This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Polte, T. Articles by Schröder, H. Search for Related Content PubMed PubMed Citation Articles by Polte, T. Articles by Schröder, H. Related Collections Cell signalling/signal transduction Gene regulation Growth factors/cytokines Oxidant stress Endothelium/vascular type/nitric oxide

(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:1209.)
© 2000 American Heart Association, Inc.

Vascular Biology

Heme Oxygenase-1 Is a cGMP-Inducible Endothelial Protein and Mediates the Cytoprotective Action of Nitric Oxide

Tobias Polte; Aida Abate; Phyllis A. Dennery; Henning Schröder

From the Department of Pharmacology and Toxicology, School of Pharmacy, Martin Luther University, Halle, Germany, and the Department of Pediatrics (P.A.D.), Stanford University School of Medicine, Stanford, Calif.

Correspondence to Dr Henning Schröder, School of Pharmacy, Martin Luther University, Wolfgang-Langenbeck-Str. 4, 06099 Halle (Saale), Germany. E-mail schroeder{at}pharmazie.uni-halle.de

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Abstract—Inducible heme oxygenase (HO-1) has recently been recognized as an antioxidant and cytoprotective gene. By use of Western blotting, cell viability analysis, and antisense technique, the present study investigates the involvement of HO-1 in endothelial protection induced by the clinically used nitric oxide (NO) donor molsidomine (specifically, its active metabolite 3-morpholinosydnonimine [SIN-1]) and the second messenger cGMP. In bovine pulmonary artery endothelial cells, SIN-1 and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) at 1 to 100 µmol/L induced the synthesis of HO-1 protein in a concentration-dependent fashion up to 3-fold over basal levels. HO-1 induction by SIN-1 was inhibited in the presence of the NO scavenger phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide and the soluble guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one. 8-Bromo-cGMP (1 to 100 µmol/L) and dibutyryl cGMP (1 to 100 µmol/L) as well as the activator of particulate guanylyl cyclase atrial natriuretic peptide (1 to 100 nmol/L) produced increases in HO-1 protein similar to those produced by SIN-1. SIN-1 and 8-bromo-cGMP increased heme oxygenase activity (bilirubin formation). Cytoprotection by NO donors was abrogated in the presence of the heme oxygenase inhibitor tin protoporphyrin IX. Pretreatment of cells with a phosphorothioate-linked HO-1 antisense oligonucleotide prevented protection by SIN-1 or 8-bromo-cGMP against tumor necrosis factor- cytotoxicity, whereas sense and scrambled HO-1 were without effect under these conditions. Our results show for the first time that HO-1 is a cGMP-sensitive endothelial gene and establish conclusively a causal relationship between HO-1 induction and endothelial protection by the NO/cGMP system. By targeting cytoprotective HO-1, NO donors may therefore be expected to induce antioxidant, antiatherogenic, and anti-inflammatory effects.

Key Words: cGMP • cytoprotection • endothelial cells • heme oxygenase-1 • nitric oxide

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Although the role for nitric oxide (NO) as a cytotoxic effector molecule of the immune surveillance system is clearly established,¹ recent evidence demonstrates a cytoprotective function for NO at lower nontoxic concentrations. Cellular protection by NO may occur independently of its vasodilatory effect and is therefore not necessarily a consequence of increased blood or nutrient flow to tissues under cytotoxic stress. Thus, NO was reported to reduce cellular damage inflicted by oxidant stress in endothelial cells and fibroblasts.^{2 3} Previous investigations from our laboratory demonstrated that NO induces long-term protection against endothelial injury caused by tumor necrosis factor (TNF)-.^{4 5}

The basic mechanisms of NO-dependent cytoprotection are diverse and include direct neutralization of the superoxide radical^{2 6} or inhibition of proapoptotic enzymes, such as caspase-3–like proteins, through S-nitrosylation.^{7 8} These actions are cGMP independent; ie, they do not require increased activity of soluble guanylyl cyclase, the target enzyme of NO in many biological systems.⁷ By contrast, endothelial protection afforded by NO against the deleterious effects of proinflammatory cytokines has clearly been shown to be cGMP dependent.^{4 9} Similarly, cGMP has emerged as a crucial mediator of cytoprotection in the central nervous system and the immune system.^{10 11 12 13 14 15} In endothelial cells, cellular resistance to oxidant damage or TNF- toxicity has a delayed onset when induced through the NO/cGMP pathway, and some studies have shown that it can be prevented by pretreatment with cycloheximide, indicating upregulation of protective proteins.^{9 16 17}

Growing evidence points to the central role of heme oxygenase-1 (HO-1, heat shock protein 32) as an inducible stress gene that confers cytoprotection through the generation of vasodilatory CO and antioxidant bilirubin.^{18 19} In addition to its general anti-inflammatory function,²⁰ HO-1 specifically prevents chronic rejection or arteriosclerosis of transplants, according to recent investigations,^{21 22} and enhances the resistance of pancreatic islet cells to cytokine-mediated injury.²³ Moreover, the first human case of HO-1 deficiency, which has been reported to be due to a genetic disorder, shows severe persistent endothelial damage and increased tissue vulnerability to oxidant injury besides growth retardation and anemia.²⁴

Although induction of HO-1 by NO has been reported in endothelial and vascular smooth muscle cells, the underlying signaling pathway, particularly with regard to a possible participation of cGMP, is unclear.^{25 26 27} Moreover, whether among the considerable number of NO-sensitive protective genes, such as bcl-2, heat shock protein 70, ferritin, and others,^{8 16 28 29} HO-1 makes a significant contribution to NO-mediated cellular protection has not been addressed; ie, a causal link between the increased cellular defense after NO treatment and the induction of HO-1 protein synthesis is still missing. Our aim, therefore, was to explore the role of cGMP in NO-dependent HO-1 induction and to assess the protective function of HO-1 in endothelial cells by using an antisense technique and the clinically used donor of NO, molsidomine (specifically, its active metabolite 3-morpholinosydnonimine [SIN-1]).

