Loss of cardiac myocytes due to apoptosis is considered to be a significant factor contributing, in part, to graft dysfunction and failure in acute cardiac rejection. A better understanding of the pathways leading to apoptosis may provide avenues to improve graft function and prevent graft failure. We used a variety of interventions, each shown to decrease histologic rejection of cardiac grafts. The new findings are that this study for the first time showed that both reactive oxygen and reactive nitrogen species contribute as signaling molecules causing activation of caspase 3 protease and apoptosis in acute cardiac allograft rejection.
Generally, it is believed that inflammatory cytokines such as TNF-α or IFN-γ, which are known to be produced during acute allograft rejection, are able to cause apoptosis of isolated cardiac myocytes in culture. Reactive oxygen species are known to be increased after exposure of cardiac myocytes to cytokines in culture and are also believed to play a significant role in graft failure in cardiac-transplant rejection. In this context, antioxidants such as N
-acetylcysteine and dithiothreitol and a NOS inhibitor were shown to inhibit DNA cleavage and staining for apoptosis in neonate rat cardiac myocytes stimulated ex vivo
with a combination of TNF-α, IL-1β, and IFN-γ (31
Surprisingly, no subsequent studies to our knowledge have evaluated signaling molecules of apoptosis, such as the role of reactive oxygen species, by using either qualitative or quantitative end points under in vivo
conditions of acute cardiac rejection. A role of reactive oxygen species in our model of acute cardiac rejection was implicated in the initial portions of this study by the findings that the antioxidant, vitamin C, or free radical spin-trap agent, PBN, prevented activation of caspase 3. Neither of these two agents has previously been evaluated to prevent cardiac apoptosis after inflammatory stimuli or alloimmune activation in myocardium in vivo.
In the case of PBN, this treatment was associated with a downregulation in iNOS, whereas no changes in iNOS were seen with vitamin C administration. Collectively, these studies indicate for the first time that reactive oxygen species play a role in caspase 3 activation in acute cardiac allograft rejection. The role of reactive oxygen supports our previous study that used another antioxidant, a vitamin E analogue, showing attenuation of caspase-3 activation in this model (17
The findings with vitamin C were conflicting owing to blockade of caspase 3 activation, yet without effect to limit TUNEL staining. We surmise that this could be explained by caspase 3–dependent and caspase 3–independent pathways for producing apoptosis. The action of vitamin C in biology is complex, and it displays both antioxidant and prooxidant effects (7
), which may be due, in part, to the presence or absence of free iron, which determines its beneficial and detrimental actions in biologic systems under inflammatory conditions.
More specifically to address the role of reactive oxygen species (specifically superoxide anion radicals), we performed additional studies by using a cell-permeable SOD mimetic, MnTmPyP. This intervention prevented activation of caspase 3 but without having any action on iNOS expression. This suggests evidence for the first time that the superoxide anion radical plays a significant role in caspase 3 activation in acute cardiac allograft rejection.
Another portion of the study was to determine the role of reactive nitrogen species, specifically the role of NO derived from iNOS on caspase 3 activation. A role of iNOS in cytokine-induced apoptosis in isolated rat neonate cardiac myocytes was implicated previously (1
), but the role of iNOS in apoptosis in acute allograft rejection is incompletely understood. In a previous mouse cardiac transplant model, nitric oxide (NO) derived from iNOS of recipients was implicated as contributing to activation of caspase 3 in iNOS+/+ vs.
). A limitation of this study is that the absence of iNOS in recipients does not eliminate the contributions of bulk NO in the graft due to NO derived from iNOS upregulation occurring within cardiac myocytes per se
of the donor. Indeed, apoptosis could be eliminated in acute cardiac allograft rejection only if iNOS was deleted in both donor and recipient mice (29
In an earlier study using pyrimidylimidazole-based iNOS inhibitors, a decreased number of TUNEL-positive apoptotic cells was observed by this treatment strategy, suggesting a role for iNOS (31
). But this intervention decreased iNOS protein expression as well suggesting an additional impact of this iNOS inhibitor secondary to decreases in inflammatory signaling. Downregulation of iNOS protein by these inhibitors has been confirmed in HEK 293 cells as well (13
In the present study, we used the iNOS inhibitor, l-NIL, at a dose that decreased histologic rejection. We showed that the increase in functional caspase 3 protease activity in cardiac allografts was prevented by treatment with l-NIL. This treatment ablated the increase in plasma NO levels but had no effect on iNOS protein levels, suggesting that this intervention inhibited NO production by iNOS but not its expression. Therefore, we conclude that NO derived from iNOS plays a significant role as a signaling molecule for caspase 3 activation in acutely rejecting cardiac allografts. These findings were consistent with the other data showing that treatment with l-NIL also blocked the allograft-induced apoptosis.
