We report here the results of our studies on the protective effects of HO-1 against I/R injury in steatotic rat livers. The principal findings of this work are as follows: (a) CoPP or gene therapy–induced HO-1 overexpression prevented I/R insult in ex vivo models of hepatic cold ischemia followed by reperfusion or syngeneic OLT; (b) HO-1 overexpression, as documented by Western blot analysis, improved liver function, preserved hepatocyte integrity, and decreased inflammatory MNC infiltration, with resultant prolongation of survival after transplantation; and (c) treatment with ZnPP, the HO-1 inhibitor, abolished these beneficial effects, documenting the direct involvement of HO-1 in protection against I/R injury. By demonstrating, for the first time to our knowledge, that exogenous HO-1 induction prevents severe I/R insult in fatty livers, our results provide the rationale for novel therapeutic approaches to maximize organ use and function through the safer use of marginal steatotic livers.
To test our hypothesis that stress-induced upregulation of HO-1 reduces I/R insult in steatotic rat livers, we have chosen 2 distinct HO-1–inducing approaches. First, donor rats were pretreated with CoPP (5 mg/kg intraperitoneally), a regimen that increases HO-1 protein levels in rat livers by 250% in a rat sandwich ELISA (R. Buelow, unpublished data). In our present study, a single dose of CoPP induced a prolonged increase in HO-1 expression that lasted more than 100 days after OLT. Second, because infusion of CoPP in high doses may modulate other heme enzymes such as nitric oxide synthase (NOS) and guanylate cyclase (28
), we have also used Ad-based gene delivery to provide “proof of principle” and to selectively upregulate HO-1 expression in prospective liver donors. Indeed, as recently shown in a hyperoxia-induced lung injury rat model (30
), our Western blot analysis confirmed increased HO-1 protein expression in the ex vivo I/R model using Ad-HO-1–transduced rat steatotic livers.
Ischemia/reperfusion is markedly increased in steatotic livers compared with normal livers, and reactive oxygen species have been shown to contribute, at least in part, to this event (31
). Moreover, the antioxidant defenses of hepatocytes in steatotic livers are decreased in comparison with normal livers (32
). Exogenous upregulation of HO-1 in our study prevented or significantly decreased hepatic injury in 2 clinically relevant and well-defined ex vivo rat fatty liver models of cold ischemia followed by reperfusion or syngeneic OLT. The beneficial effects in the ex-vivo I/R-injury model were reflected by the ability of exogenously upregulated HO-1 to improve portal vein blood flow, increase bile production, and depress sGOT levels, all well-accepted parameters of hepatic function (6
). Portal blood flow is mostly affected by resistance in the graft caused by lobular ballooning, hepatocyte swelling, and sinusoidal congestion. In this ex vivo perfusion model, the improved portal venous blood flow represents less hepatocyte injury and lobular disarray in the liver rather than the endothelium-dependant vasodilatory effects of carbon monoxide. In the in vivo liver isotransplant model, enhanced HO-1 expression improved animal survival from 40% in untreated controls to about 80% after CoPP treatment or local Ad-HO-1 gene delivery, an ultimate test for the liver function. Collectively, these results are consistent with the ability of HO-1 to protect cells from oxidative injury (33
Upregulation of HO-1 inhibits inflammatory responses (36
) consistent with our present immunohistochemical findings of markedly decreased MNC infiltration in CoPP-pretreated liver isografts, as compared with untreated controls. Whereas it is still unclear how HO-1 upregulation influenced graft infiltration, several possibilities can be envisioned: (a) decreased production of cytokines and chemokines by infiltrating cells may have prevented local cell sequestration at the graft site; (b) reduced cytokine levels could diminish endothelial cell activation; and (c) HO-1 may have influenced directly endothelial cell activation. Indeed, in our ongoing real-time RT-PCR studies, markedly diminished expression of mRNA coding for Th1-type IFN-γ/IL-2 as well as Th2-type IL-10 was readily detectable in HO-1-overexpressed and functioning liver isografts despite previous I/R injury (F. Amersi, unpublished data). Moreover, consistent with our preliminary findings in the I/R steatotic OLT model, we have recently shown that CoPP treatment suppressed both proliferative responses and elaboration of TNF-α, IFN-γ, and IL-10 in a murine cardiac allograft model (14
). CoPP-mediated in vivo effects, such as HO-1 upregulation, inhibition of T-cell proliferation, cytokine elaboration, T- and NK-cell cytotoxicity, and prolongation of allograft survival (14
) were all observed after treatment with an immunomodulatory MHC Class I–derived peptide (D2702.75-84) (33
). Most recently, we have also shown that a rationally designed HO-1–inducing immunomodulatory peptide (RDP 1258) inhibited rat renal transplant vasculopathy (36
). Moreover, in agreement with the current report, our ongoing studies show that CoPP-triggered HO-1 overexpression in small-bowel donors may decrease preservation/reperfusion injury and improve survival of both the animal and of the transplanted bowel (39
). Clearly, our present findings support the idea that HO-1 upregulation does not associate just with exogenous immunosuppression, but it may well represent an essential component of stress-mediated immunomodulation. To prove this hypothesis, we are currently generating HO-1 transgenic rats to use as liver donors and/or recipients in our hepatic I/R injury models.
