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1.  Heme Oxygenase-1/Carbon Monoxide 
Heme oxygenase-1 (HO-1), a ubiquitous inducible stress-response protein, serves a major metabolic function in heme turnover. HO activity cleaves heme to form biliverdin-IXα, carbon monoxide (CO), and iron. Genetic experiments have revealed a central role for HO-1 in tissue homeostasis, protection against oxidative stress, and in the pathogenesis of disease. Four decades of research have witnessed not only progress in elucidating the molecular mechanisms underlying the regulation and function of this illustrious enzyme, but also have opened remarkable translational applications for HO-1 and its reaction products. CO, once regarded as a metabolic waste, can act as an endogenous mediator of cellular signaling and vascular function. Exogenous application of CO by inhalation or pharmacologic delivery can confer cytoprotection in preclinical models of lung/vascular injury and disease, based on anti-apoptotic, anti-inflammatory, and anti-proliferative properties. The bile pigments, biliverdin and bilirubin, end products of heme degradation, have also shown potential as therapeutics in vascular disease based on anti-inflammatory and anti-proliferative activities. Further translational and clinical trials research will unveil whether the HO-1 system or any of its reaction products can be successfully applied as molecular medicine in human disease.
PMCID: PMC2742746  PMID: 19617398
carbon monoxide; bilirubin; heme oxygenase-1; lung injury
2.  Scalable production of biliverdin IXα by Escherichia coli 
BMC Biotechnology  2012;12:89.
Biliverdin IXα is produced when heme undergoes reductive ring cleavage at the α-methene bridge catalyzed by heme oxygenase. It is subsequently reduced by biliverdin reductase to bilirubin IXα which is a potent endogenous antioxidant. Biliverdin IXα, through interaction with biliverdin reductase, also initiates signaling pathways leading to anti-inflammatory responses and suppression of cellular pro-inflammatory events. The use of biliverdin IXα as a cytoprotective therapeutic has been suggested, but its clinical development and use is currently limited by insufficient quantity, uncertain purity, and derivation from mammalian materials. To address these limitations, methods to produce, recover and purify biliverdin IXα from bacterial cultures of Escherichia coli were investigated and developed.
Recombinant E. coli strains BL21(HO1) and BL21(mHO1) expressing cyanobacterial heme oxygenase gene ho1 and a sequence modified version (mho1) optimized for E. coli expression, respectively, were constructed and shown to produce biliverdin IXα in batch and fed-batch bioreactor cultures. Strain BL21(mHO1) produced roughly twice the amount of biliverdin IXα than did strain BL21(HO1). Lactose either alone or in combination with glycerol supported consistent biliverdin IXα production by strain BL21(mHO1) (up to an average of 23. 5mg L-1 culture) in fed-batch mode and production by strain BL21 (HO1) in batch-mode was scalable to 100L bioreactor culture volumes. Synthesis of the modified ho1 gene protein product was determined, and identity of the enzyme reaction product as biliverdin IXα was confirmed by spectroscopic and chromatographic analyses and its ability to serve as a substrate for human biliverdin reductase A.
Methods for the scalable production, recovery, and purification of biliverdin IXα by E. coli were developed based on expression of a cyanobacterial ho1 gene. The purity of the produced biliverdin IXα and its ability to serve as substrate for human biliverdin reductase A suggest its potential as a clinically useful therapeutic.
PMCID: PMC3534565  PMID: 23176158
Biliverdin IXα; Heme oxygenase; Escherichia coli; HO1; Bilirubin; Anti-inflammatory; Biliverdin reductase; Bioreactor
3.  Heme Oxygenase, Inflammation, and Fibrosis: The Good, the Bad, and the Ugly? 
Upon injury, prolonged inflammation and oxidative stress may cause pathological wound healing and fibrosis, leading to formation of excessive scar tissue. Fibrogenesis can occur in most organs and tissues and may ultimately lead to organ dysfunction and failure. The underlying mechanisms of pathological wound healing still remain unclear, and are considered to be multifactorial, but so far, no efficient anti-fibrotic therapies exist. Extra- and intracellular levels of free heme may be increased in a variety of pathological conditions due to release from hemoproteins. Free heme possesses pro-inflammatory and oxidative properties, and may act as a danger signal. Effects of free heme may be counteracted by heme-binding proteins or by heme degradation. Heme is degraded by heme oxygenase (HO) that exists as two isoforms: inducible HO-1 and constitutively expressed HO-2. HO generates the effector molecules biliverdin/bilirubin, carbon monoxide, and free iron/ferritin. HO deficiency in mouse and man leads to exaggerated inflammation following mild insults, and accumulating epidemiological and preclinical studies support the widely recognized notion of the cytoprotective, anti-oxidative, and anti-inflammatory effects of the activity of the HO system and its effector molecules. In this review, we address the potential effects of targeted HO-1 induction or administration of HO-effector molecules as therapeutic targets in fibrotic conditions to counteract inflammatory and oxidative insults. This is exemplified by various clinically relevant conditions, such as hypertrophic scarring, chronic inflammatory liver disease, chronic pancreatitis, and chronic graft rejection in transplantation.
