AJP - Heart Journal of Neurophysiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 291: H2980-H2986, 2006. First published July 28, 2006; doi:10.1152/ajpheart.01173.2005
0363-6135/06 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/6/H2980    most recent
01173.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Duranski, M. R.
Right arrow Articles by Lefer, D. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Duranski, M. R.
Right arrow Articles by Lefer, D. J.

Genetic overexpression of eNOS attenuates hepatic ischemia-reperfusion injury

Mark R. Duranski,1 John W. Elrod,1 John W. Calvert,1 Nathan S. Bryan,2 Martin Feelisch,2 and David J. Lefer1

1Department of Medicine, Division of Cardiology, and Department of Pathology, Albert Einstein College of Medicine, Bronx, New York; and 2Department of Medicine, Boston University Medical Center, Whitaker Cardiovascular Institute, Boston, Massachusetts

Submitted 4 November 2005 ; accepted in final form 18 July 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Previous studies have shown that endothelial nitric oxide (NO) synthase (eNOS)-derived NO is an important signaling molecule in ischemia-reperfusion (I-R) injury. Deficiency of eNOS-derived NO has been shown to exacerbate injury in hepatic and myocardial models of I-R. We hypothesized that transgenic overexpression of eNOS (eNOS-TG) would reduce hepatic I-R injury. We subjected two strains of eNOS-TG mice to 45 min of hepatic ischemia and 5 h of reperfusion. Both strains were protected from hepatic I-R injury compared with wild-type littermates. Because the mechanism for this protection is still unclear, additional studies were performed by using inhibitors and activators of both soluble guanylyl cyclase (sGC) and heme oxygenase-1 (HO-1) enzymes. Blocking sGC with 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and HO-1 with zinc (III) deuteroporphyrin IX-2,4-bisethyleneglycol (ZnDPBG) in wild-type mice increased hepatic I-R injury, whereas pharmacologically activating these enzymes significantly attenuated I-R injury in wild-type mice. Interestingly, ODQ abolished the protective effects of eNOS overexpression, whereas ZnDPBG had no effect. These results suggest that hepatic protection in eNOS-TG mice may be mediated in part by NO signaling via the sGC-cGMP pathway and is independent of HO-1 signal transduction pathways.

nitric oxide; heme oxygenase-1; soluble guanyly cyclase; phosphodiesterase type 5 inhibition; endothelial nitric oxide synthase

HEPATIC ISCHEMIA-REPERFUSION (I-R) injury occurs in many clinical settings, including liver resection, liver transplantation, hemorrhagic shock, septic shock, and cardiogenic shock (15). Liver transplantation has been on the rise over the last fifteen years, prompting an increasing need to better understand the pathogenic mechanisms of hepatic I-R injury and to develop novel means of hepatic protection.

One therapeutic approach, of recent interest, to minimize I-R injury, is the enhancement of nitric oxide (NO) bioavailability. NO is a product of the oxidative conversion of L-arginine to citrulline via the enzyme endothelial NO synthase (eNOS). NO is of physiological importance because of its involvement in cardiovascular homeostasis. NO fulfills many physiological roles including acting as a vasodilator by relaxing vascular smooth muscle cells (17, 29), inhibiting platelet aggregation (30), leuckocyte adhesion (25), and general inflammation (27).

Although the role of NO in hepatic I-R injury has been controversial with a number of studies (3) that cite the protective and deleterious effects of NO therapy, it is now generally accepted that eNOS-derived NO is cytoprotective in I-R injury (4, 37). Studies (24, 37) have shown that eNOS significantly contributes to the cytoprotection of hepatic tissue against I-R injury and that injury is exacerbated in eNOS-deficient mice (16, 19). In correlation, eNOS overexpression has been reported (20) to reduce I-R injury.

The aim of the present study was to investigate the effects of chronic genetic overexpression of eNOS on the severity of hepatic I-R injury. We hypothesized that the enhanced production of NO would protect the ischemic liver via an NO-mediated pathway. In additional studies, we utilized pharmacological agents to investigate the cytoprotective mechanisms related to genetic overexpression of eNOS.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals and Reagents

1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) was purchased from Alexis Biochemicals (San Diego, CA) and utilized as an inhibitor of soluble guanylyl cyclase (sGC) (14). It was administered intraperitoneally at a dose of 30 mg/kg at 22.5 min of ischemia, dissolved in a volume of 100 µl DMSO.

3-(5'-Hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1), an sGC activator (13), was obtained from Alexis Biochemicals. It was dissolved in DMSO and diluted in normal saline, and 100 µl were injected intraperitoneally at a dose of 20 mg/kg at 22.5 min into ischemia.

Zinc (III) deuteroporphyrin IX-2,4-bisethyleneglycol (ZnDPBG) is an inhibitor of heme oxygenase-1 (HO-1) activity (6). It was purchased from Alexis Biochemicals. It was dissolved in DMSO and diluted in normal saline, and 100 µl were injected intraperitoneally at a dose of 10 mg/kg at 22.5 min into ischemia.

Cobalt (III) protoporphyrin IX chloride (CoPP), an activator of HO-1, was acquired from Alexis Biochemicals. Dissolved in normal saline, it was injected intraperitoneally 22.5 min into ischemia at a dose of 5 mg/kg.

