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(Circulation Research. 2000;86:1224.)
© 2000 American Heart Association, Inc.

Integrative Physiology

Prevention of Hypoxia-Induced Pulmonary Hypertension by Enhancement of Endogenous Heme Oxygenase-1 in the Rat

Helen Christou, Toshisuke Morita, Chung-Ming Hsieh, Hideo Koike, Burak Arkonac, Mark A. Perrella, Stella Kourembanas

From the Division of Newborn Medicine, Department of Pediatrics (H.C., T.M., H.K., S.K.), Children’s Hospital, Harvard Medical School, and Cardiovascular Biology Laboratory, Pulmonary and Critical Care Division (C.-M.H., B.A., M.A.P.), Brigham and Women’s Hospital, Harvard School of Public Health, Boston, Mass.

Correspondence to Stella Kourembanas, MD, Children’s Hospital, 300 Longwood Ave, Enders 9, Boston, MA 02115. E-mail kourembanas{at}hub.tch.harvard.edu

	Abstract

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Abstract—We investigated the role of heme oxygenase (HO)–1 in the development of hypoxia-induced pulmonary hypertension. HO catalyzes the breakdown of heme to the antioxidant bilirubin and the vasodilator carbon monoxide. Hypoxia is a potent but transient inducer of HO-1 in vascular smooth muscle cells in vitro and in the lung in vivo. By using agonists of HO-1, we sustained a high expression of HO-1 in the lungs of rats for 1 week. We report that this in vivo enhancement of HO-1 in the lung prevented the development of hypoxic pulmonary hypertension and inhibited the structural remodeling of the pulmonary vessels. The mechanism(s) underlying this effect may involve a direct vasodilating and antiproliferative action of endogenous carbon monoxide, as well as an indirect effect of carbon monoxide on the production of vasoconstrictors. These results provide evidence that enhancement of endogenous adaptive responses may be used to prevent hypoxia-induced pulmonary hypertension.

Key Words: vascular remodeling • gene expression • hypoxia

	Introduction

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Heme oxygenase (HO) is the rate-limiting enzyme in the degradation of heme to biliverdin and subsequently to bilirubin. The reaction catalyzed by HO is the major source of carbon monoxide (CO) in the body, which is increasingly recognized as a physiologically important molecule rather than a toxic waste product.¹ There are 3 known isoforms of HO. HO-1 is inducible,² whereas HO-2 and HO-3 are constitutively expressed.^{3 4} HO-3 is highly homologous to HO-2 but has not been characterized fully. HO-2 is the predominant form expressed in the central nervous system, where CO is thought to act as a neurotransmitter via the soluble guanylate cyclase/cGMP pathway.⁵ HO-1, the inducible form of the enzyme, is highly expressed in erythropoietic tissues, where its function is heme degradation, but it is also expressed in vascular smooth muscle cells (SMCs),⁶ where its role may include regulation of vascular tone. Several lines of investigation provide evidence that CO may be a physiological regulator of cellular interactions in the vasculature, acting as a direct and indirect vasodilator; directly, CO acts via activation of soluble guanylate cyclase and elevation of cGMP, as in rat aortic and coronary vascular SMC preparations^{6 7} as well as in dog femoral, carotid, and coronary arteries.⁸ Indirectly, SMC-derived CO may cause SMC relaxation by inhibiting the hypoxic induction of the vasoconstrictors endothelin-1 (ET-1) and platelet-derived growth factor-B (PDGF-B) in adjacent endothelial cells.⁹ In addition to its vasodilatory actions, CO was shown to inhibit SMC proliferation by regulating the cell cycle–specific transcription factor E2F-1,¹⁰ as well as the expression of the mitogens ET-1 and PDGF-BB in culture.⁹

