Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Electroporation-mediated delivery of the FER gene in the resolution of trauma-related fatal pneumonia

Gene Therapy volume 23, pages 785–796 (2016)Cite this article

Subjects

Abstract

Injured patients with lung contusion (LC) are at risk of developing bacterial pneumonia (PNA) followed by sepsis and death. A recent genome-wide association study (GWAS) showed FER gene expression positively correlating with survival rates among individuals with above conditions. We sought to determine whether electroporation (EP)-mediated delivery of FER gene could indeed improve survival, in a lethal model of combined LC and PNA. C57BL/6 mice sustained unilateral LC, which preceded a 500 Klebsiella colony forming unit (CFU) inoculation by 6 h. In-between these insults, human FER plasmid (pFER) was introduced into the lungs followed by eight EP pulses applied externally (10 ms at 200 V cm−1). Control groups included EP of empty vector (pcDNA3) or Na+/K+-ATPase genes (pPump) and no treatment (LC+PNA). We recorded survival, histology, lung mechanics, bronchial alveolar lavage (BAL) fluid, FER and inflammatory gene expression and bacteriology. The data show that 7-day survival was significantly improved by pFER compared with control groups. pFER increased BAL monocytes and activated antibacterial response genes (nitric oxide synthase (NOS), Fizz). pFER treatment showed decreased lung and blood Klebsiella counts reaching, in some cases, complete sterilization. In conclusion, FER gene delivery promoted survival in LC+PNA mice via recruitment of activated immune cells, improving efficiency of bacterial clearance within contused lung.

Access through your institution
Buy or subscribe

This is a preview of subscription content, access via your institution

Access options

Access through your institution

Buy this article

  • Purchase on SpringerLink
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Figure 9
Figure 10

Similar content being viewed by others

References

  1. Raghavendran K, Davidson BA, Woytash JA, Helinski JD, Marschke CJ, Manderscheid PA et al. The evolution of isolated bilateral lung contusion from blunt chest trauma in rats: cellular and cytokine responses. Shock 2005; 24: 132–138.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Dolgachev VA, Yu B, Reinke JM, Raghavendran K, Hemmila MR . Host susceptibility to gram-negative pneumonia after lung contusion. J Trauma Acute Care Surg 2012; 72: 614–622; discussion 22-23.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Machado-Aranda D, Wang Z, Yu B, Suresh MV, Notter RH, Raghavendran K . Increased phospholipase A2 and lyso-phosphatidylcholine levels are associated with surfactant dysfunction in lung contusion injury in mice. Surgery 2013; 153: 25–35.

    Article  PubMed  Google Scholar 

  4. Suresh MV, Ramakrishnan SK, Thomas B, Machado-Aranda D, Bi Y, Talarico N et al. Activation of hypoxia-inducible factor-1alpha in type 2 alveolar epithelial cell is a major driver of acute inflammation following lung contusion. Crit Care Med 2014; 42: e642–e653.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Machado-Aranda D, V Suresh M, Yu B, Dolgachev V, Hemmila MR, Raghavendran K . Alveolar macrophage depletion increases the severity of acute inflammation following nonlethal unilateral lung contusion in mice. J Trauma Acute Care Surg 2014; 76: 982–990.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Rubenfeld GD, Caldwell E, Peabody E, Weaver J, Martin DP, Neff M et al. Incidence and outcomes of acute lung injury. N Engl J Med 2005; 353: 1685–1693.

    Article  CAS  PubMed  Google Scholar 

  7. Villar J, Sulemanji D, Kacmarek RM . The acute respiratory distress syndrome: incidence and mortality, has it changed? Curr Opin Crit Care 2014; 20: 3–9.

    Article  PubMed  Google Scholar 

  8. Battle CE, Hutchings H, Evans PA . Risk factors that predict mortality in patients with blunt chest wall trauma: a systematic review and meta-analysis. Injury 2012; 43: 8–17.