Sildenafil citrate 1-({3-[6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo(4,3-d)pyrimidin-5-yl]-4-ethoxyphenyl}sulfonyl)-4-methylpiperazine citrate is a highly selective inhibitor of phosphodiesterase type 5 (PDE-5). It was dissolved in saline and administered intraperitoneally at a dose of 2 mg/kg at 22.5 min into ischemia.

Animals

eNOS-TG mice. In our present studies, we used two different strains of eNOS-TG mice. One mouse, developed in Kobe, Japan (Kobe eNOS-TG), was developed on a C57BL/6 background and contained the murine prepoendothelin-1 promoter (GenBank accession no. U07982) and bovine eNOS cDNA (GenBank accession no. M99057) (20).

A second mouse contained the human eNOS gene and was developed in Rotterdam, The Netherlands (RT eNOS-TG) (20). eNOS synthase cDNA, which was donated by Dr. S. Janssens of Leuven, Belgium, was used as a probe in isolating the gene from a homemade human genomic cosmid library (18).

All control mice, i.e., wild-type (WT), used in these experiments were nontransgenic littermates of the eNOS-TG mice. All transgenic strains had been backcrossed onto the c57B6 background for a minimum of 10 generations. Male mice in the study were utilized at 8–12 wk of age and were maintained on a normal rodent chow diet. All experimental mouse procedures were approved by the Institute for Animal Care and Use committee at Albert Einstein College of Medicine and conformed to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 86-23, Revised 1996) and with federal and state regulations.

Western Blot Analysis of eNOS

Hepatic tissue lysates were centrifuged to remove any particulate, and protein concentration of the cleared lysate was measured by using the Bio-Rad DC protein assay. Equal amounts of protein (40 µg) were loaded into each lane and separated on a 7% polyacrilamide gel. Protein was transferred to polyvinylidene difluoride overnight at 30 V and then blocked in 5% milk in Tris-buffered saline Tween 20 (TBST) at room temperature for 3 h. Membranes were washed three times with TBST and then incubated with mouse-anti-eNOS (1:4,000, BD Transduction) in 5% BSA TBST overnight at 4°C. Membranes were then washed three times with TBST and then incubated with horseradish peroxidase (HRP)-linked anti-mouse secondary (Amersham) at 1:2,000 in 5% BSA TBST at room temperature for 3 h. Membranes were then washed 3 times with TBST, incubated with enhanced chemiluminescence (ECL) reagents (Amersham), and then exposed to film. Membranes used to detect eNOS were then stripped and incubated overnight with mouse-anti-beta-actin (as a loading control) at 1:2,000 in 5% BSA TBST at 4°C. Membranes were then washed and incubated with HRP-linked anti-mouse secondary at 1:2,000 in 5% BSA TBST at room temperature for 3 h, washed and incubated with ECL reagents, and then exposed to film. Densitometric analysis was performed using ImageJ software from the NIH.

NO Analysis of Hepatic Tissue

Hepatic lobes were flushed completely free of blood by ex vivo perfusion with air-equilibrated PBS supplemented with 10 mM N-ethylmaleimide (No. 23030, Pierce) and 2.5 mM EDTA (No. BP120, Fisher). Tissue nitroso compounds were quantified by using group-specific reductive denitrosation by iodine-iodide with subsequent detection of the NO liberated by gas-phase chemiluminescence as previously described (11). NO-heme was determined by parallel injection of replicate aliquots of tissue homogenates into a solution of 0.05 M ferricyanide in PBS at pH 7.5 and at 37°C. This method employs one-electron oxidation rather than reduction to achieve denitrosation, with the liberated NO being quantified by gas-phase chemiluminescence, and has been previously described in detail (5). Nitrate and nitrite concentrations were quantified by ion chromatography (ENO20 Analyzer; Eicom, Kyoto, Japan) (31).

Hepatic cGMP Assay

Hepatic cGMP comparisons between WT and eNOS-TG mice were performed by Cayman Chemical EIA Service using a competitive ELISA kit (cGMP EIA Kit, No. 581021, Ann Arbor, MI). All samples were corrected with protein concentration.

Blood Pressure and Heart Rate Analysis

Aortic blood pressures (systolic, diastolic, and mean) and heart rate were measured in WT, Kobe eNOS-TG, and RT eNOS-TG mice in the conscious state using radiotelemetry techniques. Mice were surgically implanted with the PA-C10 radiotelemeter pressure transducers (DSI, St. Paul, MN), and the catheters were placed in the aorta as previously described (10). Measurements were conducted six times at each 24-h cycle over a 48-h sampling period.

Hepatic I-R Protocol

The hepatic I-R protocol has been described previously (9). Mice were anesthetized by ketamine (100 mg/kg) and xylazine (8 mg/kg) mixed in solution (0.9% normal saline) and administered intraperitoneally. A midline laparotomy incision was made to expose the liver. Mice were injected with heparin (100 U/kg ip) to prevent clotting before reperfusion. The left median and lateral lobes of the liver were made ischemic by clamping the hepatic artery and the portal vein using a microaneurysm clamp. This procedure results in a segmental (70%) ischemia so as to prevent mesenteric venous congestion by allowing portal decompression throughout the right and caudate lobes. The liver was then placed back in its original position for 45 min and kept moist with gauze dampened with 0.9% normal saline. Core body temperature was monitored using a rectal temperature probe, and a heat lamp was utilized to maintain body temperature at 37 ± 0.4°C. Hepatic ischemia was induced for 45 min for all mice before the clamp was removed, and the liver was reperfused for 5 h. At this point, the mouse was anesthetized again as previously described. Blood was collected by using a 20-gauge needle inserted into the inferior vena cava. Blood was collected into a microtainer serum separator tube. Sample was placed on ice for 15 min and centrifuged for 12 min at 14,000 revolutions per min. Serum was then analyzed for transaminases using a commercially available assay kit.

