Abolished BMC transplantation-induced cardiac function recovery by HMGB1-inhibition.

<p>Cardiac parameters were measured by echocardiography (<b>A–D</b>) and catheterization (<b>E–H</b>) on day 28 after each treatment. Cardiac function was improved by BMC transplantation (BMC group) compared to the PBS injection control (CON group), while this effect was eliminated by antibody neutralization of HMGB1 (AB group), but not by control IgG administration (IgG group). LVFAC, left ventricular fractional area change; ECA, endocardial area. *:<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 = 8∼10 in each group.</p

Histological findings of BMMNC retention 5 min after IC injection.

Recipient hearts were collected at 5 minutes after IC injection of 8x106 BMMNC that had been labeled with PKH67. A and B represent low- and high-magnification images of Isolectin-B4 staining, respectively (scale bar = 100 μm [A] and 30 μm [B]). Representative micro images from n = 4 hearts are presented. All donor BMMNC (green) retained in the heart were found to be entrapped in isolation and within the coronary microvasculature. Blue signals for nuclei (DAPI); red for endothelial cells (Isolectin-B4).</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

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

Relationship of cell-surface proteins and retention of BMMNC.

<p>The expression profiles of cell-surface proteins, including integrin and selectin ligand, on pre-injection BMMNC and BMMNC in the coronary effluent (collected over the five-minute duration post-IC injection of 8x10⁶ BMMNC) were compared by flow cytometric analysis. No difference was found in any of the surface proteins investigated (n = 6 hearts were studied, unpaired T-test), suggesting that these cell-surface proteins are not critical for retention in normal hearts.</p

Different distribution of retained BMMNC between ventricular layers.

<p>At 5 minutes after IC injection of 8x10⁶ PKH67-labeled BMMNC, the number of donor cells within the endocardium-side myocardium, central myocardium and epicardium-side myocardium were counted in cross-section of immunohistolabelling samples. The concentration of cells progressively increased from the epicardium to the endocardium (n = 4, <i>p</i><0.0001, One-way ANOVA followed by Bonferroni post-hoc test).</p

Quantitative analysis of initial retention of BMMNC after IC injection.

(A) After IC injection of three different doses (1, 8, 40×106) of rat BMMNC in to a rat heart, the numbers of BMMNC in the coronary effluent in every minute were counted (Fig 1), from which their proportions to the total BMMNC exited in the coronary effluent over the first 5 minutes were calculated. It was observed that over 90% of the cells that were lost into the coronary effluent exited the heart within the first minute of injection. The proportion of cells lost did not differ between the doses (n = 8 hearts studied in each dose, p>0.99, Two-way repeated measures ANOVA). (B) Coronary flow rate was not altered by BMMNC injection using these three doses (n = 8 in each dose, p = 0.22, Two-way repeated measures ANOVA). The slight increase in volume during the first minute was due to injection of 3 ml of cell suspension. (C,D) Absolute numbers and rates of donor BMMNC retained in the heart were calculated. A larger number of injected cells resulted in a larger number of retained cells in the hearts (C, n = 8 in each dose, pp6 group, †p6 group). On the other hand, the proportion of retained cells of the total injected cell numbers was unchanged among the three doses (D; n = 8 in each dose, p = 0.67, One-Way ANOVA).</p

Histological findings of BMMNC retention 60 min after IC injection.

<p>Recipient hearts were collected at 60 minutes after IC injection of 8x10⁶ BMMNC that had been labeled stained with PKH67. <b>A</b> and <b>B</b> represent low- and high-magnification micro images of Isolectin-B4 staining, respectively (scale bar = 100 μm [A] and 30 μm [B]). Representative micro images from n = 4 hearts are presented. Retained BMMNC continued to be localized in the microvasculature in isolation. No donor cell (green) was observed to have extravasated out of the coronary vasculature. Blue signals for nuclei (DAPI); red for endothelial cells (Isolectin-B4).</p

Exit of BMMNC from the heart after IC injection.

<p>After IC injection of three different doses (1, 8, 40×10⁶) of rat BMMNC in to rat hearts using the Langendorff <i>ex-vivo</i> perfusion model, the numbers of BMMNC in the coronary effluent in every minute were counted. The majority of BMMNC injected exited from the heart into coronary effluent in 1 minute after IC injection of any cell dose studied. The pattern of cells exited from the heart did not differ between the doses (n = 8 different hearts studied in each dose, <i>p>0</i>.<i>99</i>, Two-way repeated measure ANOVA). Upper panel for 1×10⁶ BMMNC injection; Middle panel for 8×10⁶ BMMNC injection; Lower panel for 40×10⁶ BMMNC injection.</p