Protective role of heme oxygenase in the blood vessel wall during atherogenesis.

Several lines of evidence suggest that antioxidant processes and (or) endogenous antioxidants inhibit proatherogenic events in the blood vessel wall. Heme oxygenase (HO), which catabolizes heme to biliverdin, carbon monoxide, and catalytic iron, has been shown to have such antioxidative properties. The HO-1 isoform of heme oxygenase is ubiquitous and can be increased several fold by stimuli that induce cellular oxidative stress. Products of the HO reaction have important effects: carbon monoxide is a potent vasodilator, which is thought to play a role in modulation of vascular tone; biliverdin and its by-product bilirubin are potent antioxidants. Although HO induction results in an increase in catalytic free iron release, the enhancement of intracellular ferritin protein through HO-1 has been reported to decrease the cytotoxic effects of iron. Oxidized LDL has been shown to increase HO-1 expression in endothelial and smooth muscle cell cultures, and during atherogenesis. Further evidence of HO-1 expression associated with atherogenesis has been demonstrated in human, murine and rabbit atherosclerotic lesions. Moreover, genetic models of HO deficiency suggest that the actions of HO-1 are important in modulating the severity of atherosclerosis. Recent experiments in gene therapy using the HO gene suggest that interventions aimed at HO in the vessel wall could provide a novel therapeutic approach for the treatment or prevention of atherosclerotic disease.     

Non-heme induction of heme oxygenase-1 does not alter cellular iron metabolism

The catabolism of heme is carried out by members of the heme oxygenase (HO) family. The products of heme catabolism by HO-1 are ferrous iron, biliverdin (subsequently converted to bilirubin), and carbon monoxide. In addition to its function in the recycling of hemoglobin iron, this microsomal enzyme has been shown to protect cells in various stress models. Implicit in the reports of HO-1 cytoprotection to date are its effects on the cellular handling of heme/iron. However, the limited amount of uncommitted heme in non-erythroid cells brings to question the source of substrate for this enzyme in non-hemolytic circumstances. In the present study, HO-1 was induced by either sodium arsenite (reactive oxygen species producer) or hemin or overexpressed in the murine macrophage-like cell line, RAW 264.7. Both of the inducers elicited an increase in active HO-1; however, only hemin exposure caused an increase in the synthesis rate of the iron storage protein, ferritin. This effect of hemin was the direct result of the liberation of iron from heme by HO. Cells stably overexpressing HO-1, although protected from oxidative stress, did not display elevated basal ferritin synthesis. However, these cells did exhibit an increase in ferritin synthesis, compared with untransfected controls, in response to hemin treatment, suggesting that heme levels, and not HO-1, limit cellular heme catabolism. Our results suggest that the protection of cells from oxidative insult afforded by HO-1 is not due to the catabolism of significant amounts of cellular heme as thought previously.     

Backbone assignments of the apo and Zn(II) protoporphyrin IX-bound states of the soluble form of rat heme oxygenase-1

In nature, heme is a prosthetic group that is universally used as a cofactor for heme proteins. It is necessary for the execution of fundamental biological processes including electron transfer, oxidation and metabolism. However, free heme is toxic to cells, because of its capability to enhance oxidative stress, hence its cellular concentration is strictly regulated through multiple mechanisms. Heme oxygenase (HO) serves as an irreplaceable member in the heme degradation system. It is a ubiquitous protein, existing in many species including mammals, higher plants, and interestingly, certain pathogenic bacteria. In the HO reaction, HO catalyzes oxidative cleavage of heme to generate biliverdin and release carbon monoxide and ferrous iron. Because of the beneficial effects of these heme catabolism products, HO plays a key role in iron homeostasis and in defense mechanism against oxidative stress. HO is composed of an N-terminal structured region and a C-terminal membrane-bound region. Furthermore, the soluble form of HO, which is obtainable by excision of the membrane-bound region, retains its catalytic activity. Here, we present the backbone resonance assignments of the soluble form (residues 1–232) of HO-1 in the free and Zn(II) protoporphyrin IX (ZnPP)-bound states, and analyzed the structural differences between the states. ZnPP is a potent enzyme inhibitor, and the ZnPP-bound structure of HO-1 mimics the heme-bound structure. These assignments provide the structural basis for a detailed investigation of the HO-1 function.     

