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Tissue factor–bearing exosome secretion from human mechanically stimulated bronchial epithelial cells in vitro and in vivo  Jin-Ah Park, PhD, Asma S.

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Presentation on theme: "Tissue factor–bearing exosome secretion from human mechanically stimulated bronchial epithelial cells in vitro and in vivo  Jin-Ah Park, PhD, Asma S."— Presentation transcript:

1 Tissue factor–bearing exosome secretion from human mechanically stimulated bronchial epithelial cells in vitro and in vivo  Jin-Ah Park, PhD, Asma S. Sharif, BS, Daniel J. Tschumperlin, PhD, Laurie Lau, PhD, Rachel Limbrey, MD, Peter Howarth, MD, Jeffrey M. Drazen, MD  Journal of Allergy and Clinical Immunology  Volume 130, Issue 6, Pages (December 2012) DOI: /j.jaci Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Compressive stress induces TF production from human bronchial epithelial cells. A-C, E, TF mRNA expression (Fig 1, A), intracellular TF (Fig 1, B), and secreted TF (Fig 1, C and E) in response to compressive stress (mean ± SD, n = 3). Loading controls: E-Cadherin (Fig 1, B) and Coomassie staining of SDS-PAGE (Fig 1, C and E). D, TF measured by ELISA in the CM (mean ± SD, n = 3). SDS-PAGE, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. *P < .05 versus control. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Secretion of TF elicited by compressive stress is independent of TF transcription or protein synthesis. A-C, TF mRNA expression (Fig 2, A), intracellular TF protein (Fig 2, B), or TF secretion (Fig 2, C) was determined in the absence or presence of inhibitors (ACTD, Actinomycin D; CHX, cycloheximide). Loading controls: E-Cadherin in Fig 2, B, and Coomassie staining of SDS-PAGE in Fig 2, C. Comp., Compressive; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Veh., vehicle. *P < .05 versus control, **P < .01 versus compressive stress alone. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Human bronchial epithelial cells release exosomes in response to compressive stress. A, Detection of exosomes from compressed NHBECs by TEM. B, Western blot analysis of TF, exosome markers (GAPDH, actin, annexin V, and EGFR), and nonexosomal proteins (Lamin A/C, E-cadherin, and beta 5 integrin). C, Coomassie staining of SDS-PAGE gel corresponding to the blots shown in Fig 3, B. EGFR, Epidermal growth factor receptor; PCNA, proliferation nuclear antigen; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; WCL, whole cell lysates. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Inhibition of PKC activity attenuates compressive stress–induced TF secretion. A, TF secretion in the presence of increasing concentration of Bis 1. B and C, TF mRNA expression (*P < .05 vs control) (means ± SEM, n = 4) and TF secretion (*P < .01 vs control) (means ± SEM, n = 3) in the presence of absence of Bis 1 (1 μmol/L). Comp., Compressive; Veh., vehicle. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 TF is elevated in human BALF from patients with asthma and is detected in exosomes. A, TF in human BALF (mean ± SD, n = 5 per condition). **P = versus healthy controls. B, Human BALF-derived exosomes detected by TEM. C, TF and exosome markers in exosomes isolated from the BALF of asthmatic patients (using the 3 asterisked samples in Fig 5, A). EGFR, Epidermal growth factor receptor. Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 TF localized in the airway epithelium is associated with the diagnosis of asthma. A and B, Immunohistochemical staining of TF in sections of EBBs from a healthy control (Fig 6, A) and an asthmatic volunteer (Fig 6, B). C, Immunohistocehmical staining by using an isotype control antibody. D, The percentage of epithelial cells stained for TF in healthy controls (n = 5) and asthmatic subjects (n = 10). Journal of Allergy and Clinical Immunology  , DOI: ( /j.jaci ) Copyright © 2012 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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