Liver Enzyme Determination

Serum samples were analyzed for alanine aminotransferase (ALT) using Infinity ALT (GPT) reagent purchased from Thermo Electron. This transaminase is liver specific and is released during injury.

Statistical Analyses

Data were analyzed by Student's t-test or one-way ANOVA with post-Tukey analysis where appropriate using Prism software (San Diego, CA). Data are reported as means ± SE. P values < 0.05 were considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Characterization of eNOS-TG Mice

Western blot analysis of hepatic protein lysate revealed a substantial increase in eNOS protein in both Kobe (bovine eNOS-TG) and RT (human eNOS-TG) mice (Fig. 1A). Calculation of relative optical density (Fig. 1B) demonstrated that the Kobe eNOS-TG mouse had an approximate sixfold increase in hepatic eNOS expression. Likewise, the RT eNOS-TG mouse was found to have an approximate sevenfold increase in hepatic eNOS protein expression.


Figure 1
View larger version (27K):
[in this window]
[in a new window]
  		Fig. 1. Endothelial nitric oxide (NO) synthesis (eNOS) protein expression in transgenic (TG) mice. A: Western blot analysis of wild-type (WT), eNOS-TG [developed in Kobe, Japan (Kobe)], and eNOS-TG [developed in Rotterdam (RT), The Netherlands] hepatic tissue samples displayed the typical 140-kDa band. B: calculation of eNOS band optical density revealed ~6-fold increase in expression in Kobe eNOS-TG samples, whereas RT eNOS-TG samples revealed ~7-fold increase in hepatic eNOS protein expression. ***P < 0.001 vs. WT, numbers inside bars represent number of animals in each group.

 
Ion chromatographic analysis of plasma nitrite and nitrate in RT eNOS-TG mice revealed significant (P < 0.05) increases compared with that in WT littermates (Fig. 2). Plasma nitrite was increased by 44%, and in correlation plasma nitrate was increased by 47% in RT eNOS-TG mice. Hepatic nitrite levels were also significantly increased from 0.685 ± 0.131 µM in WT mice to 2.058 ± 0.193 µM in eNOS-TG mice (Fig. 3A, P < 0.001). Gas-phase chemiluminescence revealed that hepatic nitroso levels were 2.2-fold higher in RT eNOS-TG mice vs. WT littermates (Fig. 3B, P < 0.01). Hepatic NO-Heme levels were also found to be increased, although not significantly, in eNOS-TG compared with those in nontransgenic control mice (Fig. 3C).


Figure 2
View larger version (11K):
[in this window]
[in a new window]
  		Fig. 2. Plasma NO metabolites in RT eNOS-TG mice. A: plasma nitrite levels in RT eNOS-TG mice. B: plasma nitrate levels in RT eNOS-TG mice. *P < 0.05 vs. WT; numbers inside bars represent number of animals in each group.

 

Figure 3
View larger version (17K):
[in this window]
[in a new window]
  		Fig. 3. Hepatic tissue NO metabolites in RT eNOS-TG mice. A: hepatic nitrite levels in RT eNOS-TG mice. B: hepatic nitroso levels in RT eNOS-TG mice. C: hepatic NO-heme levels in RT eNOS-TG mice. **P < 0.01 vs. WT, ***P < 0.001 vs. WT; numbers inside bars represent number of animals in each group.

 
Hepatic cGMP Levels in eNOS-TG Mice

Quantitative ELISA analysis of hepatic lysate revealed a 60% increase in cGMP from 13.34 ± 1.30 pmol/mg protein in WT mice to 21.32 ± 4.17 pmol/mg protein in RT eNOS-TG mice.

Hemodynamic Analysis in eNOS-TG Mice

WT, Kobe eNOS-TG, and RT eNOS-TG mice were implanted with radiotelemetry pressure transducers to assess arterial blood pressure and heart rate (Table 1). eNOS-TG (Kobe and RT) mice displayed no significant differences in heart rate, mean arterial blood pressure, systolic blood pressure, or diastolic blood pressure compared with those in WT control mice.


View this table:
[in this window]
[in a new window]
  		Table 1. Systemic hemodynamics in wild-type and eNOS transgenic mice

 
eNOS Overexpression Attenuates Hepatic I-R Injury

The systemic overexpression of bovine eNOS (Kobe eNOS-TG) significantly decreased hepatic tissue injury after 45 min of ischemia followed by 5 h of reperfusion (Fig. 4). Hepatic tissue injury was evaluated by serum ALT (Fig. 4A). WT control animals displayed a mean serum ALT level of 718.6 ± 54.37 U/l versus the Kobe eNOS-TG animals whose mean serum ALT level was 197.0 ± 48.38 U/l (P < 0.001 vs. WT).


