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(The FASEB Journal. 2000;14:418-429.)
© 2000 FASEB

Inhibition of the Rac1 GTPase protects against nonlethal ischemia/reperfusion-induced necrosis and apoptosis in vivo

MICHITAKA OZAKI*, SHAILESH S. DESHPANDE*, PIAMSOOK ANGKEOW*, JOHN BELLAN*, CHARLES J. LOWENSTEIN*, MARY C. DINAUER{dagger}, PASCAL J. GOLDSCHMIDT-CLERMONT{ddagger} and KAIKOBAD IRANI*1

* Division of Cardiology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA;
{dagger} Indiana University School of Medicine, Indianapolis, Indiana 46202, USA; and
{ddagger} The Heart and Lung Institute, Ohio State University, Columbus, Ohio 43210, USA

1Correspondence: The Johns Hopkins University School of Medicine, Ross 1023, 720 Rutland Ave., Baltimore, MD 21205, USA. E-mail: kirani{at}jhmi.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reperfusion of ischemic tissue results in the generation of reactive oxygen species that contribute to tissue injury. The sources of reactive oxygen species in reperfused tissue are not fully characterized. We hypothesized that the small GTPase Rac1 mediates the oxidative burst in reperfused tissue and thereby contributes to reperfusion injury. In an in vivo model of mouse hepatic ischemia/reperfusion injury, recombinant adenoviral expression of a dominant negative Rac1 (Rac1N17) completely suppressed the ischemia/reperfusion-induced production of reactive oxygen species and lipid peroxides, activation of nuclear factor-kappa B, and resulted in a significant reduction of acute liver necrosis. Expression of Rac1N17 also suppressed ischemia/reperfusion-induced acute apoptosis. The protection offered by Rac1N17 was also evident in knockout mice deficient for the gp91phox component of the phagocyte NADPH oxidase. This work demonstrates the crucial role of a Rac1-regulated oxidase in mediating the production of injurious reactive oxygen species, which contribute to acute necrotic and apoptotic cell death induced by ischemia/reperfusion in vivo. Targeted inhibition of this oxidase, which is distinct from the phagocyte NADPH oxidase, should provide a new avenue for in vivo therapy aimed at protecting organs at risk from ischemia/reperfusion injury.—Ozaki, M., Deshpande, S. S., Angkeow, P., Bellan, J., Lowenstein, C. J., Dinauer, M. C., Goldschmidt-Clermont, P. J., Irani, K. Inhibition of the Rac1 GTPase protects against nonlethal ischemia/reperfusion-induced necrosis and apoptosis in vivo.


Key Words: reactive oxygen species • adenovirus • gene therapy


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
ISCHEMIA/REPERFUSION INJURY IS a serious concern in a variety of clinical circumstances including organ transplantation (1) , myocardial infarction (2) , and stroke (3) . In these clinical settings, prolonged ischemia, followed by reperfusion, results in the generation of reactive oxygen species (ROS) (4 5 6 7) . Although the mechanisms of ischemia/reperfusion-induced tissue damage are complex, involving the production of cytokines and the subacute infiltration by inflammatory cells into the ischemic tissue (8) , production of ROS by the ischemic tissue itself during the reperfusion phase is a major factor contributing to acute tissue injury (5 , 9) . ROS produced during reperfusion can initiate a series of cellular events that eventually lead to inflammation, necrosis, and/or apoptosis (10 11 12) . ROS can directly result in oxidative damage of lipids, proteins, and nucleic acids (13) . In addition, ROS, acting as second messengers, can act on specific redox-sensitive signaling pathways such as those regulated by nuclear factor-kappa B (NF-{kappa}B) and activator protein-1 (AP1), that are involved in the inflammatory response and apoptotic cell death (14 , 15) .

The intracellular enzymatic sources responsible for the ischemia/reperfusion-induced production of ROS have not been fully characterized. Previous work has shown that xanthine oxidase and the mitochondrial electron transport chain are among important sources of ROS produced during reperfusion (9 , 16 , 17) . However, it is also evident these enzymatic sources of ROS cannot by themselves completely account for the acute phase of ischemia/reperfusion-induced injury in vivo. This is particularly true for xanthine oxidase, as exemplified by studies demonstrating that ischemia/reperfusion injury can occur in organs known to be deficient for this enzyme (18) , and lack of protection offered by xanthine oxidase inhibitors in certain models of reperfusion injury (19 20 21 22 23) . Such studies have called into question the role of this enzyme in reperfusion injury (24) . Moreover, it is also clear that ROS produced by neutrophils infiltrating ischemic tissue contribute little or none to acute reperfusion injury in vivo (25) . Based on these findings, we hypothesized that there exists, within ischemic tissue itself, a hitherto unrecognized, very important source of ROS that contributes to ischemia/reperfusion injury in vivo.

