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Preservation of Microvascular Integrity in Murine Orthotopic Tracheal Allografts by Clopidogrel

Heim, Christian, MD1; Khan, Mohammad Afzal, PhD2; von Silva-Tarouca, Benjamin, VMD1; Kuckhahn, Annika1; Stamminger, Thomas, MD3; Ramsperger-Gleixner, Martina, PhD1; Nicolls, Mark R., MD2; Weyand, Michael, MD1; Ensminger, Stephan M., MD, DPhil4

doi: 10.1097/TP.0000000000002571
Original Basic Science—General

Background. Survival after lung transplantation is mainly limited by the development of chronic lung allograft dysfunction (CLAD). The aim of this study was to investigate if platelet inhibition by clopidogrel has a functionally relevant influence on the microvascular integrity of orthotopic tracheal allografts as an anatomic basis for the development of CLAD.

Methods. We orthotopically transplanted C57Bl/6 (H-2b) tracheas into CBA.J (H-2k) recipients who afterwards received clopidogrel (1 mg/kg). Morphometric analysis was performed by measuring epithelial height in proportion to thickness of the lamina propria (epithelium-lamina propria ratio). Tissue oxygenation was determined using a fluorescence quenching technique, and graft perfusion monitoring was performed by laser Doppler flowmetry and lectin-binding assay. Immunohistochemistry was used for detection of CD31 and inducible nitric oxide synthase while iron deposition was shown with Prussian blue reaction. Quantitative reverse transcription polymerase chain reaction analysis was used for gene expression analysis.

Results. Isografts maintained good oxygenation and perfusion throughout the experiment, while both were drastically reduced in allografts. Treatment with clopidogrel attenuated graft hypoxia and reduced loss of perfusion. Additionally, clopidogrel led to increased epithelium-lamina propria ratio while iron deposition was impaired. Gene expression analysis revealed elevated levels of angiogenic vascular endothelial growth factor in the clopidogrel group. Improved endothelial function was shown by immunohistochemistry (CD31, inducible nitric oxide synthase).

Conclusions. Continuous administration of clopidogrel significantly improved tissue oxygenation, limited microvascular leakiness, and prevented airway ischemia. These data demonstrate that clopidogrel ameliorates microvascular injury during acute airway rejection, which is a known predisposing factor for the development of CLAD.

1 Department of Cardiac Surgery, University of Erlangen-Nuremberg, Erlangen, Germany.

2 Veterans Affairs Palo Alto Health Care System/Stanford University School of Medicine, Palo Alto, CA.

3 Institute for Virology, Ulm University Medical Center, Ulm, Germany.

4 Department of Cardiovascular Surgery, UKSH, Lübeck, Germany.

Received 11 August 2018. Revision received 23 November 2018.

Accepted 30 November 2018.

The authors declare no conflicts of interest.

C.H., M.R.N., M.W., and S.M.E. participated in research design. C.H., A.K., and S.M.E. participated in the writing of the paper. C.H., M.A.K., B.v.S.-T., and A.K. participated in the performance of the research. M.A.K., T.S., and M.R.N. contributed new reagents or analytic tools. C.H., M.R.-G., and S.M.E. participated in data analysis. Research support was obtained from the ELAN and Interdisziplinäres Zentrum für Klinische Forschung (IZKF) trust of the University of Erlangen-Nuremberg and the ADUMED foundation; no other funding source was employed.

Correspondence: Christian Heim, MD, Department of Cardiac Surgery, Friedrich-Alexander University Erlangen-Nuremberg, Krankenhausstrasse 12, 91054 Erlangen, Germany. (

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