A variety of cellular stressors, such as heat shock, oxidative stress, heavy metals, and hemoproteins, induce HO-1.^{11 12 13} Therefore, a protective/antioxidant role has been proposed for HO-1, as well as its enzymatic product, bilirubin.¹⁴ We found that SMC HO-1 is also induced by hypoxia in vitro in a time-dependent fashion with an initial 6-fold induction of mRNA at 12 hours of exposure followed by return to baseline by 48 hours of continued hypoxic exposure. This induction is accompanied by increased HO-1 activity and cGMP concentrations in vitro.⁶ In the rat model of chronic hypoxia, there is a sustained induction of vasoconstrictors such as ET-1¹⁵ and angiotensin II,¹⁶ and although increased production of the vasodilator nitric oxide has been reported,^{17 18} the net balance of these agents favors active vasoconstriction and wall remodeling. We speculated that HO-1 may be transiently induced by hypoxia in the lung, representing an endogenous adaptive response and that because of its transient nature, this response is ineffective in counteracting the actions of other mediators that are persistently induced under hypoxic conditions. We thus hypothesized that sustained induction of HO-1 in the setting of hypoxia may alter this balance and prevent or ameliorate the development of pulmonary hypertension. We report here that treatment of rats with the HO-1 inducers NiCl₂ and hemin caused a sustained increased expression of HO-1 in the lung and inhibited the development of structural remodeling and pulmonary hypertension by chronic hypoxia.

	Materials and Methods

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Hypoxic Exposure and Treatment With NiCl₂ or Hemin
Adult virus-free male Sprague-Dawley rats weighing 350 to 375 g were instrumented (Zivic Miller) and transported to our animal care facility with right ventricular and aortic catheters in place. They were then exposed to normobaric hypoxia (10% oxygen environment) in a ventilated Plexiglas box for 1 week. Age- and weight-matched control rats were maintained in 21% oxygen in the same virus-free room. Four to eight animals were studied in each group. To establish the hypoxic conditions, the chamber was flushed with a gas mixture of room air and nitrogen from a liquid nitrogen reservoir as previously described.¹⁹ The chamber was opened every 2 days for 30 minutes to clean the cages, replenish food, and water and flush right ventricular and aortic catheters. The NiCl₂-treated animals were given subcutaneous injections of 250 mmol/kg of NiCl₂ in normal saline every 48 hours, whereas the control animals were injected with the same volume of saline alone. These doses were chosen after preliminary studies showed that they were effective in inducing HO-1 (data not shown). The hemin-treated animals were given intraperitoneal injections of 15 mg/kg of hemin in DMSO every 48 hours. The animals were subsequently subjected to hemodynamic measurements and then euthanized with intraperitoneal pentobarbital (75 mg/kg). Their lungs were harvested for Northern and Western analyses and histological studies. As we previously described,¹⁹ the left mainstem bronchus was ligated and the left lung was excised, flash-frozen in liquid nitrogen, and stored at -80°C until RNA or protein extraction. The pulmonary artery was infused with normal saline to clear the blood from the pulmonary vessels. For histological analysis, the pulmonary veins were subsequently ligated, and the pulmonary artery and trachea were perfused with 4% paraformaldehyde at constant pressure (100 cm H₂O for the pulmonary artery and 25 cm H₂O for the trachea) to fully distend the pulmonary blood vessels and airway, respectively. Peripheral lung specimens were then excised, fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned for morphometric analysis of pulmonary arterioles. All animal experiments were performed in accordance with National Institutes of Health (NIH) guidelines and were approved by the institutional standing committee on animals.

Northern Analysis
Total lung RNA was isolated by guanidinium isothiocyanate extraction as previously reported,²⁰ fractionated by formaldehyde gel electrophoresis, and transferred onto Duralose UV membranes (Stratagene). Fifteen micrograms of total RNA was loaded in each lane. The filters were hybridized with HO-1 and ET-1 cDNA probes specific for the rat HO-1 gene⁶ and the rat ET-1 gene.⁹ The mouse ß-actin gene²¹ was used for normalization of RNA loading. Hybridization and washes were performed using standard conditions.¹⁹ For quantification, the autoradiographs were scanned using a Phosphor Imager analyzer (Molecular Dynamics).