Rac1 belongs to the Rho family of small GTPases and regulates the production of ROS by an NADPH oxidase in phagocytic (26) as well as nonphagocytic cells (27 , 28) . Previously, Rac1 had been reported to mediate oxidant production in response to hypoxia/reoxygenation in vitro in several cells (29) . However, it is not known whether Rac1 is involved in ischemia/reperfusion-induced ROS production and injury in vivo. Moreover, a role for Rac1 in ischemia/reperfusion-induced apoptosis and inflammation has not been demonstrated. In this report we describe the importance of a Rac1-regulated oxidase in acute cellular necrosis and apoptosis and in induction of the acute inflammatory response in a mouse model of hepatic ischemia/reperfusion.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Recombinant replication-deficient adenoviruses
The replication-deficient adenovirus AdRac1N17, encoding the myc epitope-tagged dominant negative allele of Rac1, was constructed in our laboratory through homologous recombination in HEK 293 cells. Details of the construction of this virus have been described previously (30) . The Adßgal, encoding the inert bacterial LacZ gene, and AdSOD, encoding human Cu-Zn superoxide dismutase, have also been reported (31) . All viruses were amplified in HEK 293 cells, purified on double cesium gradients, and plaque-tittered.

Animal protocols and hepatic ischemia/reperfusion procedure
Male C57Black/6 mice, 6 to 8 wk, were starved overnight before use. Knockout mice, deficient for the gp91phox component of the phagocyte NADPH oxidase, have been previously described (32) . Three days before each experiment, replication-deficient recombinant adenoviruses were administered intravenously (i.v.) via the tail vein in a volume of 200 µl (2x109 pfu/body) with a 31G needle. All adenoviruses were dialyzed in 1 l of dialysis buffer (10 mM Tris pH 8.0, 1 mM MgCl2, 140 mM NaCl) for 2 h at 4°C just before use. No viruses were injected in uninfected control animals. General anesthesia was induced with inhalation anesthetic, methoxyflurane (Metofane), and heparin sulfate (100 U/kg body weight) was injected i.v. After laparotomy, all vessels (hepatic artery, portal vein, and bile duct) to the left and median liver lobes were clamped, according to a previously described method (33) . After 60 min of ischemia, these vessels were unclamped and the circulation was restored for the specified reperfusion time period before killing the animal and performing the assays on blood or liver tissue described below. Sham-operated control mice were subjected to laparotomy and closure without ischemia. All animals were handled according to uniform policies set forth by the Animal Care and Use Committee of The Johns Hopkins University.

Immunohistochemistry and Western blot analysis for Rac1 protein expression
Frozen sections of liver tissue (4 µm) were prepared. Sections were fixed in ice-cold acetone for 10 min. After blocking in 10% goat serum for 60 min at room temperature, the sections were incubated with 20 µg/ml of an antibody to the myc epitope (9E10, Santa Cruz). Washing steps and incubation with fluorescein-tagged secondary antibody were carried out according to the manufacturer’s recommendations. Images were obtained on a Zeiss fluorescence microscope. For Western blot analysis, 30 µg of whole liver protein extract was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. The antibody to the myc epitope at 2 µg/ml was used as the primary and a peroxidase-tagged sheep anti-mouse antibody as the secondary. The membrane was developed using enhanced chemiluminescence (ECL, Amersham).

Measurements of ROS generation in liver tissue
Two independent assays were used to measure ROS production in liver tissue. Lucigenin-enhanced chemiluminescence was used to detect superoxide. Lucigenin (bis-N-methylacridinium nitrate) luminesces specifically in the presence of superoxide (34) . After exposing the liver, the portal vein was cannulated with 24G catheter and ligated with 4–0 silk. The liver was perfused slowly with 1–2 ml of solution 1 [HBSS (Ca, Mg-free), 1 mM EGTA, 20 mM HEPES pH 7.4, preoxygenated and prewarmed to 37°C] and again with solution 2 (HBSS, 5 mM CaCl2, 0.05% collagenase, 20 mM HEPES pH 7.4. preoxygenated and prewarmed to 37°C) for 5–10 min until it softened. The left hepatic lobe was excised and cut into 1–2 mm pieces. After gently pipetting in solution 2 for 5 min, the homogenate was filtered through gauze and the cell suspension was centrifuged at 100 g for 3 min. The cell pellet was resuspended in HBSS, 20 mM HEPES, pH 7.4 (37°C) and used for the assay. This method of hepatocyte isolation yielded greater than 90% hepatocytes that were consistently > 80% viable, as judged by trypan blue exclusion. 1 x 105 cells were added to 1 ml of lucigenin buffer (75 µM lucigenin in HBSS, 20 mM HEPES pH 7.4). The chemiluminescent signal was measured using a luminometer (Monolight 2010) and monitored every 5 min for 30 min. The emitted light units, after subtracting a blank and integrating over 15 min, were used as a measure of superoxide production. Values are expressed as relative light units per 105 cells (RLU/105 cells), and represent mean ± SE.