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials
Bovine pulmonary artery endothelial cells (ATCC CCL 209) were obtained from the American Type Culture Collection. FBS, DMEM, and penicillin-streptomycin were obtained from GIBCO. TNF- was a gift of Knoll Deutschland GmbH (Ludwigshafen, Germany). SIN-1 was provided by Hoechst AG (Frankfurt, Germany). S-Nitroso-N-acetyl-D,L-penicillamine (SNAP), phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (PTIO), tin protoporphyrin-IX (SnPP), and 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one (ODQ) were purchased from Alexis. The chemiluminescence Western blotting kit was from Boehringer-Mannheim. The phosphorothioated oligonucleotides derived from the rat HO-1 sequence were synthesized at the Beckman Center, Stanford University, and were composed of the HO-1 transcription initiation codon and 6 bp on either side (antisense 5'-GCGCTCCATCGCGGG-3').³⁰ Negative controls were the sense oligonucleotide (5'-CCCGCGATGGAGCGC-3'), a complementary sequence to the antisense, and a random or scrambled oligonucleotide (5'-GGCCCTCTACGGGCG-3'), which contained all of the base pairs of the antisense codon in random order.³⁰ cGMP analogues, human atrial natriuretic peptide (ANP), and all other chemicals were bought from Sigma Chemical Co.

Cell Culture
Endothelial cells were maintained and subcultured in DMEM supplemented with 15% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin.³¹ The cells were grown in a humidified incubator at 37°C and 5% CO₂.

Cell Viability Analysis
Endothelial cells were seeded at 2x10⁴ cells per well in 96-well microtiter plates in 100 µL of media containing 15% FBS. After a 48-hour incubation at 37°C, the cells reached confluence, oligonucleotides were added, and the cells were then incubated for 8 hours. Cells were washed and further incubated for 6 hours in the presence of SIN-1 or 8-bromo-cGMP. PTIO or SnPP was added to the cells 10 minutes before SIN-1. Then, TNF- was given to the cells without washing out the previously added agents. Incubation at 37°C was continued for 72 hours and was followed by a cytotoxicity assay. Cell viability was measured by staining with crystal violet as previously described.^{5 32 33} This colorimetric test allows assessment of the remaining viable cells after the incubation procedure. After they were washed with PBS, cells were fixed with methanol for 10 minutes and then stained for 10 minutes with a 0.1% crystal violet solution. After 3 washes with tap water, the dye was eluted with 0.1 mol/L trisodium citrate in 50% ethanol for 10 minutes. Optical density at 630 nm was measured by use of a microtiter plate reader (Biotek EL 311s).

Western Blot Analysis
Endothelial cells were cultured in 150-mm dishes as described above. After a 24-hour incubation with control media, SIN-1, 8-bromo-cGMP, dibutyryl cGMP, or ANP, cells were washed and extracted as described previously.³¹ PTIO or ODQ was added to the cells 30 minutes before SIN-1. Protein (150 µg) was applied to SDS-PAGE. After electrophoresis, protein was transferred to a nitrocellulose membrane, and a polyclonal antibody to rat HO-1 (Stressgene) was used to identify HO-1 protein content. Antigen antibody complexes were visualized with the horseradish peroxidase chemiluminescence system according to the manufacturer’s instructions (Boehringer-Mannheim). Quantification of HO-1 protein content was performed by computer-assisted videodensitometry (Eagle Eye II-system, Stratagene).

Determination of cGMP
Cells grown to confluence in 35-mm cultured dishes were washed twice with 2 mL of a balanced salt solution containing 130 mmol/L NaCl, 5.4 mmol/L KCl, 1.8 mmol/L CaCl₂, 5.5 mmol/L glucose, and 20 mmol/L HEPES-NaOH, buffered to pH 7.3. Cells were exposed for 10 minutes at 37°C to PTIO or vehicle in the balanced solution containing 0.5 mmol/L isobutylmethylxanthine. SIN-1 or ANP was added, and the incubation was continued for another 10 minutes at 37°C. The final assay volume was 1 mL. Supernatants were aspirated, and after addition of ethanol and subsequent evaporation, cGMP levels were determined by an enzyme-linked immunoassay according to the manufacturer’s protocol (EIA kit, Cayman).

Heme Oxygenase Activity
Confluent endothelial cells in 150-mm culture dishes were incubated for 8 hours in the presence of control media, SIN-1, or 8-bromo-cGMP. The method used for the determination of heme oxygenase activity follows the protocol published by Motterlini et al.³ Briefly, after the incubation, cells were washed twice with PBS, gently scraped off the dish, and centrifuged (1000g for 10 minutes at 4°C). The cell pellet was suspended in MgCl₂ (2 mmol/L) and phosphate (100 mmol/L) buffer (pH 7.4), frozen at -70°C, thawed 3 times, and finally sonicated on ice before centrifugation at 18 000g for 10 minutes at 4°C. The supernatant (400 µL) was added to an NADPH-generating system containing 0.8 mmol/L NADPH, 2 mmol/L glucose-6-phosphate, 0.2 U glucose-6-phosphate-1-dehydrogenase, and 2 mg protein of rat liver cytosol prepared from the 105 000g supernatant fraction as a source of biliverdin reductase, potassium phosphate buffer (100 mmol/L, pH 7.4), and hemin (10 µmol/L) in a final volume of 200 µL. The reaction was conducted for 1 hour at 37°C in the dark and terminated by the addition of 1 mL chloroform. The extracted bilirubin was calculated by the difference in absorption between 464 and 530 nm with use of a quartz cuvette (extinction coefficient, 40 mmol · L^-1 · cm^-1 for bilirubin). Heme oxygenase activity was expressed as picomoles of bilirubin formed per milligram of endothelial cell protein per hour.