    Article  PubMed  Google Scholar 

  9. Taylor AP . Serious putty. The Scientist 2016; 2016: 15–16.

    Google Scholar 

  10. Hidron AI, Edwards JR, Patel J, Horan TC, Sievert DM, Pollock DA et al. NHSN annual update: antimicrobial-resistant pathogens associated with healthcare-associated infections: annual summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2006-2007. Infect Control Hosp Epidemiol 2008; 29: 996–1011.

    Article  PubMed  Google Scholar 

  11. Rautanen A, Mills TC, Gordon AC, Hutton P, Steffens M, Nuamah R et al. Genome-wide association study of survival from sepsis due to pneumonia: an observational cohort study. Lancet Respir Med 2015; 3: 53–60.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hao QL, Ferris DK, White G, Heisterkamp N, Groffen J . Nuclear and cytoplasmic location of the FER tyrosine kinase. Mol Cell Biol 1991; 11: 1180–1183.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Senis YA, Sangrar W, Zirngibl RA, Craig AW, Lee DH, Greer PA . Fps/Fes and Fer non-receptor protein-tyrosine kinases regulate collagen- and ADP-induced platelet aggregation. J Thromb Haemost 2003; 1: 1062–1070.

    Article  CAS  PubMed  Google Scholar 

  14. Sangrar W, Gao Y, Bates B, Zirngibl R, Greer PA . Activated Fps/Fes tyrosine kinase regulates erythroid differentiation and survival. Exp Hematol 2004; 32: 935–945.

    Article  CAS  PubMed  Google Scholar 

  15. Sangrar W, Gao Y, Scott M, Truesdell P, Greer PA . Fer-mediated cortactin phosphorylation is associated with efficient fibroblast migration and is dependent on reactive oxygen species generation during integrin-mediated cell adhesion. Mol Cell Biol 2007; 27: 6140–6152.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Kwok E, Everingham S, Zhang S, Greer PA, Allingham JS, Craig AW . FES kinase promotes mast cell recruitment to mammary tumors via the stem cell factor/KIT receptor signaling axis. Mol Cancer Res 2012; 10: 881–891.

    Article  CAS  PubMed  Google Scholar 

  17. Ahn J, Truesdell P, Meens J, Kadish C, Yang X, Boag AH et al. Fer protein-tyrosine kinase promotes lung adenocarcinoma cell invasion and tumor metastasis. Mol Cancer Res 2013; 11: 952–963.

    Article  CAS  PubMed  Google Scholar 

  18. Sangrar W, Shi C, Mullins G, LeBrun D, Ingalls B, Greer PA . Amplified Ras-MAPK signal states correlate with accelerated EGFR internalization, cytostasis and delayed HER2 tumor onset in Fer-deficient model systems. Oncogene 2015; 34: 4109–4117.

    Article  CAS  PubMed  Google Scholar 

  19. Rocha J, Zouanat FZ, Zoubeidi A, Hamel L, Benidir T, Scarlata E et al. The Fer tyrosine kinase acts as a downstream interleukin-6 effector of androgen receptor activation in prostate cancer. Mol Cell Endocrinol 2013; 381: 140–149.

    Article  CAS  PubMed  Google Scholar 

  20. Kawakami M, Morita S, Sunohara M, Amano Y, Ishikawa R, Watanabe K et al. FER overexpression is associated with poor postoperative prognosis and cancer-cell survival in non-small cell lung cancer. Int J Clin Exp Pathol 2013; 6: 598–612.

    CAS  PubMed Central  PubMed  Google Scholar 

  21. Lin X, Dean DA . Gene therapy for ALI/ARDS. Crit Care Clin 2011; 27: 705–718.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Machado-Aranda D, Adir Y, Young JL, Briva A, Budinger GR, Yeldandi AV et al. Gene transfer of the Na+,K+-ATPase beta1 subunit using electroporation increases lung liquid clearance. Am J Respir Crit Care Med 2005; 171: 204–211.