Hydrogen peroxide production was measured using the Amplex Red Hydrogen Peroxide Assay Kit (Molecular Probes). This assay is based on the detection of hydrogen peroxide using 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red reagent), a highly sensitive and stable probe for hydrogen peroxide (35 36 37) . Liver was perfused with 1–2 ml phosphate-buffered saline (PBS) -EDTA (preoxygenated and prewarmed to 37°C) via the portal vein. The left lobe was excised and cut into small pieces with a sharp blade. After gentle homogenization in 7 ml of cold PBS (5–7 strokes), the homogenate was diluted 1:20 in PBS and centrifuged (12,000 rpm for 30 s). One hundred microliters of supernatant was used for the assay, according to the manufacturer’s recommendations. After subtraction of blank samples, the red fluorescence signal was standardized by protein concentration and hydrogen peroxide concentration, expressed as µM/mg protein, were calculated based on a standard curve. Values represent mean ± SE.

Assays for lipid peroxidation products
Liver specimens were excised, weighed, and homogenized in detergent solution (0.1% Triton X-100, 0.05% deoxycholic acid in 0.9% NaCl), shaken gently, and placed in a cool, dark place for 10–15 min. After centrifugation, the supernatant was used for lipid peroxidation assays that measure lipid hydroperoxide (LPO-CC kit, Kamiya Biomedical Company) and malondialdehyde (MDA) (Biotech LPO-586, OXIS International). The calculated concentrations of lipid peroxidation products were normalized by protein concentration and are expressed as nmol/g protein (MDA) and µmol/g protein (LPO). Values represent mean ± SE.

Serum glutamic pyruvate transaminase (sGPT) assay
After administering inhalation anesthesia, blood was collected via the inferior vena cava, and sGPT concentration was measured colorimetrically using a GP-transaminase kit (Sigma) according to the manufacturer’s recommendations. Values were calculated based on a standard curve and are expressed in international units/liter (IU/l). Values represent mean ± SE.

Histological studies
A liver specimen was fixed in 10% buffered formalin, embedded in paraffin, and stained with hematoxylin and eosin. For TUNEL staining, sections were deparaffinized and digested with proteinase K (20 µg/ml) at 37°C for 15 min. Immunohistochemical detection of apoptosis was performed using In Situ Cell Death Detection Kit (Boehringer Mannheim) and photomicrographs were obtained.

ELISA assay for apoptosis
Liver was perfused with 1–2 ml PBS-EDTA (preoxygenated, 37°C) via the portal vein. The left lobe was excised and washed in the same solution. The specimen was stored at -135°C until use. Approximately 100 mg of liver tissue was homogenized in 1 ml of incubation buffer and kept on ice for 15 min. The supernatant of the homogenate was used in the cell death detection enzyme-linked immunoassay (ELISA) (Boehringer Mannheim), according to the manufacturer’s recommendations. Values, normalized to protein concentration, are expressed as fold change in apoptosis compared to uninfected, sham-operated, normoxic mice and represent mean ± SE.

Electophoretic mobility shift assay
Nuclear extracts from liver tissue were prepared as described previously (38) . Ten micrograms of extract was incubated with 105 cpm of a 32P-labeled {kappa}B-binding consensus oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGGC- 3') or a mutant {kappa}B oligonucleotide (5'-AGTTGAGGCGACTTTCCCAGGC- 3') for 15 min in binding buffer (10 mM Tris pH 7.4, 80 mM KCl, 5% glycerol, 1 mM DTT, 0.25 µg dIdC) at room temperature. 200x unlabeled {kappa}B oligonucleotide was added to the mixture where indicated. In supershift experiments, the extract was incubated with the antibody (1 µg of {alpha}p50; Santa Cruz) for 30 min at room temperature prior to addition of labeled oligonucleotide. Incubation mixtures were run out on a 6% polyacrylamide gel and autoradiographed.

Statistical analyses
Analysis of variance and Student’s t test (two-sided) for unpaired values were used for statistical analysis. A P value below 0.05 was considered significant. Bonferoni’s test was used for analysis of differences between multiple groups and was considered significant at a 95% confidence level. The number of animals (n) used for each experiment is indicated. Statistically nonsignificant differences are denoted by n.s.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Adenoviral expression of Rac1N17 in mouse liver
To efficiently inhibit Rac1-dependent mechanisms in mouse liver, we used a recombinant replication deficient adenovirus encoding the dominant negative form of Rac1, AdRac1N17. The Adßgal was used in control mice. Infection with AdRac1N17 achieved significant levels of Rac1N17 protein expression, as judged by immunoblotting of liver protein extract (Fig. 1A ). Immunohistochemistry of liver sections (Fig. 1B ) showed that ~50% of the hepatocytes expressed the Rac1N17 gene product.