	Results

Top Abstract Introduction Methods Results Discussion References

 
HO-1 Protein Expression
In bovine pulmonary artery endothelial cells, SIN-1 (1 to 100 µmol/L) and SNAP (1 to 100 µmol/L/L) induced the synthesis of HO-1 protein in a concentration-dependent fashion up to 3-fold over basal levels (Figures 1 and 2). HO-1 induction by SIN-1 was inhibited in the presence of the NO scavenger PTIO and the inhibitor of soluble guanylyl cyclase ODQ but remained unchanged in the presence of the superoxide scavenger superoxide dismutase (SOD, Figure 3). The membrane-permeable cGMP analogues 8-bromo-cGMP (1 to 100 µmol/L) and dibutyryl cGMP (1 to 100 µmol/L) as well as the activator of particulate guanylyl cyclase ANP (1 to 100 nmol/L) produced increases in HO-1 protein in a concentration-dependent manner and similar to increases produced by SIN-1 (Figures 4 and 5). Cells were incubated for 24 hours with control media, SIN-1, 8-bromo-cGMP, dibutyryl cGMP, or ANP. PTIO or ODQ was added to the cells 30 minutes before SIN-1. Significant elevations in HO-1 protein levels were detected as early as 4 hours after the addition of SIN-1 (100 µmol/L) to the cells. The complete time course of SIN-1–induced HO-1 protein expression was as follows: 1.1±0.1 (2 hours), 1.5±0.1 (4 hours, P<0.05), 2.0±0.2 (6 hours, P<0.05), 2.9±0.5 (12 hours, P<0.05), and 2.7±0.4 (24 hours, P<0.05). The densitometric data are mean±SEM of 3 independent experiments and represent fold induction versus control (untreated) cells (P values are for SIN-1 versus control by 2-tailed t-test). In further control experiments, TNF- alone did not alter the basal expression of HO-1 protein (not shown).

View larger version (24K): [in this window] [in a new window]   Figure 1. Effect of SIN-1 on HO-1 protein expression in endothelial cells. Cells were incubated for 24 hours with control media (CON) or SIN-1 at the concentrations indicated. Protein isolation and Western blot analysis were performed as described in Methods. The densitometric data (B) are mean±SEM of 3 separate experiments. *P<0.05 for treatment vs CON by one-way ANOVA and Bonferroni multiple comparison test. A representative Western blot analysis is shown in panel A.

View larger version (28K): [in this window] [in a new window]   Figure 2. Effect of SNAP on HO-1 protein expression in endothelial cells. Cells were incubated for 24 hours with CON or SNAP at the concentrations indicated. Protein isolation and Western blot analysis were performed as described in Methods. The densitometric data (B) are mean±SEM of 3 separate experiments. *P<0.05 for treatment vs CON by one-way ANOVA and Bonferroni multiple comparison test. A representative Western blot analysis is shown in panel A.

View larger version (28K): [in this window] [in a new window]   Figure 3. Effect of NO scavenger PTIO (30 µmol/L), guanylyl cyclase inhibitor ODQ (1 µmol/L), and SOD (50 U/mL) on SIN-1–induced HO-1 protein expression. Cells were incubated for 24 hours with CON or SIN-1. PTIO, ODQ, or SOD was added to the cells 30 minutes before SIN-1. Protein isolation and Western blot analysis were performed as described in Methods. The densitometric data (B) are mean±SEM of 3 separate experiments. *P<0.05 for treatment vs CON by one-way ANOVA and Bonferroni multiple comparison test. A representative Western blot analysis is shown in panel A.

View larger version (27K): [in this window] [in a new window]   Figure 4. Effect of 8-bromo-cGMP (8-Br-cGMP) on HO-1 protein expression in endothelial cells. Cells were incubated for 24 hours with CON or 8-Br-cGMP. Protein isolation and Western blot analysis were performed as described in Methods. The densitometric data (B) are mean±SEM of 3 separate experiments. *P<0.05 for treatment vs CON by one-way ANOVA and Bonferroni multiple comparison test. A representative Western blot analysis is shown in panel A.

View larger version (27K): [in this window] [in a new window]   Figure 5. Effect of dibutyryl cGMP (Db-cGMP) and ANP on HO-1 protein expression in endothelial cells. Cells were incubated for 24 hours with CON, Db-cGMP, or ANP. Protein isolation and Western blot analysis were performed as described in Methods. The densitometric data (B) are mean±SEM of 3 separate experiments. *P<0.05 for treatment vs CON by one-way ANOVA and Bonferroni multiple comparison test. A representative Western blot analysis is shown in panel A.

cGMP Levels
SIN-1 (1 to 100 µmol/L) and ANP (1 to 100 nmol/L) stimulated endothelial cGMP accumulation at concentrations that were also effective in HO-1 induction (Figure 6). SIN-1–induced cGMP stimulation was inhibited by PTIO (Figure 6A). Cells were exposed for 10 minutes to PTIO or vehicle in the balanced solution containing 0.5 mmol/L isobutylmethylxanthine. SIN-1 or ANP was added, and the incubation was continued for another 10 minutes. PTIO alone was without influence on basal cGMP levels under these conditions (not shown).

View larger version (14K): [in this window] [in a new window]   Figure 6. A, Effect of SIN-1 on cGMP levels in endothelial cells and its inhibition by the NO scavenger PTIO. B, Effect of ANP on cGMP levels in endothelial cells. Cells were exposed for 10 minutes to PTIO or vehicle in the balanced solution containing 0.5 mmol/L isobutylmethylxanthine. SIN-1 or ANP was added, and the incubation was continued for another 10 minutes. cGMP determinations were performed as described in Methods. *P<0.05 for treatment vs CON by one-way ANOVA and Bonferroni multiple comparison test. All data shown are mean±SEM of 6 independent observations in separate cell culture wells.

Heme Oxygenase Activity
Cells were exposed to SIN-1 or 8-bromo-cGMP for 8 hours. Heme oxygenase activity was assessed in the cell lysate by measuring formation of the heme oxygenase metabolite bilirubin. SIN-1 (0.01 to 1 mmol/L) produced a concentration-dependent increase in heme oxygenase activity up to 5-fold over basal levels (Figure 7A). Similar results were obtained when cells were incubated with 8-bromo-cGMP. A concentration-dependent stimulation of heme oxygenase activity with a maximal 3-fold increase was detected in the presence of 8-bromo-cGMP (0.01 to 1 mmol/L, Figure 7B).

View larger version (16K): [in this window] [in a new window]   Figure 7. Effect of SIN-1 (A) and 8-Br-cGMP (B) on heme oxygenase activity in endothelial cells. Cells were incubated for 8 hours with CON, SIN-1, or 8-Br-cGMP. Heme oxygenase activity was determined via bilirubin formation as described in Methods. *P<0.05 for treatment vs CON by one-way ANOVA and Bonferroni multiple comparison test. All data shown are mean±SEM of 3 independent observations in separate cell culture wells.

