Small interfering RNA targeting heme oxygenase-1 enhances ischemia-reperfusion-induced lung apoptosis

Heme oxygenase-1 (HO-1) is emerging as an important cytoprotective enzyme system in a variety of injury models. In order to optimize future therapeutic applications of HO-1, it is necessary to delineate precise functions and mechanisms as well as modes of externally regulating HO-1 expression. Investigations have been limited by difficulties with the generation of HO-1 null mice and the lack of specific HO-1 inhibitors. injury is the inciting event in acute lung failure following transplantation, surgery, and shock. In order to study the function of HO-1 in I-R-induced lung injury, we designed small interfering RNA (siRNA) sequences that effectively suppress HO-1 expression both in vitro and in vivo in an organ-specific manner. In this study we show that there is enhanced apoptosis, via increased Fas expression and caspase 3 activity, in the presence of HO-1 siRNA in endothelial cells and mouse lung during I-R injury, whereas HO-1 overexpression attenuates apoptosis. To the best of our knowledge, we are the first to demonstrate that lung-specific siRNA delivery can be achieved by intranasal administration without the need for viral vectors or transfection agents in vivo , thereby obviating potential concerns of toxicity if siRNA technology is to have clinical application in the future.


Introduction
HO-1 is one of three isoforms of HO, the rate-limiting enzyme in the degradation of heme to biliverdin and eventually, to bilirubin. HO-1 expression is induced in multiple cell types and organs in response to injury. This induction is postulated to have protective properties, however, the mechanisms remain elusive (1)(2)(3)(4). HO-2 is primarily constitutive and has been found to be important in the central nervous system (5). The function of HO-3 is yet unknown.
Synthetic heme analogues such as protoporphyrins that competitively inhibit the activity of all HO isoforms are commonly used to study HO-1 function but are limited by the lack of specificity and can have the paradoxical effect of upregulating HO-1 protein expression (6,7). The HO inhibitor tin mesoporphyrin is approved by the Federal Drug Administration for the treatment of hyperbilirubinemia but, again lacks specificity (8). The recent emergence of siRNA technology to silence mammalian genes allows for highly specific analysis of gene function and has potential clinical application. Among the limited number of reports on siRNA administration in vivo, all use systemic delivery, transfection chemicals or viral vectors and none have been organ-specific, to the best of our knowledge (9)(10)(11)(12)(13). We demonstrate that intranasal siRNA delivery, without a vector or transfection agent, has lung-specificity and that HO-1 potently regulates lung apoptosis. Lung-specific siRNA will not only be a useful tool to study gene function but may have therapeutic applications for a wide range of lung diseases. Furthermore, HO siRNA may become the basis of modulating severe hyperbilirubinemia of newborns and the severe jaundice of Crigler Najjar Type I patients, where there is excessive bilirubin formation and for whom specific therapy does not currently exist. Transfection of siRNA duplexes in vitro and in vivo. PAEC were seeded into 6-or 12-well plates one day prior to transfection using DMEM tissue culture medium supplemented with 10% FBS, without antibiotics. At the time of transfection with siRNA, the cells were 50-60% confluent.
Oligofectamine Reagent (Invitrogen, Carsbad, CA, USA) was used as the transfection agent and cells were then incubated for 6 hours. Next, 30% FBS DMEM medium was added to reach a final concentration of 10% FBS in the wells. Cells were exposed to A-R 24 hours after transfection. For in vivo studies, each mouse was anesthetized with methoxyflurane and then given intranasal HO-1 siRNA (2 mg/kg body weight) or equivalent doses of nonspecific control siRNA duplex or recombinant adenovirus containing rat HO-1 cDNA (Ad-HO-1, a generous gift from Dr. Leo Otterbein, University of Pittsburgh) or the recombinant adenovirus containing the β-galactosidase gene (Ad-X-LacZ, purchased from BD Biosciences Clontech, Palo Alto, CA, USA) in a volume of 50 µl.
Exponentially growing PA317 packaging cells were used for transfection and preparation of viral particles. Individual G418-resistant clones were selected and initial viral titer assays were measured by infecting NIH-3T3 cells as previously described (18). PA317/LSN-HHO-1 and the empty viral control cells, PA317/LXSN, were grown until subconfluence. The supernatants were harvested and used to infect PAEC. After selection with G418 a stably transfected cell line of PAEC overexpressing human HO-1, designated LSN/HO-1, and a retroviral vector control cell line, LXSN, were obtained.
Western blot analysis. As previously described (14) protein was extracted from cell or lung tissue lysates, electrotransfered, and then immunoblotted with monoclonal HO-1 antibody (Stressgen Biotechnologies, Victoria, BC, Canada). Detection was performed with Phototope-HRP western detection system (Cell Signaling Technology, Beverly, MA, USA). Equivalent Mouse lung sections were subjected to terminal deoxynucleo-tidyltransferase dUTP nick end-labeling (TUNEL) assay using the in situ cell death detection kit (Roche Molecular Biochemicals, Indianapolis, IN, USA) as previously described (19). Statistics. Data are expressed as mean ± S.E. and analyzed by student's t-test. Significant difference was accepted at P<0.05.

HO-1 siRNA inhibits A-R induced HO-1 protein expression in PAEC.
We have previously shown that mouse lungs subjected to I-R injury or endothelial cells exposed to A-R induce HO-1 expression (14). However, the function of HO-1 during lung I-R injury is unknown. In order to delineate the role of HO-1 in lung I-R injury, we sought to knock down HO-1 induction in vitro and in vivo using HO-1 siRNA. We designed five siRNA sequences (1 to 5) directed against rodent HO-1, according to the methods of Elbashir et al. (20), and transfected them into PAEC.
We determined that sequence 4 was the most effective in inhibiting A-R-induced HO-1 protein expression (Fig. 1a). The silencing effect of sequence 4 on HO-1 protein induction during A-R was dose-dependent, whereas incremental doses of non-specific siRNA had no effect on HO-1 expression (

HO-1 overexpression in PAEC and mouse lung.
In order to contrast the effects of HO-1 siRNA with HO-1 overexpression, we used human HO-1 gene in a replication-defective retroviral vector, which has been previously described (18)

HO-1 modulates apoptosis in PAEC and mouse lung during A-R and I-R injury, respectively.
Apoptosis is a pivotal mechanism of I-R-induced organ injury (21)(22)(23). We demonstrated the biological effect of HO-1 on apoptosis in PAEC during A-R and in mouse lung during I-R. When On the other hand, HO-1 siRNA, especially if directed against both the exogenous human HO-1 vector and endogenous rodent HO-1, dramatically increased caspase 3 activity in PAEC during A-R (4.6±0.2%) (Figure 5c, lane 7).

Discussion
Our studies are the first to utilize the highly specific technology of siRNA to directly demonstrate that HO-1 has a dramatic effect on apoptosis during lung I-R injury, via Fas and caspase 3- In our present study, intranasal administration of HO-1 siRNA, in the absence of transfection agents, had significant lung-specific effects in modulating apoptosis. We observed the presence of HO-1 siRNA diffusely in the lung airway and alveoli. In the near future the use of tissue-specific siRNA may become feasible (with pol II rather than pol III promoters), opening up the possibility of targeting specific lung cell types. The ability to apply siRNA in an organ or cell-specific manner will be of paramount interest not only for Iung diseases but also for a broad range of clinical processes.