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  		Figure 1. Adenoviral expression of Rac1N17 in mouse liver. A) Western blot analysis of whole liver protein extract from uninfected, Adßgal-infected, and AdRac1N17-infected mice. An antibody to the myc epitope was used to detect myc-tagged Rac1N17. Protein loading was assessed by immunoblotting against {alpha}-tubulin. B) Immunofluorescence (100x magnification) of liver sections from mice infected with Adßgal (a) and AdRac1N17 (b), using an antibody to the myc epitope. Each animal was infected with 2 x 109 PFU. Expression was assessed 72 h after infection with adenoviruses. Inset (630x magnification) demonstrates the intracellular expression of Rac1N17.

Expression of Rac1N17 suppresses ischemia/reperfusion-induced ROS production and lipid peroxidation in mouse liver
Most studies have suggested that the superoxide anion is the primary oxidant species generated with ischemia/reperfusion (4 , 39) . We therefore used a chemiluminescence-based assay that uses lucigenin as the substrate as a measure of superoxide production in our model of hepatic ischemia/reperfusion. This lucigenin-enhanced chemiluminescence assay has previously been shown to be specific for superoxide (34) . Ischemia/reperfusion resulted in an early burst and a late peak of superoxide generation (Fig. 2A ). The early burst, which occurred 5 min into reperfusion, represents ROS produced by the ischemic tissue itself (5) . In contrast, the late peak, which occurred 8–24 h into reperfusion and was higher in magnitude than the early peak, is mainly due to ROS produced by inflammatory cells, primarily neutrophils, infiltrating the reperfused tissue. This was borne out in mice lacking the gp91phox component of the phagocyte NADPH oxidase. In these knockout mice (gp91phoxKO), superoxide production at 5 min of reperfusion was not different than that seen in wild-type mice (gp91phoxKO 18210±240; wild-type 16989±1216; n=3, P = n.s.), showing that neutrophils have a minimal, if any, role in superoxide production during the immediate-early phase of reperfusion. This finding is consistent with other reports examining the role of neutrophils in reperfusion injury (40) . In contrast, hepatic superoxide levels after 8 h of reperfusion were markedly lower in the gp91phox knockout mice as compared to wild-type mice (gp91phoxKO 2636±25; wild-type 36653±2712; n=3, P<0.01), strongly implicating neutrophil involvement in superoxide production during this phase of reperfusion.



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  		Figure 2. Inhibition of ischemia/reperfusion-induced ROS production in mouse liver by expression of Rac1N17. A) Time course of ischemia/reperfusion-induced superoxide production in wild-type and gp91phoxKO mice. B) Effect of Rac1N17 on ischemia/reperfusion-induced immediate early superoxide production in wild-type and gp91phoxKO mice. C) Effect of Rac1N17 on ischemia/reperfusion-induced immediate early hydrogen peroxide production. Reperfusion times indicate in vivo reperfusion. Results are representative of two independent experiments.

Having established the kinetics of ischemia/reperfusion-induced superoxide production and that the early phase of superoxide generation was independent of the inflammatory response, we next sought to investigate the role of Rac1 in regulating this early burst of superoxide produced by hepatic tissue. ROS production at 5 min after reperfusion was measured in mice infected with Adßgal and compared to those infected with AdRac1N17 (Fig. 2B ). When compared to uninfected mice, livers of mice infected with Adßgal had no appreciable increase in ROS production, both at baseline (Adßgal 7817±1106; uninfected 5446±204; n=3, P = n.s.) and with ischemia/reperfusion (Adßgal 19428±264; uninfected 16989±1216; n=3, P = n.s.). Expression of Rac1N17 resulted in complete suppression of the early burst of reperfusion-induced superoxide production when compared to uninfected mice and mice infected with Adßgal (AdRac1N17 5293±350; uninfected 16989±1216; Adßgal 19428±264; n=3, P<0.01). To confirm the identity of the primary oxidant species produced during reperfusion and the validity of our assay for detecting superoxide, parallel experiments were conducted in mice infected with AdSOD, an adenovirus encoding the Cu-Zn form of superoxide dismutase. Similar to Rac1N17, expression of SOD in mice liver achieved complete suppression of ischemia/reperfusion-induced increase in lucigenin chemiluminescence when compared with uninfected or Adßgal-infected controls (AdSOD 7190±161; uninfected 16989±1216; Adßgal 19428±264; n=3, P<0.01).