Heme oxygenase-1 inhibits TNF-alpha-induced apoptosis in cultured fibroblasts

Heme oxygenase (HO)-1 catalyzes the oxidative cleavage of heme to yield equimolar amounts of biliverdin, iron, and carbon monoxide. HO-1 is a stress response protein, the induction of which is associated with protection against oxidative stress. The mechanism(s) of protection is not completely elucidated, although it is suggested that one or more of the catalytic by-products provide antioxidant functions either directly or indirectly. The involvement of reactive oxygen species in apoptosis raised the question of a possible role for HO-1 in programmed cell death. Using the tetracycline-regulated expression system, we show here that conditional overexpression of HO-1 prevents tumor necrosis factor-alpha-induced apoptosis in murine L929 fibroblasts. Inhibition of apoptosis was not observed in the presence of tin protoporphyrin, a specific inhibitor of HO activity, and in cells overexpressing antisense HO-1. Interestingly, exogenous administration of a low concentration of carbon monoxide also prevented tumor necrosis factor-alpha-induced apoptosis in L929 fibroblasts. Inhibition of tumor necrosis factor-alpha-induced apoptosis by HO-1 overexpression was reversed by 1H-(1,2, 4)oxadiazolo(4,3-a)quinoxalin-1-one, an inhibitor of guanylate cyclase, which is a target enzyme for carbon monoxide. Taken together, our data suggest that the antiapoptotic effect of HO-1 may be mediated via carbon monoxide.     

Concurrent expression of heme oxygenase-1 and p53 in human retinal pigment epithelial cell line

Heme oxygenase-1 (HO-1) is a stress-responsive protein that is known to regulate cellular functions such as cell proliferation, inflammation, and apoptosis. Here, we investigated the effects of HO activity on the expression of p53 in the human retinal pigment epithelium (RPE) cell line ARPE-19. Cobalt protoporphyrin (CoPP) induced the expression of both HO-1 and p53 without significant toxicity to the cells. In addition, the blockage of HO activity with the iron chelator DFO or with HO-1 siRNA inhibited the CoPP-induced expression of p53. Similarly, zinc protoporphyrin (ZnPP), an inhibitor of HO, suppressed p53 expression in ARPE-19 cells, although ZnPP increased the level of HO-1 protein while inhibiting HO activity. Also, CoPP-induced p53 expression was not affected by the formation of reactive oxygen species (ROS). Based on these results, we conclude that HO activity is involved in the regulation of p53 expression in a ROS-independent mechanism, and also suggest that the expression of p53 in ARPE-19 cells is associated with heme metabolites such as biliverdin/bilirubin, carbon monoxide, and iron produced by the activity of HO.     

Amplifying the fluorescence of bilirubin enables the real-time detection of heme oxygenase activity

Heme oxygenases (HO) are the rate-limiting enzymes in the degradation of heme to equimolar amounts of antioxidant bile pigments, the signaling molecule carbon monoxide, and ferric iron. The inducible form HO-1 confers protection on cells and tissues that mediates beneficial effects in many diseases. Consequently, measurement of the enzymatic activity is vital in the investigation of the regulatory role of HO. Here we report that the fluorescence characteristics of bilirubin in complex with serum albumin can be used for the real-time detection of HO activity in enzymatic kinetics measurements. We characterized the enzymatic activity of a truncated human HO-1 and measured the HO activity for various cell types and organs, in either the basal naive or the HO-1-induced state. The bilirubin-dependent increase in fluorescence over time monitored by this assay facilitates a very fast, sensitive, and reliable measurement of HO activity. Our approach offers the basis for a highly sensitive high-throughput screening, which provides, inter alia, the opportunity to discover new therapeutic HO-1-inducing agents.     

Modulation of heme oxygenase in tissue injury and its implication in protection against gastrointestinal diseases.

Heme oxygenase (HO) is the rate-limiting enzyme in the catabolism of heme, followed by production of biliverdin, free iron and carbon monoxide (CO). There are three isoforms of HO: HO-1 is highly inducible, whereas HO-2 and HO-3 are constitutively expressed. In addition to heme, a variety of nonheme compounds, including heavy metals, cytokines, endotoxins and heat shock stress are strong inducers of HO-1 expression. Many studies indicated that induction of HO-1 is associated with a protective response due to the removal of free heme, which is shown to be toxic. However, recent studies demonstrated that the expression of HO-1 in response to different inflammatory mediators could contribute in part to the resolution of inflammation and have protective effects on brain, liver, kidney and lung against injuries. These beneficial effects seem to be due to the production of bile pigment biliverdin and bilirubin that is a potent antioxidant, as well as the release of iron and CO. However, there are few studies concerning the relationship between HO-1 and inflammation as well as injury in the gut. Interestingly, a preliminary study implicated that induction of HO-1 expression in a colonic damage model induced by trinitrobenzene sulfonic acid played a critical protective role, indicating that activation of HO-1 could act as a natural defensive mechanism to alleviate inflammation and tissue injury in the gastrointestinal tract.     

