Extracellular High Mobility Group Box 1 Plays a Role in the Effect of Bone Marrow Mononuclear Cell Transplantation for Heart Failure

Transplantation of unfractionated bone marrow mononuclear cells (BMCs) repairs and/or regenerates the damaged myocardium allegedly due to secretion from surviving BMCs (paracrine effect). However, donor cell survival after transplantation is known to be markedly poor. This discrepancy led us to hypothesize that dead donor BMCs might also contribute to the therapeutic benefits from BMC transplantation. High mobility group box 1 (HMGB1) is a nuclear protein that stabilizes nucleosomes, and also acts as a multi-functional cytokine when released from damaged cells. We thus studied the role of extracellular HMGB1 in the effect of BMC transplantation for heart failure. Four weeks after coronary artery ligation in female rats, syngeneic male BMCs (or PBS only as control) were intramyocardially injected with/without anti-HMGB1 antibody or control IgG. One hour after injection, ELISA showed that circulating extracellular HMGB1 levels were elevated after BMC transplantation compared to the PBS injection. Quantitative donor cell survival assessed by PCR for male-specific sry gene at days 3 and 28 was similarly poor. Echocardiography and catheterization showed enhanced cardiac function after BMC transplantation compared to PBS injection at day 28, while this effect was abolished by antibody-neutralization of HMGB1. BMC transplantation reduced post-infarction fibrosis, improved neovascularization, and increased proliferation, while all these effects in repairing the failing myocardium were eliminated by HMGB1-inhibition. Furthermore, BMC transplantation drove the macrophage polarization towards alternatively-activated, anti-inflammatory M2 macrophages in the heart at day 3, while this was abolished by HMGB1-inhibition. Quantitative RT-PCR showed that BMC transplantation upregulated expression of an anti-inflammatory cytokine IL-10 in the heart at day 3 compared to PBS injection. In contrast, neutralizing HMGB1 by antibody-treatment suppressed this anti-inflammatory expression. These data suggest that extracellular HMGB1 contributes to the effect of BMC transplantation to recover the damaged myocardium by favorably modulating innate immunity in heart failure

microRNA-150 Regulates Mobilization and Migration of Bone Marrow-Derived Mononuclear Cells by Targeting

The interaction between chemokine receptor type 4 (CXCR4) and its ligand, stromal cell-derived factor (SDF)-1, plays an important role in stem cell mobilization and migration in ischemic tissues. MicroRNAs (miRs) are key regulators of stem cell function and are involved in regulation of stem cell survival and differentiation to adopt different cell lineages. In this study, we show that ischemia inhibits the expression of miR-150 in BM-derived mononuclear cells (MNC) and activates its target Cxcr4 gene. Our results show that miR-150/CXCR4 cascade enhances MNC mobilization and migration. By using mouse acute myocardial infarction (MI) model, we found that MNCs in peripheral blood (PB) were increased significantly at day 5 after AMI as compared to control group and the number of CXCR4 positive MNCs both in bone marrow (BM) and PB was also markedly increased after MI. Analysis by microarray-based miRNA profiling and real-time PCR revealed that the expression of miR-150 which targets Cxcr4 gene as predicted was significantly downregulated in BM-MNCs after MI. Abrogation of miR-150 markedly increased CXCR4 protein expression suggesting its target gene. To show that miR-150 regulates MNC mobilization, knockdown of miR-150 in BM-MNCs by specific antisense inhibitor resulted in their higher migration ability in vitro as compared to scramble-transfected MNCs. Furthermore, in vivo BM transplantation of MNCs lacking miR-150 expression by lentiviral vector into the irradiated wild type mice resulted in the increased number of MNCs in PB after AMI as compared to control. In conclusion, this study demonstrates that ischemia mobilizes BM stem cells vi

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

Compression Stockings Suppressed Reduced Muscle Blood Volume and Oxygenation Levels Induced by Persistent Sitting

This study quantitatively analyzed the effects of 3 h of constant sitting on skeletal muscle oxygenation in the lower extremities, using near-infrared time-resolved spectroscopy (NIRTRS). The effects of compression stockings were also evaluated. Eleven healthy men (age, 30.0 &#177; 6.7 years) maintained their knee joints at 90&#176; flexion during 3 h of constant sitting and wore a compression stocking on either the right or left leg. The side the stocking was worn was chosen randomly. Subsequently, leg circumference and extracellular water were measured. After 3 h of sitting, both factors increased significantly in uncompressed limbs. Furthermore, intracellular water and muscle oxygenation had significantly decreased. In contrast, extracellular water had not increased in the limbs wearing compression stockings. Furthermore, the increased circumference of compressed limbs was significantly smaller than that of uncompressed limbs. Decreases in oxygenated hemoglobin and total hemoglobin were significantly smaller in compressed limbs than in uncompressed limbs (oxy-Hb; p = 0.021, total-Hb; p = 0.013). Three hours of sitting resulted in decreased intracellular water and increased extracellular water in the lower extremities, leading to reduced blood volume and oxygenation levels in skeletal muscle. Compression stockings successfully suppressed these negative effects

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