Metabolite Carbon Monoxide Research Articles

Heme Oxygenase-1 and Its Metabolites Carbon Monoxide and Biliverdin, but Not Iron, Exert Antiviral Activity against Porcine Circovirus Type 3.

Porcine circovirus type 3 (PCV3) is a newly discovered pathogen that causes porcine dermatitis and nephropathy syndrome (PDNS)-like clinical signs, multisystemic inflammation, and reproductive failure. Heme oxygenase-1 (HO-1), a stress-inducible enzyme, exerts protective functions by converting heme into carbon monoxide (CO), biliverdin (BV), and iron. However, the effects of HO-1 and its metabolites on PCV3 replication remain unknown. In this study, experiments involving specific inhibitors, lentivirus transduction, and small interfering RNA (siRNA) transfection revealed that active PCV3 infection reduced HO-1 expression and that the expression of HO-1 negatively regulated virus replication in cultured cells, depending on its enzymatic activity. Subsequently, the effects of the HO-1 metabolites (CO, BV, and iron) on PCV3 infection were investigated. The CO inducers (cobalt protoporphyrin IX [CoPP] or tricarbonyl dichloro ruthenium [II] dimer [CORM-2]) mediate PCV3 inhibition by generating CO, and this inhibition is reversed by hemoglobin (Hb; a CO scavenger). The inhibition of PCV3 replication by BV depended on BV-mediated reactive oxygen species (ROS) reduction, as N-acetyl-l-cysteine affected PCV3 replication while reducing ROS production. The reduction product of BV, bilirubin (BR), specifically promoted nitric oxide (NO) generation and further activated the cyclic GMP/protein kinase G (cGMP/PKG) pathway to attenuate PCV3 infection. Both the iron provided by FeCl3 and the iron chelated by deferoxamine (DFO) with CoPP treatment failed to affect PCV3 replication. Our data demonstrate that the HO-1-CO-cGMP/PKG, HO-1-BV-ROS, and HO-1-BV-BR-NO-cGMP/PKG pathways contribute crucially to the inhibition of PCV3 replication. These results provide important insights regarding preventing and controlling PCV3 infection. IMPORTANCE The regulation of host protein expression by virus infection is the key to facilitating self-replication. As an important emerging pathogen of swine, clarification of the interaction between PCV3 infection and the host enables us to understand the viral life cycle and pathogenesis better. Heme oxygenase-1 (HO-1) and its metabolites carbon monoxide (CO), biliverdin (BV), and iron have been demonstrated to involve a wealth of viral replications. Here, we, for the first time, demonstrated that HO-1 expression decreases in PCV3-infected cells and negatively regulates PCV3 replication and that the HO-1 metabolic products CO and BV inhibit PCV3 replication by the CO- or BV/BR/NO-dependent cGMP/PKG pathway or BV-mediated ROS reduction, but the iron (the third metabolic product) does not. Specifically, PCV3 infection maintains normal proliferation by downregulating HO-1 expression. These findings clarify the mechanism by which HO-1 modulates PCV3 replication in cells and provide important targets for preventing and controlling PCV3 infection.

Pharmacological Targeting of Heme Oxygenase-1 in Osteoarthritis.

Osteoarthritis (OA) is a common aging-associated disease that clinically manifests as joint pain, mobility limitations, and compromised quality of life. Today, OA treatment is limited to pain management and joint arthroplasty at the later stages of disease progression. OA pathogenesis is predominantly mediated by oxidative damage to joint cartilage extracellular matrix and local cells such as chondrocytes, osteoclasts, osteoblasts, and synovial fibroblasts. Under normal conditions, cells prevent the accumulation of reactive oxygen species (ROS) under oxidatively stressful conditions through their adaptive cytoprotective mechanisms. Heme oxygenase-1 (HO-1) is an iron-dependent cytoprotective enzyme that functions as the inducible form of HO. HO-1 and its metabolites carbon monoxide and biliverdin contribute towards the maintenance of redox homeostasis. HO-1 expression is primarily regulated at the transcriptional level through transcriptional factor nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2), specificity protein 1 (Sp1), transcriptional repressor BTB-and-CNC homology 1 (Bach1), and epigenetic regulation. Several studies report that HO-1 expression can be regulated using various antioxidative factors and chemical compounds, suggesting therapeutic implications in OA pathogenesis as well as in the wider context of joint disease. Here, we review the protective role of HO-1 in OA with a focus on the regulatory mechanisms that mediate HO-1 activity.