The results of our experiments in which ZnPP-mediated inhibition of HO-1 negated beneficial effects seen in vivo after CoPP treatment or Ad-HO-1 gene transfer endorse the hypothesis that the mechanism behind protection against hepatic I/R injury involves HO-1 induction rather than modulation of other biochemical pathways that may protect hepatocytes from oxidative injury. In other studies rat epithelial cells transfected with HO-1 cDNA exhibited marked increases of HO-1 protein and resistance against hyperoxia, whereas inhibition of HO-1 with tin protoporphyrin (SnPP) ablated protection against hyperoxia (40
). Similarly, in rodent models of renal failure, elevated HO-1 expression reduced tissue injury (41
). Again, protection could be reversed by SnPP-induced inhibition of HO-1. In animal models of inflammatory disease, enhanced HO-1 activity downregulated inflammation, whereas inhibition of HO exacerbated the inflammatory response (37
). In septic shock models, increased HO-1 activity protected animals from LPS-induced death (43
). Consistent with these observations, HO-1–deficient mice suffer from progressive chronic inflammatory disease and are extremely sensitive to stressful injury (44
). Similar observations were made with the first known human case of HO-1 deficiency (45
The mechanisms behind HO-1–mediated cytoprotection remain unclear. However, all of the end products of heme degradation, including biliverdin, bilirubin, and CO, are known to modulate immune effector functions (46
). Biliverdin has also been shown to inhibit human complement in vitro (46
). Bilirubin inhibits human lymphocyte responses, including PHA-induced proliferation, IL-2 production, and antibody-dependent and -independent cell-mediated cytotoxicity (44
). Moreover, because of the heme protein nature of NOS, induction of HO-1 is likely to modulate nitric oxide (NO) production, an important effector molecule involved in inflammation and immune regulation (49
). Indeed, HO-1 upregulation correlates with increased production of NO, which in turn may inhibit lymphocyte proliferation following CoPP therapy, as in our present studies. On the other hand, NO is also known to induce HO-1 expression. This effect may be of significance because CO directly inhibits NOS activity by binding to the heme moiety of the NOS enzyme and thus downregulating NO production. Like NO, CO contributes to endothelium-dependent vasodilatation and inhibits platelet aggregation by elevating intracellular cGMP levels (50
). The deleterious effects of hyperoxia are thought to be mediated by reactive oxygen species (ROS). Both biliverdin and bilirubin are efficient peroxyl radical scavengers that inhibit lipid peroxidation (51
). Bilirubin scavenges peroxyl radicals as efficiently as α-tocopherol, which is regarded as the most potent antioxidant of lipid peroxidation. On the other hand, oxygen radicals may trigger cascade of antiapoptotic events, including those that involve activation of bcl-2 protooncogene. Indeed, as shown by us (15
) and others (13
), increased expression of bcl-2 may represent one of the mechanisms by which increased HO-1 expression may promote protection against tissue injury. All these factors point to a complex picture of putative regulatory interactions between the HO system and the host cytokine network set in motion through the biological activity of heme degradation products.
In conclusion, CoPP- or gene therapy–induced HO-1 overexpression protects against severe I/R injury in steatotic rat liver models of ex vivo cold ischemia followed by reperfusion or OLT. To our knowledge, this is the first report that documents the potential utility of HO-1 in increasing the donor transplant pool through modulation of marginal steatotic livers or conditions of prolonged ischemia. Our findings raise the possibility of refined new treatment regimens in OLT that may ultimately improve the overall success of liver transplantation.