Endothelial Cell Viability
Hemin, the established activator of HO-1 expression increased the number of surviving cells in TNF–-treated cultures in a concentration-dependent fashion (Figure 8). Endothelial protection by NO donors was abrogated in the presence of the heme oxygenase inhibitor SnPP (Figure 9). Pretreatment of cells with a phosphorothioate-linked HO-1 antisense oligonucleotide prevented protection by SIN-1 (Figure 10) or 8-bromo-cGMP (Figure 11) against TNF- cytotoxicity, whereas sense and scrambled HO-1 were without effect under these conditions. In control experiments, antisense HO-1 showed a similar specificity in blocking HO-1 protein induction by SIN-1 (see insert Figure 10). Cells were incubated with HO-1 oligonucleotides for 8 hours and were then washed. PTIO or SnPP was added to the cells 10 minutes before SIN-1 and was not washed out (Figure 6). Cells were further incubated in the presence of 8-bromo-cGMP or SIN-1 for 6 hours. TNF- was given to the cells without washing out 8-bromo-cGMP or SIN-1. Incubation was continued for 72 hours and was followed by a viability assay. SIN-1, 8-bromo-cGMP, oligonucleotides, or PTIO alone had no significant effect on cell viability under these conditions (not shown).

View larger version (13K): [in this window] [in a new window]   Figure 8. Effect of hemin on TNF-–mediated toxicity in endothelial cells. Cells were incubated with CON or hemin for 6 hours. TNF- was given to the cells without washing out hemin. Incubation was continued for 72 hours and was followed by a viability assay, which was carried out as described in Methods. *P<0.05 for treatment vs CON by one-way ANOVA and Bonferroni multiple comparison test. All data shown are mean±SEM of 6 independent observations in separate cell culture wells.

View larger version (17K): [in this window] [in a new window]   Figure 9. Effect of heme oxygenase inhibitor SnPP on endothelial protection induced by SIN-1 (A) or SNAP (B). Cells were incubated with CON, SIN-1, or SNAP for 6 hours. SnPP was added to the cells 10 minutes before the NO donors. TNF- was given to the cells without washing out the NO donors or SnPP. Incubation was continued for 72 hours and was followed by a viability assay, which was carried out as described in Methods. *P<0.05 for treatment vs CON by one-way ANOVA and Bonferroni multiple comparison test. All data shown are mean±SEM of 6 independent observations in separate cell culture wells.

View larger version (16K): [in this window] [in a new window]   Figure 10. Effect of antisense (AS), sense (S), and scrambled (Scr) HO-1 (all oligonucleotides at 20 ng/mL), NO scavenger PTIO (30 µmol/L), and guanylyl cyclase inhibitor ODQ (1 µmol/L) on SIN-1–induced protection against TNF- toxicity in endothelial cells. Cells were incubated with HO-1 oligonucleotides for 8 hours and were then washed. PTIO or ODQ was added to the cells 10 minutes before SIN-1 and was not washed out. Then, cells were further incubated in the presence of SIN-1 for 6 hours. TNF- was given to the cells without washing out SIN-1 and PTIO/ODQ. Incubation was continued for 72 hours and was followed by a viability assay, which was carried out as described in Methods. *P<0.05 for treatment vs CON by one-way ANOVA and Bonferroni multiple comparison test. #P<0.05 for treatment with SIN-1 and oligonucleotides vs SIN-1 alone (-) by one-way ANOVA and Bonferroni multiple comparison test. The following treatments were significantly different (P<0.05) from non–TNF-–treated cells (open bar): CON, AS+SIN-1, PTIO+SIN-1, and ODQ+SIN-1 (one-way ANOVA and Bonferroni multiple comparison test). All data shown are mean±SEM of 6 independent observations in separate cell culture wells. Insert shows effect of AS HO-1 and S HO-1 on HO-1 induction by SIN-1. Incubation procedures were as in Figure 3 and above. Blot is representative of 3 separate experiments.

View larger version (14K): [in this window] [in a new window]   Figure 11. Effect of AS, S, and Scr HO-1 (all oligonucleotides at 20 ng/mL) on protection induced by 8-Br-cGMP against TNF- toxicity in endothelial cells. Cells were incubated with HO-1 oligonucleotides for 8 hours and washed. Then, cells were further incubated in the presence of 8-Br-cGMP for 6 hours. TNF- was given to the cells without washing out 8-Br-cGMP. Incubation was continued for 72 hours and was followed by a viability assay, which was carried out as described in Methods. *P<0.05 for treatment vs CON by one-way ANOVA and Bonferroni multiple comparison test. #P<0.05 for treatment with 8-Br-cGMP and oligonucleotides vs 8-Br-cGMP alone (-) by one-way ANOVA and Bonferroni multiple comparison test. All treatments with TNF- (solid bars) were significantly different (P<0.05) from non–TNF-–treated cells (open bar) by one-way ANOVA and Bonferroni multiple comparison test. All data shown are mean±SEM of 6 independent observations in separate cell culture wells.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study shows, for the first time, that cGMP is a crucial mediator in the endothelial regulation of HO-1 expression and that endothelial protection afforded by the NO/cGMP system is causally related to the induction of HO-1.

In the present study, TNF-–induced endothelial toxicity serves as a model to study long-term protection from oxidant stress. TNF- is a proinflammatory cytokine with a direct toxic effect on the endothelium, resulting in endothelial lesions that favor thrombus formation and atherogenesis. This direct necrotic effect of TNF- is caused, to a substantial degree, by the formation of reactive oxygen species.^{34 35} Moreover, the crucial role of oxidant stress in TNF-–mediated cellular injury was previously established by using the same endothelial cell type as used in the present study.³⁶

SIN-1, the active metabolite of the antianginal drug molsidomine, was used as an NO-releasing and HO-1–inducing agent in the present study. Although under certain conditions (eg, in the absence of biological material or in pure aqueous solutions) SIN-1 is known to generate superoxide or peroxynitrite rather than NO,³⁷ data from a number of sources, including ours, have clearly shown that SIN-1 in intact cells (ie, in the presence of electron acceptors other than oxygen) functions as a true donor of NO.^{6 16 38} Moreover, and in agreement with this, we report in the present study that HO-1 induction by SIN-1 is inhibited by the NO scavenger PTIO³⁹ but not by SOD. That NO plays a key role as an active SIN-1 metabolite is further evident from our observation that SIN-1 produced a significant cGMP increase, which was also susceptible to inhibition by PTIO.