Reversal of HO-1 related cytoprotection with increased expression is due to reactive iron.

It is often postulated that the cytoprotective nature of heme oxygenase (HO-1) explains the inducible nature of this enzyme. However, the mechanisms by which protection occurs are not verified by systematic evaluation of the physiological effects of HO. To explain how induction of HO-1 results in protection against oxygen toxicity, hamster fibroblasts (HA-1) were stably transfected with a tetracycline response plasmid containing the full-length rat HO-1 cDNA construct to allow for regulation of gene expression by varying concentrations of doxycycline (Dox). Transfected cells were exposed to hyperoxia (95% O(2)/5% CO2) for 24 h and several markers of oxidative injury were measured. With varying concentrations of Dox, HO activity was regulated between 3- and 17-fold. Despite cytoprotection with low (less than fivefold) HO activity, high levels of HO-1 expression (greater than 15-fold) were associated with significant oxygen cytotoxicity. Levels of non-heme reactive iron correlated with cellular injury in hyperoxia whereas lower levels of heme were associated with cytoprotection. Cellular levels of cyclic GMP and bilirubin were not significantly altered by modification of HO activity, precluding a substantial role for activation of guanylate cyclase by carbon monoxide or for accumulation of bile pigments in the physiological consequences of HO-1 overexpression. Inhibition of HO activity or chelation of cellular iron prior to hyperoxic exposure decreased reactive iron levels in the samples and significantly reduced oxygen toxicity. We conclude that there is a beneficial threshold of HO-1 overexpression related to the accumulation of reactive iron released in the degradation of heme. Therefore, despite the ready induction of HO-1 in oxidant stress, accumulation of reactive iron formed makes it unlikely that exaggerated expression of HO-1 is a cytoprotective response.     

Role of haem oxygenase-1 in microbial host defence

Haem oxygenase (HO)-1 is a cytoprotective enzyme that plays a critical role in defending the body against oxidant-induced injury during inflammatory processes. HO catalydes the degradation of haem 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 haem 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 defence. This microreview will address our present understanding of HO-1 and its functional significance in a variety of microbial infections.     

Heme oxygenase-1 induction may explain the antioxidant profile of pentaerythrityl trinitrate

The organic nitrate pentaerythrityl tetranitrate (PETN) is known to exert long-term antioxidant and antiatherogenic effects by as yet unidentified mechanisms. In cultured endothelial cells derived from human umbilical vein, the active PETN metabolite PETriN (0.01-1 mM) increased heme oxygenase (HO)-1 mRNA and protein levels in a concentration-dependent fashion. HO-1 induction was accompanied by a marked increase in catalytic activity of the enzyme as reflected by enhanced formation of carbon monoxide and bilirubin. Pretreatment with PETriN or bilirubin at low micromolar concentrations protected endothelial cells from hydrogen peroxide-mediated toxicity. HO-1 induction and endothelial protection by PETriN were not mimicked by isosorbide dinitrate, another long-acting nitrate. The present study demonstrates that PETriN stimulates mRNA and protein expression as well as enzymatic activity of the antioxidant defense protein HO-1 in endothelial cells. Increased HO-1 expression and ensuing formation of cytoprotective bilirubin may contribute to and explain the specific antioxidant and antiatherogenic actions of PETN.     

Neuroprotective role of heme-oxygenase 1 against iodoacetate-induced toxicity in rat cerebellar granule neurons: Role of bilirubin

Heme oxygenase (HO) catalyses the breakdown of heme to iron, carbon monoxide and biliverdin, the latter being further reduced to bilirubin. A protective role of the inducible isoform, HO-1, has been described in pathological conditions associated with the production of reactive oxygen species (ROS). The aim of this study was to investigate the role of HO-1 in the neurotoxicity induced by iodoacetate (IAA) in primary cultures of cerebellar granule neurons (CGNs). IAA, an inhibitor of the glycolysis pathway, reduces cell survival, increases ROS production and enhances HO-1 expression in CGNs. Furthermore, the induction of HO-1 expression by cobalt protoporphyrin (CoPP) prevented cell death and ROS production induced by IAA, whereas the inhibition of HO activity with tin mesoporphyrin exacerbated the IAA-induced neurotoxicity. The protective effect elicited by CoPP was reproduced by bilirubin addition, suggesting that this molecule may be involved in the protective effect of HO-1 induction in this experimental model.     