To confirm the role of Rac1 in ischemia/reperfusion-induced ROS production and to determine whether oxidant species other than superoxide were being generated with ischemia/reperfusion, we measured ROS production in mouse liver by an independent, fluorescence-based assay designed specifically to detect hydrogen peroxide (36) (Fig. 2C ). Infection with Adßgal did not lead to an increase in hepatic peroxide levels. Ischemia/reperfusion led to a two- to threefold increase in hydrogen peroxide concentration in Adßgal-infected mice livers (normoxia 22.3±0.2; ischemia/reperfusion 65.5±1.1; n=3, P<0.01). Similar to the effect of Rac1N17 on superoxide production, expression of Rac1N17 resulted in complete suppression of the ischemia/reperfusion-induced increase in hydrogen peroxide generation (normoxia 20.4±0.4; ischemia/reperfusion 23.2±0.5; n=5, P = n.s.).

ROS can lead to tissue injury through direct oxidative damage of cellular proteins, lipids, and DNA. Malondialdehyde and lipid hydroperoxide are two lipid peroxidation products that are elevated during ischemia/reperfusion injury (13) . We therefore asked whether suppression of ischemia/reperfusion-induced ROS production by Rac1N17 translated into a reduction of lipid peroxidation products (Fig. 3A, B ). Compared to uninfected and Adßgal-infected mice, hepatic tissue of mice infected with AdRac1N17 showed a significant decrease in both reperfusion-induced lipid hydroperoxide (AdRac1N17 1.45±0.14; uninfected 7.02±1.04; Adßgal 6.26±1.06; n=3, P<0.01) and malondialdehyde (AdRac1N17 32±0.5; uninfected 48.6±1.4; Adßgal 53.3±0.3; n=3, P<0.01) accumulation.



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  		Figure 3. Inhibition of ischemia/reperfusion-induced lipid peroxides in mouse liver by expression of Rac1N17. Effect of Rac1N17 on A) malondialdehyde and B) lipid hydroperoxide formation after ischemia/reperfusion. Results are representative of two independent experiments.

The Rac1-regulated oxidase responsible for ROS production in hepatic tissue is distinct from the phagocyte NADPH oxidase
Rac is known to be essential for the production of ROS in neutrophils through the regulation of a plasma membrane NADPH oxidase within such cells (26) . We were intrigued by the possibility that this oxidase present in hepatic cells may be responsible for the ischemia/reperfusion-induced ROS production. To test this possibility, we assessed the role of Rac1 in ischemia/reperfusion-induced superoxide production in gp91phox knockout mice (Fig. 2B ). The early burst in superoxide production (5 min after reperfusion) seen in uninfected mice, and which is indicative of endogenous hepatic superoxide generation, was completely inhibited in mice infected with AdRac1N17 (AdRacN17 3333±346; uninfected 18210±439; n=3, P<0.01). This indicates that gp91phox is not a component of the Rac1-regulated enzymatic system responsible for ischemia/reperfusion-induced intrinsic hepatic superoxide production.

Inhibition of Rac1 leads to suppression of ischemia/reperfusion-induced acute liver necrosis and apoptosis
Since ROS are thought to be primary mediators of ischemia/reperfusion-induced tissue injury, the ability of Rac1N17 to suppress this injury in the liver was assessed. Serum glutamic pyruvate transaminase (sGPT), a marker of hepatocellular injury, was measured 1 and 8 h after reperfusion (Fig. 4A ). Adenoviral infection alone led to a predictable increase in sGPT levels (uninfected 40.8±5.1; Adßgal 117.5±16.5; n=3, P<0.05), which is probably a reflection of acute toxic and cytotoxic T cell responses (41) . Ischemia/reperfusion resulted in a further severalfold rise in sGPT in both uninfected and Adßgal-infected mice. Expression of Rac1N17 resulted in a marked reduction in sGPT levels (AdRac1N17 633±90; Adßgal 1612±165; n=3, P<0.01). The magnitude of this reduction was the same as that seen in mice infected with AdSOD (AdSOD 421±112; Adßgal 1612±165; n=3, P<0.01), suggesting that a Rac1-regulated enzymatic system was primarily responsible for superoxide-induced acute hepatocellular injury. Somewhat surprisingly, mice infected with AdRac1N17 continued to show reduced levels of sGPT, even at 8 h after reperfusion, compared to Adßgal-infected controls (AdRac1N17 723±16; Adßgal 1473±15; n=3, P<0.05). In concert with the data on superoxide production, Rac1N17 suppressed ischemia/reperfusion-induced sGPT rise to the same extent in gp91phox knockout mice as in their wild-type littermates (gp91phoxKO 614±37; wild-type 723±16; n=3, P = n.s.). To further demonstrate the role of Rac1 in ischemia/reperfusion-induced hepatic necrosis, histological sections were stained with H&E and necrotic areas were qualitatively assessed (Fig. 4B ). Liver sections from normoxic, sham-operated mice showed no necrosis. Ischemia followed by 8 h of reperfusion led to a marked induction of histologically-identifiable necrosis in uninfected and Adßgal-infected mice. These necrotic areas were extensive in their distribution. Mice infected with AdRac1N17 showed a significant reduction in extent of ischemia/reperfusion-induced necrosis. The degree of necrosis, as judged by histological sections, correlated with the sGPT levels measured 8 h after reperfusion.