Western Analysis
Frozen lung tissue was homogenized in lysis buffer (containing, in mmol/L, NaCl 150, Tris [pH 7.5] 30, and PMSF 1; 0.25 mol/L sucrose; 5 µg/mL leupeptin; and 1.9 µg/mL aprotinin), and protein concentrations were determined with a dye-binding assay (Bio-Rad) using BSA as standard. Fifty micrograms of total lung protein were prepared in sample buffer and boiled at 95°C for 5 minutes. Samples were subjected to electrophoresis in a 12% SDS polyacrylamide gel for 1 hour at 25 mA. The gel was transferred electrophoretically onto polyvinylidene difluoride membranes (Millipore Corp), and Western blotting was performed using primary rabbit polyclonal antibody against rat HO-1 (1:1000 dilution) purchased from StressGen. For detection of signal, we used an enhanced chemiluminescence detection kit from Amersham, and for quantification, we used Fotolook and NIH Image Software programs.

Hemodynamic Measurements
Hemodynamic measurements were performed at the end of the experimental period of 1 week in conscious rats. Systolic and mean right ventricular pressure (RVP) and mean arterial blood pressure (MAP) were continuously recorded for 2 minutes using MacLab monitoring equipment from CB Sciences, Inc. Results are reported in mm Hg. The position of the catheters was confirmed after the animals were euthanized.

Morphometric Analysis
Six-micrometer lung sections were stained with hematoxylin and eosin and examined with light microscopy. At least 5 representative pulmonary arterioles chosen from 3 different sections from each animal were independently examined by 2 of the investigators (H.C. and B.A.), who were not aware of the origin of the lung sections. The images of the arterioles were captured and analyzed using NIH Image software.²² Blood vessels were identified according to the accompanying airway, and intra-acinar vessels were chosen. Percentage medial thickness was determined by dividing the area occupied by the medial muscular layer by the total cross-sectional area of the arteriole. This method was used to account for uneven medial thickness and areas that had obliquely sectioned pulmonary arterioles.

Measurement of Circulating cGMP Concentrations
Blood specimens were collected in EDTA tubes and centrifuged, and plasma was separated and stored at –80°C until assayed. Amprep SAX minicolumns were used to extract cGMP from plasma samples, and plasma cGMP concentrations were measured in a single batch by radioimmunoassay (Amersham) as previously described.²³ The sensitivity of the assay was 0.5 fmol/tube, and the antibody cross-reacted fully with cGMP and <0.001% with other adenosine or guanosine phosphates. Intra-assay coefficient of variation was 5%. cGMP concentrations are expressed as pmol/L.

Statistical Methods
The nonparametric ANOVA (Kruskal-Wallis) test and Dunn multiple-comparisons tests were used to compare median values among groups, respectively, and differences were considered significant if P<0.05.

	Results

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In Vivo Induction of HO-1 mRNA and Protein in the Lung
We first examined whether hypoxia regulates HO-1 expression in vivo as it does in vitro. Similar to our in vitro observations in cultured SMCs,⁶ lung HO-1 mRNA was transiently induced by hypoxia in vivo but to a much lesser degree (2- to 3-fold in total lung versus 7-fold induction in cultured SMCs). After exposure of rats to hypoxia, we found that lung HO-1 mRNA levels increased 2- to 3-fold at 9 hours of hypoxia and returned to almost basal levels by 48 hours of hypoxic exposure (Figure 1A). In preliminary experiments, we used a number of known HO-1 agonists^{24 25 26} at various concentrations to assess their effectiveness in inducing and sustaining high levels of HO-1 mRNA in the lung in vivo. After treatment of animals with NiCl₂ (250 mmol/kg every 48 hours) or hemin (15 mg/kg every 48 hours), we found a mild induction of HO-1 mRNA in the lung after 48 hours (1.4- to 2.3-fold; data not shown). We found a more marked increase (average of 3-fold above baseline) after 1 week of treatment with these inducers under hypoxia (Figure 1B). This correlated with a 4- to 5-fold induction of HO-1 protein levels in the lungs of hypoxic animals treated with NiCl₂ or hemin compared with the normoxic controls (Figure 1C). As expected, HO-1 protein levels in the lung had returned to baseline after 1 week of hypoxic exposure in the absence of NiCl₂ or hemin treatment (Figure 1C).

