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Abstract Background Ischemia/reperfusion induced innate immune injury is inescapable in solid organ transplantation. Prolonged cold ischemia exacerbates the primary manifestation of late graft rejection, allograft vasculopathy (AV). The relationship between prolonged cold ischemia and late graft events is unclear and the subject of this study. Methods Aortic interposition transplants were performed between fully disparate mice treated with CyclosporineA. Allografts were exposed to 20 min or 60 min of cold ischemia and harvested between 1 d-6 wk. Lesion size, smooth muscle cells (SMC), neutrophils (N∅), and CD8+ T cells were quantified. Results Early SMC loss was identical in both groups. When compared to 20 min cold ischemia, grafts exposed to 60 min exhibited greater early N∅ influx, greater SMC proliferation but fewer medial SMC at 1 wk and 2 wk. Subsequently, earlier and greater CD8+ T cell infiltration were seen in the 60 min group with larger lesions at every time point. Conclusions These data suggest that the larger neointimal lesions in grafts exposed to 60 min cold ischemia result from enhanced early innate immune events resulting in impaired SMC recovery and subsequent increased adaptive immune response.

Abstract Background Cardiac allograft vasculopathy (AV) is a pathological process of vascular remodeling leading to late graft loss following cardiac transplantation. While there is consensus that AV is alloimmune mediated, and evidence that the most important alloimmune target is medial smooth muscle cells (SMC), the role of the innate immune response in the initiation of this disease is still being elucidated. As ischemia reperfusion (IR) injury plays a pivotal role in the initiation of AV, we hypothesize that IR enhances the early innate response to cardiac allografts. Methods Aortic transplants were performed between fully disparate mouse strains (C3H/HeJ and C57BL/6), in the presence of therapeutic levels of Cyclosporine A, as a model for cardiac AV. Neutrophils were depleted from some recipients using anti-PMN serum. Grafts were harvested at 1,2,3,5d and 1,2wk post-transplant. Ultrastructural integrity was examined by transmission electron microscopy. SMC and neutrophils were quantified from histological sections in a blinded manner. Results Grafts exposed to cold ischemia, but not transplanted, showed no medial SMC loss and normal ultrastructural integrity. In comparison, allografts harvested 1d post-transplant exhibited > 90% loss of SMC (p < 0.0001). SMC partially recovered by 5d but a second loss of SMC was observed at 1wk. SMC loss at 1d and 1wk post-transplant correlated with neutrophil influx. SMC loss was significantly reduced in neutrophil depleted recipients (p < 0.01). Conclusions These novel data show that there is extensive damage to medial SMC at 1d post-transplant. By depleting neutrophils from recipients it was demonstrated that a portion of the SMC loss was mediated by neutrophils. These results provide evidence that IR activation of early innate events contributes to the etiology of AV.

Background: Ischemia/reperfusion induced innate immune injury is inescapable in solid organ transplantation. Prolonged cold ischemia exacerbates the primary manifestation of late graft rejection, allograft vasculopathy (AV). The relationship between prolonged cold ischemia and late graft events is unclear and the subject of this study. Methods: Aortic interposition transplants were performed between fully disparate mice treated with CyclosporineA. Allografts were exposed to 20 min or 60 min of cold ischemia and harvested between 1 d-6 wk. Lesion size, smooth muscle cells (SMC), neutrophils (NØ), and CD8+ T cells were quantified. Results: Early SMC loss was identical in both groups. When compared to 20 min cold ischemia, grafts exposed to 60 min exhibited greater early NØ influx, greater SMC proliferation but fewer medial SMC at 1 wk and 2 wk. Subsequently, earlier and greater CD8+ T cell infiltration were seen in the 60 min group with larger lesions at every time point. Conclusions: These data suggest that the larger neointimal lesions in grafts exposed to 60 min cold ischemia result from enhanced early innate immune events resulting in impaired SMC recovery and subsequent increased adaptive immune response. [ABSTRACT FROM AUTHOR]

