Low expression of monocyte-macrophage markers in the populations of podoplanin-positive cells in the infarcted myocardium.

<p>(<b>A</b>,<b>B</b>) Thin cardiac sections were indirectly immunolabeled with MOMA-2 (<b>A</b>) or F4/80 (<b>B</b>) antibodies and counterstained with hematoxylin and eosin. Time after MI is indicated. Spleen sections were included as positive controls. Areas in rectangles with the corresponding numbers are shown at a higher magnification in the adjacent insets. Note the presence of cells immunoreactive for MOMA-2 or F4/80 (arrows) in the inflamed epicardium (2 days, insets 2), myocardial interstitium (2 weeks, insets 2) and spleen, but not in the BZ of necrotic myocardium (2 days, insets 1) or the maturing scar (2 weeks, insets 1). (<b>C</b>) Flow-cytometry analysis of the F4/80 or CD11b expression in the podoplanin-positive cohorts populating the hearts of non-infarcted (NO), sham-operated (SHAM) and infarcted (MI) mice at 2 days after surgery. Graphs displaying individual values and the respective means (left), as well as representative scatterplots (right) are shown. Data represent frequency of double-positive cells within podoplanin-labeled populations (calculated as % cells in gate 2 out of the sum of cells in gate 1 and gate 2).</p

Expression of PDGFRα and PDGFRβ in podoplanin-positive populations.

<p>Thin cardiac sections were indirectly immunolabeled with podoplanin (red) and either PDGFRα (<b>A</b>-<b>D</b>; green), PDGFRβ (<b>E</b>-<b>H</b>; green), or PDGFRβ and Prox-1 (<b>I</b>; green and grey, respectively) antibodies. Nuclei, blue. In <b>A</b>-<b>H</b>, time after MI is indicated. Areas in rectangles are shown in the adjacent images for each color channel and merged. In <b>A</b>-<b>D</b>, podoplanin is frequently co-stained with PDGFRα at every time point; examples are indicated by white arrowheads. In <b>E</b>, podoplanin-positive cells are mostly PDGFRβ-negative, as exemplified by yellow arrowheads. In <b>F</b>-<b>H</b>, PDGFRβ distinctly co-stained with podoplanin. In <b>C</b> and <b>G</b>, quantitative image analyses (graphs, lower panels) of the fraction of podoplanin-expressing cells co-labeled with PDGFRα (<b>C</b>) or PDGFRβ (<b>G</b>) are shown at indicated times after MI. Data represent mean and SD of the % double-positive cells out of total podoplanin-positive cells; n = 5 image fields per group. By one-way ANOVA for PDGFRβ, *P < 0.0001 for 2 days vs. 2 weeks or 1 month; ns, not-significant for 1 month vs. 2 weeks. In <b>I</b>, arrows point to the examples of Prox-1 staining in the nuclei of PDGFRβ-positive podoplanin-negative BVs.</p

Phenotype of podoplanin-positive cells in the fibrotic tissue.

<p>Thin cardiac sections were indirectly immunolabeled with antibodies that recognize podoplanin (red) and either fibronectin and vimentin (<b>A</b>; green and grey, respectively), VEGFR-2 and CD34 (<b>B</b> and <b>C</b>; green and grey, respectively), or α-SMA (<b>D</b>; grey). Nuclei, blue. Time after MI is indicated. Areas in rectangles are shown at higher magnifications in the adjacent images for each color channel. Note that vimentin (<b>A</b>) or α-SMA (<b>D</b>) labeling is rarely detectable in podoplanin-expressing cells (examples are pointed by yellow arrowheads). In <b>B</b> and <b>C</b>, the podoplanin-presenting cells show minimal VEGFR-2 labeling. At 2 days after MI, the podoplanin-bearing cells mostly do not co-stain with CD34 (exemplified by yellow arrowheads). Starting 2 weeks after MI, the CD34 staining is present in irregular capillary-like structures (examples are indicated by white arrows). In <b>C</b>, quantitative image analysis demonstrating changes in the podoplanin co-labeling with CD34 at indicated times after MI is included in the graph (lower panel). Data represent mean and SD of the co-localization coefficient; n = 5–6 image fields per group. By one-way ANOVA, *P < 0.02 for 2 weeks vs. 2 days; **P < 0.0001 for 1 month vs. 2 days; ns, not-significant for 1 month vs. 2 weeks.</p

Variable expression of Prox-1 and VEGFR-3 in podoplanin-positive cells in the infarcted heart.

<p>Thin cardiac sections were indirectly immunolabeled with antibodies that recognize podoplanin (red) and either Prox-1 (<b>A</b>-<b>D</b>; green) or VEGFR-3 (<b>E</b>-<b>H</b>; green). Nuclei, blue. Time after MI is indicated. Areas in rectangles are shown in the adjacent images for each color channel and merged. White arrows indicate examples of Prox-1 or VEGFR-3 labeling in the lymphatic endothelial cells of CLVs, and white arrowheads point to the examples of Prox-1 or VEGFR-3 staining in podoplanin-expressing interstitial cells. Yellow arrowheads exemplify instances of podoplanin-positive cells in which Prox-1 or VEGFR-3 expression was undetectable. Note the consistent detection of Prox-1 and VEGFR-3 in CLVs, and the heterogeneity in Prox-1 and VEGFR-3 labeling intensity in podoplanin-stained cells not organized into vessels. In <b>C</b>, quantitative image analysis (graph, lower panel) of the fraction of podoplanin-expressing cells co-labeled with Prox-1 is shown at indicated times after MI. Data represent mean and SD of the % double-positive cells out of all podoplanin-positive cells in the imaging field; n = 6–9 image fields per group. By one-way ANOVA, no significant changes between the groups. In <b>G</b>, quantitative image analysis (graph, lower panel) of the podoplanin co-labeling with VEGFR-3 is shown. Data represent mean and SD of the co-localization coefficient measured at indicated times after MI; n = 4–7 image fields per group. By one-way ANOVA, **P = 0.002 for 2 weeks vs. 2 days; *P = 0.009 for 1 month vs. 2 days; ns, not-significant for 1 month vs. 2 weeks.</p

CLVs in the forming and mature scar.

<p>Thick cardiac sections were indirectly immunolabeled with the mix of LYVE-1 and podoplanin antibodies (<b>A</b>,<b>B</b>; red) and α-SMA antibody (<b>B</b>; grey), and co-stained with isolectin GS-IB4 (<b>A</b>,<b>B</b>; green). NO, non-operated. Time after MI is indicated. Corresponding single channel images are included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173927#pone.0173927.s003" target="_blank">S2 Fig</a>. CLVs are recognized by the staining with podoplanin and LYVE-1. In <b>A</b>, note the changes in the abundance and distribution of the vessels and LYVE-1 and podoplanin immunolabeled cells at different stages of infarct healing. In <b>B</b>, α-SMA-positive cells are apparent in the fibrotic tissue and the coating of large vessels.</p

Perivascular localization of the podoplanin-expressing cells.

<p>Thin cardiac sections were indirectly immunolabeled with podoplanin (red) and VEGFR-2 (green) antibodies. Nuclei, blue. Time after MI is indicated. Areas in rectangles are shown at a higher magnification in the adjacent images for each color channel and merged. Note the podoplanin-positive cells encircling the VEGFR-2-labeled BVs.</p