View larger version (22K): [in this window] [in a new window]   Figure 1. Effect of hypoxia and treatment with HO-1 inducers on HO-1 mRNA and protein levels. A, Time course of HO-1 mRNA induction in rat lungs by hypoxia. Relative HO-1 mRNA levels were normalized to ß-actin mRNA. Lane 1, normoxic control; lanes 2 to 5, hypoxic exposure for 3 hours (lane 2), 9 hours (lane 3), 15 hours (lane 4), and 48 hours (lane 5). A representative Northern blot is shown on the upper panel, and mean relative mRNA levels from 5 or 6 animals studied in each group are indicated on the graph (lower panel). Mean mRNA levels at 9 and 15 hours of hypoxia (bars 3 and 4) are significantly higher compared with all others (bars 1, 2, and 5) (P<0.05). B, Lung HO-1 mRNA levels in normoxic and hypoxic animals treated with HO-1 inducers. Relative HO-1 mRNA levels were compared after 1 week of treatment and normalized to ß-actin mRNA. Lane 1, normoxic animals; lane 2, hypoxic animals; lane 3, hypoxic animals treated with hemin; lane 4, hypoxic animals treated with NiCl2. A representative Northern blot is shown on the upper panel, and the mean±SEM of 4 to 5 animals studied in each group is summarized on the graph (bottom). Mean values for hemin and NiCl2 (bars 3 and 4, respectively) are significantly different from normoxia or hypoxia alone (bars 1 and 2) (P<0.05). C, Lung HO-1 protein levels in normoxic and hypoxic animals treated with the HO-1 inducers. These are normalized to levels of ß-actin protein in the 4 groups of animals. Lane 1, normoxia; lane 2, hypoxia for 1 week; lane 3, hypoxia plus NiCl2 treatment for 1 week; lane 4, hypoxia plus hemin treatment for 1 week. Representative Western analysis from 4 to 5 animals in each condition is shown, and mean±SEM are summarized on the graph. Mean values for NiCl2 and hemin (bars 3 and 4) are significantly different from normoxic or hypoxic controls (bars 1 and 2) (P<0.05).

Prevention of Hypoxia-Induced Pulmonary Hypertension
We measured RVP as an indicator of pulmonary artery pressure in conscious rats. Systolic RVP in normoxic rats was 7.5±2.1 mm Hg (mean±SEM, n=5) (Figure 2A). As expected, the hypoxic animals developed pulmonary hypertension after 1 week of exposure to 10% oxygen. Their systolic RVP was 39.7±9.8 mm Hg (mean±SEM, n=5), which was significantly higher than that of the normoxic controls (P=0.0071). In contrast, the hypoxic animals treated with NiCl₂ did not develop pulmonary hypertension after 1 week of hypoxic exposure. Their systolic RVP was 10.5±3 mm Hg (mean±SEM, n=4), which was not statistically different from the normoxic controls.

View larger version (16K): [in this window] [in a new window]   Figure 2. Systolic RVP and MAP measurements. A, Mean±SEM for systolic RVP measurements in conscious rats after 1-week exposure to the experimental conditions of hypoxia (n=5), normoxia (n=5), and hypoxia plus NiCl2 (n=4). **P<0.05 compared with normoxia. B, Mean and SEM are shown for MAP of hypoxic animals (n=4), normoxic controls (n=5), and hypoxic animals treated with NiCl2 (n=5). **P<0.05 compared with hypoxia.

Effect of HO-1 Induction on Systemic Circulation
To determine whether the effect of HO-1 induction was restricted to the pulmonary circulation or affected the systemic circulation, we measured MAP in conscious rats. MAP was not significantly affected by a 1-week exposure to hypoxia (128.2±4.9 mm Hg in normoxia versus 131±6.1 mm Hg in hypoxia, P>0.05, n=5 per group). Treatment with NiCl₂ in the setting of hypoxia led to a small but significant reduction in MAP (108.6±6.6 mm Hg, P<0.05, n=5) (Figure 2B). Treatment of normoxic animals with NiCl₂ did not affect MAP significantly (120.2±2.3 mm Hg, n=5).