Figure 4
View larger version (10K):
[in this window]
[in a new window]
  		Fig. 4. TG overexpression of eNOS in hepatic ischemic-reperfusion (I-R) injury. A: serum alanine aminotransferase (ALT) levels in mice overexpressing the bovine eNOS gene (Kobe eNOS-TG) subjected to 45 min of ischemia and 5 h of reperfusion. B: serum ALT levels in mice overexpressing the human eNOS gene (RT eNOS-TG). ***P < 0.001 vs. WT; numbers inside bars represent number of animals in each group.

 
To further our understanding of genetic overexpression of eNOS, we looked at the effects of chronic overexpression of the human eNOS gene (RT eNOS-TG). We found that overexpression of human eNOS also protected against hepatic I-R injury, showing a 2.6-fold decrease in serum ALT (Fig. 4B). The WT control group showed a mean serum ALT level of 732.3 ± 69.37 U/l, whereas the human eNOS-TG mouse showed a mean serum ALT level of 278.5 ± 48.38 U/l (P < 0.001 vs. WT). These data clearly demonstrate that increasing the availability of NO limits the extent of I-R injury.

sGC-cGMP Pathway in Hepatic I-R Injury

sGC inhibition. To examine the downstream signaling effects of NO, we evaluated the role of sGC in hepatic I-R injury (Fig. 5). First we looked at the inhibition of sGC in WT animals. Blocking sGC with the potent inhibitor ODQ (30 mg/kg) exacerbated hepatic I-R injury in WT mice (Fig. 5A), showing a mean serum ALT level of 1,728 ± 100.6 U/l versus 1,061 ± 68.88 U/l in mice receiving vehicle (P < 0.001 vs. vehicle).


Figure 5
View larger version (21K):
[in this window]
[in a new window]
  		Fig. 5. Role of soluble guanylyl cyclase (sGC)-cGMP signaling pathway in hepatic I-R. A: serum ALT levels after liver I-R in WT mice receiving DMSO vehicle (Veh) or the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 30 mg/kg). B: serum ALT levels in WT mice receiving DMSO vehicle or sGC activator 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1, 20 mg/kg) subjected to liver I-R. C: serum ALT levels after hepatic I-R in WT mice receiving saline vehicle or sildenafil (2 mg/kg). D: serum ALT levels in eNOS-TG mice (Kobe eNOS-TG) receiving vehicle DMSO or ODQ (30 mg/kg) and subjected to hepatic I-R. *P < 0.05 and ***P < 0.001 vs. vehicle. Numbers inside bars indicate animals investigated in each group.

 
sGC activation. In contrast, stimulation of sGC in WT animals with YC-1 (20 mg/kg) significantly attenuated hepatic injury (Fig. 5B). The mean serum ALT value for vehicle was 1,061 ± 68.88 U/l compared with a mean value of 488.9 ± 93.38 U/l (P < 0.001 vs. vehicle) for the treated group. These results suggest that sGC plays a pertinent role in protecting against hepatic I-R injury.

PDE-5 inhibition. To further identify the role of the sGC-cGMP pathway in hepatic I-R injury, we utilized the potent and selective PDE-5A inhibitor sildenafil. Sildenafil was administered intraperitoneally at 22.5 min into ischemia to see if inhibiting the breakdown of cGMP would protect against hepatic I-R injury (Fig. 5C). Animals treated with sildenafil were significantly protected compared with animals receiving vehicle. The sildenafil group showed a mean serum ALT level of 210.6 ± 35.72 U/l versus 707.5 ± 61.42 U/l for the vehicle (P < 0.001). These results suggest the preservation of cGMP limits hepatic I-R injury.

eNOS overexpression and sGC inhibition. To further investigate the possible mechanism that sGC stimulation by NO leads to cytoprotection in hepatic I-R, we looked at the effect of sGC inhibition in the setting of eNOS overexpression (Fig. 5D). Inhibition of sGC in the eNOS-TG mouse increased tissue injury showing a mean serum ALT value of 511.6 ± 78.68 U/l versus vehicle (313.0 ± 28.87 U/l, P < 0.05). Although sGC inhibition abolished the protective effects of NO in the eNOS-TG mouse, these animals maintained significant protection compared with WT mice receiving ODQ (1,728 ± 100.6 U/l). Although these results suggest the involvement of sGC in NO-mediated protection (eNOS overexpression), other protective signaling pathways may also be involved.

HO-1 Pathway in Hepatic I-R Injury

HO-1 inhibition. We also investigated the possible participation of the HO-1 pathway in hepatic I-R (Fig. 6). First, we studied the effect of pharmacological inhibition with the enzyme ZnDPBG (Fig. 6A). In WT mice administered ZnDPBG, hepatic I-R injury was greatly exacerbated, displaying a mean serum ALT of 3,480 ± 1,349 U/l compared with the vehicle group with a mean ALT value of 926.3 ± 48.92 U/l (P < 0.05).


Figure 6
View larger version (18K):
[in this window]
[in a new window]
  		Fig. 6. NO-mediated hepatoprotection and heme oxygenase-1 (HO-1) signaling. A: serum ALT levels in WT mice after hepatic I-R given DMSO vehicle or the HO-1 inhibitor HO-1 zinc (III) deuteroporphyrin IX-2,4-bisethyleneglycol (ZnDPBG, 10 mg/kg). B: serum ALT levels after liver I-R in WT mice administered saline vehicle or the HO-1 activator cobalt (III) protoporphyrin IX chloride (CoPP, 5 mg/kg). C: levels of serum ALT after liver I-R in TG mice (Kobe eNOS-TG) receiving DMSO vehicle or ZnDPBG (10 mg/kg) *P < 0.05 vs. vehicle; **P < 0.01 vs. vehicle. NS, not significant. Numbers in the bars represent the number of animals used in each group.

