Effect of Bone Marrow-Derived Mesenchymal Stem Cells on Endotoxin-Induced Oxidation of Plasma Cysteine and Glutathione in Mice

Bone marrow-derived mesenchymal stem cells (BMDMSC) are emerging as a therapeutic modality in various inflammatory disease states, including acute lung injury (ALI). A hallmark of inflammation, and a consistent observation in patients with ALI, is a perturbation in the systemic redox environment. However, little is known about the effects of BMDMSC on the systemic redox status. The objective of the present study was to determine whether exogenously infused BMDMSC protect against endotoxin-induced oxidation of plasma cysteine (Cys) and glutathione (GSH) redox states. To determine the effect on the redox state if BMDMSC, mice received endotoxin intraperitoneally (1 mg/kg), followed by intravenous infusion of either 5 × 105 BMDMSC or an equal volume of saline solution. Control mice received intraperitoneal endotoxin followed by 5 × 105 lung fibroblasts given intravenously. Cys, cystine (CySS), GSH, and glutathione disulfide (GSSG) concentrations were determined by HPLC. Results showed sequential preservation of plasma Cys and GSH levels in response to BMDMSC infusion. The data show that BMDMSC infusion leads to a more reducing Cys and GSH redox state. The findings are the first to demonstrate that BMDMSC have antioxidant effects in vivo, and add to our understanding of the systemic effects of BMDMSC in lung injury

Inhibition of the CXCL12/CXCR4-axis as preventive therapy for radiation-induced pulmonary fibrosis

Background: A devastating late injury caused by radiation is pulmonary fibrosis. This risk may limit the volume of irradiation and compromise potentially curative therapy. Therefore, development of a therapy to prevent this toxicity can be of great benefit for this patient population. Activation of the chemokine receptor CXCR4 by its ligand stromal cell-derived factor 1 (SDF-1/CXCL12) may be important in the development of radiation-induced pulmonary fibrosis. Here, we tested whether MSX-122, a novel small molecule and partial CXCR4 antagonist, can block development of this fibrotic process. Methodology/Principal Findings: The radiation-induced lung fibrosis model used was C57BL/6 mice irradiated to the entire thorax or right hemithorax to 20 Gy. Our parabiotic model involved joining a transgenic C57BL/6 mouse expressing GFP with a wild-type mouse that was subsequently irradiated to assess for migration of GFP+ bone marrow-derived progenitor cells to the irradiated lung. CXCL12 levels in the bronchoalveolar lavage fluid (BALF) and serum after irradiation were determined by ELISA. CXCR4 and CXCL12 mRNA in the irradiated lung was determined by RNase protection assay. Irradiated mice were treated daily with AMD3100, an established CXCR4 antagonist; MSX-122; and their corresponding vehicles to determine impact of drug treatment on fibrosis development. Fibrosis was assessed by serial CTs and histology. After irradiation, CXCL12 levels increased in BALF and serum with a corresponding rise in CXCR4 mRNA within irradiated lungs consistent with recruitment of a CXCR4+ cell population. Using our parabiotic model, we demonstrated recruitment of CXCR4+ bone marrow-derived mesenchymal stem cells, identified based on marker expression, to irradiated lungs. Finally, irradiated mice that received MSX-122 had significant reductions in development of pulmonary fibrosis while AMD3100 did not significantly suppress this fibrotic process. Conclusions/Significance: CXCR4 inhibition by drugs such as MSX-122 may alleviate potential radiation-induced lung injury, presenting future therapeutic opportunities for patients requiring chest irradiation. © 2013 Shu et al

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Bone marrow-derived mesenchymal stem cells (BMDMSC) are emerging as a therapeutic modality in various inflammatory disease states, including acute lung injury (ALI). A hallmark of inflammation, and a consistent observation in patients with ALI, is a perturbation in the systemic redox environment. However, little is known about the effects of BMDMSC on the systemic redox status. The objective of the present study was to determine whether exogenously infused BMDMSC protect against endotoxininduced oxidation of plasma cysteine (Cys) and glutathione (GSH) redox states. To determine the effect on the redox state if BMDMSC, mice received endotoxin intraperitoneally (1 mg/kg), followed by intravenous infusion of either 5 × 10 5 BMDMSC or an equal volume of saline solution. Control mice received intraperitoneal endotoxin followed by 5 × 10 5 lung fibroblasts given intravenously. Cys, cystine (CySS), GSH, and glutathione disulfide (GSSG) concentrations were determined by HPLC. Results showed sequential preservation of plasma Cys and GSH levels in response to BMDMSC infusion. The data show that BMDMSC infusion leads to a more reducing Cys and GSH redox state. The findings are the first to demonstrate that BMDMSC have antioxidant effects in vivo, and add to our understanding of the systemic effects of BMDMSC in lung injury

