http://hdl.handle.net/10027/7817 Sun, 19 May 2013 09:18:41 GMT 2013-05-19T09:18:41Z http://hdl.handle.net/10027/8573 Improvement of cardiac function in mouse myocardial infarction after transplantation of epigenetically-modified bone marrow progenitor cells Rajasingh, Johnson; Thangavel, Jayakumar; Siddiqui, Mohammad R.; Gomes, Ignatius; Gao, Xiao-pei; Kishore, Raj; Malik, Asrar B. Objective To study usefulness of bone marrow progenitor cells (BPCs) epigenetically altered by chromatin modifying agents in mediating heart repair after myocardial infarction in mice. Methods and Results We tested the therapeutic efficacy of bone marrow progenitor cells treated with the clinically-used chromatin modifying agents Trichostatin A (TSA, histone deacetylase inhibitor) and 5Aza-2-deoxycytidine (Aza, DNA methylation inhibitor) in a mouse model of acute myocardial infarction (AMI). Treatment of BPCs with Aza and TSA induced expression of pluripotent genes Oct4, Nanog, Sox2, and thereafter culturing these cells in defined cardiac myocyte-conditioned medium resulted in their differentiation into cardiomyocyte progenitors and subsequently into cardiac myocytes. Their transition was deduced by expression of repertoire of markers: Nkx2.5, GATA4, cardiotroponin T, cardiotroponin I, α-sarcomeric actinin, Mef2c and MHC-α. We observed that the modified BPCs had greater AceH3K9 expression and reduced histone deacetylase1 (HDAC1) and lysine-specific demethylase1 (LSD1) expression compared to untreated BPCs, characteristic of epigenetic changes. Intra-myocardial injection of modified BPCs after AMI in mice significantly improved left ventricular function. These changes were ascribed to differentiation of the injected cells into cardiomyocytes and endothelial cells. Conclusion Treatment of BPCs with Aza and TSA converts BPCs into multipotent cells, which can then be differentiated into myocyte progenitors. Transplantation of these modified progenitor cells into infarcted mouse hearts improved left ventricular function secondary to differentiation of cells in the niche into myocytes and endothelial cells. © 2011 Rajasingh et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. DOI: 10.1371/journal.pone.0022550. Fri, 22 Jul 2011 05:00:00 GMT http://hdl.handle.net/10027/8573 2011-07-22T05:00:00Z http://hdl.handle.net/10027/8521 Sphingosine Kinase 1 Mediation of Expression of the Anaphylatoxin Receptor C5L2 Dampens the Inflammatory Response to Endotoxin Bachmaier, Kurt; Guzman, Edgar; Kawamura, Takeshi; Gao, Xiaopei; Malik, Asrar B. The complement anaphylatoxin C5a has a pathogenetic role in endotoxin-induced lung inflammatory injury by regulating phagocytic cell migration and activation. Endotoxin and C5a activate the enzyme sphingosine kinase (Sphk) 1 to generate the signaling lipid sphingosine-1-phosphate (S1P), a critical regulator of phagocyte function. We assessed the function of Sphk1 and S1P in experimental lung inflammatory injury and determined their roles in anaphylatoxin receptor signaling and on the expression of the two C5a receptors, C5aR (CD88) and C5L2, on phagocytes. We report that Sphk1 gene deficient (Sphk12/2) mice had augmented lung inflammatory response to endotoxin compared to wild type mice. Sphk1 was required for C5a-mediated reduction in cytokine and chemokine production by macrophages. Moreover, neutrophils from Sphk12/2 mice failed to upregulate the anaphylatoxin receptor C5L2 in response to LPS. Exogenous S1P restored C5L2 cell surface expression of Sphk12/2 mouse neutrophils to wild type levels but had no effect on cell surface expression of the other anaphylatoxin receptor, CD88. These results provide the first genetic evidence of the crucial role of Sphk1 in regulating the balance between expression of CD88 and C5L2 