The regulatory role of cGMP can be derived from the stimulatory effect of membrane-permeable cGMP analogues on HO-1 protein synthesis and is further highlighted by our finding that ODQ, the inhibitor of soluble guanylyl cyclase,⁴⁰ attenuates HO-1 induction by the NO donor SIN-1. Whereas Immenschuh et al⁴¹ clearly showed cGMP to activate HO-1 transcription in hepatocytes, other authors were not able to demonstrate a signaling function of cGMP in endothelial HO-1 regulation.³ In that latter study, however, the only experimental approach to explore the involvement of cGMP was assessing a possible stimulatory effect of 8-bromo-cGMP on HO-1 catalytic activity.³ It is known that membrane permeability of, and cellular responsiveness to, cyclic nucleotide analogues may vary despite the existence of specific cGMP binding sites,⁴² which could be why cGMP sensitivity of HO-1 regulation was not detected in previous reports.^{3 43} The present study, in addition to showing HO-1 induction by exogenous cGMP and its inhibition by ODQ, provides a third line of evidence for the signaling role of cGMP in endothelial HO-1 expression by including experiments with ANP, a direct activator of particulate guanylyl cyclase. ANP at nanomolar concentrations that were previously shown to result in increased particulate guanylyl cyclase activity and cellular cGMP in kidney cells^{44 45} led to a robust elevation of cGMP accumulation and HO-1 protein expression in endothelial cells. This is the first time that ANP has been linked to HO-1 expression. Beyond a generally improved defense against oxidant injury, induction of an anti-inflammatory protein by activators of particulate guanylyl cyclase may have specific clinical implications because (1) ANP and related compounds reduce drug-induced nephrotoxicity in transplant recipients,^{46 47} and (2) increased HO-1 expression is considered to protect against the chronic rejection of transplants.^{19 20 21} In the present study, however, HO-1 induction by ANP is mainly interpreted as a crucial observation that substantiates cGMP sensitivity of endothelial HO-1 expression. That HO-1 may contain cyclic nucleotide–responsive elements is also suggested by recent investigations of HO-1 regulation in smooth muscle and liver tissue.^{27 48}

To find out whether HO-1 induction and cytoprotection by NO/cGMP are causally related, the present study uses an antisense oligonucleotide to HO-1. Antisense HO-1 attenuated endothelial protection against TNF- by SIN-1 and 8-bromo-cGMP, whereas after treatment with sense or scrambled HO-1, the surviving cell fractions remained unaltered. In additional control experiments, the same specificity of antisense HO-1 was found for inhibition of SIN-1–induced HO-1 protein expression. SIN-1 and 8-bromo-cGMP stimulated heme oxygenase activity, ie, bilirubin formation. Moreover, endothelial protection by NO donors was abrogated in the presence of the heme oxygenase inhibitor SnPP.^{18 20} Thus, increased expression of HO-1 by SIN-1 and its causal relation to ensuing cytoprotection is also reflected at the level of enzymatic activity. These results, in our view, conclusively demonstrate the decisive role that HO-1 plays in mediating endothelial protection by the NO/cGMP system.

The pathway, through which cGMP induces HO-1 and as a consequence endothelial resistance to cytotoxic stress, could be a direct one, ie, via cGMP-sensitive transcription factors, such as activator protein-1.⁴⁹ Alternatively, cGMP may act through secondary increases in cAMP, which we reported previously to occur in endothelial cells in response to NO donors and which are possibly due to cGMP-elicited inhibition of cAMP breakdown.⁵ Because activator protein-1 and cAMP-responsive elements have been identified in the promoter region of HO-1,^{27 41 48 50} HO-1 induction by NO/cGMP may be regulated through different mechanisms, depending on species and tissue.

We have shown, for the first time, that cGMP is a regulator of HO-1 expression in endothelial cells. Moreover, according to our data, induction of the defense protein HO-1 is clearly responsible for the endothelial protection afforded by the NO/cGMP system against the cytotoxic effects of TNF-. Thus, HO-1 is an important cellular target of NO donors, with clinical implications for the therapy or prevention of atherosclerosis and inflammatory diseases.

	Acknowledgments

 
This study was supported by the Deutsche Forschungsgemeinschaft (Schr 298/8-2). The authors would like to thank Drs Roberta Foresti and Roberto Motterlini, Northwick Park Institute for Medical Research, UK, and Dr Guang Yang, Department of Pediatrics, Stanford University, California, for very helpful discussions and advice with regard to the measurement of heme oxygenase activity and expression.

Received May 5, 1999; accepted January 4, 2000.

	References

Top Abstract Introduction Methods Results Discussion References

 
1. Moncada S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol Rev. 1991;43:109–142.[Medline] [Order article via Infotrieve]

2. Wink DA, Hanbauer I, Krishna MC, DeGraff W, Gamson J, Mitchell JB. Nitric oxide protects against cellular damage and cytotoxicity from reactive oxygen species. Proc Natl Acad Sci U S A. 1993;90:9813–9817.[Abstract/Free Full Text]

3. Motterlini R, Foresti R, Intaglietta M, Winslow RM. NO-mediated activation of heme oxygenase: endogenous cytoprotection against oxidative stress to endothelium. Am J Physiol. 1996;270:H107–H114.[Abstract/Free Full Text]

4. Polte T, Oberle S, Schröder H. Nitric oxide protects endothelial cells from tumor necrosis factor--mediated cytotoxicity: possible involvement of cyclic GMP. FEBS Lett. 1997;409:46–48.[Medline] [Order article via Infotrieve]

5. Polte T, Schröder H. Cyclic AMP mediates endothelial protection by nitric oxide. Biochem Biophys Res Commun. 1998;251:460–465.[Medline] [Order article via Infotrieve]

6. Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and the ugly. Am J Physiol.. 1996;271:C1424–C1437.[Abstract/Free Full Text]

7. Dimmeler S, Haendeler J, Nehls M, Zeiher AM. Suppression of apoptosis by nitric oxide via inhibition of interleukin-1beta-converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like proteases. J Exp Med. 1997;185:601–607.[Abstract/Free Full Text]

8. Kim YM, Talanian RV, Billiar TR. Nitric oxide inhibits apoptosis by preventing increases in caspase-3-like activity via two distinct mechanisms. J Biol Chem. 1997;272:31138–31148.[Abstract/Free Full Text]

9. Shen YH, Wang XL, Wilcken DEL. Nitric oxide induces and inhibits apoptosis through different pathways. FEBS Lett. 1998;433:125–131.[Medline] [Order article via Infotrieve]

10. Genaro AM, Hortelano S, Alvarez A, Martinez C, Bosca L. Splenic B lymphocyte programmed cell death is prevented by nitric oxide release through mechanisms involving sustained Bcl-2 levels. J Clin Invest. 1995;95:1884–1890.

