It is well established that prolonging CI leads to poorer short and long term graft survival [12, 18, 21]. Despite this evidence, the lack of donor availability is driving longer ischemic times. We have previously shown that there is an early and profound loss of medial SMC post transplant in an aortic transplant model . In this study, we examine the mechanisms responsible for the deleterious effect of prolonged CI on the development of AV. We used an aortic interposition model with clinically relevant levels of CNI immunosuppression (C2 levels approximating human levels) to ablate acute adaptive immune rejection events and reveal mechanistic detail of both early innate and late adaptive rejection responses. Clear evidence liking innate and adaptive immune responses can be invoked to develop a hypothesis by which prolonged CI could worsen AV by increasing the early innate inflammatory response and consequently augmenting the adaptive immune response . We explored this hypothesis in our current set of experiments.
The two ischemic times we chose were designed to mimic the clinical situation, with 20 min in cold saline being the standard cold ischemic time in our laboratory and 60 min in cold saline being considered our upper limit for immediate graft function. Moreover, we were able to show pathologic differences between the two time points . No doubt these time points could be adjusted upward by the use of preservation solution but they provided useful validated markers of long term graft function.
We first confirmed that 60 min CI would exacerbate vascular lesion formation in our model since this has not been previously demonstrated in models using CNI immunosuppression. Although it was not expected that CNI immunosuppression would dramatically alter early innate events (1 d-1 wk), the effects of CNI on the adaptive responses that are ultimately responsible for lesion formation are profound . In this first section we examined lesion progression between 3-6 wk post-transplant. Our findings in this series of experiments confirmed that lesion formation was earlier and more robust in the 60 min CI group compared to the 20 min CI group.
IR damage to vascular tissue is widely held to be the pivotal event in the initiation of AV [22, 23]. The specific target of that damage, however, is unclear and somewhat controversial. For example, Gohra and co-workers, using a rat aortic transplant model, demonstrated nearly complete endothelial denudation at 1 d post-transplant . Similarly, Lai et al. showed endothelial destruction beginning at 1 d post-transplant in a rat heterotopic heart transplant model . These data have suggested that early innate endothelial damage is the driving force behind the initiation of AV.
In contrast, more recent evidence has suggested that loss of medial SMC by late adaptive immune responses plays a dominant role in the development of AV . For instance, we have demonstrated that CD8+ T cells, Fas/FasL interactions, and IFN-γ expression, contribute to the late loss of medial SMC and the subsequent formation of the neointimal lesion . However, the influence of early innate responses in the medial compartment early post transplant, on the subsequent development of AV has not been explored. Given the relationship between late SMC loss and AV, it would be plausible to link the early loss of SMC to this complex process. As such, we hypothesized that prolonged CI would lead to enhanced innate immune response to the graft, with impaired medial SMC viability, resulting in exacerbation of AV.
To explore this hypothesis we compared medial SMC numbers in grafts exposed to 20 vs. 60 min CI. There was a similar, immediate and dramatic loss of medial SMC at 1 d in both groups. Interestingly, by 1 wk, levels of medial SMC recovery demonstrated a remarkable divergence between the two ischemic groups. Medial SMC numbers recovered to near normal levels in the grafts exposed to 20 min CI, whereas the grafts exposed to 60 min CI showed markedly impaired medial SMC recovery.
To examine the mechanism behind the differences in SMC recovery between the two groups, we first examined N∅ influx. As predicted, there was substantial N∅ influx into the grafts by 1 d post transplant in both groups. A significantly increased N∅ infiltration was observed in the prolonged CI group at the 1 d time point, but this increased influx was not observed at any of the other time points examined (3 d, 5 d, 1 wk). In both groups, N∅ numbers followed the same pattern, in that at 1 wk there was a resurgence of medial N∅ infiltration which was similar in magnitude in both groups. The relationship of the increased N∅ influx at 1 d to later events, namely impaired SMC recovery, enhanced T cell influx and more robust lesion formation, is unclear. It may be that the enhanced early (1 d) N∅ influx amplifies the recruitment of other innate cells that exert downstream effects of the developing response. This could involve the elastin fragmentation in the media observed in this study and others.
To explore the nature of the impaired recovery we examined the possibility that medial SMC in the 60 min CI grafts demonstrate impaired ability to activate proliferation programs. Using a Ki-67 proliferation assay, we quantified the percentage of proliferating SMC within the media of the grafts. Surprisingly, the grafts exposed to 60 min CI had a significantly higher percentage of proliferating SMC than the 20 min group, suggesting the activation of a robust SMC repair mechanism among the SMC spared from the loss at 1 d. Despite this robust repair response in the 60 min CI grafts, it was not adequate to fully repopulate the media with SMC. In contrast, despite a lower percentage of proliferating SMC detected in the 20 min CI grafts, and a similar starting number of medial SMC at 1 d post transplant, the 20 min CI grafts did exhibit successful media repopulation. This strongly suggests that the proliferating donor medial SMC in the 60 min CI grafts are being killed by innate or adaptive immune elements. The mechanism of SMC death has yet to be elucidated. At 1 wk and 2 wk apoptotic cells (using TUNEL) were evident in the media but the numbers were similar between the 20 min and 60 min groups (data not shown). This leads us to believe that the loss of SMC may be due to necrosis but further studies are needed to fully rule out apoptosis.
The principle adaptive alloimmune response under CNI immunosuppression is mediated by CD8+ T cells . We have previously shown that CD8+ T cells infiltrate the aortic grafts by 5 wk post transplant in the presence of CNI immunosuppression [19, 20]. Our data here demonstrates that increased CI led to an earlier and more robust appearance of CD8+ T cells into the media. Although the early and greater influx of cells of the adaptive response would account for the earlier and more robust lesion formation, they appear too late to account for the reduced recovery of SMC in the media. This implicates other cells of the innate response, for instance macrophages, in the impairment of recovery of medial SMC in the 60 min CI group and, potentially in the earlier initiation of the adaptive response. Further work in this area will be required to confirm this hypothesis.