in phagocytes. S1P-mediated up-regulation of C5L2 is a novel therapeutic target for mitigating endotoxin-induced lung inflammatory injury. © 2012 Bachmaier et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. doi: 10.1371/journal.pone.0030742 Sun, 01 Jan 2012 06:00:00 GMT http://hdl.handle.net/10027/8521 2012-01-01T06:00:00Z http://hdl.handle.net/10027/8490 The redox‐sensitive cation channel TRPM2 modulates phagocyte ROS production and inflammation Di, Anke; Gao, Xiao‐Pei; Qian, Feng; Kawamura, Takeshi; Han, Jin; Hecquet, Claudie; Ye, Richard D; Vogel, Stephen M; Malik, Asrar B The NADPH oxidase activity of phagocytes and its generation of reactive oxygen species (ROS) is critical for host‐defense, but ROS overproduction can also lead to inflammation and tissue injury. Here we report that TRPM2, a nonselective and redox‐sensitive cation channel, inhibited ROS production in phagocytic cells and prevented endotoxin‐induced lung inflammation in mice. TRPM2‐deficient mice challenged with endotoxin (lipopolysaccharide) showed an enhanced inflammatory response and lower survival relative to that of wild‐type mice challenged with endotoxin. TRPM2 functioned by dampening NADPH oxidase–mediated ROS production through depolarization of the plasma membrane in phagocytes. As ROS also activate TRPM2, our findings establish a negative feedback mechanism for the inactivation of ROS production through inhibition of the membrane potential–sensitive NADPH oxidase. © 2011 by Nature Publishing Group, Nature Immunology doi:10.1038/ni.2171 Sat, 01 Jan 2011 06:00:00 GMT http://hdl.handle.net/10027/8490 2011-01-01T06:00:00Z http://hdl.handle.net/10027/8439 Src Phosphorylation of Endothelial Cell Surface ICAM-1 Mediates Neutrophil Adhesion and Contributes to the Mechanism of Lung Inflammation Liu, Guoquan; Vogel, Stephen M.; Gao, Xiaopei; Javaid, Kamran; Hu, Guochang; Danilov, Sergei M.; Malik, Asrar B.; Minshall, Richard D. Objective: To determine whether TNFα-induced Src activation and ICAM-1 phosphorylation rapidly increases endothelial cell adhesivity and PMN sequestration independent of de novo ICAM-1 synthesis. Methods and Results: TNFα exposure of mouse lungs for 5 min produced a 3-fold increase in 125I-anti-ICAM-1 mAb binding and 111In oxine-labeled PMN sequestration as well as Src activation, ICAM-1 Tyr518 phosphorylation, and pTyr518-ICAM-1 co-immunoprecipitation with actin. The response was absent in Nox2-/- lungs or following Src inhibition. In COS-7 cells transfected with wild-type (WT), phospho-defective (Y518F), or phospho-mimicking (Y518D) mouse ICAM-1 cDNA constructs, TNFα increased the Bmax of YN1/1.7.4 anti-ICAM-1 mAb binding to WT-ICAM-1 but not to Y518F-ICAM-1 indicating increased binding avidity secondary to ICAM-1 phosphorylation. This effect was mimicked by expression of the Y518DICAM- 1 mutant. TNFα also increased the staining intensity and cell surface clustering of YN1/1.7.4 mAb-labeled WT-ICAM-1 that co-localized with F-actin which was not observed with Y518F-ICAM-1 but was recapitulated with Y518D-ICAM-1. Finally, overexpression of ICAM-1 in mouse lungs significantly increased LPS-induced transvascular albumin leakage and bronchoalveolar lavage PMN counts at 2 and 24 hrs after LPS inhalation compared to lungs expressing Y518F ICAM-1 mutant. Conclusions: Src-dependent phosphorylation of endothelial cell ICAM-1 Tyr518 induces PMN adhesion by promoting ICAM-1 clustering which we propose mediates rapid-phase lung vascular accumulation of PMNs during inflammation. Post print version of article may differ from published version. The definitive version is available through American Heart Association at DOI: 10.1161/ATVBAHA.110.222208 Wed, 01 Jun 2011 05:00:00 GMT http://hdl.handle.net/10027/8439 2011-06-01T05:00:00Z