    Article  PubMed  Google Scholar 

  23. Machado-Aranda DA, Suresh MV, Yu B, Raghavendran K . Electroporation-mediated in vivo gene delivery of the Na(+)/K(+)-ATPase pump reduced lung injury in a mouse model of lung contusion. J Trauma Acute Care Surg 2012; 72: 32–39; discussion 9–40.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Weiss DJ . Delivery of gene transfer vectors to lung: obstacles and the role of adjunct techniques for airway administration. Mol Ther 2002; 6: 148–152.

    Article  CAS  PubMed  Google Scholar 

  25. Somiari S, Glasspool-Malone J, Drabick JJ, Gilbert RA, Heller R, Jaroszeski MJ et al. Theory and in vivo application of electroporative gene delivery. Mol Ther 2000; 2: 178–187.

    Article  CAS  PubMed  Google Scholar 

  26. Mir LM . Electroporation-based gene therapy: recent evolution in the mechanism description and technology developments. Methods Mol Biol 2014; 1121: 3–23.

    Article  CAS  PubMed  Google Scholar 

  27. Dean DA, Machado-Aranda D, Blair-Parks K, Yeldandi AV, Young JL . Electroporation as a method for high-level nonviral gene transfer to the lung. Gene Ther 2003; 10: 1608–1615.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Mutlu GM, Machado-Aranda D, Norton JE, Bellmeyer A, Urich D, Zhou R et al. Electroporation-mediated gene transfer of the Na+,K+ -ATPase rescues endotoxin-induced lung injury. Am J Respir Crit Care Med 2007; 176: 582–590.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Machado-Aranda DA, Suresh MV, Yu B, Raghavendran K . Electroporation-mediated in vivo gene delivery of the Na+/K+-ATPase pump reduced lung injury in a mouse model of lung contusion. J Trauma Acute Care Surg 2012; 72: 32–39; discussion 9–40.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Matute-Bello G, Downey G, Moore BB, Groshong SD, Matthay MA, Slutsky AS et al. An official American Thoracic Society workshop report: features and measurements of experimental acute lung injury in animals. Am J Respir Cell Mol Biol 2011; 44: 725–738.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Amato MB, Meade MO, Slutsky AS, Brochard L, Costa EL, Schoenfeld DA et al. Driving pressure and survival in the acute respiratory distress syndrome. N Engl J Med 2015; 372: 747–755.

    Article  CAS  PubMed  Google Scholar 

  32. Raghavendran K, Notter RH, Davidson BA, Helinski JD, Kunkel SL, Knight PR . Lung contusion: inflammatory mechanisms and interaction with other injuries. Shock 2009; 32: 122–130.

    Article  PubMed  PubMed Central  Google Scholar 

  33. Zisman DA, Kunkel SL, Strieter RM, Tsai WC, Bucknell K, Wilkowski J et al. MCP-1 protects mice in lethal endotoxemia. J Clin Invest 1997; 99: 2832–2836.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Raghavendran K, Davidson BA, Mullan BA, Hutson AD, Russo TA, Manderscheid PA et al. Acid and particulate-induced aspiration lung injury in mice: importance of MCP-1. Am J Physiol Lung Cell Mol Physiol 2005; 289: L134–L143.