PMCID: PMC3345581  PMID: 22586396
heme oxygenase; heme; HO-effector molecules; fibrosis; therapy
4.  Heme Oxygenase-1, a Critical Arbitrator of Cell Death Pathways in Lung Injury and Disease 
Increases in cell death by programmed (ie., apoptosis, autophagy) or non-programmed mechanisms (ie., necrosis) occur during tissue injury, and may contribute to the etiology of several pulmonary or vascular disease states. The low molecular weight stress protein heme oxygenase-1 (HO-1) confers cytoprotection against cell death in various models of lung and vascular injury by inhibiting apoptosis, inflammation, and cell proliferation. HO-1 serves a vital metabolic function as the rate-limiting step in the heme degradation pathway and in the maintenance of iron homeostasis. The transcriptional induction of HO-1 occurs in response to multiple forms of chemical and physical cellular stress. The cytoprotective functions of HO-1 may be attributed to heme turnover, as well as to beneficial properties of its enzymatic reaction products: biliverdin-IXα, iron, and carbon monoxide (CO). Recent studies have demonstrated that HO-1 or CO inhibits stress-induced extrinsic and intrinsic apoptotic pathways in vitro. A variety of signaling molecules have been implicated in the cytoprotection conferred by HO-1/CO, including autophagic proteins, p38 mitogen activated protein kinase, signal transducer and activator of transcription proteins, nuclear factor-κB, phosphatydylinositol-3-kinase/Akt, and others. Enhanced HO-1 expression or the pharmacological application of HO end-products affords protection in preclinical models of tissue injury, including experimental and transplant-associated ischemia/reperfusion injury, promising potential future therapeutic applications.
PMCID: PMC3078523  PMID: 19362144
5.  Mitochondrial Localization and Function of Heme Oxygenase-1 in Cigarette Smoke–Induced Cell Death 
Cigarette smoke–induced apoptosis and necrosis contribute to the pathogenesis of chronic obstructive pulmonary disease. The induction of heme oxygenase-1 provides cytoprotection against oxidative stress, and may protect in smoking-related disease. Since mitochondria regulate cellular death, we examined the functional expression and mitochondrial localization of heme oxygenase-1 in pulmonary epithelial cells exposed to cigarette smoke extract (CSE), and its role in modulating cell death. Heme oxygenase-1 expression increased dramatically in cytosolic and mitochondrial fractions of human alveolar (A549), or bronchial epithelial cells (Beas-2b) exposed to either hemin, lipopolysaccharide, or CSE. Mitochondrial localization of heme oxygenase-1 was also observed in a primary culture of human small airway epithelial cells. Furthermore, heme oxygenase activity increased dramatically in mitochondrial fractions, and in whole cell extracts of Beas-2b after exposure to hemin and CSE. The mitochondrial localization of heme oxygenase-1 in Beas-2b was confirmed using immunogold-electron microscopy and immunofluorescence labeling on confocal laser microscopy. CSE caused loss of cellular ATP and rapid depolarization of mitochondrial membrane potential. Apoptosis occurred in Beas-2b at low concentrations of cigarette smoke extract, whereas necrosis occurred at high concentrations. Overexpression of heme oxygenase-1 inhibited CSE-induced Beas-2b cell death and preserved cellular ATP levels. Finally, heme oxygenase-1 mRNA expression was elevated in the lungs of mice chronically exposed to cigarette smoke. We demonstrate the functional compartmentalization of heme oxygenase-1 in the mitochondria of lung epithelial cells, and its potential role in defense against mitochondria-mediated cell death during CSE exposure.
PMCID: PMC1899328  PMID: 17079780
cigarette smoke; COPD; heme oxygenase-1; mitochondria
6.  Biliverdin Rescues the HO-2 Null Mouse Phenotype of Unresolved Chronic Inflammation Following Corneal Epithelial Injury 
The authors provide substantial evidence that the injured HO-2 null cornea, which experiences extensive oxidative stress, exaggerated inflammation, and impaired wound healing, can be rescued by the heme oxygenase product biliverdin, further supporting the notion that HO-2 is a critical cytoprotective system in the cornea.
The heme oxygenase system (HO-1 and HO-2) represents an intrinsic cytoprotective and anti-inflammatory pathway based on its ability to modulate leukocyte migration and to inhibit the expression of inflammatory cytokines and proteins by its products biliverdin/bilirubin and carbon monoxide. Corneal injury in HO-2 null mice leads to impaired healing and chronic inflammatory complications, including ulceration and neovascularization. The authors examined whether topically administered biliverdin can counteract the effects of HO deficiency in a corneal epithelial injury model.
HO-2 null mice were treated with biliverdin 1 hour before epithelial injury and twice a day thereafter. Reepithelialization and neovascularization were assessed by fluorescein staining and vital microscopy, respectively, and were quantified by image analysis. Inflammation was quantified by histology and Gr-1–specific immunofluorescence, and oxidative stress was assessed by DHE fluorescence.
Treatment with biliverdin accelerated wound closure, inhibited neovascularization and reduced epithelial defects. It also reduced inflammation, as evidenced by a reduction in the appearance of inflammatory cells and the expression levels of inflammatory and oxidant proteins, including KC and NOXs.
The results clearly show that biliverdin, directly or through its metabolism to bilirubin by biliverdin reductase—the expression of which is increased after injury—rescues the aberrant inflammatory phenotype, further underscoring the importance of the HO system in the cornea for the execution of an ordered inflammatory and reparative response.
