Eliminated BMC transplantation-induced tissue recovery by HMGB1-inhibition.

<p>Reduced extracellular collagen deposition (<b>A–C;</b> picrosirius red = red), increased capillary density (<b>D–F;</b> Isolectin B4 = red), and increased proliferation (<b>G–I;</b> Ki67 = red; nuclei = blue; cTnT = green) were observed in the border areas at day 28 after BMC transplantation (BMC group), compared to the PBS control (CON group). These effects were all abolished by anti-HMGB1 antibody neutralization (AB group), but not by control IgG administration (IgG group). Representative images of only BMC and AB groups are present (see <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076908#pone.0076908.s002" target="_blank">Figure S2</a></b> for additional images). Scale bars = 50 µm in <b>A, B, G, H</b> and 30 µm in <b>D, E</b>. *:<i>p</i><0.05 <i>versus</i> the CON group, ^†:<i>p</i><0.05 <i>versus</i> the BMC group, ^‡:<i>p</i><0.05 <i>versus</i> the IgG group, mean±SEM for n = 5∼7 in each group.</p

Poor donor cell survival and HMGB1 leakage after BMC transplantation.

<p>(<b>A</b>) Quantitative PCR for the male specific <i>sry</i> gene showed that the survival of male donor cells in female hearts was poor similarly in the BMC (BMC injection), IgG (BMC+control IgG injection), and AB (BMC+anti-HMGB1 antibody injection) groups at both days 3 and 28; n = 5∼7 in each point. (<b>B</b>) Clusters of DiI-labeled (red) donor BMCs were detected in the heart at day 3 after BMC transplantation. A higher magnification image of the yellow frame is shown. Green = cardiomyocytes (cTnT); blue = nuclei (DAPI). Scale bar = 300 µm. (<b>C</b>) ELISA showed that the circulating HMGB1 level was increased at 1 hour in the BMC group compared to the PBS injection control (CON group). *:<i>p</i><0.05 <i>versus</i> the CON group, mean±SEM for n = 5 each.</p

Modulation of innate immunity by BMC transplantation via released HMGB1.

<p>Accumulation of CD68⁺ pan-macrophages (<b>A</b>), CD86⁺ classically-activated pro-inflammatory M1 macrophages (<b>B</b>), and CD163⁺ alternatively-activated anti-inflammatory M2 macrophages (<b>C</b>) in the border areas at day 3 after each treatment was assessed by immunolabeling. See <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076908#pone.0076908.s003" target="_blank">Figure S3</a></b> for representative images. Myocardial expression of <i>IL-10</i> (<b>D</b>), <i>IL-1β</i> (<b>E</b>)), and <i>TNF-α</i> (<b>F</b>) at day 3 after each treatment was measured by quantitative RT-PCR. *:<i>p</i><0.05 <i>versus</i> the CON group, ^†:<i>p</i><0.05 <i>versus</i> the BMC group, mean±SEM for n = 5∼7 in each group.</p

Extracellular collagen and capillary density in the myocardium.

<p>A: Extracellular collagen deposition in the myocardium was assessed by Picrosirus red staining at day 28. Representative pictures in the peri-infarct area show a marked accumulation of extracellular collagen (red colour) in the PBS-IM and PBS-IC groups, compared to the SMB-IM and SMB-IC groups (×400). B: Representative pictures of the infarct-remote area also show reduced collagen accumulation in the SMB-IM and SMB-IC groups, compared to the PBS-IM and PBS-IC groups (×400). C and D: Collagen volume in both the peri-infarct © and infarct-remote (D) areas, analysed by computer-assisted morphometry, was significantly smaller in the SMB-IM and SMB-IC groups, compared to the corresponding PBS groups. E: Capillary number at day 28, assessed by immunoconfocal microscopy (red for vWF, blue for nuclei), appeared to be similar among the groups. F: Capillary density, expressed as the number of capillary vessels per mm², did not show a significant difference among any groups. Scale bar = 100 µm. *<i>p</i><0.05 <i>vs.</i> the PBS-IM group; ^†<i>p</i><0.05 <i>vs.</i> the PBS-IC group. <i>N</i> = 5 in each group.</p

Cardiac performance after SMB transplantation.

<p><i>n</i> = 10 in each group at each time point.</p>*<p><i>p</i><0.05 <i>vs.</i> PBS-IM.</p>†<p><i>p</i><0.05 <i>vs.</i> PBS-IC.</p

Behaviour of grafted cells in the myocardium.