Heme oxygenase 1 regulates apoptosis induced by heat stress in bovine ovarian granulosa cells via the ERK1/2 pathway.

Heat stress can inhibit follicular development in dairy cows, and thus can affect their reproductive performance. Follicular granulosa cells can synthesize estrogen, that affects the development and differentiation of follicles by apoptosis. Heme oxygenase 1 (HO-1/heat shock protein 32) plays an antiapoptotic and cytoprotective role in various cells during stress-induced apoptosis, but little is known about its definitive function in bovine (ovarian) granulosa cells (bGCs). In our study, the roles and mechanism of HO-1 on the heat stress-induced apoptosis of bGCs were studied. Our results show that the expression of HO-1 was significantly increased under heat stress. Moreover, HO-1 silencing increased apoptosis, whereas its overexpression dampened apoptosis by regulating the expression of Bax/Bcl-2 and the levels of cleaved caspase-3. In addition, HO-1 can also play a cytoprotective role by affecting estrogen levels and decomposing heme to produce biologically active metabolite carbon monoxide (CO). Meanwhile, CO significantly increased the level of HO-1, decreased Bax/Bcl-2 levels, and inhibited the activation of extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway. The apoptosis of ovarian GCs can affect the secretion of estrogen and lead to disorder of the ovarian microenvironment, thus affecting the normal function of the ovary. Our results indicate that HO-1 acts as a cytoprotective enzyme and plays a protective role in heat-induced apoptosis of bGCs. In conclusion, HO-1 and its metabolite CO inhibit the apoptosis of bGCs induced by heat stress through the ERK1/2 pathway. The results of this study provide a valuable clue for improving the fertility of heat stressed cows in summer.

Carbon monoxide and biliverdin suppress bovine viral diarrhoea virus replication.

Bovine viral diarrhoea virus (BVDV) causes significant economic losses to the cattle industry worldwide. Previously, we demonstrated that heme oxygenase-1 (HO-1) can inhibit BVDV replication via an unknown molecular mechanism. To elucidate the mechanism involved, we assess whether the HO-1 downstream metabolites carbon monoxide (CO), biliverdin (BV) and iron affect BVDV replication. We treated Madin-Darby bovine kidney (MDBK) cells with an exogenous CO donor, CORM-2. We found that CORM-2 but not its inactive form (iCORM-2) inhibited BVDV replication in a dose-dependent and time duration-dependent manner, suggesting a CO-specific mediation of the CORM-2 antiviral effect. Direct incubation of BVDV with high-dose CORM-2 reduced virus titres, suggesting that CORM-2 attenuates BVDV growth by both physically inactivating virus particles in the extracellular environment and affecting intracellular BVDV replication, but mainly via an intracellular mechanism. Exogenous BV treatment, both post-infection and co-incubation with BVDV, inhibited BVDV replication in a dose-dependent manner, indicating that BV has potent antiviral activity against BVDV. Direct incubation of BVDV with BV had no significant effect on virus titres, indicating that BV is not virucidal and attenuates BVDV growth by affecting intracellular BVDV replication. Furthermore, BV was found to affect BVDV penetration but not attachment. However, increased iron via addition of FeCl3 did not interfere with BVDV replication. Collectively, the results of the present study demonstrate that the HO-1 metabolites BV and CO, but not iron, inhibit BVDV replication. These findings not only provide new insights into the molecular mechanism of HO-1 inhibition of BVDV replication but also suggest potential new control measures for future BVDV infection.

The Heme Metabolite Carbon Monoxide Facilitates KSHV Infection by Inhibiting TLR4 Signaling in Endothelial Cells.

