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. Scale bars?=?50 m.(TIF) pone.0076908.s003.tif (1.0M) GUID:?49940B0C-017E-4111-BE04-09F95E7FCCBA Abstract 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 (E)-Alprenoxime 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 Rabbit Polyclonal to ITGA5 (L chain, Cleaved-Glu895) 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 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 (E)-Alprenoxime 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 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. Introduction Transplantation of stem or progenitor cells is an emerging approach to repair and/or regenerate damaged myocardium undergoing adverse ventricular remodeling. Unfractionated bone marrow mononuclear cells (BMCs) contain several kinds of stem/progenitor cells and are the most frequently used donor cell type in clinical cell therapy to the heart [1]. The therapeutic effect of BMC transplantation in not only acute myocardial infarction (MI) but also post-MI chronic heart failure (ischemic cardiomyopathy) has been confirmed in animal and human studies [1]C[3]. Because injected BMCs do not vigorously differentiate to functioning cardiomyocytes or vascular cells gene in these samples was assessed by real-time polymerase chain reaction (PCR; Prism 7900HT, Applied Biosystems). The levels were normalized to the DNA amount using the autosomal single copy gene, oesteopontin. The number of surviving donor cells was estimated by correcting the relative expression using a standard curve as previously described [3], [17], [22]. Measurements of Myocardial Gene Expression Total RNA was extracted from the whole LV samples and assessed for myocardial expression of and by quantitative RT-PCR (Prism 7900HT, Applied Biosystems) as previously described [22]. TaqMan primers and probes were purchased from Applied Biosystems. Expression was normalized using gene demonstrated that donor cell survival after BMC transplantation was poor similarly in the BMC, AB, and IgG groups; below 10% at day (E)-Alprenoxime 3, further decreasing to below 1% by day 28 ( Figure 1A ). Histological analysis detected islet-like clusters of donor cells at day 3 after BMC transplantation ( Figure (E)-Alprenoxime 1B ). ELISA showed that the circulating extracellular HMGB1 level was 2.5-fold elevated one hour after BMC transplantation, compared to the PBS injection control ( Figure 1C ). Open in a separate window Figure 1 Poor donor cell survival and HMGB1 leakage after BMC transplantation.(A) Quantitative PCR for the male specific gene showed that the survival of male donor cells in female hearts was poor similarly (E)-Alprenoxime 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?=?57 in each point. (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 m. (C) ELISA showed that the circulating HMGB1 level was increased at 1 hour in the BMC group compared to the PBS injection control (CON group). *:the CON group, meanSEM for n?=?5 each. Abolished BMC Transplantation-induced Cardiac Function Recovery by HMGB1-inhibition Four weeks after BMC transplantation (BMC group), echocardiography and cardiac catheterization consistently demonstrated that both systolic and diastolic LV function, in terms of LV fractional area change, max and min dP/dt, and systolic pressure, was improved compared to the control (CON group; Figure 2 ). Enlargement of LV systolic endocardial area in the control group was attenuated by BMC transplantation. Of note,.