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

Platelets from Asthmatic Individuals Show Less Reliance on Glycolysis.

Asthma, a chronic inflammatory airway disease, is typified by high levels of TH2-cytokines and excessive generation of reactive nitrogen and oxygen species, which contribute to bronchial epithelial injury and airway remodeling. While immune function plays a major role in the pathogenesis of the disease, accumulating evidence suggests that altered cellular metabolism is a key determinant in the predisposition and disease progression of asthma. Further, several studies demonstrate altered mitochondrial function in asthmatic airways and suggest that these changes may be systemic. However, it is unknown whether systemic metabolic changes can be detected in circulating cells in asthmatic patients. Platelets are easily accessible blood cells that are known to propagate airway inflammation in asthma. Here we perform a bioenergetic screen of platelets from asthmatic and healthy individuals and demonstrate that asthmatic platelets show a decreased reliance on glycolytic processes and have increased tricarboxylic acid cycle activity. These data demonstrate a systemic alteration in asthma and are consistent with prior reports suggesting that oxidative phosphorylation is more efficient asthmatic individuals. The implications for this potential metabolic shift will be discussed in the context of increased oxidative stress and hypoxic adaptation of asthmatic patients. Further, these data suggest that platelets are potentially a good model for the monitoring of bioenergetic changes in asthma

Platelets show less reliance on glycolysis in asthma.

<p>(A) Oxygen consumption trace for healthy (open squares) and asthmatic (filled squares). Basal rate is shown and arrows denote the addition of oligomycin A (oligo), FCCP, 2-deoxyglucose (2DG) and rotenone. (B) Quantification of basal rate, proton leak, ATP-linked respiration and non-mitochondrial oxygen consumption in platelets from healthy (open bars) and asthmatic (filled bars) subjects (calculated from traces such as those shown in panel A). (C) Quantification of oxygen consumption rate after the addition of 2-DG in healthy (open bars) and asthmatic (filled bars) platelets. (D) Changes in OCR as a function of ECAR in healthy (control; open circles) and asthmatic (Asthma; filled squares) platelets basally and after the addition of 2-DG. Arrows depict the shift after addition of 2-DG. Data are means ± SEM. #p<0.05. n = 12 for asthma, n = 13 for control.</p

Asthmatics show airflow obstruction compared to healthy controls.

<p>Airflow obstruction as measured by (A) FEV1% predicted and (B) FEV1/FVC in asthmatic individuals compared with healthy control subjects. #p<0.05.</p

Platelets show no change in mitochondrial number and morphology in asthma.

<p>(A-B) Representative electron micrograph of platelet from (A) healthy control and (B) asthma (scale bars: 1 μm). (C) Mitochondrial DNA copy number per platelet in platelets from healthy and asthmatic individuals. n = 7 per group.</p

Platelets show decreased glycolytic rate in asthma.

<p>(A) Extracellular acidification rate (ECAR) trace in asthmatic (filled squares) and healthy controls (open squares) basally, after the addition of oligomycin A, FCCP and 2-DG. (B) Quantification of ECAR basally, after oligomycin A addition and 2DG treatment in healthy controls (open bars) and asthmatic platelets (filled bars). (C-D) ECAR basally and after oligomycin A addition for platelets from each (C) healthy control subject and (D) asthmatic individuals. (E) Quantification of the difference in ECAR from basal to oligomycin A addition in healthy (control)(white bar) and asthmatic individuals (black bar). (F) The change in ECAR as a function of OCR in healthy (open circles) and asthmatic (filled squares) platelets basally and after the addition of oligomycin A. Arrows show the shift in ECAR/OCR after oligomycin A addition. n = 12 asthma, n = 13 controls; *p<0.01; #p<0.05.</p

Features of Study Participants.

<p>Definition of abbreviations: M, male; F, female; C, Caucasian; AA, African Amirican; FEV₁, Forced expiratory volume in 1 second; FVC, Forced vital capacity;</p><p><b>*</b><i>P</i> value, asthma <i>vs</i>. controls.</p><p>Features of Study Participants.</p

Greater TCA Cycle Activity in Asthma.

<p>(A) ATP content in control (white bars) and asthmatic (black bars) individuals basally and after inhibition of glycolysis by 2-DG. (B) Western analyses of aconitase expression in platelets. Asthmatic individials (lanes 4−6) had similar aconitase protein expression in platelets to healthy controls (lanes 1−3) by western blot. (C) Activity of aconitase, succinate dehydrogenase and citrate synthase in platelets from asthmatics (black bars) as a fold change of the activity of control platelets (white bars). n = 13 controls, n = 10 asthmatics. *p<0.01; #p<0.05.</p