Kaposi sarcoma herpesvirus (KSHV) is the etiologic agent of Kaposi sarcoma (KS) and certain rare B cell lymphoproliferative disorders. KSHV infection of endothelial cells (EC) in vitro increases expression of the inducible host-encoded enzyme heme oxygenase-1 (HO-1), which is also strongly expressed in KS tumors. HO-1 catalyzes the rate-limiting step in the conversion of heme into iron, biliverdin and the gasotransmitter carbon monoxide (CO), all of which share anti-apoptotic, anti-inflammatory, pro-survival, and tumorigenic activities. Our previous work has shown that HO-1 expression in KSHV-infected EC is characterized by a rapid yet transient induction at early times post-infection, followed by a sustained upregulation co-incident with establishment of viral latency. These two phases of expression are independently regulated, suggesting distinct roles for HO-1 in the virus life cycle. Here, we investigated the role of HO-1 during acute infection, prior to the onset of viral gene expression. The early infection phase involves a series of events that culminate in delivery of the viral genome to the nucleus. Primary infection also leads to activation of host innate immune effectors, including the pattern recognition receptor TLR4, to induce an antiviral response. It has been shown that TLR4-deficient EC are more susceptible to KSHV infection than wild-type controls, suggesting an important inhibitory role for TLR4 in the KSHV life cycle. TLR4 signaling is in turn subject to regulation by several virus-encoded immune evasion factors. In this report we identify HO-1 as a host protein co-opted by KSHV as part of a rapid immune evasion strategy. Specifically, we show that early HO-1 induction by KSHV results in increased levels of endogenous CO, which functions as a TLR4 signaling inhibitor. In addition, we show that CO-mediated inhibition of TLR4 signaling leads to reduced expression of TLR4-induced antiviral genes, thus dampening the host antiviral response and facilitating KSHV infection. Conversely, inhibition of HO-1 activity decreases intracellular CO, enhances the host antiviral response and inhibits KSHV infection. In conclusion, this study identifies HO-1 as a novel innate immune evasion factor in the context of KSHV infection and supports HO-1 inhibition as a viable therapeutic strategy for KS.

Molecular mechanisms and therapeutic implications of the carbon monoxide/hmox1 and the hydrogen sulfide/CSE pathways in the prevention of pre-eclampsia and fetal growth restriction

The exact aetiology of preeclampsia is unknown, but there is a good association with an imbalance in angiogenic growth factors and abnormal placentation (Ahmad and Ahmed, 2004). The incidence of preeclampsia is reduced by a third in smokers, but not in snuff users. Soluble Flt-1 (sFlt-1) and soluble endoglin (sEng) are increased prior to the clinical onset of preeclampsia. Animals exposed to high circulating levels of sFlt-1 and sEng elicit severe preeclampsia-like symptoms. Smokers have reduced circulating sFlt-1 and cigarette smoke extract decreases sFlt-1 release from placental villous explants. An anti-inflammatory enzyme, heme oxygenase-1 (HO-1) and its metabolite carbon monoxide (CO), inhibit sFlt-1 and sEng release. Women with preeclampsia exhale less CO than women with normal pregnancies and HO expression decreases as the severity of preeclampsia increases. In contrast, sFlt-1 levels increase with increasing severity. More importantly, chorionic villous sampling from women at eleven weeks gestation shows that HO-1 mRNA expression is decreased in women who go on to develop preeclampsia. Collectively, these facts provide compelling evidence to support the proposition that the pathogenesis of preeclampsia is largely due to loss of HO activity. This results in an increase in inflammation and excessive elevation of the two key anti-angiogenic factors responsible for the clinical signs of preeclampsia. The identification of a protective role for HO-1 in pregnancy, offers HO/CO pathway as a target for the treatment of preeclampsia. The cardiovascular drugs, statins, stimulate HO-1 expression and inhibit sFlt-1 release in vivo and in vitro, suggesting statins have the potential to ameliorate preeclampsia. The StAmP trial is underway to address this and if positive, its outcome will lead to the very first therapeutic intervention to prolong affected pregnancies. Hydrogen sulphide (H2S), a gaseous messenger produced mainly by cystathionineY-lyase (CSE), is pro-angiogenic vasodilator (Yang et al., 2008; Papapetropoulos et al., 2009). We hypothesized that a reduction in CSE activity may alter the angiogenic balance in pregnancy and induce abnormal placentation and maternal hypertension. Plasma levels of H2S were significantly decreased in preeclamptic women (p<-0.01), which was associated with reduced CSE message and protein expression in human placenta as determined by real - time PCR and immunohistochemistry. Inhibition of CSE activity by DL-propargylglycine (PAG) in first trimester (8-12weeks gestation) human placental explants had reduced placenta growth factor (PIGF) production as assessed by ELISA and inhibited trophoblast invasion in vitro. Endothelial CSE knockdown by siRNA transfection increased the endogenous release of soluble fms-Like tyrosine kinase-1 (sFlt-1) and soluble endoglin, (sEng) from human umbilical vein endothelial cells while adenoviral-mediated CSE overexpression inhibited their release. Administration of PAG to pregnant mice induced hypertension, liver damage, and promoted abnormal labyrinth vascularisation in the placenta and decreased fetal growth. Finally, a slow releasing, H2S-generating compound, GYY4137, inhibited circulating sFlt-1 and sEng levels and restored fetal growth that was compromised by PAG-treatment demonstrating that the effect of CSE inhibitor was due to inhibition of H2S production. These results imply that endogenous H2S is required for healthy placental vasculature and a decrease in of CSE/ H2S activity may contribute to the pathogenesis of preeclampsia. This work was supported by grants from the Medical Research Council (G0601295 and G0700288) and Aston University StrategicFunds.