PMCID: PMC3109026  PMID: 21345995
7.  Cytoprotective role of heme oxygenase-1 and heme degradation derived end products in liver injury 
World Journal of Hepatology  2013;5(10):541-549.
The activation of heme oxygenase-1 (HO-1) appears to be an endogenous defensive mechanism used by cells to reduce inflammation and tissue damage in a number of injury models. HO-1, a stress-responsive enzyme that catabolizes heme into carbon monoxide (CO), biliverdin and iron, has previously been shown to protect grafts from ischemia/reperfusion and rejection. In addition, the products of the HO-catalyzed reaction, particularly CO and biliverdin/bilirubin, have been shown to exert protective effects in the liver against a number of stimuli, as in chronic hepatitis C and in transplanted liver grafts. Furthermore, the induction of HO-1 expression can protect the liver against damage caused by a number of chemical compounds. More specifically, the CO derived from HO-1-mediated heme catabolism has been shown to be involved in the regulation of inflammation; furthermore, administration of low concentrations of exogenous CO has a protective effect against inflammation. Both murine and human HO-1 deficiencies have systemic manifestations associated with iron metabolism, such as hepatic overload (with signs of a chronic hepatitis) and iron deficiency anemia (with paradoxical increased levels of ferritin). Hypoxia induces HO-1 expression in multiple rodent, bovine and monkey cell lines, but interestingly, hypoxia represses expression of the human HO-1 gene in a variety of human cell types (endothelial cells, epithelial cells, T cells). These data suggest that HO-1 and CO are promising novel therapeutic molecules for patients with inflammatory diseases. In this review, we present what is currently known regarding the role of HO-1 in liver injuries and in particular, we focus on the implications of targeted induction of HO-1 as a potential therapeutic strategy to protect the liver against chemically induced injury.
PMCID: PMC3812456  PMID: 24179613
Heme oxygenases; Bilirubin; Hepatitis C; Kupffer cells; Polymorphisms; Immunoregulatory; Hypoxia; Liver ischemia
8.  Heme Oxygenase in the Regulation of Vascular Biology: From Molecular Mechanisms to Therapeutic Opportunities 
Antioxidants & Redox Signaling  2011;14(1):137-167.
Heme oxygenases (HOs) are the rate-limiting enzymes in the catabolism of heme into biliverdin, free iron, and carbon monoxide. Two genetically distinct isoforms of HO have been characterized: an inducible form, HO-1, and a constitutively expressed form, HO-2. HO-1 is a kind of stress protein, and thus regarded as a sensitive and reliable indicator of cellular oxidative stress. The HO system acts as potent antioxidants, protects endothelial cells from apoptosis, is involved in regulating vascular tone, attenuates inflammatory response in the vessel wall, and participates in angiogenesis and vasculogenesis. Endothelial integrity and activity are thought to occupy the central position in the pathogenesis of cardiovascular diseases. Cardiovascular disease risk conditions converge in the contribution to oxidative stress. The oxidative stress leads to endothelial and vascular smooth muscle cell dysfunction with increases in vessel tone, cell growth, and gene expression that create a pro-thrombotic/pro-inflammatory environment. Subsequent formation, progression, and obstruction of atherosclerotic plaque may result in myocardial infarction, stroke, and cardiovascular death. This background provides the rationale for exploring the potential therapeutic role for HO system in the amelioration of vascular inflammation and prevention of adverse cardiovascular outcomes. Antioxid. Redox Signal. 14, 137–167.
HO Expression
Heme oxygenase-1
Mitogen-activated protein kinases
Nrf2 and Bach1
Activator protein-1
Biliverdin reductase
Heme oxygenase-2
HO By-Products
Carbon monoxide
Ferrous iron and ferritin
BV and BR
Effect of HO-1 on Vascular Inflammation
Cytokines, chemokines, and mediators
Endothelial cells
Control of Vascular Diseases by HO-1=CO
Ischemic diseases
Diabetes mellitus
Therapeutic potential of HO-1=CO in vascular diseases
Effect of HO-1 on Angiogenesis
Cross-talk between the HO-1=CO and NOS=NO pathways
Effect of HO-1=CO pathway on vascular homeostasis
Regulation of VEGF expression by HO-1=CO
Restenosis and vasculopathy
PMCID: PMC2988629  PMID: 20624029
9.  Role of heme oxygenase-1 in microbial host defense 
Cellular microbiology  2008;11(2):199-207.
Heme oxygenase (HO)-1 is a cytoprotective enzyme that plays a critical role in defending the body against oxidant-induced injury during inflammatory processes. HO catalyzes the degradation of heme to carbon monoxide (CO), biliverdin, and ferrous iron. Biliverdin is converted to bilirubin, a potent endogenous antioxidant. CO has a number of biological functions, including anti-inflammatory properties. In various models of disease, HO-1 is known to play a critical role by ameliorating the pathological consequences of injury. In many of these models, the beneficial effects of HO-1 and its products of heme catabolism are by suppressing an inflammatory response. However, when investigating diseases due to microbial infections, inhibition of the inflammatory response could disrupt the ability of the immune system to eradicate an invading pathogen. Thus, questions remain regarding the role of HO-1 in microbial host defense. This microreview will address our present understanding of HO-1 and its functional significance in a variety of microbial infections.