 
HO-1 activation. HO-1 activation was also examined pharmacologically using the compound CoPP. Tissue injury was significantly decreased (2.8-fold) in animals receiving CoPP (Fig. 6B). Serum ALT levels measured 195.4 ± 45.56 U/l in the treated group and 554.3 ± 88.05 U/l in the vehicle group (P < 0.01 vs. vehicle). These results suggest the HO-1 pathway is involved in limiting the extent of I-R injury.

eNOS overexpression and HO-1 inhibition. Inhibition of HO-1 in the eNOS-TG mouse was investigated to determine whether NO-mediated protection was dependent on HO-1 signaling (Fig. 6C). Our findings did not show a significant difference between the ZnDPBG group and the vehicle group. The mean serum ALT concentrations for the treated versus nontreated groups were 441.4 ± 94.61 U/l and 336.6 ± 59.86 U/l (P = not significant), respectively. These data suggest that the cytoprotective effects of NO in this experimental model system are independent of HO-1 signaling.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study we found that genetic overexpression of eNOS protects against hepatic I-R injury. We investigated two distinct strains of eNOS-overexpressing mice. One mouse (Kobe eNOS-TG) featured the bovine eNOS gene and a second mouse featured the human eNOS gene (RT eNOS-TG). In both mouse models, we observed a 2.5- to 3.5-fold decrease in serum ALT levels after 45 min of ischemia and 5 h of reperfusion. These results, which support our initial hypothesis, were expected because Lefer and colleagues (20) had previously observed a reduction of myocardial infarct size in both mouse models after subjecting them to myocardial I-R. Whereas eNOS overexpression was found to be associated with elevated levels of NO-related metabolites in blood and liver and higher concentrations of hepatic cGMP, enhanced systemic NO formation was not accompanied by significant differences in heart rate and blood pressure, rendering hemodynamic changes an unlikely contributor to eNOS-mediated protection against hepatic I-R injury.

The soluble isoform of guanylyl cyclase is an important cellular target of NO. Thus, to further investigate the protective role of eNOS overexpression in hepatic I-R, a series of pharmacological approaches aimed at modulating cGMP availability was used to assess the suspected involvement of the NO-cGMP pathway. First, we looked at the downstream effects of direct pharmacological inhibition of sGC by ODQ. NO binds to the heme group of sGC, resulting in enzyme activation and accumulation of the second messenger cGMP. ODQ oxidizes the heme group of sGC from the ferrous to the ferric form, to which NO has a poorer binding affinity (32), preventing enzyme stimulation. Direct sGC inhibition increased serum ALT levels in WT animals after hepatic I-R, suggesting that sGC function plays a pivotal role in attenuating I-R injury.

We next examined the effects of sGC activation with YC-1 on the severity of hepatic I-R injury in WT animals. This compound allosterically binds to sGC and instigates a conformational change, resulting in an enhancement of stimulation by NO (13). In addition, YC-1 can slow down sGC deactivation by maintaining the association of NO with sGC for a longer time period (2). YC-1 stimulation of sGC attenuated hepatic injury in WT mice subjected to 45 min of ischemia followed by 5 h of reperfusion.

In an attempt to further define the role of the sGC-cGMP pathway, we looked at the direct effects of preventing cGMP breakdown using the potent PDE-5A inhibitor sildenafil. Mice treated with sildenafil were significantly protected against hepatic I-R injury. Taken together, these data suggest that the NO-sGC-cGMP axis plays a critical role in limiting the extent of hepatic I-R injury.

The pharmacological inhibition of sGC was next utilized to investigate the downstream pathways responsible for hepatocellular protection in eNOS-TG mice. ODQ partially reduced the protective effects of eNOS overexpression. These data suggest that hepatic cytoprotection in the eNOS-TG mouse may involve the sGC-cGMP pathway. However, NO-mediated hepatoprotection was not completely reduced with ODQ, suggesting that additional pathways contribute to the protection provided by eNOS overexpression.

In agreement with our results, many recent reports have cited the protective effects of the sGC-cGMP pathway in hepatic I-R injury. In a study (1) of endotoxin-induced hepatic failure in mice, two NO donors and two cGMP analogs were shown to inhibit apoptosis, but the protective effects of the NO donors were abolished by a sGC inhibitor, suggesting that the protection was dependent on cGMP production. In further support of the notion that the sGC-cGMP pathway is protective in hepatic I-R, a study utilizing a rat model (7) found the cGMP analog (8-bromo-cGMP) to protect the ischemic liver. Furthermore, preserving endogenous cGMP levels with sildenafil has been shown to protect against myocardial I-R injury in numerous animal models (26). These aforementioned studies along with our current findings point to the sGC-cGMP pathway as being critical in NO-mediated cytoprotection.