Prevention of Pulmonary Vascular Remodeling
Increased thickness of pulmonary arterioles due to SMC hypertrophy and hyperplasia is the structural hallmark of pulmonary hypertension.²⁷ As shown in Figure 3A, pulmonary arterioles in normoxic animals were thin, whereas after 1 week of hypoxic exposure they developed increased medial thickness characteristic of pulmonary hypertension (Figure 3B). In contrast, the hypoxic animals treated with NiCl₂ (Figure 3C) or hemin (Figure 3D) had markedly reduced vascular remodeling and the medial thickness of their pulmonary arterioles was comparable with that of normoxic controls. Quantification of these structural changes in several lung sections of all of the animals exposed to the 3 different conditions revealed significantly increased medial thickness of pulmonary arterioles in hypoxic animals compared with normoxic controls (49% versus 26%, P<0.05), but essentially unchanged from baseline normoxic medial thickness in the hypoxic animals treated with NiCl₂ (26% versus 36%; NS) (Figure 4).

View larger version (106K): [in this window] [in a new window]   Figure 3. Effects of hypoxia and HO-1 agonists on distal lung vascular remodeling. A, Morphology of pulmonary arterioles by H&E staining of peripheral lung sections from a normoxic control animal. B, Representative pulmonary arteriole of animals exposed to hypoxia for 1 week. C and D, Representative arterioles from lungs of hypoxic animals that were treated with NiCl2 and hemin, respectively. Original magnification x400.

View larger version (15K): [in this window] [in a new window]   Figure 4. Quantification of pulmonary arteriolar medial thickness. Mean±SEM percentage of medial pulmonary arteriolar thickness is shown for normoxic control rats (n=4), hypoxic animals (n=5), and hypoxic animals treated with NiCl2 (n=5). **P<0.05 compared with hypoxia.

Effect of HO-1 Induction in Circulating Concentrations of cGMP
CO is known to activate guanylyl cyclase and increase cGMP levels in SMCs.⁶ cGMP in turn mediates CO-induced effects such as vasodilation and inhibition of ET-1, PDGF-B, and E2F-1 transcription.^{9 10} To determine whether treatment with HO-1 inducers leads to increased levels of biologically active CO, we measured circulating cGMP concentrations in the animals. We found that treatment with NiCl₂ led to significantly higher circulating cGMP concentrations compared with control animals under normoxia (26.2 versus 8.7 pmol/mL, respectively, P<0.05) (Figure 5). In the hypoxic setting, treatment with NiCl₂ also increased circulating cGMP levels, but the difference did not reach statistical significance compared with hypoxic controls (15.5 versus 9.3 pmol/mL, respectively, P>0.05). There was no difference in cGMP levels between normoxic and hypoxic animals (8.7 versus 9.3 pmol/mL, respectively, P>0.05).

View larger version (13K): [in this window] [in a new window]   Figure 5. Circulating cGMP levels in rats after exposure to experimental conditions for 1 week. Mean±SEM are shown. Column 1, normoxia (n=8); column 2, NiCl2 treatment for 1 week in normoxia (n=5); column 3, hypoxia for 1 week (n=6); column 4, treatment with NiCl2 for 1 week in hypoxia (n=6). **P<0.05 compared with normoxia.

	Discussion

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Regulation of pulmonary vascular tone and homeostasis involves a number of vasoconstrictors, relaxing agents, and growth factors that are released from different cell types in the vasculature and interact through feedback loops. Hypoxia is known to regulate these cellular interactions mainly by modifying gene expression.²⁸ In culture, hypoxia induces the expression of vasoconstrictors such as ET-1 and PDGF-B^{21 29} and inhibits the production of the vasodilator NO via inhibition of NO synthase gene expression.³⁰ Hypoxia thus may shift the balance of vasoactive mediators toward agents promoting vasoconstriction and structural remodeling, as seen in pulmonary hypertension. In this study, we found that HO-1, which catalyzes the production of CO and bilirubin, is transiently induced by hypoxia in the lung in vivo. We demonstrated that treatment with NiCl₂ or hemin, each of which is an inducer of HO-1, enhanced and sustained high levels of HO-1 expression in the lung and led to increased HO enzymatic activity with increased levels of circulating cGMP. In the hypoxic setting, these inducers protected rats from developing pulmonary hypertension and prevented the accompanying structural remodeling of pulmonary arterioles. An earlier study by Sacerdoti et al,³¹ using tin to induce HO, prevented the development of systemic hypertension in spontaneously hypertensive rats. The mechanism(s) implicated in that study included depletion of heme, the substrate of HO, resulting in decreased hemoproteins such as renal cytochrome P-450 and reduced free arachidonic acid metabolites that have prohypertensive properties. It is possible that increased levels of CO, the enzymatic product of HO, may have also contributed to the reduction in blood pressure in that animal model.