PMCID: PMC3080039  PMID: 19016784
10.  Degradation of Heme in Gram-Negative Bacteria: the Product of the hemO Gene of Neisseriae Is a Heme Oxygenase 
Journal of Bacteriology  2000;182(23):6783-6790.
A full-length heme oxygenase gene from the gram-negative pathogen Neisseria meningitidis was cloned and expressed in Escherichia coli. Expression of the enzyme yielded soluble catalytically active protein and caused accumulation of biliverdin within the E. coli cells. The purified HemO forms a 1:1 complex with heme and has a heme protein spectrum similar to that previously reported for the purified heme oxygenase (HmuO) from the gram-positive pathogen Corynebacterium diphtheriae and for eukaryotic heme oxygenases. The overall sequence identity between HemO and these heme oxygenases is, however, low. In the presence of ascorbate or the human NADPH cytochrome P450 reductase system, the heme-HemO complex is converted to ferric-biliverdin IXα and carbon monoxide as the final products. Homologs of the hemO gene were identified and characterized in six commensal Neisseria isolates, Neisseria lactamica, Neisseria subflava, Neisseria flava, Neisseria polysacchareae, Neisseria kochii, and Neisseria cinerea. All HemO orthologs shared between 95 and 98% identity in amino acid sequences with functionally important residues being completely conserved. This is the first heme oxygenase identified in a gram-negative pathogen. The identification of HemO as a heme oxygenase provides further evidence that oxidative cleavage of the heme is the mechanism by which some bacteria acquire iron for further use.
PMCID: PMC111422  PMID: 11073924
11.  Heme Oxygenase-1: Its Therapeutic Roles in Inflammatory Diseases 
Heme oxygenase (HO)-1 is an inducible enzyme that catalyzes the first and rate-limiting step in the oxidative degradation of free heme into ferrous iron, carbon monoxide (CO), and biliverdin (BV), the latter being subsequently converted into bilirubin (BR). HO-1, once expressed during inflammation, forms high concentrations of its enzymatic by-products that can influence various biological events, and this expression is proven to be associated with the resolution of inflammation. The degradation of heme by HO-1 itself, the signaling actions of CO, the antioxidant properties of BV/BR, and the sequestration of ferrous iron by ferritin all concertedly contribute to the anti-inflammatory effects of HO-1. This review focuses on the anti-inflammatory mechanisms of HO-1 actions and its roles in inflammatory diseases.
PMCID: PMC2803295  PMID: 20107533
heme oxygenase-1; carbon monoxide; bilirubin/biliverdin; inflammation; nuclear factor E2-related factor-2; mitogen-activated protein kinase
12.  Integrative Survival Response Evoked by Heme Oxygenase-1 and Heme Metabolites 
Heme oxygenase (HO) catalyzes the rate-limiting step in heme degradation to produce carbon monoxide (CO), iron, and biliverdin. Biliverdin is subsequently converted to bilirubin by its reductase, and iron is recycled for heme synthesis. The inducible HO isoform, HO-1, is involved in the protection of multiple tissues and organs. The mechanism of protective actions of HO-1 has not been completely elucidated, but recent evidence suggests that one or more of heme metabolites can mediate the protective effects of HO-1. Particularly, CO mimics the antioxidant, anti-inflammatory, anti-apoptotic and antiproliferative actions of HO-1. Many of these effects of CO depend on the production of cyclic guanosine monophosphate (cGMP), and the modulation of mitogen-activated protein kinase (MAPK) pathways. The transcription factors, including nuclear factor E2-related factor-2 (Nrf2), and their upstream kinases, including MAPK pathway, play an important regulatory role in HO-1 expression by dietary antioxidants and drugs. This review attempts to concisely summarize the molecular and biochemical characteristics of HO-1, with a discussion on the mechanisms of signal transduction and gene regulation that mediate the induction of HO-1 by dietary antioxidants and drugs. In addition, the cytoprotective roles of HO-1 shall be discussed from the perspective of each of the metabolic by-products.
PMCID: PMC2386522  PMID: 18545641
heme oxygenase; antioxidant; anti-inflammation; anti-apoptosis; antiproliferation
13.  Therapeutic Potential of Heme Oxygenase-1/Carbon Monoxide in Lung Disease 
Heme oxygenase (HO), a catabolic enzyme, provides the rate-limiting step in the oxidative breakdown of heme, to generate carbon monoxide (CO), iron, and biliverdin-IXα. Induction of the inducible form, HO-1, in tissues is generally regarded as a protective mechanism. Over the last decade, considerable progress has been made in defining the therapeutic potential of HO-1 in a number of preclinical models of lung tissue injury and disease. Likewise, tissue-protective effects of CO, when applied at low concentration, have been observed in many of these models. Recent studies have expanded this concept to include chemical CO-releasing molecules (CORMs). Collectively, salutary effects of the HO-1/CO system have been demonstrated in lung inflammation/acute lung injury, lung and vascular transplantation, sepsis, and pulmonary hypertension models. The beneficial effects of HO-1/CO are conveyed in part through the inhibition or modulation of inflammatory, apoptotic, and proliferative processes. Recent advances, however, suggest that the regulation of autophagy and the preservation of mitochondrial homeostasis may serve as additional candidate mechanisms. Further preclinical and clinical trials are needed to ascertain the therapeutic potential of HO-1/CO in human clinical disease.
