Neutrophils are responsible for impaired medial smooth muscle cell recovery and exaggerated allograft vasculopathy in aortic allografts exposed to prolonged cold ischemia

Background: Ischemia and reperfusion injury is critical in allograft vasculopathy (AV) development. We have shown that neutrophil-mediated medial smooth muscle cell (SMC) loss precedes AV and that prolonged cold ischemia (CI) impairs medial SMC recovery and accelerates AV development. We hypothesize that neutrophils (NØs) are responsible for failed medial SMC recovery that precedes AV. Methods: Aortic transplants were performed between fully disparate C3H/HeJ murine donors and wild-type C57BL/6 (WT B6), B6.129S7-Rag1 (Rag1 ^-/-; intact innate but no adaptive immunity), and B6.129S-Cybb (NOX2^-/-; NØ loss-of-function) recipients under cyclosporine A immunosuppression. Grafts were exposed to 20 or 60 minutes CI before transplant and harvested at 1 day, 2 weeks, and 8 weeks after transplant. Some WT B6 recipients were treated with remote ischemic pre-conditioning (rIPC). Grafts were assessed for medial SMCs, NØs, and lesion area. Results: The 60-minute vs 20-minute CI grafts exhibited reduced SMC recovery at 2 weeks in WT B6 and Rag1^-/- recipients (WT B6: p = 0.0009; Rag1^-/-: p = 0.0006). NØ influx was greater in Rag1^-/- recipients of 60-minute vs 20-minute CI grafts at 1 day (p = 0.0002). The difference in 2-week medial SMC recovery between ischemia groups was abrogated in NOX2^-/- recipients. At 8 weeks, NOX2^-/- and rIPC recipients of 60-minute CI grafts exhibited smaller neointimal lesions than B6 recipients (NOX2 ^-/-: p = 0.0009; rIPC: p = 0.0005). Conclusions: Impaired medial SMC recovery in murine aortic allografts at 2 weeks occurs in the absence of adaptive immunity. Enhanced medial SMC recovery and reduced neointimal lesion formation in NOX2^-/- and rIPC recipients of 60-minute CI grafts suggest a causal role for NØs in impaired medial SMC repopulation and the development of AV.