BMP6 attenuates oxidant injury in HK-2 cells via Smad-dependent HO-1 induction

Oxidative stress is involved in a variety of kidney diseases, and heme oxygenase 1 (HO-1) induction is a protective response to oxidative stress. Downregulation of bone morphogenetic protein 6 (BMP6) is associated with renal damage in intrauterine growth-restricted newborns. However, it is unknown whether BMP6 has a renoprotective effect or HO-1 induction property. In this study, we demonstrate that BMP6 effectively protects renal proximal tubule cells (HK-2) against hydrogen peroxide (H₂O₂)-induced cell injury. BMP6 also increased HO-1 gene expression and activity of HO. Inhibition of de novo gene expression, the HO inhibitor ZnPPIX, HO-1 knockdown, or the carbon monoxide (CO) scavenger hemoglobin attenuated the cytoprotective effect of BMP6, whereas HO-1 constitutive expression, the HO-1 inducer hemin, or the hemin metabolites bilirubin and CO ameliorated H₂O₂-induced cell injury. Stimulation of HK-2 cells with BMP6 activated Smad signaling but not mitogen-activated protein kinases. In addition, BMP6-mediated induction of HO-1 expression and increase in HO activity were inhibited by Smad5 knockdown. Furthermore, deletion or mutation of the Smad-binding element in the HO-1 promoter also inhibited BMP6-induced luciferase activity. In summary, these findings suggest that induction of HO-1 through a Smad-dependent mechanism is responsible for the cytoprotective effect of BMP6 in H₂O₂-mediated renal cell injury.     

Catalytically inactive heme oxygenase-2 mutant is cytoprotective

Heme oxygenase (HO) catalyzes the rate-limiting step in heme degradation, producing iron, carbon monoxide, and bilirubin/biliverdin. HO consists of two isozymes: HO-1, which is an oxidative stress-response protein, and HO-2, which is constitutively expressed. HO-2 accounts for most HO activity within the nervous system. Its posttranslational modifications and/or interactions with other proteins make HO-2 a unique regulator of cellular homeostasis. Our previous results revealed that brain infarct volume was enlarged in HO-2 knockout mice. A similar neuroprotective role of HO-2 was shown using primary cortical neurons. To better understand the neuroprotective mechanism of HO-2, we used a catalytically inactive mutant, HO-2H45A, and investigated its cellular effects in response to hemin and hydrogen peroxide-induced cytotoxicity. We observed that HO-2WT overexpression in the HEK293 cell lines became less sensitive to hemin, whereas the inactive mutant HO-2H45A was more sensitive to hemin as compared to control. Interestingly, HO-2WT- and HO-2H45A-overexpressing cells were both protected against H2O2-induced oxidative stress and had less oxidatively modified proteins as compared to control cells. These data indicate that when HO-2 cannot metabolize the prooxidant heme, more cytotoxicity is found, whereas, interestingly, the catalytically inactive HO-2H45A was also able to protect cells against oxidative stress injury. These results suggest the multiplicity of action of the HO-2 protein itself.     

Celastrol suppresses IFN-gamma-induced ICAM-1 expression and subsequent monocyte adhesiveness via the induction of heme oxygenase-1 in the HaCaT cells

Celastrol, a quinone methide triterpenoid derived from the medicinal plant Tripterygium wilfordii, possesses various biological activities such as anti-oxidant, anti-tumor, and anti-inflammatory activities. In this study, we examined the suppressive effect of celastrol on IFN-γ-induced expression of ICAM-1 and the molecular mechanism responsible for these activities. We found that celastrol induced mRNA and protein expression of heme oxygenase-1 (HO-1) in the human keratinocyte cell line HaCaT. Treatment of HaCaT cells with tin protoporphyrin IX (SnPP), a specific inhibitor of HO-1, reversed the suppressive effect of celastrol on IFN-γ-induced protein and mRNA expression of ICAM-1. HO-1 knockdown using small interfering RNA (siRNA) led to reverse inhibition of IFN-γ-induced up-regulation of ICAM-1 by celastrol. In addition, SnPP reversed suppression of IFN-γ-induced promoter activity of ICAM-1 by celastrol. Furthermore, blockage of HO-1 activity by SnPP and HO-1 siRNA reversed the inhibitory effect of celastrol on IFN-γ-induced adhesion of monocytes to keratinocytes. These results suggest that celastrol may exert anti-inflammatory responses by suppressing IFN-γ-induced expression of ICAM-1 and subsequent monocyte adhesion via expression of HO-1 in the keratinocytes.     