The mechanism(s) by which sustained HO-1 expression prevented hypoxic pulmonary hypertension remain to be elucidated. These may include direct vasodilatory and antiproliferative effects of CO, the product of HO-1, as well as antioxidant properties of bilirubin, also released from the breakdown of heme by HO-1. It is likely that reactive oxygen species may contribute to the pathogenesis of hypoxic pulmonary hypertension and bilirubin may limit this process. Indeed, the antioxidant activity of bilirubin is significantly enhanced under low oxygen conditions (2% O₂) compared with that at 20% O₂.¹⁴ Alternatively, the increased local production of CO in the lung may lead to increased lung cGMP levels, which are known to mediate the vasodilator and antiproliferative effects of CO. In our in vitro studies, CO/cGMP was shown to inhibit the cell cycle–specific transcription factor E2F-1; to reduce SMC proliferation in culture¹⁰ ; and to suppress the hypoxic induction of ET-1, PDGF-B, and vascular endothelial growth factor by endothelial cells.^{9 32} CO interferes with cell cycle progression directly at the G₁/S phase via its effect on E2F-1 transcription, as well as indirectly at the G₀/G₁ phase via its effect on mitogen transcription (ET-1 and PDGF-B). Similar mechanisms may limit medial SMC hyperplasia in vivo.

We found increased circulating cGMP levels in response to treatment with HO-1 inducers under normoxia, demonstrating that the increase in HO-1 protein resulted in enhanced biologic activity. A suppression of the NO pathway may explain the absence of a more dramatic increase in cGMP levels under hypoxia when HO-1 inducers were used. Indeed, endothelial cell–derived NO is the molecule classically described to control cGMP content in the vasculature under basal conditions. In cases of vascular injury such as pulmonary hypertension, endothelial cell–derived NO production may be reduced,^{33 34} leading to decreased cGMP.²³ We speculate that vascular SMC-derived CO may represent a compensatory response of the vasculature during hypoxia when NO production is suppressed. NO and CO share common properties of vasodilation and have been reported to inhibit SMC growth as well as to regulate gene expression. Both were shown to inhibit the DNA-binding activity of hypoxia-inducible factor-1,^{32 35} a key transcription factor in the hypoxic signaling pathway. It is thus reasonable to speculate that a critical balance of NO and CO is required for the maintenance of vascular tone and vessel wall integrity under physiological as well as pathophysiological conditions of hypoxia. The unraveling of the molecular mechanisms underlying hypoxia-induced vascular changes is likely to lead to the development of novel approaches in the management of disorders characterized by abnormal pulmonary vascular tone and structure such as pulmonary hypertension.

	Acknowledgments

 
H.C. was supported by the National Institute of Child Health and Human Development Child Health Research Center New Project Development Funds as a Janeway Scholar. M.A.P. was supported by an American Heart Association Grant-in-Aid, by NIH Grant K08HL03194, and by Bristol-Myers Squibb Pharmaceutical Research Institute. S.K. was supported by the American Heart Association and by NIH Grants R01 HL55454 and SCOR IP50 HL 56398. We thank Jessica Johnson for her expert assistance in the preparation of the manuscript.