    Article  CAS  PubMed  Google Scholar 

  35. Suresh MV, Yu B, Machado-Aranda D, Bender MD, Ochoa-Frongia L, Helinski JD et al. Role of macrophage chemoattractant protein 1 in acute inflammation following lung contusion. Am J Respir Cell Mol Biol 2012; 46: 797–806.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Neumann E, Schaefer-Ridder M, Wang Y, Hofschneider PH . Gene transfer into mouse lyoma cells by electroporation in high electric fields. EMBO J 1982; 1: 841–845.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Emr BM, Roy S, Kollisch-Singule M, Gatto LA, Barravecchia M, Lin X et al. Electroporation-mediated gene delivery of Na+,K+ -ATPase, and ENaC subunits to the lung attenuates acute respiratory distress syndrome in a two-hit porcine model. Shock 2015; 43: 16–23.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Dean DA, Ramanathan T, Machado D, Sundararajan R . Electrical impedance spectroscopy study of biological tissues. J Electrostat 2008; 66: 165–177.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Calautti E, Cabodi S, Stein PL, Hatzfeld M, Kedersha N, Paolo Dotto G . Tyrosine phosphorylation and src family kinases control keratinocyte cell-cell adhesion. J Cell Biol 1998; 141: 1449–1465.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Kim L, Wong TW . Growth factor-dependent phosphorylation of the actin-binding protein cortactin is mediated by the cytoplasmic tyrosine kinase FER. J Biol Chem 1998; 273: 23542–23548.

    Article  CAS  PubMed  Google Scholar 

  41. Kim J, Ogata Y, Feldman RA . Fes tyrosine kinase promotes survival and terminal granulocyte differentiation of factor-dependent myeloid progenitors (32D) and activates lineage-specific transcription factors. J Biol Chem 2003; 278: 14978–14984.

    Article  CAS  PubMed  Google Scholar 

  42. Qi W, Ebbert KV, Craig AW, Greer PA, McCafferty DM . Absence of Fer protein tyrosine kinase exacerbates endotoxin induced intestinal epithelial barrier dysfunction in vivo. Gut 2005; 54: 1091–1097.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Oh MA, Choi S, Lee MJ, Choi MC, Lee SA, Ko W et al. Specific tyrosine phosphorylation of focal adhesion kinase mediated by Fer tyrosine kinase in suspended hepatocytes. Biochim Biophys Acta 2009; 1793: 781–791.

    Article  CAS  PubMed  Google Scholar 

  44. Putzke AP, Rothman JH . Repression of Wnt signaling by a Fer-type nonreceptor tyrosine kinase. Proc Natl Acad Sci USA 2010; 107: 16154–16159.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Khajah M, Andonegui G, Chan R, Craig AW, Greer PA, McCafferty DM . Fer kinase limits neutrophil chemotaxis toward end target chemoattractants. J Immunol 2013; 190: 2208–2216.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Craig AW, Greer PA . Fer kinase is required for sustained p38 kinase activation and maximal chemotaxis of activated mast cells. Mol Cell Biol 2002; 22: 6363–6374.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Xu G, Craig AW, Greer P, Miller M, Anastasiadis PZ, Lilien J et al. Continuous association of cadherin with beta-catenin requires the non-receptor tyrosine-kinase Fer. J Cell Sci 2004; 117 (Pt 15): 3207–3219.

    Article  CAS  PubMed  Google Scholar 

  48. McCafferty DM, Craig AW, Senis YA, Greer PA . Absence of Fer protein-tyrosine kinase exacerbates leukocyte recruitment in response to endotoxin. J Immunol 2002; 168: 4930–4935.

    Article  CAS  PubMed  Google Scholar 

  49. Zirngibl RA, Senis Y, Greer PA . Enhanced endotoxin sensitivity in fps/fes-null mice with minimal defects in hematopoietic homeostasis. Mol Cell Biol 2002; 22: 2472–2486.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Vacik J, Dean BS, Zimmer WE, Dean DA . Cell-specific nuclear import of plasmid DNA. Gene Ther 1999; 6: 1006–1014.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Young JL, Dean DA . Electroporation-mediated gene delivery. Adv Genet 2015; 89: 49–88.

    Article  PubMed  Google Scholar 

  52. Hoth JJ, Hudson WP, Brownlee NA, Yoza BK, Hiltbold EM, Meredith JW et al. Toll-like receptor 2 participates in the response to lung injury in a murine model of pulmonary contusion. Shock 2007; 28: 447–452.