PMCID: PMC3296197  PMID: 22518295
14.  The Role of Heme Oxygenase-1 in Pulmonary Disease 
Heme oxygenase (HO)-1, the inducible isoform of heme oxygenase, is a cytoprotective enzyme that plays a central role in the defense against oxidative and inflammatory insults in the lung. HO-1 catalyzes the degradation of heme, a potent oxidant, into biliverdin, iron, and carbon monoxide (CO). These downstream products of heme catabolism have recently been found to mediate the antioxidant, antiapoptotic, antiproliferative, vasodilatory, and anti-inflammatory properties of HO-1. Although absence of HO-1 is rare in humans, a number of HO-1 promoter polymorphisms have been identified that may influence HO-1 expression in vivo and lead to disease states. This review will summarize studies that implicate HO-1 and heme metabolites in the pathophysiology of pulmonary disease and discuss recent advances in the therapeutic applications of HO-1.
PMCID: PMC2176110  PMID: 16980551
HO-1; polymorphism; ARDS; pulmonary hypertension; COPD
15.  Cerebroprotective functions of HO-2 
Current pharmaceutical design  2008;14(5):443-453.
The constitutive isoform of heme oxygenase, HO-2, is highly expressed in the brain and in cerebral vessels. HO-2 functions in the brain have been evaluated using pharmacological inhibitors of the enzyme and HO-2 gene deletion in in vivo animal models and in cultured cells (neurons, astrocytes, cerebral vascular endothelial cells). Rapid activation of HO-2 via post-translational modifications without upregulation of HO-2 expression or HO-1 induction coincides with the increase in cerebral blood flow aimed at maintaining brain homeostasis and neuronal survival during seizures, hypoxia, and hypotension. Pharmacological inhibition or gene deletion of brain HO-2 exacerbates oxidative stress induced by seizures, glutamate, and inflammatory cytokines, and causes cerebral vascular injury. Carbon monoxide (CO) and bilirubin, the end products of HO-catalyzed heme degradation, have distinct cytoprotective functions. CO, by binding to a heme prosthetic group, regulates the key components of cell signaling, including BKCa channels, guanylyl cyclase, NADPH oxidase, and the mitochondria respiratory chain. Cerebral vasodilator effects of CO are mediated via activation of BKCa channels and guanylyl cyclase. CO, by inhibiting the major components of endogenous oxidantgenerating machinery, NADPH oxidase and the cytochrome C oxidase of the mitochondrial respiratory chain, blocks formation of reactive oxygen species. Bilirubin, via redox cycling with biliverdin, is a potent oxidant scavenger that removes preformed oxidants. Overall, HO-2 has dual housekeeping cerebroprotective functions by maintaining autoregulation of cerebral blood flow aimed at improving neuronal survival in a changing environment, and by providing an effective defense mechanism that blocks oxidant formation and prevents cell death caused by oxidative stress.
PMCID: PMC3587165  PMID: 18289071
Heme oxygenase; cerebral protection; cerebrovascular disease; oxidative stress; seizures; carbon monoxide; bilirubin
16.  Heme oxygenase-1: a novel therapeutic target for gastrointestinal diseases 
Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in the catabolism of heme, followed by production of biliverdin, free iron and carbon monoxide (CO). HO-1 is a stress-responsive protein induced by various oxidative agents. Recent studies demonstrate that the expression of HO-1 in response to different inflammatory mediators may contribute to the resolution of inflammation and has protective effects in several organs against oxidative injury. Although the mechanism underlying the anti-inflammatory actions of HO-1 remains poorly defined, both CO and biliverdin/bilirubin have been implicated in this response. In the gastrointestinal tract, HO-1 is shown to be transcriptionally induced in response to oxidative stress, preconditioning and acute inflammation. Recent studies suggest that the induction of HO-1 expression plays a critical protective role in intestinal damage models induced by ischemia-reperfusion, indomethacin, lipopolysaccharide-associated sepsis, trinitrobenzene sulfonic acid, and dextran sulfate sodium, indicating that activation of HO-1 may act as an endogenous defensive mechanism to reduce inflammation and tissue injury in the gastrointestinal tract. In addition, CO derived from HO-1 is shown to be involved in the regulation in gastro-intestinal motility. These in vitro and in vivo data suggest that HO-1 may be a novel therapeutic target in patients with gastrointestinal diseases.