Carbon Monoxide Promotes Proliferation of Uterine Natural Killer Cells and Remodeling of Spiral Arteries in Pregnant Hypertensive Heme Oxygenase-1 Mutant Mice

Heme Oxygenase-1 (HO-1) and its metabolite carbon monoxide (CO) promote implantation and placentation. Pregnancy disorders such as preeclampsia and intrauterine growth restriction are linked to both HO-1 diminution and impaired remodeling of maternal spiral arteries (SAs). Here, we investigated whether CO is able to prevent preeclampsia and intrauterine growth restriction through the modulation of uterine natural killer (uNK) cells that are necessary for initiation of SA remodeling. Hmox1(+/-) or Hmox1(-/-) implantations presented fewer uNK cell numbers and lower expression of uNK-related angiogeneic factors compared with Hmox1(+/+) sites. Quantitative histology revealed that Hmox1(+/-) and Hmox1(-/-) implantations had shallow SA development that was accompanied by intrauterine growth restriction and gestational hypertension. Application of CO at low dose during early to midgestation prevented intrauterine growth restriction in Hmox1(+/-) mothers, this being associated with enhanced in situ proliferation of uNK cells and normalization of angiogenic parameters. Most importantly, CO improved SA remodeling and normalized blood pressure, ensuring a proper fetal growth. Thus, CO emerges as a key molecular player in pregnancy success by modulating uNK cells, which results in promotion of SA remodeling, adequate fetal support/growth, and prevention of hypertension.

Carbon monoxide alleviates ethanol-induced oxidative damage and inflammatory stress through activating p38 MAPK pathway