Regulation of heme oxygenase expression by cyclopentenone prostaglandins

Prostaglandins (PGs) originate from the degradation of membranar arachidonic acid by cyclooxygenases (COX-1 and COX-2). The prostaglandin actions in the nervous system are multiple and have been suggested to play a significant role in neurodegenerative disorders. Some PGs have been reported to be toxic and, interestingly, the cyclopentenone PGs have been reported to be cytoprotective at low concentration and could play a significant role in neuronal plasticity. They have been shown to be protective against oxidative stress injury; however, the cellular mechanisms of protection afforded by these PGs are still unclear. It is postulated that the cascade leading to neuronal cell death in acute and chronic neurodegenerative conditions, such as cerebral ischemia and Alzheimer's disease, would be mediated by free radical damage. We tested the hypothesis that the neuroprotective action of cyclopentanone could be caused partially by an induction of heme oxygenase 1 (HO-1). We and others have previously reported that modulation of HO total activity may well have direct physiological implications in stroke and in Alzheimer's disease. HO acts as an antioxidant enzyme by degrading heme into iron, carbon monoxide, and biliverdin that is rapidly converted into bilirubin. Using mouse primary neuronal cultures, we demonstrated that PGs of the J series induce HO-1 in a dose-dependent manner (0, 0.5, 5, 10, 20, and 50 micro g/ml) and that PGJ(2) and dPGJ(2) were more potent than PGA(2), dPGA(2), PGD(2), and PGE(2). No significant effects were observed for HO-2 and actin expression. In regard to HO-3 expression found in rat, with its protein deducted sequence highly homologous to HO-2, no detection was observed in HO-2(-/-) mice, suggesting that HO-3 protein would not be present in mouse brain. We are proposing that several of the protective effects of PGJ(2) could be mediated through beneficial actions of heme degradation and its metabolites. The design of new mimetics based on the cyclopentenone structure could be very useful as neuroprotective agents and be tested in animal models of stroke and Alzheimer's disease.     

Pentoxifylline protects L929 fibroblasts from TNF-alpha toxicity via the induction of heme oxygenase-1

Tumor necrosis factor-alpha (TNF-alpha) is recognized as a principal mediator of a variety of inflammatory conditions. Pentoxifylline (PTX), which can inhibit cellular TNF-alpha synthesis, also attenuates the toxic effect of TNF-alpha. However, the mechanism underlying PTX-induced cytoprotection is unknown. Heme oxygenase 1 (HO-1) is an enzyme which degrades heme into biliverdin, free iron, and carbon monoxide (CO). This enzyme has recently been shown to have anti-inflammatory and cytoprotective effects. In this study, we investigated whether protection by PTX against TNF-alpha-mediated toxicity could be related to its ability to induce HO-1 expression and HO activity in L929 cells. PTX in the range of 0.1-1.0mM significantly induced HO-1 expression and the resulting HO activity. Pre-incubation of L929 cells with either PTX or the HO activator hemin resulted in the protection of the cells against TNF-alpha-mediated toxicity. Zinc protoporphyrin, a specific HO competitive inhibitor, abrogated the protective effect of PTX. Hemoglobin, a scavenger of CO, reversed the protective effect of PTX. A cytoprotection comparable to PTX was observed when the cells were treated with the CO-releasing compound tricarbonyldichlororuthenium(II) dimer. These results suggest that HO-1 expression and the ensuing formation of the HO metabolite CO may be a novel pathway by which PTX protects L929 cells from TNF-alpha-mediated toxicity.     

Facilitated angiogenesis induced by heme oxygenase-1 gene transfer in a rat model of hindlimb ischemia

Heme oxygenase-1 (HO-1) is an inducible form of heme oxygenase that catabolizes heme to carbon monoxide, biliverdin, and ferrous iron. We have investigated whether HO-1 can induce angiogenic effects in vivo. Rats were subjected to a bolus injection of either wild type adenovirus (ad-wt) or adenovirus encoding HO-1 (ad-HO-1) through the right femoral artery, which was then removed immediately. HO-1 gene transfer resulted in about a sixfold increase in HO-1 protein levels as compared to the non-treated animals. The increase in both blood flow and capillary density was significantly greater in the ischemic hindlimbs that had been injected with ad-HO-1 than in those injected with ad-wt. These angiogenic effects of ad-HO-1 infection could be completely abolished by treating the animals with the HO inhibitor, zinc protoporphyrin, indicating that they were specifically due to the expression of HO-1. Thus, HO-1 gene transfer improves the blood flow in ischemic hindlimb, at least in part, via angiogenesis facilitated by the induction of this molecule.