PMCID: PMC3045685  PMID: 21373265
Bach1; bilirubin; carbon monoxide; heme oxygenase; indomethacin; Nrf2; ulcerative colitis
17.  Curcumin-Induced Heme Oxygenase-1 Expression Prevents H2O2-Induced Cell Death in Wild Type and Heme Oxygenase-2 Knockout Adipose-Derived Mesenchymal Stem Cells 
Mesenchymal stem cell (MSC) administration is a promising adjuvant therapy to treat tissue injury. However, MSC survival after administration is often hampered by oxidative stress at the site of injury. Heme oxygenase (HO) generates the cytoprotective effector molecules biliverdin/bilirubin, carbon monoxide (CO) and iron/ferritin by breaking down heme. Since HO-activity mediates anti-apoptotic, anti-inflammatory, and anti-oxidative effects, we hypothesized that modulation of the HO-system affects MSC survival. Adipose-derived MSCs (ASCs) from wild type (WT) and HO-2 knockout (KO) mice were isolated and characterized with respect to ASC marker expression. In order to analyze potential modulatory effects of the HO-system on ASC survival, WT and HO-2 KO ASCs were pre-treated with HO-activity modulators, or downstream effector molecules biliverdin, bilirubin, and CO before co-exposure of ASCs to a toxic dose of H2O2. Surprisingly, sensitivity to H2O2-mediated cell death was similar in WT and HO-2 KO ASCs. However, pre-induction of HO-1 expression using curcumin increased ASC survival after H2O2 exposure in both WT and HO-2 KO ASCs. Simultaneous inhibition of HO-activity resulted in loss of curcumin-mediated protection. Co-treatment with glutathione precursor N-Acetylcysteine promoted ASC survival. However, co-incubation with HO-effector molecules bilirubin and biliverdin did not rescue from H2O2-mediated cell death, whereas co-exposure to CO-releasing molecules-2 (CORM-2) significantly increased cell survival, independently from HO-2 expression. Summarizing, our results show that curcumin protects via an HO-1 dependent mechanism against H2O2-mediated apoptosis, and likely through the generation of CO. HO-1 pre-induction or administration of CORMs may thus form an attractive strategy to improve MSC therapy.
PMCID: PMC4227200  PMID: 25299695
adipose-derived mesenchymal stem cells; oxidative stress; apoptosis; heme oxygenase; carbon monoxide
18.  Heme oxygenase-1 is a modulator of inflammation and vaso-occlusion in transgenic sickle mice 
Journal of Clinical Investigation  2006;116(3):808-816.
Transgenic sickle mice expressing βS hemoglobin have activated vascular endothelium that exhibits enhanced expression of NF-κB and adhesion molecules that promote vascular stasis in sickle, but not in normal, mice in response to hypoxia/reoxygenation. Sickle mice hemolyze rbcs in vivo as demonstrated by increased reticulocyte counts, plasma hemoglobin and bilirubin, and reduced plasma haptoglobin. The heme content is elevated in sickle organs, which promotes vascular inflammation and heme oxygenase-1 expression. Treatment of sickle mice with hemin further increases heme oxygenase-1 expression and inhibits hypoxia/reoxygenation–induced stasis, leukocyte-endothelium interactions, and NF-κB, VCAM-1, and ICAM-1 expression. Heme oxygenase inhibition by tin protoporphyrin exacerbates stasis in sickle mice. Furthermore, treatment of sickle mice with the heme oxygenase enzymatic product carbon monoxide or biliverdin inhibits stasis and NF-κB, VCAM-1, and ICAM-1 expression. Local administration of heme oxygenase-1 adenovirus to subcutaneous skin increases heme oxygenase-1 and inhibits hypoxia/reoxygenation–induced stasis in the skin of sickle mice. Heme oxygenase-1 plays a vital role in the inhibition of vaso-occlusion in transgenic sickle mice.
PMCID: PMC1366501  PMID: 16485041
19.  Targeted suppression of HO-2 gene expression impairs the innate anti-inflammatory and repair responses of the cornea to injury 
Molecular Vision  2011;17:1144-1152.
Heme oxygenase (HO)-2 is highly expressed in the corneal epithelium and is a component of the heme oxygenase system that represents an intrinsic cytoprotective and anti-inflammatory system based on its ability to modulate leukocyte migration and to inhibit expression of inflammatory cytokines and proteins via its products biliverdin/bilirubin and carbon monoxide (CO). We have shown that in HO-2 null mice epithelial injury leads to unresolved corneal inflammation and chronic inflammatory complications including ulceration, perforation and neovascularization. In this study, we explore whether a localized corneal suppression of HO-2 is sufficient for disrupting the innate anti-inflammatory and repair capability of the cornea.
Silencing hairpin RNA (shRNA) against HO-2 was administered subconjunctivally (100 ng/eye) as well as topically (100 ng/eye) starting one day before corneal epithelial debridement and once daily, thereafter. The corneal epithelium was removed using an Alger Brush in anesthetized mice. Re-epithelialization was assessed by fluorescein staining using a dissecting microscope and image analysis. Inflammatory response was quantified by myeloperoxidase activity. Levels of mRNA were measured by RT–PCR.
Local injection of HO-2-specific shRNA led to a 50% reduction in corneal HO-2 mRNA. Administration of HO-2-specific shRNA delayed corneal re-epithelialization when compared with the control shRNA-treated group by 14%, 20%, and 12% at days 3, 4, and 7 after injury, respectively (n=18–24). The observed delay in the wound repair process in HO-2 shRNA treated mice was accompanied by a threefold and 3.5 fold increase in the neovascular response at days 4 and 7 after injury. Further, local knockdown of HO-2 lead to an aberrant chronic inflammatory response, as shown by presence of high numbers of inflammatory cells still present in the cornea at day 7 after injury; 1.04±0.45×106 in HO-2 knockdown mice versus 0.14±0.03×106 inflammatory cells in control mice. Matrix metalloproteinase-2 (MMP-2) but not MMP-9 increased following injury and remained elevated in the injured corneas of the HO-2 shRNA-treated eyes.