Slow-Release Nanoparticle-Encapsulated Delivery System for Laryngeal Injection

Objectives/Hypothesis: There is a need for a slow-release system for local delivery of therapeutics to the larynx. Most therapeutic substances, such as steroids or chemotherapeutic agents that are injected into the larynx are cleared rapidly. Repeated laryngeal injection of these substances at short intervals is impractical. Injectable encapsulated poly(lactide-coglycolide) (PLGA) nanoparticles offer a potential slow-release delivery system for biologically active substances in the larynx. Study Design: Controlled animal study. Methods: PLGA nanoparticles were fabricated using a double emulsion method and were loaded with Texas Red-dextran (NPTR), hepatocyte growth factor (NPHGF), and bovine serum albumin (NPBSA). In vitro release of NPTR, NPBSA, and NPHGF was determined over approximately 2 weeks to assess potential duration of PLGA nanoparticle delivery. In vivo release of NPTR was assessed in a murine vocal fold injection model. The transcriptional effect of NPHGF on procollagen was measured in vitro to assess whether released growth factor retained functionality. Results: In vitro release kinetics demonstrated slow release of NPTR, NPBSA, and NPHGF over 12 to 14 days. In vitro NPTR release correlated with in vivo results. In vivo presence of NPTR occurred up to 7 days compared to 1 day for Texas Red control. In addition, NPHGF ameliorated transforming growth factor-beta induced procollagen in vitro in 3T3 fibroblast cells. Conclusions: The results demonstrate the potential utility of nanoparticle encapsulation as an effective method for long-term delivery of specific drugs and biologically active substances to the larynx

CXCR4 antagonists inhibit radiation-induced PF.

<p>Mice underwent hemithoracic irradiation (20-Gy) and treated with CXCR4 antagonists or their corresponding vehicle controls (n=10/group). <i>A</i>, Lungs were evaluated histologically after trichrome staining (dark bars) or by CT scan (light bars) at 20-weeks post-irradiation. Histologic evaluation was scored (0-12) in each lung as described in the Methods section. CT scan evaluation was scored as the percent volume of lung with density>-200 HU. In all assessments, the vehicle controls values were normalized to 100% with drug-treated values expressed relative to corresponding controls to evaluate the efficacy of the CXCR4 antagonists in attenuating radiation-induced PF. Error bar represents SEM. * indicates statistically significant difference (p<0.05) compared with vehicle controls. <i>B</i>, Representative micrographs of trichrome staining are shown in 4X and 40X magnification.</p

CXCL12 and CXCR4 expression in lungs of mice after 20 Gy radiation.

<p>Graphs of CXCL12 levels (ng/ml) (n=5 or 6) measured by ELISA in (<i>A</i>) serum and (<i>B</i>) BALF as well as (<i>C</i>) CXCL12 and (<i>D</i>) CXCR4 normalized mRNA levels (n=5) measured by RNase protection assay in lungs at 0, 1, 3, 7, 14 and 28-days post-irradiation (whole thorax) are shown. Error bars represent SEM. * indicates statistically significant difference (p<0.05) compared with value at day-0. </p

Baclofen, a GABABR agonist, ameliorates immune-complex mediated acute lung injury by modulating pro-inflammatory mediators.

Immune-complexes play an important role in the inflammatory diseases of the lung. Neutrophil activation mediates immune-complex (IC) deposition-induced acute lung injury (ALI). Components of gamma amino butyric acid (GABA) signaling, including GABA B receptor 2 (GABABR2), GAD65/67 and the GABA transporter, are present in the lungs and in the neutrophils. However, the role of pulmonary GABABR activation in the context of neutrophil-mediated ALI has not been determined. Thus, the objective of the current study was to determine whether administration of a GABABR agonist, baclofen would ameliorate or exacerbate ALI. We hypothesized that baclofen would regulate IC-induced ALI by preserving pulmonary GABABR expression. Rats were subjected to sham injury or IC-induced ALI and two hours later rats were treated intratracheally with saline or 1 mg/kg baclofen for 2 additional hours and sacrificed. ALI was assessed by vascular leakage, histology, TUNEL, and lung caspase-3 cleavage. ALI increased total protein, tumor necrosis factor α (TNF-α and interleukin-1 receptor associated protein (IL-1R AcP), in the bronchoalveolar lavage fluid (BALF). Moreover, ALI decreased lung GABABR2 expression, increased phospho-p38 MAPK, promoted IκB degradation and increased neutrophil influx in the lung. Administration of baclofen, after initiation of ALI, restored GABABR expression, which was inhibited in the presence of a GABABR antagonist, CGP52432. Baclofen administration activated pulmonary phospho-ERK and inhibited p38 MAPK phosphorylation and IκB degradation. Additionally, baclofen significantly inhibited pro-inflammatory TNF-α and IL-1βAcP release and promoted BAL neutrophil apoptosis. Protective effects of baclofen treatment on ALI were possibly mediated by inhibition of TNF-α- and IL-1β-mediated inflammatory signaling. Interestingly, GABABR2 expression was regulated in the type II pneumocytes in lung tissue sections from lung injured patients, further suggesting a physiological role for GABABR2 in the repair process of lung damage. GABABR2 agonists may play a potential therapeutic role in ALI