = macrophage
 cells -          Wt = wild type animals -          Arg2^-/-^ = Arg2^-/-^
 animals -          Y = young mice Representative blot regions used for
 visualization in the associated publication are indicated with a red
 dotted outline. **Figure7-source_data3.zip** (TIFF image files) This
 folder contains the original TIFF image files used for quantification of
 protein expression shown in the related analysis. In the folder the user
 will find sub-folder with following structure: -          ARG2 - raw
 membrane images for ARG2 -          IL1β - raw membrane images for IL1β
 -          Tubulin - raw membrane images for Tubulin loading control These
 raw images provide the underlying data for the quantifications reported in
 the manuscript. **Figure7-_figure_supplement_1-source_data1.zip**
 (GraphPad Prism file) This file contains experimental data underlying
 analyses of RAW 264.7 mouse macrophage-cardiomyocyte crosstalk in vitro.
 The datasets include measurements of ARG2 and IL1β protein levels in RAW
 cells under LPS stimulation and *Arg2* gene silencing. In addition, the
 file includes data on cardiomyocyte responses to macrophage-conditioned
 media, evaluated by the percentage of TUNEL-positive cells as an indicator
 of apoptosis. The file includes multiple data tables, each corresponding
 to a different experimental measurement. For each dataset, experimental
 groups are organized in separate columns. Experimental groups: -         
 *Lacz* = control rAd expressing shRNA targeting *Lacz* -          *Arg2* =
 rAd expressing shRNA targeting *Arg2* -          C = control condition
 -          LPS = LPS stimulation -          CM = conditioned media
 Contents of the data tables: -         
 Figure7-figure_supplement1-source_data_1B: protein level of ARG2 across
 groups -          Figure7-figure_supplement1-source_data_1C: protein level
 of IL-1β across groups -         
 Figure7-figure_supplement1-source_data_1E: : % of TUNEL positive
 cardiomyocytes cells Each table is accompanied by a simple graph generated
 in GraphPad Prism to visualize the data distribution.
 **Figure7-_figure_supplement_1-source_data2.zip** (PDF file) This file
 contains uncropped western blot membranes used for quantification of
 protein levels. The proteins analysed include ARG2, IL1β and the
 respective loading control Tubulin. Each image includes: -          The
 relevant experimental group or condition -          The molecular weight
 marker -          The full, uncropped membrane image Experimental groups:
 -          Lacz = control rAd expressing shRNA targeting *Lacz* -         
 Arg2 = rAd expressing shRNA targeting *Arg2* -          LPS = LPS
 stimulation Representative blot regions used for visualization in the
 associated publication are indicated with a red dotted outline.
 **Figure7-_figure_supplement_1-source_data3.zip** (TIFF image files) This
 folder contains the original TIFF image files used for quantification of
 protein expression shown in the related analysis. In the folder the user
 will find sub-folder with following structure: -          ARG2 - raw
 membrane images for ARG2 -          IL1β - raw membrane images for IL-1β
 -          Tubulin - raw membrane images for Tubulin loading control These
 raw images provide the underlying data for the quantifications reported in
 the manuscript **Figure_7_–*figure_supplement_2*-_source_data1.zip**
 (GraphPad Prism file) This dataset contains the experimental data
 underlying the analyses of ARG2 and NOS2 in the regulation of IL-1β
 production in macrophages. The data include both protein and mRNA
 expression analyses performed in human THP1 macrophage-like cells, and
 protein levels analysis performed using mouse bone-marrow-derived
 macrophages (BMDMs) under LPS stimulation. The file includes multiple data
 tables, each corresponding to a different experimental measurement. For
 each dataset, experimental groups are organized in separate columns.
 Experimental groups: -          WT = wild type THP1 or BMDM cells
 -          *Arg2^-/-^* = *Arg2* knockout THP1 -          *NOS2^-/-^* =
 *NOS2* knockout BMDMs -          LPS = LPS stimulation The table is
 accompanied by a simple graph generated in GraphPad Prism to visualize the
 data distribution. **Figure_7_–*figure_supplement_2*-_source_data2.zip**
 (PDF file) This file contains uncropped western blot membranes used for
 quantification of protein levels. The proteins analysed include NOS2, ARG2
 and IL-1β with the respective loading controls (Tubulin or GAPDH). Each
 image includes: -          The relevant experimental group or condition
 -          The molecular weight marker -          The full, uncropped
 membrane image Experimental groups: -          THP1^wt^ = wild type THP1
 cells -          THP1^Arg2-/-^ = *Arg2* knockout THP1 cells -          MØ
 = mouse BMDM macrophage cells -          MØ^wt^ = wild type BMDMs
 -          MØ^NOS2-/-^ = *NOS2^-/-^* BMDMs -          LPS = LPS
 stimulation Representative blot regions used for visualization in the
 associated publication are indicated with a red dotted outline.