Corneal knockdown of HO-2 via local administration of HO-2-specific shRNA leads to delayed re-epithelialization, increased neovascularization and an aberrant inflammatory response similar to what is observed in the HO-2 null mouse. The elevated MMP-2 expression may contribute to the increase in neovascularization in corneas in which HO-2 expression is suppressed.
PMCID: PMC3087447  PMID: 21552471
Biochemistry  2007;46(23):6822-6829.
Hemoglobin digestion in the midgut of hematophagous animals results in the release of its prosthetic group, heme, which is a pro-oxidant molecule. Heme enzymatic degradation is a protective mechanism that has been described in several organisms, including plants, bacteria, and mammals. This reaction is catalyzed by heme oxygenase and results in formation of carbon monoxide, ferrous ion, and biliverdin IXα. During digestion, a large amount of a green pigment is produced and secreted into the intestinal lumen of A. aegypti adult females. In the case of another blood-sucking insect, the kissing-bug Rhodnius prolixus, we have recently shown that heme degradation involves a complex pathway that generates dicysteinyl-biliverdin IX gamma. The light absorption spectrum of the Aedes purified pigment was similar to biliverdin, but its mobility on a reverse-phase chromatography column suggested a compound less hydrophobic than biliverdin IXα. Structural characterization by ESI-MS revealed that the mosquito pigment is the α isomer of biliverdin bound to two glutamine residues by an amide bond. This biglutaminyl-biliverdin is formed by oxidative cleavage of the heme porphyrin ring followed by two subsequent additions of glutamine residues to the biliverdin IXα. The role of this pathway in the adaptation of this insect vector to a blood-feeding habit is discussed.
PMCID: PMC2763637  PMID: 17508725
heme; heme oxygenase; biliverdin; detoxification; Aedes aegypti
21.  The Haptoglobin-CD163-Heme Oxygenase-1 Pathway for Hemoglobin Scavenging 
The haptoglobin- (Hp-) CD163-heme oxygenase-1 (HO-1) pathway is an efficient captor-receptor-enzyme system to circumvent the hemoglobin (Hb)/heme-induced toxicity during physiological and pathological hemolyses. In this pathway, Hb tightly binds to Hp leading to CD163-mediated uptake of the complex in macrophages followed by lysosomal Hp-Hb breakdown and HO-1-catalyzed conversion of heme into the metabolites carbon monoxide (CO), biliverdin, and iron. The plasma concentration of Hp is a limiting factor as evident during accelerated hemolysis, where the Hp depletion may cause serious Hb-induced toxicity and put pressure on backup protecting systems such as the hemopexin-CD91-HO pathway. The Hp-CD163-HO-1 pathway proteins are regulated by the acute phase mediator interleukin-6 (IL-6), but other regulatory factors indicate that this upregulation is a counteracting anti-inflammatory response during inflammation. The heme metabolites including bilirubin converted from biliverdin have overall an anti-inflammatory effect and thus reinforce the anti-inflammatory efficacy of the Hp-CD163-HO-1 pathway. Future studies of animal models of inflammation should further define the importance of the pathway in the anti-inflammatory response.
PMCID: PMC3678498  PMID: 23781295
22.  Carbon monoxide: present and future indications for a medical gas 
Gaseous molecules continue to hold new promise in molecular medicine as experimental and clinical therapeutics. The low molecular weight gas carbon monoxide (CO), and similar gaseous molecules (e.g., H2S, nitric oxide) have been implicated as potential inhalation therapies in inflammatory diseases. At high concentration, CO represents a toxic inhalation hazard, and is a common component of air pollution. CO is also produced endogenously as a product of heme degradation catalyzed by heme oxygenase enzymes. CO binds avidly to hemoglobin, causing hypoxemia and decreased oxygen delivery to tissues at high concentrations. At physiological concentrations, CO may have endogenous roles as a signal transduction molecule in the regulation of neural and vascular function and cellular homeostasis. CO has been demonstrated to act as an effective anti-inflammatory agent in preclinical animal models of inflammation, acute lung injury, sepsis, ischemia/reperfusion injury, and organ transplantation. Additional experimental indications for this gas include pulmonary fibrosis, pulmonary hypertension, metabolic diseases, and preeclampsia. The development of chemical CO releasing compounds constitutes a novel pharmaceutical approach to CO delivery with demonstrated effectiveness in sepsis models. Current and pending clinical evaluation will determine the usefulness of this gas as a therapeutic in human disease.
PMCID: PMC3604600  PMID: 23525151
Acute lung injury; Carbon monoxide; Heme oxygenase (decyclizing); Reperfusion injury; Sepsis
23.  Ribavirin induced hemolysis: A novel mechanism of action against chronic hepatitis C virus infection 
World Journal of Gastroenterology : WJG  2014;20(43):16184-16190.