11. Sciorati C, Rovere P, Ferrarini M, Heltai S, Manfredi AA, Clementi E. Autocrine nitric oxide modulates CD95-induced apoptosis in gammadelta T lymphocytes. J Biol Chem. 1997;272:23211–23215.[Abstract/Free Full Text]

12. Moro MA, FernandezTome P, Leza JC, Lorenzo P, Lizasoain I. Neuronal death induced by SIN-1 in the presence of superoxide dismutase: protection by cyclic GMP. Neuropharmacology. 1998;37:1071–1079.[Medline] [Order article via Infotrieve]

13. Thippeswamy T, Morris R. Cyclic guanosine 3',5'-monophosphate-mediated neuroprotection by nitric oxide in dissociated cultures of rat dorsal root ganglion neurones. Brain Res. 1997;774:116–122.[Medline] [Order article via Infotrieve]

14. Pantazis NJ, West JR, Dai D. The nitric oxide-cyclic GMP pathway plays an essential role in both promoting cell survival of cerebellar granule cells in culture and protecting the cells against ethanol neurotoxicity. J Neurochem. 1998;70:1826–1838.[Medline] [Order article via Infotrieve]

15. Estevez AG, Spear N, Thompson JA, Cornwell RL, Radi R, Barbeito L, Beckman JS. Nitric oxide-dependent production of cGMP supports the survival of rat embryonic motor neurons cultured with brain-derived neurotrophic factor. J Neurosci. 1998;18:3708–3714.[Abstract/Free Full Text]

16. Oberle S, Schröder H. Ferritin may mediate SIN-1-induced protection against oxidative stress. Nitric Oxide. 1997;1:308–314.[Medline] [Order article via Infotrieve]

17. Kim YM, Bergonia H, Lancaster JR Jr. Nitrogen oxide-induced autoprotection in isolated rat hepatocytes. FEBS Lett. 1995;374:228–232.[Medline] [Order article via Infotrieve]

18. Maines MD. The heme oxygenase system: a regulator of second messenger gases. Annu Rev Pharmacol Toxicol. 1997;37:517–554.[Medline] [Order article via Infotrieve]

19. Platt JL, Nath KA. Heme oxygenase: protective gene or Trojan horse. Nat Med. 1998;4:1364–1365.[Medline] [Order article via Infotrieve]

20. Willis D, Moore AR, Frederick R, Willoughby DA. Heme oxygenase: a novel target for the modulation of the anti-inflammatory response. Nat Med. 1996;2:87–90.[Medline] [Order article via Infotrieve]

21. Hancock WW, Buelow R, Sayegh MH, Turka LA. Antibody-induced transplant arteriosclerosis is prevented by graft expression of anti-oxidant and anti-apoptotic genes. Nat Med. 1998;4:1392–1396.[Medline] [Order article via Infotrieve]

22. Soares MP, Lin Y, Anrather J, Csizmadia E, Takigami K, Sato K, Grey ST, Colvin RB, Choi AM, Poss KD, Bach FH. Expression of heme oxygenase-1 can determine cardiac xenograft survival. Nat Med. 1998;4:1073–1077.[Medline] [Order article via Infotrieve]

23. Ye J, Laychock SG. A protective role for heme oxygenase expression in pancreatic islets exposed to interleukin-1 beta. Endocrinology. 1998;139:4155–4163.[Abstract/Free Full Text]

24. Yachie A, Niida Y, Wada T, Igarashi N, Kaneda H, Toma T, Ohta K, Kasahara Y, Koizumi S. Oxidative stress causes enhanced endothelial cell injury in human heme oxygenase-1 deficiency. J Clin Invest. 1999;103:129–135.[Medline] [Order article via Infotrieve]

25. Yee EL, Pitt BR, Billiar TR, Kim YM. Effect of nitric oxide on heme metabolism in pulmonary artery endothelial cells. Am J Physiol. 1996;271:L512–L518.[Abstract/Free Full Text]

26. Durante W, Kroll MH, Christodoulides N, Peyton KJ, Schafer AI. Nitric oxide induces heme oxygenase-1 gene expression and carbon monoxide production in vascular smooth muscle cells. Circ Res. 1997;80:557–564.[Abstract/Free Full Text]

27. Durante W, Christodoulides N, Cheng K, Peyton KJ, Sunahara RK, Schafer AI. cAMP induces heme oxygenase 1 gene expression and carbon monoxide production in vascular smooth muscle. Am J Physiol. 1997;273:H317–H323.[Abstract/Free Full Text]

28. Dimmeler S, Zeiher AM. Nitric oxide and apoptosis: another paradigm for the double-edged role of nitric oxide. Nitric Oxide. 1997;1:275–281.[Medline] [Order article via Infotrieve]

29. Nishikawa M, Takeda K, Sato EF, Kuroki T, Inoue M. Nitric oxide regulates energy metabolism and Bcl-2 expression in intestinal epithelial cells. Am J Physiol. 1998;274:G797–G801.[Abstract/Free Full Text]

30. Dennery PA, Sridhar KJ, Lee CS, Wong HE, Shokoohi V, Rodgers PA, Spitz DR. Heme oxygenase-mediated resistance to oxygen toxicity in hamster fibroblasts. J Biol Chem. 1997;272:14937–14942.[Abstract/Free Full Text]

31. Oberle S, Polte T, Abate A, Podhaisky H-P, Schröder H. Aspirin increases ferritin synthesis in endothelial cells: a novel antioxidant pathway. Circ Res. 1998;82:1016–1020.[Abstract/Free Full Text]

32. Flick DA, Gifford GE. Comparison of in vitro cell cytotoxic assays for tumor necrosis factor. J Immunol Methods. 1984;68:167–175.[Medline] [Order article via Infotrieve]