 **Figure_7_–*figure_supplement_2*-_source_data3.zip** (TIFF image files)
 This folder contains the original TIFF image files used for quantification
 of protein expression shown in the related analysis. In the folder the
 user will find 2 sub-folders with following structure: 1.      Figure 7
 -figure supplement 2A - raw membrane images for ARG2, NOS2 and Tubulin
 2.      Figure 7 -figure supplement 2D: a.      ARG2 - raw membrane images
 for ARG2 b.      GAPDH - raw membrane images for GAPDH c.      IL-1β - raw
 membrane images for IL-1β d.      NOS2 - raw membrane images for NOS2
 These raw images provide the underlying data for the quantifications
 reported in the manuscript **Figure8-source_data1.zip** (GraphPad Prism
 file) This file contains quantitative data underlying analyses of the
 crosstalk between splenic macrophages and cardiac fibroblasts and the
 cell-autonomous effects of ARG2 in cardiac fibroblasts. The data were
 collected from multiple in vitro experiments evaluating fibroblast
 responses to macrophage-conditioned media and to *Arg2* modulation. The
 file includes multiple data tables, each corresponding to a different
 experimental measurement. For each dataset, experimental groups are
 organized in separate columns. Labels to interpret experimental groups:
 -          WT = wild type animals -          KO = *Arg2^-/-^* animals
 -          O = old animals -          CM = conditioned media -          C
 = control -          *LacZ* = transfection with rAd-CMV-Control -         
 *Arg2+* = transfection with rAd-CMV-*Arg2* -          TEMPO = mito-TEMPO
 used to dampen mitochondrial-derived ROS -          *Lacz*-Sil = control
 rAd expressing shRNA targeting Lacz -          *Arg2*-Sil = rAd expressing
 shRNA targeting Arg2 -          N = Normoxia -          H = Hypoxia
 Contents of the data tables: -          Figure 8A: Hydroxyproline levels
 across groups -          Figure 8B: expression levels of *Fib *across
 groups -          Figure 8C: expression levels of *Col3a* across groups
 -          Figure 8D: expression levels of *Tgfb1* across groups
 -          Figure 8E: Hydroxyproline levels across groups -         
 Figure 8G: expression levels of *Arg2* across groups -          Figure 8H:
 protein levels of Vimentin across groups -          Figure 8I: expression
 levels of *Col3a* across groups -          Figure 8K: MitoSox signal
 detected in fibroblasts under the reported conditions -          Figure
 8L: expression levels of *Col3a* in fibroblasts under the reported
 conditions -          Figure 8P: MitoSox signal detected in fibroblasts
 under the reported conditions Each table is accompanied by a simple graph
 generated in GraphPad Prism to visualize the data distribution.