<p>A: Immunohistolabelling for GFP with haematoxylin counterstaining detected GFP-positive (brown) cell-clusters disrupting the myocardial structure in the SMB-IM group at day 3. B: In higher magnification of the outlined area in A, the cell-clusters consisted of GFP-positive and negative cells. C: Confocal micrograph (green for GFP, red for CD45, blue for nuclei) in the SMB-IM group at day 3 shows that GFP-positive cells are surrounded by a number of GFP-negative CD45-positive cells. D: Confocal micrograph (green for GFP, red for Cx43) in the SMB-IM group at day 3 shows the lack of gap junctions between GFP-positive cells and adjacent GFP-negative cardiomyocytes. E: At day 28 in the SMB-IM group, a small number of GFP-positive cells are surrounded by fibrous tissues. F: Confocal micrograph (green for GFP, red for Cx43) in the SMB-IM group at day 28 shows no gap-junctions between the GFP-positive cells and adjacent GFP-negative cardiomyocytes. G: At day 3 in the SMB-IC group, GFP-positive cells were widely disseminated without disrupting the myocardial structure. Please note that it is difficult to see individual disseminated cells in the low magnification image. H: Higher magnification of the outlined areas in G shows GFP-positive cells with less myocardial damage and inflammation, compared to the SMB-IM group. I: Confocal micrograph (green for GFP, red for CD45, blue for nuclei) shows less accumulation of CD45-positive cells surrounding the GFP-positive cells compared to the SMB-IM group. J: Confocal micrograph (green for GFP, red for Cx43) in the SMB-IC group at day 3 does not show any gap-junctions between the GFP-positive cells and cardiomyocytes. K: At day 28 in the SMB-IC group, a small number of GFP-positive cells were detected. L: Confocal micrograph (green for GFP, red for Cx43) shows the only example of a GFP-positive cell with cardiomyocyte-like morphology forming gap-junctions with a native cardiomyocyte (arrows) in the SMB-IC group. Scale bar = 500 µm in A and F, 50 µm in B–E and G–L.</p

Size of native cardiomyocytes.

<p>The size of native cardiomyocytes, isolated by enzymatic digestion at day 28 after cell transplantation, was assessed. Cardiomyocytes in the SMB-IM and SMB-IC groups were significantly smaller than those in the PBS-IM group, but still larger than those from the normal hearts. <i>n</i> = 5 in the PBS-IM, SMB-IM and SMB-IC groups; <i>n</i> = 6 in the Normal group. *<i>p</i><0.05 <i>vs.</i> the Normal group; ^†<i>p</i><0.05 <i>vs.</i> the PBS-IM group.</p

Spontaneous and induced occurrence of arrhythmias.

<p>A: The hourly number of spontaneous VPC was calculated as an average of the VPC number detected over the 24-hour continuous ECG recording by telemetry. VPC were frequently detected at day 1 only in the SMB-IM group with a peak at day 3. The VPC number in the SMB-IM group decreased by day 14, but a small number of VPC were persistent until day 84. In contrast, in the SMB-IC group, VPC rarely occurred until day 7, while a small number of VPC were observed between day 14 and 84. Both the PBS-IM or PBS-IC groups rarely showed VPC throughout the study. <i>n</i> = 7 in the PBS-IM and PBS-IC groups; <i>n</i> = 8 in the SMB-IM and SMB-IC groups at each time point. *<i>p</i><0.05 <i>vs.</i> the PBS-IM group; ^†<i>p</i><0.05 <i>vs.</i> the PBS-IC group at each time point. B: Representative ECG of spontaneous VT in the SMB-IM group. C and D: Representative ECGs of induced VT by isoproterenol administration under Langendorff isolated-heart perfusion in the SMB-IM (C) and SMB-IC groups (D). Scale bar = 500 msec.</p

Isoproterenol-induced arrhythmias.

<p><i>n</i> = 7 in the PBS-IM and PBS-IC groups; <i>n</i> = 8 in the SMB-IM and SMB-IC groups.</p>*<p><i>p</i><0.05 <i>vs.</i> baseline value.</p>†<p><i>p</i><0.05 <i>vs.</i> the PBS-IM group.</p>‡<p><i>p</i><0.05 <i>vs.</i> the PBS-IC group.</p

Survival of grafted cells in the myocardium.

<p>Time-course of donor-derived cell number present in the native LV was estimated by real-time PCR for the Y-chromosome specific gene, <i>Sry</i>. Both the SMB-IM and SMB-IC groups showed a small number of donor-derived cells at day 3, which further decreased by day 28. <i>n</i> = 5 in each group at each time point.</p