P. L. Jernigan1, R. S. Hoehn1, J. Sutton1, E. Midura1, T. Johannigman1, C. C. Caldwell1, M. J. Edwards1, E. Gulbins1,2, T. A. Pritts1 1University Of Cincinnati,Department Of Surgery,Cincinnati, OH, USA 2University Of Duisburg-Essen,Department Of Molecular Biology,Essen, NORTH RHINE-WESTPHALIA, Germany
Introduction: Transfusion with older packed red blood cell (pRBC) units during trauma resuscitation is associated with lung injury and increased mortality. Our recent work implicates pRBC-derived microparticles (MPs) in this process. Ceramide, a cell membrane-based sphingolipid, can be formed by the enzyme acid sphingomyelinase (Asm) and act as a signaling agent to alter cell membranes. The potential role of the Asm/ceramide system in mediating microparticle formation and lung injury from stored pRBC units is unknown. We hypothesized that inhibiting Asm in stored pRBC units could decrease the harmful effects of pRBC-derived MPs.
Methods: Murine pRBC units were prepared from healthy donors, treated with the Asm inhibitor Amitriptyline (AT; up to 250 µM) or PBS (vehicle) and stored under standard blood banking conditions for up to 14 days. MPs were isolated and counted with Nanoparticle Tracking Analysis immediately prior to use. Healthy mice underwent hemorrhage and resuscitation that included equal concentrations of MPs isolated from AT- or vehicle-treated pRBC units and lungs were collected for analysis. To study specific effects of MPs on endothelial cells, murine hemangioendothelioma (EOMA) cells were incubated for thirty minutes with equal numbers of MPs from AT- or vehicle-treated pRBCs. Cells were then fixed and stained for the tight junction proteins ZO-1 and occludin.
Results: Treatment with amitriptyline reduced Asm activity and ceramide levels in MPs isolated from the pRBC units. Mice that underwent hemorrhage followed by resuscitation with MPs isolated from vehicle-treated pRBC units demonstrated increased inflammatory cell recruitment to the lung, alveolar wall thickening, and decreased pulmonary ZO-1 and ZO-2 expression relative to baseline, consistent with acute lung injury. These changes were significantly attenuated in mice resuscitated with equal numbers of MPs from AT-treated pRBC units. EOMA cells treated with MPs from aged vehicle-treated pRBCs showed a significant reduction in the tight junction proteins ZO-1 (Figure A) and occludin relative to untreated cells or those treated with MPs from fresh (unstored) pRBCs, consistent with tight junction disruption. This decrease in ZO-1 and occludin was abrogated in cells treated with MPs isolated from AT-treated pRBCs (Figure B and C).
Conclusion: Microparticles shed by stored pRBCs mediate lung injury after resuscitation, with disruption of endothelial cell tight junctions. Treating stored pRBCs with the Asm inhibitor Amitriptyline mitigates this effect. These data suggest that Asm inhibition may ameliorate harmful aspects of pRBC storage and reduce lung injury after transfusion.