 **Figure8-source_data2.zip** (PDF file) This file contains uncropped
 western blot membranes used for quantification of protein levels. The
 proteins analysed include Vimentin, ARG2 and HIF-1α with the respective
 loading controls (Tubulin or Gapdh). Each image includes: -          The
 relevant experimental group or condition -          The molecular weight
 marker -          The full, uncropped membrane image Experimental groups:
 -          CMV *Lacz* = transfection with rAd-CMV-Control -          CMV
 *Arg2* = transfection with rAd-CMV-*Arg2* -          Hypoxia = culturing
 under hypoxic conditions (1% oxygen level) -          M Kidney = mouse
 kidney protein extract used as positive control Representative blot
 regions used for visualization in the associated publication are indicated
 with a red dotted outline. **Figure8-source_data3.zip** (TIFF image files)
 This folder contains the original TIFF image files used for quantification
 of protein expression shown in the related analysis. In the folder the
 user will find 2 sub-folders with following structure: 1.      Figure 8F:
 a.      ARG2 - raw membrane images for ARG2 b.      GAPDH - raw membrane
 images for GAPDH c.      Vimentin - raw membrane images for Vimentin
 2.      Figure 8M - raw membrane images for ARG2, Hif1α and Tubulin These
 raw images provide the underlying data for the quantifications reported in
 the manuscript **Figure_8_–*figure_supplement_1*-source_data1.zip**
 (GraphPad Prism file) This file contains quantitative data underlying
 analyses of the crosstalk between splenic macrophages and cardiac
 fibroblasts and the cell-autonomous effects of ARG2 in cardiac
 fibroblasts. The data were collected from multiple in vitro experiments
 evaluating fibroblast responses to macrophage-conditioned media and to
 ARG2 modulation. The file includes multiple data tables, each
 corresponding to a different experimental measurement. For each dataset,
 experimental groups are organized in separate columns. Labels to interpret
 experimental groups: -          WT = wild type animals -          KO =
 Arg2-/- animals -          Y = young animals -          O = old animals
 Contents of the data tables: -          Figure8-figure_supplement1-A:
 Hydroxyproline levels across groups -         
 Figure8-figure_supplement1-B: expression levels of *Mmp2 *across groups
 -          Figure8-figure_supplement1-C: expression levels of *Mmp9*
 across groups -          Figure8-figure_supplement1-D: expression levels
 of *Col1a1* across groups Each table is accompanied by a simple graph
 generated in GraphPad Prism to visualize the data distribution.
 **Figure9-source_data1.zip** (GraphPad Prism file) This file contains
 quantitative data underlying analyses of endothelial-to-mesenchymal
 transition (EndMT) in human umbilical vein endothelial cells (HUVECs)
 exposed to conditioned media (CM) from splenic macrophages of young and
 old mice, either Wt or *Arg2^-/-^*. The experiments investigate how aging
 and *Arg2* expression in macrophages regulate EndMT marker expression in
 endothelial cells. The file includes multiple data tables, each
 corresponding to a different experimental measurement. For each dataset,
 experimental groups are organized in separate columns. Experimental
 groups: -          WT = wild type animals -          KO = *Arg2^-/-
 ^*animals -          Y = young animals -          O = old animals
 -          C = control -          IRA = cells treated with IL receptor
 antagonist -          CM = conditioned media Contents of the data tables:
 -          Figure 9B: protein levels of ARG2 across groups -         
 Figure 9C: protein levels of N-Cadherin across groups -          Figure
 9D: protein levels of Vimentin across groups -          Figure 9E: protein
 levels of VE-Cadherin across groups -          Figure 9G: protein levels
 of ARG2 across groups -          Figure 9H: protein levels of N-Cadherin
 across groups -          Figure 9I: protein levels of Vimentin across
 groups -          Figure 9J: protein levels of VE-Cadherin across groups
 Each table is accompanied by a simple graph generated in GraphPad Prism to
 visualize the data distribution. **Figure9-source_data2.zip** (PDF file)
 These files contain uncropped western blot membranes used for
 quantification of protein levels. The proteins analysed include ARG2,
 Vimentin, N-Cadherin and VE-Cadherin with the respective loading control
 Gapdh. Each image includes: -          The relevant experimental group or
 condition -          The molecular weight marker -          The full,
 uncropped membrane image Experimental groups: -          WT = wild type
 animals -          Arg2^-/-^ = Arg2^-/- ^animals -          Young = young
 animals -          Old = old animals -          MØ-CM = conditioned media
 collected from splenic macrophages -          IRA = cells treated with IL
 receptor antagonist Representative blot regions used for visualization in
 the associated publication are indicated with a red dotted outline.