To address the nature of additional protective pathways, we next investigated the potential role of HO-1 in hepatic I-R injury. Heme oxygenase is the rate-limiting step in the oxidative degradation of heme. The heme breakdown products carbon monoxide (21) and biliverdin (12) have been shown to be protective in I-R injury. HO-1 belongs to the class of heat shock proteins, and its expression is quickly induced in many cell types in response to oxidative insults, such as in I-R injury (33).

We first examined the effects of pharmacologically inhibiting the HO-1 enzyme in WT mice utilizing ZnDPBG. ZnDPBG is a metalloporphyrin that has previously been shown to selectively inhibit liver heme oxygenase at very low concentrations (6). We found that inhibition of HO-1 significantly increased hepatocellular injury by 3.8-fold after hepatic I-R. In agreement with these findings, pharmacological stimulation of HO-1 with the compound CoPP (22) significantly decreased by 2.8-fold serum ALT levels in the liver after 45 min of ischemia and 5 h of reperfusion. These results suggest that HO-1 does indeed play a role in limiting the extent of hepatic I-R injury.

We next sought to determine whether the hepatoprotection observed after I-R injury in eNOS-TG mice also involved the HO-1 pathway. We subjected the eNOS-TG mouse to 45 min of ischemia and 5 h of reperfusion after administration of the HO-1 inhibitor ZnDPBG and found no significant difference in serum ALT levels compared with those in the vehicle group. These data demonstrate that NO-mediated hepatoprotection seen in the eNOS-TG mouse is likely independent of HO-1-mediated signaling.

Extensive evidence (8, 23, 35) exists showing that HO-1 is protective in I-R injury, although the mechanism of this still remains unclear. Our data are congruent with existing findings regarding the actions of this enzyme during I-R. It has been reported that HO-1 activity within the liver decreases leukocyte interaction with the endothelium (38), possibly by suppressing the induction of adhesion molecules (36). HO-1 has also been reported to suppress inflammatory cytokine pathways involved in hepatic I-R injury (34). These findings suggest that HO-1 is central in limiting the inflammatory response in hepatocytes. Inhibition of this enzyme has also been shown to increase the magnitude and duration of apoptotic cell death (28), whereas overexpression has been shown (23) to limit apoptosis after I-R injury. However, although we found HO-1 to be protective in WT animals, it seemed to play no role in the protection seen in eNOS-TG mice. This would suggest that HO-1 is not featured in the eNOS-derived NO cytoprotective pathways during hepatic I-R injury.

In conclusion, we have shown that eNOS overexpression significantly attenuates hepatic I-R injury. Utilizing pharmacological modulation, we investigated the role of sGC-cGMP and HO-1 pathways in I-R injury. We found that both pathways were prominent in limiting the extent of I-R injury in WT mice, but only the sGC-cGMP pathway was found to play a role in the protection resulting from eNOS overexpression. These results suggest that NO-mediated hepatoprotection in I-R injury involves sGC but not HO-1. More research is needed to better define the protective mechanism of eNOS-derived NO in hepatic I-R injury.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
These studies were supported by National Heart, Lung, and Blood Institute Grant 2RO1-HL-6049 (to D. J. Lefer) and American Diabetes Association Grant 7-04-RA-59 (to D. J. Lefer).