Stress-inducible protein heme oxygenase-1(HO-1) is well-appreciative to counteract oxidative damage and inflammatory stress involving the pathogenesis of alcoholic liver diseases (ALD). The potential role and signaling pathways of HO-1 metabolite carbon monoxide (CO), however, still remained unclear. To explore the precise mechanisms, ethanol-dosed adult male Balb/c mice (5.0g/kg.bw.) or ethanol-incubated primary rat hepatocytes (100mmol/L) were pretreated by tricarbonyldichlororuthenium (II) dimmer (CORM-2, 8mg/kg for mice or 20μmol/L for hepatocytes), as well as other pharmacological reagents. Our data showed that CO released from HO-1 induction by quercetin prevented ethanol-derived oxidative injury, which was abolished by CO scavenger hemoglobin. The protection was mimicked by CORM-2 with the attenuation of GSH depletion, SOD inactivation, MDA overproduction, and the leakage of AST, ALT or LDH in serum and culture medium induced by ethanol. Moreover, CORM-2 injection or incubation stimulated p38 phosphorylation and suppressed abnormal Tnfa and IL-6, accompanying the alleviation of redox imbalance induced by ethanol and aggravated by inflammatory factors. The protective role of CORM-2 was abolished by SB203580 (p38 inhibitor) but not by PD98059 (ERK inhibitor) or SP600125 (JNK inhibitor). Thus, HO-1 released CO prevented ethanol-elicited hepatic oxidative damage and inflammatory stress through activating p38 MAPK pathway, suggesting a potential therapeutic role of gaseous signal molecule on ALD induced by naturally occurring phytochemicals.

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.

New insights into the etiology of preeclampsia: identification of key elusive factors for the vascular complications

The incidence of preeclampsia is reduced by a third in smokers, but not in snuff users. Soluble Flt-1 (sFlt-1) and soluble endoglin (sEng) are increased prior to the clinical onset of preeclampsia. Animals exposed to high circulating levels of sFlt-1 and sEng elicit severe preeclampsia-like symptoms. Smokers have reduced circulating sFlt-1 and cigarette smoke extract decreases sFlt-1 release from placental villous explants. An anti-inflammatory enzyme, heme oxygenase-1 (HO-1) and its metabolite carbon monoxide (CO), inhibit sFlt-1 and sEng release. Women with preeclampsia exhale less CO than women with normal pregnancies and HO expression decreases as the severity of preeclampsia increases. In contrast, sFlt-1 levels increase with increasing severity. More importantly, chorionic villous sampling from women at eleven weeks gestation shows that HO-1 mRNA expression is decreased in women who go on to develop preeclampsia. Collectively, these facts provide compelling evidence to support the proposition that the pathogenesis of preeclampsia is largely due to loss of HO activity. This results in an increase in inflammation and excessive elevation of the two key anti-angiogenic factors responsible for the clinical signs of preeclampsia. These findings provide strong evidence for a protective role of HO-1 in pregnancy and identify HO as a target for the treatment of preeclampsia. The cardiovascular drugs, statins, stimulate HO-1 expression and inhibit sFlt-1 release in vivo and in vitro, thus, they have the potential to ameliorate early onset preeclampsia. The StAmP trial is underway to address this and if positive, its outcome will lead to the very first therapeutic intervention to prolong affected pregnancies.

Carbon monoxide is a poison … to microbes! CO as a bactericidal molecule

Inflammation and immunity result in a wide range of disease processes, including chronic obstructive pulmonary disease, ischemia-reperfusion injury, atherosclerosis, vascular thrombosis and sepsis. Heme oxygenase-1 (HO-1) is a key enzyme that is indispensable for the temporal and spatial regulation of host response and, together with its essential metabolite carbon monoxide (CO), is crucial for maintaining homeostasis, inhibition of inflammation and the preservation of function and life. The biology of HO-1 is being discussed in this review series by Soares and colleagues and thus will not be reviewed here. Rather we will complement the HO-1 overview with a comprehensive discussion of CO as perhaps the one product of HO-1 that has been most studied. Of the numerous physiologic effects observed with CO, in the past five years it has become apparent that CO has been ascribed an additional novel role as a 'bactericidal agent'. Its role in the maintenance of homeostasis remains intact; however, the designation necessitates the paradoxical induction of the inflammatory response and binding to hemoproteins in order to restore homeostasis and sustain life. In this article, we review and discuss reports that have propelled and challenged the paradoxical use of CO, once viewed as a toxic molecule, now as a host defense molecule agent against microbes.