 **Figure9-source_data3.zip** (TIFF image files) This folder contains the
 original TIFF image files used for quantification of protein expression
 shown in the related analysis. In the folder the user will find 2
 sub-folders with following structure: 1.      Figure 9A: a.      ARG2 -
 raw membrane images for ARG2 b.      GAPDH - raw membrane images for GAPDH
 c.      Vimentin - raw membrane images for Vimentin d.      N-CAD - raw
 membrane images for N-Cadherin e.      V-CAD - raw membrane images for
 VE-Cadherin 2.      Figure 9F: a.      ARG2 - raw membrane images for ARG2
 b.      GAPDH - raw membrane images for GAPDH c.      Vimentin - raw
 membrane images for Vimentin d.      N-CAD - raw membrane images for
 N-Cadherin e.      V-CAD - raw membrane images for VE-Cadherin These raw
 images provide the underlying data for the quantifications reported in the
 manuscript **Figure10-source_data1.zip** (GraphPad Prism file) This file
 contains quantitative data underlying the analysis of cardiac functional
 recovery following global ischemia/reperfusion (I/R) injury in *ex-vivo*
 Langendorff-perfused hearts from aged female mice. The experiment
 evaluates the impact of *Arg2* gene ablation on myocardial performance
 during reperfusion. The file includes multiple data tables, each
 corresponding to a different experimental measurement. For figure 10B each
 dataset column (e.g. Figure10B_DataSource1 – A:Y1 to A:Y5) shows the time
 course (in minutes) of the recovery from I/R injury of either WT or
 *Arg2^-/-^* animals. Experimental groups: -          WT = wild type
 animals -          KO = *Arg2^-/-^* animals -          O = old animals
 Contents of the data tables: -          Table 10B_1: recovery of left
 ventricular developed pressure (LVDP) after ischemia / reperfusion in
 ex-vivo Langendorf experiment across groups -          Table 10B_2:
 recovery of left ventricular end-diastolic pressure (LVEDP) after ischemia
 / reperfusion in ex-vivo Langendorf experiment across groups -         
 Table 10B_3: recovery of maximal rate of contraction of LV (dP/dT max)
 after ischemia / reperfusion in ex-vivo Langendorf experiment across
 groups -          Table 10B_4: recovery of relaxation rate of LV (dP/dT
 min) after ischemia / reperfusion in ex-vivo Langendorf experiment across
 groups -          Table 10D: Infarct size across groups Each table is
 accompanied by a simple graph generated in GraphPad Prism to visualize the
 data distribution. **Figure_10_–*figure_supplement_1*-_source_data_1.zip**
 (GraphPad Prism file) This file contains quantitative data underlying the
 analysis of myocardial injury following short-duration global
 ischemia/reperfusion (I/R) in ex vivo Langendorff-perfused hearts from
 female mice. The experiment evaluates the extent of infarction in Wt and
 *Arg2^-/-^* animals after a mild I/R protocol. Experimental groups:
 -          WT = wild type animals -          KO = *Arg2^-/-^* animals
 -          O = old animals Contents of the data tables: -         
 Figure10-figure_supplement1B: Infarct size across groups
 **Figure_10_–*figure_supplement_2*-_source_data_1.zip** (GraphPad Prism
 file) This file contains quantitative data underlying the analysis of
 cardiac functional recovery following global ischemia/reperfusion (I/R)
 injury in *ex-vivo* Langendorff-perfused hearts from aged male mice. The
 experiment evaluates the impact of *Arg2* gene ablation on myocardial
 performance during reperfusion. The file includes multiple data tables,
 each corresponding to a different experimental measurement. For
 Figure10-figure_supplement2-B each dataset column (e.g.
 Figure10-figure_supplement2-B_1 – A:Y1 to A:Y5) shows the time course (in
 minutes) of the recovery from I/R injury of either WT or *Arg2^-/-^*
 animals. Experimental groups: -          WT = wild type animals -         
 KO = *Arg2^-/-^* animals -          O = old animals Contents of the data
 tables: -          Figure10-figure_supplement2-B_1: recovery of left
 ventricular developed pressure (LVDP) after ischemia / reperfusion in
 ex-vivo Langendorf experiment across groups -         
 Figure10-figure_supplement2-B_2: recovery of left ventricular
 end-diastolic pressure (LVEDP) after ischemia / reperfusion in ex-vivo
 Langendorf experiment across groups -         
 Figure10-figure_supplement2-B_3: recovery of maximal rate of contraction
 of LV (dP/dT max) after ischemia / reperfusion in ex-vivo Langendorf
 experiment across groups -          Figure10-figure_supplement2-B_4:
 recovery of relaxation rate of LV (dP/dT min) after ischemia / reperfusion
 in ex-vivo Langendorf experiment across groups -         
 Figure10-figure_supplement2-D: Infarct size across groups Each table is
 accompanied by a simple graph generated in GraphPad Prism to visualize the
 data distribution.