miR-150 targets Cxcr4 and regulates MNC migration.

<p>(A) A putative target site of miR-150 highly conserved in the <i>Cxcr4</i> mRNA 3′-UTR as predicted by computational analysis. (B) miR-150 expression was decreased in BM-MNCs after AMI as validated by real-time PCR. (C) CXCR4 protein expression in MNCs significantly increased by transfection with miR-150 inhibitor. (D) BM-MNCs isolated from mice with LAD ligation enhanced migration capacity of BM-MNCs in response to SDF-1α as evaluated by transwell migration system. Transfection of wild type MNCs with anti-miR-150 also increased the number of migrating cells.</p

AMI increases the numbers of BM-MNCs and CXCR4 positive MNCs.

<p>(A) MNCs were isolated at 1, 3 and 5 days after left anterior descending artery ligation (LAD) and analyzed for molecular characterization. (B) The number of PB-MNCs decreased at one day after LAD ligation and increased gradually up to 5 days (n≥10 in each groups). (C) The percentage of CXCR4 positive cells in MNCs increased in both PB and BM after LAD ligation as compared to control. (n = 8 in control group and n = 6 in AMI group). (D) Densitometric analysis of CXCR4 positive MNCs counted after LAD ligation.</p

Lentivirus-mediated knockdown of miR-150 augments CXCR4 expression and mobilization of MNCs.

<p>(A) Structures of Lenti-miR-150 inhibitor and Lenti-Sc which contains mCherry reporter gene (red signal), (B) Titeration and transduction efficiency of lentivirus-mediated miR-150 inhibition in MNCs was evaluated by mCherry signal under microsope. (C) Lentivirus-mediated knockdown of miR-150 elevated CXCR4 protein expression in MNCs. (D) Number of BM-MNCs mobilized to PB was markedly increased in peripheral circulation of BN transplantation of mice which received MNCs lacking miR-150.</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

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

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