Negative Regulation of Soluble Flt-1 and Soluble Endoglin Release by Heme Oxygenase-1

Preeclampsia is characterized clinically by hypertension and proteinuria. Soluble Flt-1 (sFlt-1; also known as soluble vascular endothelial growth factor receptor-1 [VEGFR-1]) and soluble endoglin (sEng) are elevated in preeclampsia, and their administration to pregnant rats elicits preeclampsia-like symptoms. Heme oxygenase-1 (HO-1) and its metabolite carbon monoxide (CO) exert protective effects against oxidative stimuli. Thus, we hypothesized that HO-1 upregulation may offer protection against preeclampsia by inhibiting sFlt-1 and sEng release. Preeclamptic villous explants secreted high levels of sFlt-1 and sEng. Adenoviral overexpression of HO-1 in endothelial cells inhibited VEGF-mediated sFlt-1 release and interferon-gamma- and tumor necrosis factor-alpha-induced sEng release, whereas HO-1 inhibition potentiated sFlt-1 and sEng production from endothelial cells and placental villous explants. Consistent with these findings, mice lacking HO-1 produced higher levels of sFlt-1 and sEng compared with wild-type mice. Using selective ligands (VEGF-E and placental growth factor) and a receptor-specific inhibitor (SU-1498), we demonstrated that VEGF-induced sFlt-1 release was VEGFR-2 dependent. Furthermore, CO-releasing molecule-2 (CORM-2) or CO decreased sFlt-1 release and inhibited VEGFR-2 phosphorylation. Treatment of endothelial cells with statins upregulated HO-1 and inhibited the release of sFlt-1, whereas vitamins C and E had no effect. The present study demonstrates that the HO-1/CO pathway inhibits sFlt-1 and sEng release, providing compelling evidence for a protective role of HO-1 in pregnancy, and identifies HO-1 as a novel target for the treatment of preeclampsia.

Quercetin, but not rutin and quercitrin, prevention of H 2O 2-induced apoptosis via anti-oxidant activity and heme oxygenase 1 gene expression in macrophages

In the present study, we examine the protective mechanism of quercetin (QE) on oxidative stress-induced cytotoxic effect in RAW264.7 macrophages. Results of Western blotting show that QE but not its glycoside rutin (RUT) and quicitrin-induced HO-1 protein expression in a time- and dose-dependent manner, and HO-1 protein induced by QE was blocked by an addition of cycloheximide or actinomycin D. Induction of HO-1 gene expression by QE was accompanied by inducing ERKs, but not JNKs or p38, proteins phosphorylation. Addition of PD98059, but not SB203580 or SP600125, significantly attenuates QE-induced HO-1 protein and mRNA expression associated with blocking the expression of phosphorylated ERKs proteins. H 2O 2 addition reduces the viability of cells by MTT assay, and appearance of DNA ladders, hypodiploid cells, and an increase in intracellular peroxide level was detected. Addition of QE, but not QI or RUT, significantly reduced the cytotoxic effect induced by H 2O 2 associated with blocking the production of intracellular peroxide, DNA ladders, and hypodiploid cells. QE protection of cells from H 2O 2-induced apoptosis was significantly suppressed by adding HO inhibitor SnPP or ERKs inhibitor PD98059. Additionally, QE protects cells from H 2O 2-induced a decrease in the mitochondrial membrane potential and a release of cytochrome c from mitochondria to cytosol by DiOC6 and Western blotting assay, respectively. Activation of apoptotic proteins including the caspase 3, caspase 9, PARP, D4-GDI proteins was identified in H 2O 2-treated cells by Western blotting and enzyme activity assay, and that was significantly blocked by an addition of QE, but not RUT and QI. Furthermore, HO-1 catalytic metabolites carbon monoxide (CO), but not Fe 2+, Fe 3+, biliverdin or bilirubin, performed protective effect on cells from H 2O 2-induced cell death with an increase in HO-1 protein expression and ERKs protein phosphorylation. These data suggest that induction of HO-1 protein may participate in the protective mechanism of QE on oxidative stress (H 2O 2)-induced apoptosis, and reduction of intracellular ROS production and mitochondria dysfunction with blocking apoptotic events were involved. Differential anti-apoptotic effect between QE and its glycosides RUT and QI via distinct HO-1 protein induction was also delineated.