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  		Figure 4. Rac1N17 suppresses ischemia/reperfusion-induced acute necrotic liver injury. A) The effect of Rac1N17 on sGPT levels in wild-type and gp91phoxKO mice 1 and 8 h after reperfusion. Results are representative of two independent experiments. B) H&E-stained liver sections (100x magnification) from a) uninfected, sham-operated normoxia, b) uninfected + ischemia/reperfusion, c) Adßgal-infected + ischemia/reperfusion, d) AdRac1N17-infected + ischemia/reperfusion mice. Ischemia was for 1 h, followed by 8 h of reperfusion. Arrows indicate necrotic areas surrounded by normal tissue.

In addition to having direct toxicity on cellular macromolecules, ROS are also known to influence redox-sensitive signaling pathways (14 , 15) . Such pathways regulate the inflammatory response and apoptotic cell death seen with ischemia/reperfusion (10 , 11) (12) . We therefore examined the role of Rac1 in mediating ischemia/reperfusion-induced acute apoptotic cell death in mouse liver. Histological sections of liver tissue 8 h after reperfusion were examined by the TUNEL method to detect apoptotic cells (Fig. 5A ). Ischemia/reperfusion led to an induction of apoptosis, as evidenced by the appearance of regions of TUNEL-positive cells. Distribution of the TUNEL-positive regions was similar to that of the necrotic areas. Expression of Rac1N17 markedly decreased the extent of ischemia/reperfusion-induced apoptotic areas when compared to uninfected or Adßgal-infected controls. The reduction in apoptosis was manifested as a decrease in the size and number of regions of TUNEL-positive cells. Comparison with uninfected, sham-operated controls showed that infection with AdRac1N17 did not result in a complete inhibition of ischemia/reperfusion-induced apoptosis. Quantification of apoptosis and confirmation of the results of the TUNEL assay were obtained with an apoptosis ELISA that measures cytoplasmic DNA-histone fragments (Fig. 5B ). This assay demonstrated that ischemia/reperfusion resulted in a 10-fold induction of apoptosis in mice liver compared to normoxic sham-operated controls. Expression of Rac1N17 resulted in a 60% reduction of ischemia/reperfusion-induced hepatocellular apoptosis (Adßgal 10.0±0.18; AdRac1N17 4.13±1.23; n=4, P<0.01). The protective effect of Rac1N17 against ischemia/reperfusion-induced apoptosis was apparent even at 24–36 h into reperfusion (data not shown), although the magnitude of the effect was not as striking as that seen at 8 h after reperfusion.



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  		Figure 5. Rac1N17 suppresses ischemia/reperfusion-induced acute liver apoptosis. A) TUNEL staining of liver sections (100x magnification) from a) uninfected, sham-operated normoxia, b) uninfected + ischemia/reperfusion, c) Adßgal-infected + ischemia/reperfusion, d) AdRac1N17 + ischemia/reperfusion mice. Insets (630x magnification) show presence or absence of TUNEL-positive nuclei. Ischemia was for 1 h, followed by 8 h of reperfusion. B) Quantification of ischemia/reperfusion-induced hepatocellular apoptosis in uninfected, Adßgal-infected, and AdRac1N17-infected mice, using a cell death ELISA. Results are representative of two independent experiments.

Rac1 regulates ischemia/reperfusion-induced NF-{kappa}B activation
Activation of the transcription factor NF-{kappa}B has been strongly implicated in ischemia/reperfusion injury. NF-{kappa}B is believed to mediate reperfusion injury via the induction of various {kappa}B-driven inflammatory genes. Not coincidentally, NF-{kappa}B activation is sensitive to the redox state of the cell in many in vitro and in vivo systems (30 , 42) . We were therefore interested in the role of Rac1 on NF-{kappa}B activation in our model of ischemia/reperfusion. Electrophoretic mobility shift assay was used to detect the DNA binding activity of NF-{kappa}B in vivo (Fig. 6 ). Under normoxic conditions, infection with Adßgal or AdRac1N17 had no appreciable effect on NF-{kappa}B DNA binding activity as compared to uninfected mice. Ischemia/reperfusion resulted in a profound increase in hepatic NF-{kappa}B DNA binding activity in both uninfected and Adßgal-infected mice. In striking contrast, mice infected with AdRac1N17 exhibited almost no induction of NF-{kappa}B activity when subjected to ischemia/reperfusion. One hour of ischemia alone also led to a small increase in NF-{kappa}B DNA binding activity. An antibody against the p50 subunit of NF-{kappa}B was used to further delineate the composition of NF-{kappa}B responsible for the ischemia/reperfusion-induced increase in DNA binding activity. Supershifted bands showed that the activated NF-{kappa}B consisted of the p50 subunit. This shows that Rac1 is an important part of the mechanism that leads to NF-{kappa}B activation and thus contributes to the inflammatory response that occurs in reperfused tissue.