Received February 4, 2000; accepted April 20, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Marks GS, Brien JF, Nakatsu K, McLaughlin BE. Does carbon monoxide have a physiological function? Trends Pharmacol Sci. 1991;12:185–188.[Medline] [Order article via Infotrieve]
2. Shibahara S, Muller R, Taguchi H, Yoshida T. Cloning and expression of cDNA for rat heme oxygenase. Proc Natl Acad Sci U S A. 1985;82:7865–7869.[Abstract/Free Full Text]
3. Trakshel GM, Kutty RK, Maines MD. Purification and characterization of the major constitutive form of testicular heme oxygenase. J Biol Chem. 1986;261:11131–11137.[Abstract/Free Full Text]
4. McCoubrey WK, Huang TJ, Maines MD. Isolation and characterization of a cDNA from the rat brain that encodes hemoprotein heme oxygenase-3. Eur J Biochem. 1997;247:725–732.[Medline] [Order article via Infotrieve]
5. Verma A, Hirsch DJ, Glatt CE, Ronnett GV, Snyder SH. Carbon monoxide: a putative neural messenger. Science. 1993;259:381–384.[Abstract/Free Full Text]
6. Morita T, Perrella MA, Lee M-E, Kourembanas S. Smooth muscle cell-derived carbon monoxide is a regulator of vascular cGMP. Proc Natl Acad Sci U S A. 1995;92:1475–1479.[Abstract/Free Full Text]
7. Ramos KS, Lin H, McGrath JJ. Modulation of cyclic monophosphate levels in cultured aortic smooth muscle cells by carbon monoxide. Biochem Pharmacol. 1989;38:1368–1370.[Medline] [Order article via Infotrieve]
8. Vedernikov YP, Graser T, Vanin AF. Similar endothelium-independent arterial relaxation by carbon monoxide and nitric oxide. Biomed Biochim Acta. 1989;48:601–603.[Medline] [Order article via Infotrieve]
9. Morita T, Kourembanas S. Endothelial cell expression of vasoconstrictors and growth factors is regulated by smooth muscle cell-derived carbon monoxide. J Clin Invest. 1995;96:2676–2682.
10. Morita T, Mitsialis SA, Koike H, Liu Y, Kourembanas S. Carbon monoxide controls the proliferation of hypoxic vascular smooth muscle cells. J Biol Chem. 1997;272:32804–32809.[Abstract/Free Full Text]
11. Shibahara S. Regulation of heme oxygenase gene expression. Semin Hematol. 1988;25:370–376.[Medline] [Order article via Infotrieve]
12. Maines MD. Heme oxygenase: function, multiplicity, regulatory mechanisms, and clinical applications. FASEB J. 1988;2:2557–2568.[Abstract]
13. Rodgers PA, Stevenson DK. Developmental biology of heme oxygenase. Clin Perinatol. 1990;17:275–291.[Medline] [Order article via Infotrieve]
14. Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN. Bilirubin is an antioxidant of possible physiological importance. Science. 1987;235:1043–1046.[Abstract/Free Full Text]
15. Elton TS, Oparil S, Taylor GR, Hicks PH, Yang RH, Jin H, Chen YF. Normobaric hypoxia stimulates endothelin-1 gene expression in the rat. Am J Physiol. 1992;263:R1260–R1264.[Abstract/Free Full Text]
16. Morrell NW, Danilov SM, Satyan KB, Morris KG, Stenmark KR. Right ventricular angiotensin converting enzyme activity and expression is increased during hypoxic pulmonary hypertension. Cardiovasc Res. 1997;34:393–403.[Abstract/Free Full Text]
17. Le Cras TD, Tyler RC, Horan MP, Morris KG, Tuder RM, McMurtry IF, Johns RA, Abman SH. Effects of chronic hypoxia and altered hemodynamics on endothelial nitric oxide synthase expression in the adult rat lung. J Clin Invest. 1998;101:795–801.[Medline] [Order article via Infotrieve]
18. Xue C, Rengasamy A, Le Cras TD, Koberna PA, Dailey GC, Johns RA. Distribution of NOS in normoxic vs hypoxic rat lung: upregulation of NOS by chronic hypoxia. Am J Physiol. 1994;267:L667–L678.[Abstract/Free Full Text]