It is interesting that MnTmPyP and l
-NIL individually blocked caspase 3 activity, suggesting a role of both NO and superoxide in the development of apoptosis in acute cardiac rejection. Thus, increases in superoxide could decrease NO levels by reacting with NO to form peroxynitrite. In this context, we found that MnTmPyP increased plasma NO levels, and that this prevented activation of caspase 3 activity. TUNEL staining also confirmed effects of MnTmPyP to abolish downstream effects of caspase 3 activation on apoptosis. This shows that apoptosis is not likely due to a direct effect of NO but rather to peroxynitrite as the signaling molecule. Peroxynitrite is formed from the reaction of NO and superoxide, producing a potent oxidizing and nitrating species. This has potential significance in that addition of peroxynitrite to normal isolated rat neonate cardiac myocytes causes apoptosis (1
). That peroxynitrite formation occurred in our models was shown previously by the detection of nitrotyrosine, a footprint of peroxynitrite, in rejecting cardiac allografts (18
). Consistent with this are other previous studies from our laboratory showing that treatment with a peroxynitrite-decomposition catalyst inhibited the increase in poly(ADP)ribose seen in acutely rejecting cardiac allografts in vivo
). In this regard, a downstream effect of caspase 3 is to cleave poly(ADP)ribose synthase, thereby increasing levels of poly(ADP)ribose. Thus, it is possible that both inhibition of NO and superoxide by these interventions could act upstream to prevent peroxynitrite formation, which in turn could mediate activation of caspase 3 proteolytic activity and the subsequent effects of apoptosis in acute cardiac allograft rejection.
With one exception, all treatment regimens produced near or complete blockade of caspase 3 activation or TUNEL staining under conditions in which each regimen inhibited histologic rejection scores. However, the effect on rejection scores was not completely inhibited by every drug intervention. This could be explained by the fact that apoptosis is only one facet of the overall process of graft rejection and that the histologic scoring by definition includes aspects, such as grades of inflammatory cell infiltration, hemorrhage, and necrosis, that are not considered in the apoptosis analysis (28
). In this context, it was reported previously in a cardiac rejection model that a greater correlation exists between apoptotic myocytes and left ventricular pressure development than with histologic rejection scores (21
Pathways of caspase 3 activation in other posttransplant model systems
As indicated earlier, this is the first study to examine the role of NO and superoxide in signaling caspase 3 activation in acute cardiac allograft rejection. Interestingly, it was recently shown that SOD1 overexpression inhibited caspase 3 activity as well as caspase 9 activity but not caspase 8 activity at 4
h of reperfusion in a model of ischemic reperfusion injury in isogeneic cardiac transplants (32
). These findings suggest a role of superoxide produced in ischemia–reperfusion preferentially on caspase 9–dependent activation in cardiac transplants. The conventional paradigm would be that activation of caspase 3 via
caspase 8 and caspase 9 would reflect alloimmune-mediated (CD8, Fas-Fas ligand, TNF) vs.
free radical–mediated (superoxide) pathways, respectively. It is important to underscore that the focus of the study was on prolonged ischemia with cold saline storage conditions rather than traditional organ-preserving solutions to study the effects on apoptosis after revascularization by using isogeneic cardiac transplants and its latent effects on chronic vasculopathy. The study did not address the separate issue of the effect on alloimmune activation–induced caspase 3 activation in transplanted hearts, so it is difficult to relate these results with the new findings in our model because of the dissimilarities in design.
We believe that the impact of ischemia–reperfusion injury and caspase 3 activation via
reactive oxygen-mediated caspase 8 activation would not be contextually relevant to our model. The reasons are that we used short ischemic times and cold University of Wisconsin preserving solutions. Under our conditions, no obvious signs of ischemia–reperfusion injury appear in the posttransplant period. This was shown by findings in our cardiac allografts that inactivation of cardiac aconitase activity, which is very sensitive to inhibition by superoxide and well known to be inactivated after ischemia–reperfusion in heart, was not found until posttransplant day 4 (23
). We also found no significant increase in iNOS protein expression in Western blots of cardiac allografts before posttransplant day 4 (26
). Also, we previously reported that NF-κB is not activated at both 2 and 24
h of revascularization in our model of acute cardiac allograft rejection (27
). This indicates a negligible role of reactive oxygen-induced ischemia–reperfusion injury in our model. Thus, it is unlikely that ROS-mediated apoptosis as a result of revascularization could be expected if this redox-sensitive transcription factor were not also activated. Finally, it is important to note that all of our drug treatments commenced long after revascularization and surgeries had been completed. Therefore, our interventions do not affect caspase 3 activation and apoptosis due to ischemia–reperfusion per se
, yet they were clearly able to block apoptosis in allografts.
Only one published study examined caspase 8 and caspase 9 activation in any model of acute cardiac rejection (33
). This study, conducted in rabbits, showed equivalent increases in both caspase 8 and 9, which would argue against a preferential caspase 8 pathway in acute rejection via
alloimmune responses (i.e.
, CD8, Fas-Fas ligand, TNF). Another study showed that CD8 depletion did not change activation of either caspase 3 or 8 in hepatic allograft rejection (20
). Finally, in a model of acute cardiac rejection (4
), genetic mutations of Fas and Fas ligand that resulted in nonfunctional Fas/FasL-mediated pathways caused no change in the extent of apoptosis, leading the authors to conclude that the Fas-mediated pathway (i.e.
, caspase 8–mediated pathway) is not essential for acute cardiac rejection.
Collectively, based on these findings and those derived from other experimental models (5
), the issue that reactive oxygen is solely a mediator of caspase 9 activation cannot be supported, as caspase 8 is also activated. Likewise, interrupting Fas-mediated activation is now known to inhibit both caspase 8 and caspase 9 activation. Thus, growing evidence suggests that considerable cross-talk occurs between the traditional understood pathways of activation of caspase 8 and caspase 9 by reactive oxygen species. Because of these limitations and the concept of nontraditional pathways of caspase activation, it is clear that future studies are warranted to unravel the various complex pathways leading to apoptosis in cardiac transplants, including the role of ischemic storage times, acute rejection, and chronic rejection.