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  		Figure 6. Rac1N17 inhibits ischemia/reperfusion-induced NF-{kappa}B activation. Electrophoretic mobility shift assay for NF-{kappa}B DNA binding. All viruses were used at 2 x 109 PFU/animal. Specific binding to the consensus {kappa}B sequence was confirmed with the use of a mutant {kappa}B oligonucleotide and unlabeled excess {kappa}B oligonucleotide (comp 200x). Presence of the p50 unit in the NF-{kappa}B band was determined by supershift analysis using an antibody to this unit. Supershifted bands are indicated by arrows. Results are representative of two independent experiments.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The role of ROS in ischemia/reperfusion-induced organ damage is well established. Many studies have demonstrated that inhibition of ROS production or scavenging of ROS offers protection against tissue injury incurred during ischemia/reperfusion. Some of these studies have used chemical or enzymatic scavengers of ROS (42) , whereas others have aimed at inhibiting the source(s) responsible for the production of ROS (39 , 43) . With regards to the latter, attention has primarily been directed toward xanthine oxidase and the mitochondrial electron transport chain as intracellular sources of ROS. In this report, we demonstrate for the first time that a cellular oxidase regulated by the small GTP-binding protein Rac1 is also a very important source of ischemia/reperfusion-induced oxidant stress in vivo.

Using recombinant adenoviruses, we show that expression of Rac1N17 completely inhibits ischemia/reperfusion-induced superoxide production in mouse liver. Inhibition of superoxide production is equivalent to that seen with the overexpression of SOD, suggesting that a Rac1-regulated enzymatic system(s) is the main source(s) of superoxide in our model of ischemia/reperfusion. Because of recent criticism regarding the use of lucigenin-enhanced chemiluminescence as a modality for detecting superoxide (44) , we validated our findings about the production of ROS in our model, using an independent complementary assay that specifically detects hydrogen peroxide. Identical to our observations using lucigenin-enhanced chemiluminescence, ischemia/reperfusion-induced hydrogen peroxide production was also completely abolished in livers expressing Rac1N17. This is consistent with the fact that hydrogen peroxide is a dismutation product of superoxide. Using both assays, we also detected basal ROS levels under normoxic conditions. Since ROS are involved in many cellular functions (45) , this basal ROS production may be necessary for growth or other cellular functions of hepatocytes and/or may be due to low levels of ROS production from tissue phagocytes present in the liver, such as Kupffer cells.

The role of the phagocyte NADPH oxidase in our model of ischemia/reperfusion injury is well illustrated using the gp91phoxKO animals. Immediate early reperfusion-induced superoxide production (5 min after reperfusion) and early reperfusion injury (sGPT levels 1 h after reperfusion) in the gp91phoxKO mice were no different than in their control littermates, demonstrating that phagocytes are not important in early reperfusion injury. At 8 h after reperfusion, superoxide production in liver tissue was markedly lower in the gp91phoxKO mice than in their wild-type littermates, showing the important role of phagocytic cells in infiltrating liver tissue and producing ROS at this period of reperfusion. sGPT levels continued to remain elevated at this time in the gp91phoxKO mice despite a reduction in superoxide levels, a reflection of the fact that tissue injury lags behind ROS production. Suppression of immediate early superoxide production and acute reperfusion injury in the gp91phoxKO mice, by expression of Rac1N17, illustrates the importance of an endogenous Rac1-regulated oxidase distinct from the phagocyte NADPH oxidase in reperfusion injury. Thus, our findings suggest that complete inhibition of reperfusion injury, which includes both the acute and late phase components, would require not only inhibition of neutrophil function, but also suppression of a tissue Rac1-dependent oxidase, and are consistent with other reports showing the same (46) .

Inhibition of acute tissue necrosis was not complete with expression of Rac1N17 despite complete suppression of endogenous hepatic reperfusion-induced ROS production. This suggests that ROS production is only one mechanism by which ischemia/reperfusion results in injury. This possibility is supported by our findings that expression of Cu-Zn SOD also achieved only incomplete suppression of acute reperfusion injury, despite completely inhibiting reperfusion-induced superoxide production. The subcellular localization of the ROS produced may be important in determining the extent of injury. Thus, expression of Cu-Zn SOD or Rac1N17 that scavenge or inhibit cytosolic ROS may not be sufficient to completely abrogate ROS-mediated injury. Scavenging of mitochondrial ROS produced during reperfusion, by expression of mitochondrial-specific Mn-SOD, may prove complementary to Rac1N17 in protecting against acute reperfusion injury. Using overexpression of Mn-SOD alone, such a strategy has been successful in partially inhibiting ischemia/reperfusion injury (42) .