Hepatitis C virus (HCV) is not usually cleared by our immune system, leading to the development of chronic hepatitis C infection. Chronic HCV induces the production of various cytokines, predominantly by Kupffer cells (KCs), and creates a pro-inflammatory state in the liver. The chronic dysregulated production of interferon (IFN) and other cytokines by KCs also promotes innate immune tolerance. Ribavirin (RBV) monotherapy has been shown to decrease inflammation in liver of patients with chronic hepatitis C. Sustained virological response (SVR) is significantly higher when IFN is combined with RBV in chronic HCV (cHCV) infection. However, the mechanism of their synergy remains unclear. Previous theories have attempted to explain the anti-HCV effect based on direct action of RBV alone on the virus or on the immune system; however, these theories have serious shortcomings. We propose that hemolysis, which universally occurs with RBV therapy and which is considered a limiting side effect, is precisely the mechanism by which the anti-HCV effect is exerted. Passive hemolysis results in anti-inflammatory/antiviral actions within the liver that disrupt the innate immune tolerance, leading to the synergy of RBV with IFN-α. Ribavirin-induced hemolysis floods the hepatocytes and KCs with heme, which is metabolized and detoxified by heme oxygenase-1 (HMOX1) to carbon monoxide (CO), biliverdin and free iron (which induces ferritin). These metabolites of heme possess anti-inflammatory and antioxidant properties. Thus, HMOX1 plays an extremely important anti-oxidant, anti-inflammatory and cytoprotective role, particularly in KCs and hepatocytes. HMOX1 has been noted to have anti-viral effects in hepatitis C infected cell lines. Additionally, it has been shown to enhance the response to IFN-α by restoring interferon-stimulated genes (ISGs). This mechanism can be clinically corroborated by the following observations that have been found in patients undergoing RBV/IFN combination therapy for cHCV: (1) SVR rates are higher in patients who develop anemia; (2) once anemia (due to hemolysis) occurs, the SVR rate does not depend on the treatment utilized to manage anemia; and (3) ribavirin analogs, such as taribavirin and levovirin, which increase intrahepatic ribavirin levels and which produce lesser hemolysis, are inferior to ribavirin for treating cHCV. This mechanism can also explain the observed RBV synergy with direct antiviral agents. This hypothesis is testable and may lead to newer and safer medications for treating cHCV infection.
PMCID: PMC4239506  PMID: 25473172
Chronic hepatitis C; Therapy; Ribavirin; Hemolysis; Heme oxygenase-1
24.  Leydig cell–derived heme oxygenase-1 regulates apoptosis of premeiotic germ cells in response to stress 
Stress-induced downregulation of spermatogenesis remains poorly understood. This study examined the induction of heme oxygenase-1 (HO-1), a carbon monoxide–generating inducible enzyme, in modulation of spermatogenesis. Rats were exposed to cadmium chloride (CdCl2), a stressor causing oligozoospermia, and HO-1–induction was monitored by following HO isozyme expression. CdCl2-treated testes increased HO-1 activity and suppressed microsomal cytochromes P450, which are required for steroidogenesis. CdCl2-elicited HO-1 occurred mostly in Leydig cells and coincided with CO generation, as judged by bilirubin-IXα immunoreactivity. Under these circumstances, germ cells in peripheral regions of seminiferous tubules exhibited apoptosis; laser flow cytometry revealed that these apoptotic cells involve diploid and tetraploid germ cells, suggesting involvement of spermatogonia and primary spermatocytes in CdCl2-elicited apoptosis. Pretreatment with zinc protoporphyrin-IX, an HO inhibitor, but not copper protoporphyrin-IX, which does not block the enzyme, attenuated the CdCl2-induced apoptosis. Such antiapoptotic effects of zinc protoporphyrin-IX were repressed by supplementation of dichloromethane, a CO donor. Upon CdCl2-treatment, both Sertoli cells and the germ cells upregulated Fas ligand; this event was also suppressed by zinc protoporphyrin-IX and restored by dichloromethane. Thus, Leydig cells appear to use HO-1–derived CO to trigger apoptosis of premeiotic germ cells and thereby modulate spermatogenesis under conditions of stress.
PMCID: PMC150871  PMID: 11854317
25.  Physiological significance of heme oxygenase in hypertension 
The last decade has witnessed an explosion in the elucidation of the role that the heme oxygenase system plays in human physiology. This system encompasses not only the heme degradative pathway, including heme oxygenase and biliverdin reductase, but also the products of heme degradation, carbon monoxide, iron, and biliverdin/bilirubin. Their role in diabetes, inflammation, heart disease, hypertension, transplantation, and pulmonary disease are areas of burgeoning research. The research has focused not only on heme itself but also on its metabolic products as well as endogenous compounds involved in a vast number of genetic and metabolic processes that are affected when heme metabolism is perturbed. It should be noted, however, that although the use of carbon monoxide and biliverdin/bilirubin as therapeutic agents has been successful, these agents can be toxic at high levels in tissue, e.g., kernicterus. Care must be used to ensure that when these compounds are used as therapeutic agents their deleterious effects are minimized or avoided. On balance, however, the strategies to target heme oxygenase-1 as described in this review offer promising therapeutic approaches to clinicians for the effective management of hypertension and renal function. The approaches detailed may prove to be seminal in the development of a new therapeutic strategy to treat hypertension.
PMCID: PMC2745554  PMID: 19027871
Heme oxygenase; Hypertension; Carbon monoxide; Bilirubin; Adiponectin

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