19. Christou H, Yoshida A, Arthur V, Morita T, Kourembanas S. Increased vascular endothelial growth factor production in the lungs of rats with hypoxia-induced pulmonary hypertension. Am J Respir Cell Mol Biol. 1998;18:768–776.[Abstract/Free Full Text]
20. Chomczynski P, Sacchi N. Single-step method of RNA isolation by guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156–159.[Medline] [Order article via Infotrieve]
21. Kourembanas S, Hannan RL, Faller DV. Oxygen tension regulates the expression of the platelet-derived growth factor-B chain gene in human endothelial cells. J Clin Invest. 1990;86:670–674.
22. Shi C, Lee W-S, Russell ME, Zhang D, Fletcher DL, Newell JB, Haber E. Hypercholesterolemia exacerbates transplant arteriosclerosis via increased neointimal smooth muscle cell accumulation: studies in apolipoprotein E knockout mice. Circulation. 1997;96:2722–2728.[Abstract/Free Full Text]
23. Christou H, Adatia I, Van Marter LJ, Kane JW, Thompson JE, Stark AR, Wessel DL, Kourembanas S. Effect of inhaled nitric oxide on endothelin-1 and cyclic guanosine 5'-monophosphate plasma concentrations in newborn infants with persistent pulmonary hypertension. J Pediatr. 1997;130:603–611.[Medline] [Order article via Infotrieve]
24. Kappas A, Maines MD. Tin: a potent inducer of heme oxygenase in kidney. Science. 1976;192:60–62.[Abstract/Free Full Text]
25. Sunderman FWJ, Reid MC, Bibeau LM, Linden JV. Nickel induction of microsomal heme oxygenase activity in rodents. Toxicol Appl Pharmacol. 1983;68:87–95.[Medline] [Order article via Infotrieve]
26. Sunderman FWJ. Metal induction of heme oxygenase. Ann N Y Acad Sci. 1987;514:65–80.[Abstract]
27. Jones RC, Reid LM. Structural basis of pulmonary hypertension. Curr Pulmonol. 1987;8:175–210.
28. Kourembanas S, Morita T, Liu Y, Christou H. Mechanisms by which oxygen regulates gene expression and cell-cell interaction in the vasculature. Kidney Int. 1997;51:438–443.[Medline] [Order article via Infotrieve]
29. Kourembanas S, Marsden PA, McQuillan LP, Faller DV. Hypoxia induces endothelin gene expression and secretion in cultured human endothelium. J Clin Invest. 1991;88:1054–1057.
30. McQuillan LP, Leung GK, Marsden PA, Kostyk SK, Kourembanas S. Hypoxia inhibits expression of eNOS via transcriptional and posttranscriptional mechanisms. Am J Physiol. 1994;267:H1921–H1927.[Abstract/Free Full Text]
31. Sacerdoti D, Escalante B, Abraham NG, McGiff JC, Levere RD, Schwartzman ML. Treatment with tin prevents the development of hypertension in spontaneously hypertensive rats. Science. 1989;243:388–390.[Abstract/Free Full Text]
32. Liu Y, Christou H, Morita T, Laughner E, Semenza GL, Kourembanas S. Carbon monoxide and nitric oxide suppress the hypoxic induction of vascular endothelial growth factor gene via the 5' enhancer. J Biol Chem. 1998;273:15257–15262.[Abstract/Free Full Text]
33. Vosatka RJ, Kashyap S, Trifiletti RR. Arginine deficiency accompanies persistent pulmonary hypertension of the newborn. Biol Neonate. 1994;66:65–70.[Medline] [Order article via Infotrieve]
34. Dollberg S, Warner BW, Myatt L. Urinary nitrite and nitrate concentrations in patients with idiopathic persistent pulmonary hypertension of the newborn and effect of extracorporeal membrane oxygenation. Pediatr Res. 1995;37:31–34.[Medline] [Order article via Infotrieve]
35. Sogawa K, Numayama-Tsuruta K, Ema M, Abe M, Abe H, Fujii-Kuriyama Y. Inhibition of hypoxia-inducible factor 1 activity by nitric oxide donors in hypoxia. Proc Natl Acad Sci U S A. 1998;95:7368–7373.[Abstract/Free Full Text]

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