   ACKNOWLEDGMENTS
 
We thank Michael Hicks and James J. M. Greer for invaluable technical expertise in conducting these studies.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. J. Lefer, Albert Einstein College of Medicine, Div. of Cardiology, 1300 Morris Park Ave., Bronx, NY 10461 (e-mail: dlefer{at}aecom.yu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Akahori M, Yamada S, Takeyama N, and Tanaka T. Nitric oxide ameliorates actinomycin D/endotoxin-induced apoptotic liver failure in mice. J Surg Res 85: 286–293, 1999.[CrossRef][Web of Science][Medline]
  2. Bellamy TC and Garthwaite J. Pharmacology of the nitric oxide receptor, soluble guanylyl cyclase, in cerebellar cells. Br J Pharmacol 136: 95–103, 2002.[CrossRef][Web of Science][Medline]
  3. Billiar TR and Harbrecht BG. Resolving the nitric oxide paradox in acute tissue damage. Gastroenterology 113: 1405–1407, 1997.[CrossRef][Web of Science][Medline]
  4. Bolli R. Cardioprotective function of inducible nitric oxide synthase and role of nitric oxide in myocardial ischemia and preconditioning: an overview of a decade of research. J Mol Cell Cardiol 33: 1897–1918, 2001.[CrossRef][Web of Science][Medline]
  5. Bryan NS, Rassaf T, Maloney RE, Rodriguez CM, Saijo F, Rodriguez JR, and Feelisch M. Cellular targets and mechanisms of nitros(yl)ation: an insight into their nature and kinetics in vivo. Proc Natl Acad Sci USA 101: 4308–4313, 2004.[Abstract/Free Full Text]
  6. Chernick RJ, Martasek P, Levere RD, Margreiter R, and Abraham NG. Sensitivity of human tissue heme oxygenase to a new synthetic metalloporphyrin. Hepatology 10: 365–369, 1989.[Web of Science][Medline]
  7. Cottart CH, Nivet-Antoine V, Do L, Al-Massarani G, Descamps G, Xavier-Galen F, and Clot JP. Hepatic cytoprotection by nitric oxide and the cGMP pathway after ischaemia-reperfusion in the rat. Nitric Oxide 9: 57–63, 2003.[CrossRef][Web of Science][Medline]
  8. Dorman RB, Bajt ML, Farhood A, Mayes J, and Jaeschke H. Heme oxygenase-1 induction in hepatocytes and non-parenchymal cells protects against liver injury during endotoxemia (Abstract). Comp Hepatol 3, Suppl 1: S42, 2004.[CrossRef][Medline]
  9. Duranski MR, Greer JJ, Dejam A, Jaganmohan S, Hogg N, Langston W, Patel RP, Yet SF, Wang X, Kevil CG, Gladwin MT, and Lefer DJ. Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver. J Clin Invest 115: 1232–1240, 2005.[CrossRef][Web of Science][Medline]
  10. Elrod JW, Greer JJ, Bryan NS, Langston W, Szot JF, Gebregzlabher H, Janssens S, Feelisch M, and Lefer DJ. Cardiomyocyte-specific overexpression of NO synthase-3 protects against myocardial ischemia-reperfusion injury. Arterioscler Thromb Vasc Biol 26: 1517–1523, 2006.[Abstract/Free Full Text]
  11. Feelisch M, Rassaf T, Mnaimneh S, Singh N, Bryan NS, Jourd'Heuil D, and Kelm M. Concomitant S-, N-, and heme-nitros(yl)ation in biological tissues and fluids: implications for the fate of NO in vivo. FASEB J 16: 1775–1785, 2002.[Abstract/Free Full Text]
  12. Fondevila C, Shen XD, Tsuchiyashi S, Yamashita K, Csizmadia E, Lassman C, Busuttil RW, Kupiec-Weglinski JW, and Bach FH. Biliverdin therapy protects rat livers from ischemia and reperfusion injury. Hepatology 40: 1333–1341, 2004.[CrossRef][Web of Science][Medline]
  13. Friebe A and Koesling D. Mechanism of YC-1-induced activation of soluble guanylyl cyclase. Mol Pharmacol 53: 123–127, 1998.[Abstract/Free Full Text]
  14. Garthwaite J, Southam E, Boulton CL, Nielsen EB, Schmidt K, and Mayer B. Potent and selective inhibition of nitric oxide-sensitive guanylyl cyclase by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one. Mol Pharmacol 48: 184–188, 1995.[Abstract]
  15. Glantzounis GK, Salacinski HJ, Yang W, Davidson BR, and Seifalian AM. The contemporary role of antioxidant therapy in attenuating liver ischemia-reperfusion injury: a review. Liver Transpl 11: 1031–1047, 2005.[CrossRef][Web of Science][Medline]
  16. Hines IN, Kawachi S, Harada H, Pavlick KP, Hoffman JM, Bharwani S, Wolf RE, and Grisham MB. Role of nitric oxide in liver ischemia and reperfusion injury. Mol Cell Biochem 234–235: 229–237, 2002.
  17. Ignarro LJ, Buga GM, Wood KS, Byrns RE, and Chaudhuri G. Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci USA 84: 9265–9269, 1987.[Abstract/Free Full Text]
  18. Janssens SP, Simouchi A, Quertermous T, Bloch DB, and Bloch KD. Cloning and expression of a cDNA encoding human endothelium-derived relating factor/nitric oxide synthase. J Biol Chem 267: 14519–14522, 1992.[Abstract/Free Full Text]
  19. Jones SP, Girod WG, Palazzo AJ, Granger DN, Grisham MB, Jourd'Heuil D, Huang PL, and Lefer DJ. Myocardial ischemia-reperfusion injury is exacerbated in absence of endothelial cell nitric oxide synthase. Am J Physiol Heart Circ Physiol 276: H1567–H1573, 1999.[Abstract/Free Full Text]
  20. Jones SP, Greer JJ, Kakkar AK, Ware PD, Turnage RH, Hicks M, van Haperen R, de Crom R, Kawashima S, Yokoyama M, and Lefer DJ. Endothelial nitric oxide synthase overexpression attenuates myocardial reperfusion injury. Am J Physiol Heart Circ Physiol 286: H276–H282, 2004.[Abstract/Free Full Text]
  21. Kaizu T, Nakao A, Tsung A, Toyokawa H, Sahai R, Geller DA, and Murase N. Carbon monoxide inhalation ameliorates cold ischemia/reperfusion injury after rat liver transplantation. Surgery 138: 229–235, 2005.[CrossRef][Web of Science][Medline]
