*Purpose: Static cold storage (SCS) times for organs and tissues are currently limited to hours. Once outside the body, tissues lack oxygen and nutrient supply leading to electrolyte disturbances and accumulation of ROS. The resulting innate immune-driven sterile inflammation and tissue damage contribute to the incidence/severity of peri-transplant ischemia/reperfusion injury (IRI), a barrier to long-term graft survival and the acquisition of immune tolerance. This study investigates whether taming endothelial activation by interrupting P-selectin dependent leukocyte adhesion cascade (CD62-CD162), with a soluble form of a tandem-PSGL-1 (TSGL-1 Ig), may represent a novel therapeutic paradigm against IRI in VCA recipients.

*Methods: Groups of 6 to 10-week-old mice were used for syngeneic (C57BL/6 to C57BL/6) and allogeneic (Balb/c to C57BL/6) orthotopic hindlimb transplants. Hindlimb grafts were subjected to 6, 12, or 24h of SCS and flushed with TSGL-1 Ig (0.4mg/kg) after procurement and before reperfusion. In the allogeneic setting, animals received rapamycin (RPM, 1mg/kg i.p.) after recovery from surgery. Biopsies of different VCA tissue components obtained 24h after reperfusion were analyzed by histology, qRT-PCR and Western blots.

*Results: Histology showed increased leukocyte (macrophage/neutrophil) VCA infiltrate that correlated with the duration of SCS. Compared to skin, the muscle tissue was more susceptible to IR-stress. In the syn- and allogeneic setting, TSGL-1 Ig treatment did decrease relative expression of CXCL-1, CXCL-2, IL-1b, IL-6 in graft skin 24h after reperfusion. Differences were most pronounced after prolonged cold ischemia times. In the muscle tissue, however, TSGL-1 Ig treatment resulted in similar relative expression of CXCL-1, CXCL-2, IL-1b, and IL-6 in the syngeneic group, and even increased expression in the allogeneic group. Western blot-assisted analysis further revealed that P-selectin blockade increased the expression of IkBα and Nrf2 cytoprotective transcripts in the skin but not muscle at 24h after VCA reperfusion in grafts subjected to 6h and 24h SCS.

*Conclusions: Novel therapeutic paradigms mitigating ischemia/reperfusion injury are needed to reduce IR-induced tissue damage. Though beneficial in solid organ transplantation (SOT), our data indicate that disruption of endothelial CD62-CD162 signaling had a dichotomic effect on VCA tissue components. While IRI pathology was attenuated in the treated skin, muscle tissue showed similar or even increased proinflammatory phenotype at 24h post-transplantation. Further studies are warranted to fully appreciate divergent molecular mechanisms of IRI in SOT vs. VCA; and assess the therapeutic potential of TSGL-1 Ig in VCA setting.

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