Background: Cardiac allograft vasculopathy (AV) is a pathological process of vascular remodeling leading to late graft loss following cardiac transplantation. While there is consensus that AV is alloimmune mediated, and evidence that the most important alloimmune target is medial smooth muscle cells (SMC), the role of the innate immune response in the initiation of this disease is still being elucidated. As ischemia reperfusion (IR) injury plays a pivotal role in the initiation of AV, we hypothesize that IR enhances the early innate response to cardiac allografts. Methods: Aortic transplants were performed between fully disparate mouse strains (C3H/HeJ and C57BL/6), in the presence of therapeutic levels of Cyclosporine A, as a model for cardiac AV. Neutrophils were depleted from some recipients using anti-PMN serum. Grafts were harvested at 1,2,3,5d and 1,2wk post-transplant. Ultrastructural integrity was examined by transmission electron microscopy. SMC and neutrophils were quantified from histological sections in a blinded manner. Results: Grafts exposed to cold ischemia, but not transplanted, showed no medial SMC loss and normal ultrastructural integrity. In comparison, allografts harvested 1d post-transplant exhibited > 90% loss of SMC (p < 0.0001). SMC partially recovered by 5d but a second loss of SMC was observed at 1wk. SMC loss at 1d and 1wk post-transplant correlated with neutrophil influx. SMC loss was significantly reduced in neutrophil depleted recipients (p < 0.01). Conclusions: These novel data show that there is extensive damage to medial SMC at 1d post-transplant. By depleting neutrophils from recipients it was demonstrated that a portion of the SMC loss was mediated by neutrophils. These results provide evidence that IR activation of early innate events contributes to the etiology of AV. [ABSTRACT FROM AUTHOR]

Background: In this study, we evaluated the capacity of a single percutaneous injection of recombinant human bone morphogenetic protein-2 (rhBMP-2) delivered in a rapidly resorbable calcium phosphate paste (α-BSM) to accelerate bone-healing in a canine tibial osteotomy model. We hypothesized that the osteotomy sites would heal faster after percutaneous delivery of rhBMP-2/(α-BSM than they would after injection of α-BSM alone or after no treatment. Methods: Bilateral tibial osteotomy was performed and the sites were stabilized with external fixators in sixteen dogs. Four hours after the surgery, one limb of each dog was treated with a single percutaneous injection of rhBMP-2/α-BSM paste or an equal volume of α-BSM alone. There were eight limbs in each group, and the osteotomy site in the contralateral limb served as an untreated control. The results were evaluated with serial radiography and force-plate analysis at four and eight weeks after surgery and with mechanical testing and histologic examination at eight weeks after the surgery. Results: At four and eight weeks after the osteotomy and treatment, the scores for radiographic union were significantly greater for the rhBMP-2/α-BSM-treated limbs than they were for the α-BSM-treated or untreated, control limbs (p < 0.05). The callus area in the rhBMP-2/α-BSM-treated limbs was significantly greater than that in the α-BSM-treated and untreated, control limbs at four and eight weeks post injection (p < 0.05). The time-integrated vertical force for the rhBMP-2-treated limbs was significantly greater than that for their contralateral controls at four weeks and significantly greater than that for the treated and control limbs of the α-BSM-treated dogs at four and eight weeks after the surgery (p ≤ 0.05). The rhBMP-2-treated limbs were significantly stiffer in bending and in torsion (p < 0.05) compared with the α-BSM-treated and control limbs. Histologic analysis demonstrated increased bone formation and more mature bone at the osteotomy site in the rhBMP-2-treated limbs compared with that in the α-BSM-treated and control limbs. Conclusions: This study demonstrates the capacity of a single percutaneous injection of rhBMP-2 delivered in a resorbable calcium phosphate paste α-BSM) four hours after surgery to accelerate the healing of tibial osteotomy sites in a canine model. Clinical Relevance: A single percutaneous injection of rhBMP-2/α-BSM may be useful as an adjuvant treatment to accelerate healing of bone defects similar to those created in this study. [ABSTRACT FROM AUTHOR]