  22. Kappas A and Drummond GS. Control of heme and cytochrome P-450 metabolism by inorganic metals, organometals and synthetic metalloporphyrins. Environ Health Perspect 57: 301–306, 1984.[Web of Science][Medline]
  23. Katori M, Buelow R, Ke B, Ma J, Coito AJ, Iyer S, Southard D, Busuttil RW, and Kupiec-Weglinski JW. Heme oxygenase-1 overexpression protects rat hearts from cold ischemia/reperfusion injury via an antiapoptotic pathway. Transplantation 73: 287–292, 2002.[CrossRef][Web of Science][Medline]
  24. Koti RS, Tsui J, Lobos E, Yang W, Seifalian AM, and Davidson BR. Nitric oxide synthase distribution and expression with ischemic preconditioning of the rat liver. FASEB J 19: 1155–1157, 2005.[Abstract/Free Full Text]
  25. Kubes P, Suzuki M, and Granger DN. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc Natl Acad Sci USA 88: 4651–4655, 1991.[Abstract/Free Full Text]
  26. Kukreja RC, Salloum F, Das A, Ockaili R, Yin C, Bremer YA, Fisher PW, Wittkamp M, Hawkins J, Chou E, Kukreja AK, Wang X, Marwaha VR, and Xi L. Pharmacological preconditioning with sildenafil: basic mechanisms and clinical implications. Vascul Pharmacol 42: 219–232, 2005.[CrossRef][Web of Science][Medline]
  27. Laroux FS, Lefer DJ, Kawachi S, Scalia R, Cockrell AS, Gray L, Van der Heyde H, Hoffman JM, and Grisham MB. Role of nitric oxide in the regulation of acute and chronic inflammation. Antioxid Redox Signal 2: 391–396, 2000.[CrossRef][Medline]
  28. McCarter SD, Akyea TG, Lu X, Bihari A, Scott JR, Badhwar A, Dungey AA, Harris KA, Feng Q, and Potter RF. Endogenous heme oxygenase induction is a critical mechanism attenuating apoptosis and restoring microvascular perfusion following limb ischemia/reperfusion. Surgery 136: 67–75, 2004.[CrossRef][Web of Science][Medline]
  29. Palmer RM, Ferrige AG, and Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327: 524–526, 1987.[CrossRef][Medline]
  30. Radomski MW, Palmer RM, and Moncada S. Endogenous nitric oxide inhibits human platelet adhesion to vascular endothelium. Lancet 2: 1057–1058, 1987.[Web of Science][Medline]
  31. Rassaf T, Bryan NS, Kelm M, and Feelisch M. Concomitant presence of N-nitroso and S-nitroso proteins in human plasma. Free Radic Biol Med 33: 1590–1596, 2002.[CrossRef][Web of Science][Medline]
  32. Schrammel A, Behrends S, Schmidt K, Koesling D, and Mayer B. Characterization of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one as a heme-site inhibitor of nitric oxide-sensitive guanylyl cyclase. Mol Pharmacol 50: 1–5, 1996.[Abstract]
  33. Sharma HS, Das DK, and Verdouw PD. Enhanced expression and localization of heme oxygenase-1 during recovery phase of porcine stunned myocardium. Mol Cell Biochem 196: 133–139, 1999.[CrossRef][Web of Science][Medline]
  34. Tsuchihashi S, Zhai Y, Fondevila C, Busuttil RW, and Kupiec-Weglinski JW. HO-1 upregulation suppresses type 1 IFN pathway in hepatic ischemia/reperfusion injury. Transplant Proc 37: 1677–1678, 2005.[CrossRef][Web of Science][Medline]
  35. Tullius SG, Nieminen-Kelha M, Buelow R, Reutzel-Selke A, Martins PN, Pratschke J, Bachmann U, Lehmann M, Southard D, Iyer S, Schmidbauer G, Sawitzki B, Reinke P, Neuhaus P, and Volk HD. Inhibition of ischemia/reperfusion injury and chronic graft deterioration by a single-donor treatment with cobalt-protoporphyrin for the induction of heme oxygenase-1. Transplantation 74: 591–598, 2002.[Web of Science][Medline]
  36. Vachharajani TJ, Work J, Issekutz AC, and Granger DN. Heme oxygenase modulates selectin expression in different regional vascular beds. Am J Physiol Heart Circ Physiol 278: H1613–H1617, 2000.[Abstract/Free Full Text]
  37. Wang Y, Vodovotz Y, Kim PK, Zamora R, and Billiar TR. Mechanisms of hepatoprotection by nitric oxide. Ann NY Acad Sci 962: 415–422, 2002.[Web of Science][Medline]
  38. Wunder C, Brock RW, McCarter SD, Bihari A, Harris K, Eichelbronner O, and Potter RF. Inhibition of haem oxygenase activity increases leukocyte accumulation in the liver following limb ischaemia-reperfusion in mice. J Physiol 540: 1013–1021, 2002.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Appl. Physiol.Home page
W.-H. Kan, J.-T. Hsu, M. G. Schwacha, M. A. Choudhry, R. Raju, K. I. Bland, and I. H. Chaudry
Selective inhibition of iNOS attenuates trauma-hemorrhage/resuscitation-induced hepatic injury
J Appl Physiol, October 1, 2008; 105(4): 1076 - 1082.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
S. Jha, J. W. Calvert, M. R. Duranski, A. Ramachandran, and D. J. Lefer
Hydrogen sulfide attenuates hepatic ischemia-reperfusion injury: role of antioxidant and antiapoptotic signaling
Am J Physiol Heart Circ Physiol, August 1, 2008; 295(2): H801 - H806.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
S. Shiva, M. N. Sack, J. J. Greer, M. Duranski, L. A. Ringwood, L. Burwell, X. Wang, P. H. MacArthur, A. Shoja, N. Raghavachari, et al.
Nitrite augments tolerance to ischemia/reperfusion injury via the modulation of mitochondrial electron transfer
J. Exp. Med., September 3, 2007; 204(9): 2089 - 2102.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
291/6/H2980    most recent
01173.2005v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (8)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Duranski, M. R.
Right arrow Articles by Lefer, D. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Duranski, M. R.
Right arrow Articles by Lefer, D. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2006 by the American Physiological Society.