A substantial body of evidence, accumulated in recent years, points to an important functional role for HO-1 in providing cellular protection against oxidative stress. Both in vitro
and in vivo
data demonstrate that the induction of endogenous levels of HO-1 in response to oxidant injury in turn protects the cells and tissues against subsequent oxidant-induced injury (8
). The recent study by Poss and Tonegawa (26
), demonstrating the increased susceptibility of HO-1–null knockout mice to oxidative stress, further strengthens the emerging paradigm that HO-1 is indeed an important molecule in the host’s and cell’s defense against oxidant stress. In particular, our laboratory has shown that endogenous expression of HO-1 is induced both in vivo
and in vitro
in response to hyperoxia exposure and that increasing levels of HO-1 in cells can provide cytoprotection against hyperoxic injury in vitro
). We sought to examine whether HO-1 similarly can provide protection against hyperoxia in vivo.
We elected to use recombinant adenovirus to deliver exogenous HO-1 in a rat model of hyperoxia-induced lung injury. The use of adenovirus as a vector for lung-directed gene therapy has made significant progress in the area of lung biology, in particular in disease states such as cystic fibrosis. We show for the first time the feasibility of delivering exogenous HO-1 by gene transfer into the lung with successful expression of HO-1 mRNA and protein. Our data indicate that pretreatment with HO-1 transgene significantly attenuates hyperoxia-induced lung injury and increases survival in response to lethal hyperoxia. Although we cannot absolutely exclude a combined vector and HO-1 effect, this is unlikely since we used vector and vehicle controls in our experiments. While 66% is a significant improvement in mortality, there is still 34% of animals that eventually succumb to the lethal concentration of oxygen exposure. It is not surprising that in a diffuse lung injury model such as hyperoxia, which encompasses and involves complex and overlapping pathways, modulation of one specific molecule would have incomplete protection against a specific stimulus. This incomplete protection in terms of survival has also been observed in models of tolerance to hyperoxia involving stress response gene products such as heat shock proteins (27
). Furthermore, in studies involving intratracheal administration of adenovirus, such as this model, there will be variability among animals in the delivered amount of virus, incorporation and expression of HO-1, and differences in the immune response of rats.
In contrast to the conclusions reached herein, a recent study by Taylor et al
) has suggested that HO-1 does not provide protection against hyperoxic lung injury. In that study, intratracheal administration of hemoglobin into rats before hyperoxia exposure induced lung-specific expression of HO-1 and ameliorated subsequent lung injury due to oxygen toxicity. Inhibition of HO enzyme activity by tin-protoporphyrin, however, neither augmented hyperoxic lung injury nor reversed the protection conferred by hemoglobin. Based on these findings, they concluded that the protective effects of hemoglobin against hyperoxia were not due to HO-1, but perhaps were caused by direct induction of ferritin by hemoglobin. Certainly, reconciliation of the contradictory results within the two studies will require additional investigations. One potential explanation, however, is that hemoglobin has pleiotropic effects and activates multiple independent protective mechanisms. If these mechanisms are redundant, inhibition of an individual protective pathway may not be sufficient to reverse the ameliorative effects of hemoglobin. This idea is consistent with the results of Taylor et al
), who observed that hemoglobin stimulated HO-1 and ferritin expression independent of HO activity. Ferritin, by virtue of its ability to sequester free iron in ferritin complexes, and thus remove a potent catalyst of hydroxyl radical formation, is proposed to protect against oxidative stress (1
). Administration of Ad5-HO-1 provides for more direct assessment of the role of HO-1 independent of other antioxidant or protective functions (Figure ). We observed that upregulating HO-1 expression directly by Ad5-HO-1 can provide protection against hyperoxia in vivo
without affecting expression of antioxidant enzymes or ferritin. Furthermore, in contrast to the effects of Ad5-HO-1 on neutrophil influx (Figure ), the ameliorative mechanism of hemoglobin apparently does not require antineutrophil or antioxidant activities. Finally, in our hands, and in contrast to the observations of Taylor et al.
), administration of tin-protoporphyrin to animals consistently augments lung tissue injury in response to various oxidants (9
), including hyperoxia (29
The precise mechanisms by which ectopic expression of HO-1 provided protection against lethal hyperoxia remains unclear. Our studies demonstrating that exogenous administration of HO-1–imparted anti-inflammatory and antiapoptotic effects are consistent with published reports of the effects of endogenous HO-1 overexpression. Willis et al.
) recently reported that induction of endogenous HO-1 imparts potent anti-inflammatory effects in vivo
, and Soares et al.
) reported that HO-1 serves as a potent antiapoptotic molecule in a model of inflammation in tissue transplantation. The by-products of HO catalysis, including CO, ferritin from released iron, and bilirubin, represent attractive candidate molecules that may play key roles in mediating the protective effects of HO-1 against oxidant-mediated lung injury. For example, Stocker et al.
) have shown that bilirubin possesses potent antioxidant properties, which also have been demonstrated in vivo
). The other formal possibility is the role of CO, a gaseous molecule with biological activities similar to nitric oxide (34
). Although the biological and physiological role of CO remains unclear in our model, the intriguing possibility that CO, a nonreactive diffusible gas, could impart biological function warrants further studies in various models of oxidative stress, including hyperoxia. Our laboratory has recently observed that exogenous CO at low concentrations can provide tolerance against hyperoxic lung injury (29
). Further studies are needed to examine the possible mechanisms by which CO mediates protection against hyperoxia.
In summary, we have demonstrated the feasibility of delivering exogenous HO-1 by an adenovirus gene therapy approach, with successful incorporation and expression of the gene product. We report here that the induction of HO-1 by exogenous delivery provided physiologic protection against lethal hyperoxia. We have also explored in this study possible mechanisms to explain the protective functions attributed to HO-1 in hyperoxia-induced lung injury. This study raises the intriguing possibility of the potential therapeutic use of HO-1, not only in lung disorders such as ARDS and sepsis, but also in other inflammatory disease states.