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