    Article  CAS  PubMed  Google Scholar 

  53. Suresh MV, Yu B, Machado-Aranda D, Bender MD, Ochoa-Frongia L, Helinski JD et al. Role of macrophage chemoattractant protein-1 in acute inflammation after lung contusion. Am J Respir Cell Mol Biol 2012; 46: 797–806.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Hao QL, Heisterkamp N, Groffen J . Isolation and sequence analysis of a novel human tyrosine kinase gene. Mol Cell Biol 1989; 9: 1587–1593.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Taddonio MA, Dolgachev V, Bosmann M, Ward PA, Su G, Wang SC et al. Influence of lipopolysaccharide-binding protein on pulmonary inflammation in gram-negative pneumonia. Shock 2015; 43: 612–619.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Dolgachev VA, Yu B, Sun L, Shanley TP, Raghavendran K, Hemmila MR . Interleukin 10 overexpression alters survival in the setting of gram-negative pneumonia following lung contusion. Shock 2014; 41: 301–310.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Dolgachev V, Thomas M, Berlin A, Lukacs NW . Stem cell factor-mediated activation pathways promote murine eosinophil CCL6 production and survival. J Leukoc Biol 2007; 81: 1111–1119.

    Article  CAS  PubMed  Google Scholar 

  58. Dolgachev V, Farooqui MS, Kulaeva OI, Tainsky MA, Nagy B, Hanada K et al. De novo ceramide accumulation due to inhibition of its conversion to complex sphingolipids in apoptotic photosensitized cells. J Biol Chem 2004; 279: 23238–23249.

    Article  CAS  PubMed  Google Scholar 

  59. Ding L, Dolgachev V, Wu Z, Liu T, Nakashima T, Wu Z et al. Essential role of stem cell factor-c-Kit signalling pathway in bleomycin-induced pulmonary fibrosis. J Pathol 2013; 230: 205–214.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We gratefully acknowledge the support of National Institutes of Health-R01HL102013 (KR). We thank Dr. JE Wilkinson (Department of Pathology/ULAM, University of Michigan Medical School) for the help with lung tissue histology scorings. We also thank Ms. Lane Kelley McCandless for her contribution in revising this manuscript.

Author contributions

VAD, RHG, MVS, BT, NT and DMA conducted experiments and wrote the manuscript. DMA, MRH, KR contributed to conceptual design, interpretation of data and critical review.

Author information

Authors and Affiliations

  1. Division of Acute Care Surgery, Department of Surgery, University of Michigan, Ann Arbor, MI, USA

    V A Dolgachev, R Goldberg, M V Suresh, B Thomas, N Talarico, M R Hemmila, K Raghavendran & D Machado-Aranda

Authors
  1. V A Dolgachev
    View author publications

    Search author on:PubMed Google Scholar

  2. R Goldberg
    View author publications

    Search author on:PubMed Google Scholar

  3. M V Suresh
    View author publications

    Search author on:PubMed Google Scholar

  4. B Thomas
    View author publications

    Search author on:PubMed Google Scholar

  5. N Talarico
    View author publications

    Search author on:PubMed Google Scholar

  6. M R Hemmila
    View author publications

    Search author on:PubMed Google Scholar

  7. K Raghavendran
    View author publications

    Search author on:PubMed Google Scholar

  8. D Machado-Aranda
    View author publications

    Search author on:PubMed Google Scholar

Corresponding author

Correspondence to D Machado-Aranda.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Dolgachev, V., Goldberg, R., Suresh, M. et al. Electroporation-mediated delivery of the FER gene in the resolution of trauma-related fatal pneumonia. Gene Ther 23, 785–796 (2016). https://doi.org/10.1038/gt.2016.58

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue date:

  • DOI: https://doi.org/10.1038/gt.2016.58

This article is cited by

Search

Advanced search

Quick links