Previous studies have shown the important role of apoptotic cell death in ischemia/reperfusion injury (10 , 11) . We also observed apoptosis in our model of ischemia/reperfusion injury, and it is interesting that the widespread distribution of the apoptotic regions was similar to that of the necrotic areas. The extent of apoptosis is somewhat surprising, but consistent with other reports showing 40–50% apoptotic cells at a similar time point after reperfusion (10) . The high percentage of TUNEL-positive cells may also represent, to some degree, the false positives seen with this assay. Nonetheless, the codistribution of necrotic and apoptotic regions, combined with the observation that expression of Rac1N17 suppressed both apoptosis and necrosis, suggests that these two forms of cell death may share common mediators. Certainly, ROS can result in both apoptosis and necrosis (47) , and the ability of Rac1N17 to inhibit ischemia/reperfusion-induced ROS production may be the underlying mechanism for its protective effect against apoptosis and necrosis.

Rac1N17 did not completely inhibit ischemia/reperfusion-induced apoptosis, implicating alternative Rac1 and ROS-independent mechanisms in this form of injury. Moreover, the maximal effect of Rac1N17 was observed at 8 h after reperfusion and diminished with time over the next 24 h (data not shown). This is not unexpected since adenoviral delivery targets Rac1N17 to hepatic tissue and not peripheral neutrophils, and therefore late neutrophil-mediated events, including apoptosis, may not be as dramatically altered as early phenomena dependent on endogenous hepatic ROS production. However, the fact that apoptosis that occurs late into reperfusion is inhibited to some extent by Rac1N17 does suggest that Rac1-dependent mechanisms in hepatic tissue are at least partially involved in promoting apoptosis initiated by infiltrating neutrophils. Such mechanisms may include, but are not limited to, regulation of leukocyte adhesion molecule expression in hepatic endothelial cells by Rac1 (48 , 49) . This is supported by our finding that Rac1 regulates ischemia/reperfusion-induced activation of NF-{kappa}B, which transcriptionally regulates the expression of several cell surface adhesion molecules. Moreover, p50 knockout mice sustain significantly reduced cerebral ischemia/reperfusion injury (50) , further suggesting that the suppression of NF-{kappa}B activation, and in particular p50-containing NF-{kappa}B dimers, by Rac1N17 may be an important mechanism through which Rac1N17 protects against reperfusion injury.

Due to inherent limitations in our assays for detecting ROS, we cannot exclude the possibility that other, previously recognized enzymatic systems and pathways such as xanthine oxidase, the mitochondrial electron transport chain, or arachidonate metabolism may also be contributing to reperfusion-induced ROS production in our model. Indeed, there is ample evidence to show that one or more of these mechanisms play an important part in reperfusion injury, depending on the tissue and species being examined (16 , 17 , 51 52 53 54) . Taken in conjunction with these previous studies, our work raises the intriguing possibility that Rac1 participates in the regulation of such enzymatic systems or pathways in vivo. In support of this, studies in vitro have shown that members of the Ras family of proteins, including Rac1, regulate mitochondrial ROS production (55) and participate in arachidonate metabolism (56) . Whatever the mechanism for the observed effect may be, this report demonstrates that, either directly or indirectly, Rac1 participates in regulating ischemia/reperfusion-induced ROS production in vivo, and is thereby crucial in promoting reperfusion injury. More important than providing another way of ameliorating ischemia/reperfusion injury, this work identifies a potentially new mechanism for the production of ROS with ischemia/reperfusion in vivo. Furthermore, since Rac1 is ubiquitously expressed and highly conserved through species, a Rac1-regulated oxidase may represent a common mechanism for the production of ROS and injury across species and in a variety of organs exposed to ischemia and reperfusion.


   ACKNOWLEDGMENTS
 
We thank R. G. Crystal and N. T. Eissa for the Adßgal and AdSOD, E. Marban and L. Becker for their constructive criticisms of the manuscript, K. Baughman and S. Dirks for their unwavering support, and P. Emig for secretarial assistance. This work was supported by the Johns Hopkins University Clinician Scientist Award (K.I.), a Maryland American Heart Association Grant-in-Aid (K.I.), an American Liver Foundation Amgen Postdoctoral Research Fellowship (M.O.), the Bernard Foundation, and an endowment from Mr., and Mrs. Abraham Weiss.


   FOOTNOTES
 
Received for publication February 18, 1999. Accepted for publication October 1, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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