33. Wink DA, Cook JA, Pacelli R, DeGraff W, Gamson J, Liebmann J, Krishna MC, Mitchell JB. The effect of various nitric oxide-donor agents on hydrogen peroxide-mediated toxicity: a direct correlation between nitric oxide formation and protection. Arch Biochem Biophys. 1996;331:241–248.[Medline] [Order article via Infotrieve]

34. Schuger L, Varani J, Marks RM, Kunkel SL, Johnson KJ, Ward PA. Cytotoxicity of tumor necrosis factor- for human umbilical endothelial cells. Lab Invest. 1989;61:62–68.[Medline] [Order article via Infotrieve]

35. Goossens V, Grooten J, De Vos K, Fiers W. Direct evidence for tumor necrosis factor-induced mitochondrial reactive oxygen intermediates and their involvement in cytotoxicity. Proc Natl Acad Sci U S A. 1995;92:8115–8119.[Abstract/Free Full Text]

36. Schröder H, Warren S, Bargetzi MJ, Torti SV, Torti FM. N-Acetyl-L-cysteine protects endothelial cells but not L929 tumor cells from tumor necrosis factor--mediated cytotoxicity. Naunyn Schmiedebergs Arch Pharmacol. 1993;347:664–666.[Medline] [Order article via Infotrieve]

37. Feelisch M, Stamler JS. Donors of nitrogen oxides. In: Feelisch M, Stamler JS, eds. Methods in Nitric Oxide Research. Chichester, NY: John Wiley & Sons Inc; 1996:71–115.

38. Singh RJ, Hogg N, Joseph J, Konorev E, Kalyanaraman B. The peroxynitrite generator, SIN-1, becomes a nitric oxide donor in the presence of electron acceptors. Arch Biochem Biophys. 1999;361:331–339.[Medline] [Order article via Infotrieve]

39. Akaike T, Maeda H. Quantitation of nitric oxide using 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO). Methods Enzymol. 1996;268:211–221.[Medline] [Order article via Infotrieve]

40. Moro MA, Russel RJ, Cellek S, Lizasoain I, Su Y, Darley-Usmar VM, Radomski MW, Moncada S. cGMP mediates the vascular and platelet actions of nitric oxide: confirmation using an inhibitor of the soluble guanylyl cyclase. Proc Natl Acad Sci U S A. 1996;93:1480–1485.[Abstract/Free Full Text]

41. Immenschuh S, Hinke V, Ohlmann A, Gifhorn-Katz S, Katz N, Jungermann K, Kietzmann T. Transcriptional activation of the haem oxygenase-1 gene by cGMP via a cAMP response element activator protein-1 element in primary cultures of rat hepatocytes. Biochem J. 1998;334:141–146.

42. Schröder H, Ney P, Woditsch I, Schrör K. Cyclic GMP mediates SIN-1-induced inhibition of human polymorphonuclear leukocytes. Eur J Pharmacol. 1990;182:211–218.[Medline] [Order article via Infotrieve]

43. Takahashi K, Hara E, Ogawa K, Kimura D, Fujita H, Shibahara S. Possible implications of the induction of human heme oxygenase-1 by nitric oxide donors. J Biochem (Tokyo). 1997;121:1162–1168.[Abstract/Free Full Text]

44. Leitman DC, Agnost VL, Catalano RM, Schröder H, Waldman SA, Bennett BM, Tuan JJ, Murad F. Atrial natriuretic peptide, oxytocin, and vasopressin increase guanosine 3',5'-monophosphate in LLC-PK1 kidney epithelial cells. Endocrinology. 1988;122:1478–1485.[Abstract/Free Full Text]

45. Schröder H, Leitman DC, Bennett BM, Waldman SA, Murad F. Glyceryl trinitrate-induced desensitization of guanylate cyclase in cultured rat lung fibroblasts. J Pharmacol Exp Ther. 1988;245:413–418.[Abstract/Free Full Text]

46. Cedidi C, Meyer M, Kuse ER, Schulz-Knappe P, Ringe B, Frei U, Pichlmayr R, Forssmann WG. Urodilatin: a new approach for the treatment of therapy-resistant acute renal failure after liver transplantation. Eur J Clin Invest. 1994;24:632–639.[Medline] [Order article via Infotrieve]

47. Gianello P, Carlier M, Jamart J, Hulhoven R, Bernheim J, Bernard A, Ketelslegers JM, Squifflet JP. Effect of 1–28 alpha-h atrial natriuretic peptide on acute renal failure in cadaveric renal transplantation. Clin Transplant. 1995;9:481–489.[Medline] [Order article via Infotrieve]

48. Immenschuh S, Kietzmann T, Hinke V, Wiederhold M, Katz N, Muller-Eberhard U. The rat heme oxygenase-1 gene is transcriptionally induced via the protein kinase a signaling pathway in rat hepatocyte cultures. Mol Pharmacol. 1998;53:483–491.[Abstract/Free Full Text]

49. Pilz RB, Suhasini M, Idriss S, Meinkoth JL, Boss GR. Nitric oxide and cGMP analogs activate transcription from AP-1-responsive promoters in mammalian cells. FASEB J. 1995;9:552–558.[Abstract]

50. Alam J, Den Z. Distal AP-1 binding sites mediate basal level enhancement and TPA induction of the mouse heme oxygenase-1 gene. J Biol Chem. 1992;267:21894–21900.[Abstract/Free Full Text]

This article has been cited by other articles:

				K. Ueda, T. Ueyama, K.-i. Yoshida, H. Kimura, T. Ito, Y. Shimizu, M. Oka, Y. Tsuruo, and M. Ichinose Adaptive HNE-Nrf2-HO-1 pathway against oxidative stress is associated with acute gastric mucosal lesions Am J Physiol Gastrointest Liver Physiol, September 1, 2008; 295(3): G460 - G469. [Abstract] [Full Text] [PDF]

				G. Alba, R. El Bekay, P. Chacon, M. E. Reyes, E. Ramos, J. Olivan, J. Jimenez, J. M. Lopez, J. Martin-Nieto, E. Pintado, et al. Heme oxygenase-1 expression is down-regulated by angiotensin II and under hypertension in human neutrophils J. Leukoc. Biol., August 1, 2008; 84(2): 397 - 405. [Abstract] [Full Text] [PDF]

				N. G. Abraham and A. Kappas Pharmacological and Clinical Aspects of Heme Oxygenase Pharmacol. Rev., March 1, 2008; 60(1): 79 - 127. [Abstract] [Full Text] [PDF]

				K. Erdmann, N. Grosser, K. Schipporeit, and H. Schroder The ACE Inhibitory Dipeptide Met-Tyr Diminishes Free Radical Formation in Human Endothelial Cells via Induction of Heme Oxygenase-1 and Ferritin J. Nutr., August 1, 2006; 136(8): 2148 - 2152. [Abstract] [Full Text] [PDF]

				H. J. Kim, I. Tsoy, M. K. Park, Y. S. Lee, J. H. Lee, H. G. Seo, and K. C. Chang Iron Released by Sodium Nitroprusside Contributes to Heme Oxygenase-1 Induction via the cAMP-Protein Kinase A-Mitogen-Activated Protein Kinase Pathway in RAW 264.7 Cells Mol. Pharmacol., May 1, 2006; 69(5): 1633 - 1640. [Abstract] [Full Text] [PDF]

				H. Schroder No Nitric Oxide for HO-1 from Sodium Nitroprusside Mol. Pharmacol., May 1, 2006; 69(5): 1507 - 1509. [Abstract] [Full Text] [PDF]

				D. Morse and A. M. K. Choi Heme Oxygenase-1: From Bench to Bedside Am. J. Respir. Crit. Care Med., September 15, 2005; 172(6): 660 - 670. [Abstract] [Full Text] [PDF]

				C. Espinola-Klein, S. Blankenberg, and T. Munzel Editorial Comment--Is Heme Oxygenase-1 a Causal Player for Plaque Stability? Stroke, September 1, 2005; 36(9): 1901 - 1903. [Full Text] [PDF]

				B. Braam, R. de Roos, H. Bluyssen, P. Kemmeren, F. Holstege, J. A. Joles, and H. Koomans Nitric Oxide-Dependent and Nitric Oxide-Independent Transcriptional Responses to High Shear Stress in Endothelial Cells Hypertension, April 1, 2005; 45(4): 672 - 680. [Abstract] [Full Text] [PDF]

				I. Lim, S. J Gibbons, G. L. Lyford, S. M. Miller, P. R. Strege, M. G. Sarr, S. Chatterjee, J. H. Szurszewski, V. H. Shah, and G. Farrugia Carbon monoxide activates human intestinal smooth muscle L-type Ca2+ channels through a nitric oxide-dependent mechanism Am J Physiol Gastrointest Liver Physiol, January 1, 2005; 288(1): G7 - G14. [Abstract] [Full Text] [PDF]

				H. Fujimoto, M. Ohno, S. Ayabe, H. Kobayashi, N. Ishizaka, H. Kimura, K.-i. Yoshida, and R. Nagai Carbon Monoxide Protects Against Cardiac Ischemia--Reperfusion Injury In Vivo via MAPK and Akt--eNOS Pathways Arterioscler Thromb Vasc Biol, October 1, 2004; 24(10): 1848 - 1853. [Abstract] [Full Text] [PDF]

				F. A. D. T. G. Wagener, H.-D. Volk, D. Willis, N. G. Abraham, M. P. Soares, G. J. Adema, and C. G. Figdor Different Faces of the Heme-Heme Oxygenase System in Inflammation Pharmacol. Rev., September 1, 2003; 55(3): 551 - 571. [Abstract] [Full Text] [PDF]

				N. Grosser and H. Schroder Aspirin Protects Endothelial Cells From Oxidant Damage Via the Nitric Oxide-cGMP Pathway Arterioscler Thromb Vasc Biol, August 1, 2003; 23(8): 1345 - 1351. [Abstract] [Full Text] [PDF]

				S. Oberle, A. Abate, N. Grosser, A. Hemmerle, H. J. Vreman, P. A. Dennery, H. T. Schneider, D. Stalleicken, and H. Schroder Endothelial Protection by Pentaerithrityl Trinitrate: Bilirubin and Carbon Monoxide as Possible Mediators Experimental Biology and Medicine, May 1, 2003; 228(5): 529 - 534. [Abstract] [Full Text] [PDF]

				A. K. Kiemer, N. Bildner, N. C. Weber, and A. M. Vollmar Characterization of Heme Oxygenase 1 (Heat Shock Protein 32) Induction by Atrial Natriuretic Peptide in Human Endothelial Cells Endocrinology, March 1, 2003; 144(3): 802 - 812. [Abstract] [Full Text] [PDF]

				J. Wang, S. Lu, P. Moenne-Loccoz, and P. R. Ortiz de Montellano Interaction of Nitric Oxide with Human Heme Oxygenase-1 J. Biol. Chem., January 17, 2003; 278(4): 2341 - 2347. [Abstract] [Full Text] [PDF]

				D. G. Peters, X.-C. Zhang, P. V. Benos, E. Heidrich-O'Hare, and R. E. Ferrell Genomic analysis of immediate/early response to shear stress in human coronary artery endothelial cells Physiol Genomics, December 26, 2002; 12(1): 25 - 33. [Abstract] [Full Text] [PDF]

				E. Rydkina, A. Sahni, D. J. Silverman, and S. K. Sahni Rickettsia rickettsii Infection of Cultured Human Endothelial Cells Induces Heme Oxygenase 1 Expression Infect. Immun., August 1, 2002; 70(8): 4045 - 4052. [Abstract] [Full Text] [PDF]

				T. J. Rabelink and E. Stroes Atherosclerosis : Defeat of the Defense? Circ. Res., March 16, 2001; 88(5): 456 - 457. [Full Text] [PDF]

				L. E. Otterbein and A. M. K. Choi Heme oxygenase: colors of defense against cellular stress Am J Physiol Lung Cell Mol Physiol, December 1, 2000; 279(6): L1029 - L1037. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Polte, T. Articles by Schröder, H. Search for Related Content PubMed PubMed Citation Articles by Polte, T. Articles by Schröder, H. Related Collections Cell signalling/signal transduction Gene regulation Growth factors/cytokines Oxidant stress Endothelium/vascular type/nitric oxide