Biliverdin Reductase Research Articles

MALDI-MS tissue imaging identification of biliverdin reductase B overexpression in prostate cancer

New biomarkers are needed to improve the specificity of prostate cancer detection and characterisation of individual tumors. In a proteomics profiling approach using MALDI-MS tissue imaging on frozen tissue sections, we identified discriminating masses. Imaging analysis of cancer, non-malignant benign epithelium and stromal areas of 15 prostatectomy specimens in a test and 10 in a validation set identified characteristic m/z peaks for each tissue type, e.g. m/z 10775 for benign epithelial, m/z 6284 and m/z 6657.5 for cancer and m/z 4965 for stromal tissue. A 10-fold cross-validation analysis showed highest discriminatory ability to separate tissue types for m/z 6284 and m/z 6657.5, both overexpressed in cancer, and a multicomponent mass peak cluster at m/z 10775-10797.4 overexpressed in benign epithelial tissue. ROC AUC values for these three masses ranged from 0.85 to 0.95 in the discrimination of malignant and non-malignant tissue. To identify the underlying proteins, prostate whole tissue extract was separated by nano-HPLC and subjected to MALDI TOF/TOF analysis. Proteins in fractions containing discriminatory m/z masses were identified by MS/MS analysis and candidate marker proteins subsequently validated by immunohistochemistry (IHC). Biliverdin reductase B (BLVRB) turned out to be overexpressed in PCa tissue. In this study on cryosections of radical prostatectomies of prostate cancer patients, we performed a MALDI-MS tissue imaging analysis and a consecutive protein identification of significant m/z masses by nano-HPLC, MALDI TOF/TOF and MS/MS analysis. We identified BLVRB as a potential biomarker in the discrimination of PCa and benign tissue, also suggesting BVR as a feasible therapeutic target.

Small molecule activators of the Nrf2-HO-1 antioxidant axis modulate heme metabolism and inflammation in BV2 microglia cells

The nuclear factor erythroid derived 2-related factor 2 (Nrf2) and the antioxidant protein heme oxygenase-1 (HO-1) are crucial components of the cellular stress response. These two systems work together to combat oxidative stress and inflammation and are attractive drug targets for counteracting different pathologies, including neuroinflammation. We aimed to identify the most effective Nrf2/HO-1 activators that modulate the inflammatory response in microglia cells. In the present study, we searched the literature and selected 56 compounds reported to activate Nrf2 or HO-1 and analyzed them for HO-1 induction at 6 and 24h and cytotoxicity in BV2 microglial cells in vitro. Approximately 20 compounds up-regulated HO-1 at the concentrations tested (5–20μM) with carnosol, supercurcumin, cobalt protoporphyrin-IX and dimethyl fumarate exhibiting the best induction/low cytotoxicity profile. Up-regulation of HO-1 by some compounds resulted in increased cellular bilirubin levels but did not augment the expression of proteins involved in heme synthesis (ALAS 1) or biliverdin reductase. Bilirubin production by HO-1 inducers correlated with their potency in inhibiting nitrite production after challenge with interferon-γ (INF-γ) or lipopolysaccharide (LPS). The compounds down-regulated the inflammatory response (TNF-α, PGE2 and nitrite) more strongly in cells challenged with INF-γ than LPS, and silencing HO-1 or Nrf2 with shRNA differentially affected the levels of inflammatory markers. These findings indicate that some small activators of Nrf2/HO-1 are effective modulators of microglia inflammation and highlight the chemical scaffolds that can serve for the synthesis of potent new derivatives to counteract neuroinflammation and neurodegeneration.

Biliverdin reductase/bilirubin mediates the anti-apoptotic effect of hypoxia in pulmonary arterial smooth muscle cells through ERK1/2 pathway

Inhibition of pulmonary arterial smooth muscle cell (PASMC) apoptosis induced by hypoxia plays an important role in pulmonary arterial remodeling leading to aggravate hypoxic pulmonary arterial hypertension. However, the mechanisms of hypoxia acting on PASMC apoptosis remain exclusive. Biliverdin reductase (BVR) has many essential biologic roles in physiological and pathological processes. Nevertheless, it is unclear whether the hypoxia-induced inhibition on PASMC apoptosis is mediated by BVR. In the present work, we found BVR majorly localized in PASMCs and was up-regulated in levels of protein and mRNA by hypoxia. Then we studied the contribution of BVR to anti-apoptotic response of hypoxia in PASMCs. Our results showed that siBVR, blocking generation of bilirubin, reversed the effect of hypoxia on enhancing cell survival and apoptotic protein (Bcl-2, procasepase-9, procasepase-3) expression, preventing nuclear shrinkage, DNA fragmentation and mitochondrial depolarization in starved PASMCs, which were recovered by exogenous bilirubin. Moreover, the inhibitory effect of bilirubin on PASMC apoptosis under hypoxic condition was blocked by the inhibitor of ERK1/2 pathway. Taken together, our data indicate that BVR contributes to the inhibitory process of hypoxia on PASMC apoptosis, which is mediated by bilirubin through ERK1/2 pathway.

Gene Expression Profiling of PC12 Cells Exposed to Manganese

Chronic exposure to Manganese (Mn) causes manganism with symptoms similar to Parkinson's disease. The objective of this study was to determine the molecular targets of Mn in PC12 cells. We applied microarray technology to identify molecular targets of Mn and RT‐PCR/Western to validate results. Our results showed that Mn altered the expression of 223 genes; 146 were up regulated and 77 were down regulated. The sub‐lethal dose range (38–300 μM MnCl2) initiated metabolic stress, an induction of enolase/aldolase and hemeoxygenase 1 (HO‐1) (7.7 fold). Concentrations greater than 300 μM caused production of H2O2 concomitant to increase of gene expression of antioxidant enzymes; biliverdin reductase, arsenite inducible RNA associated protein, dithiolethione‐inducible gene‐1 (DIG‐1) and thioredoxin reductase 1 with reduced expression of mitochondrial glutaryl‐coenzyme A dehydrogenase (GCDH). In conclusion the results suggest that Mn toxicity is primarily associated with dose dependent induction of HO‐1 and elevated production of H2O2, along with a minor loss of mitochondrial function. (Supported by NIH grant NCRR G12 RR03020 and G12 MD007582).

Conditional deletion of biliverdin reductase in macrophages determines the sensitivity to acetaminophen injury.

Biliverdin reductase (BVR) is a multifunctional enzyme, kinase, receptor as well as exhibiting transcription factor activity. We have recently elucidated mechanisms by which BV and BVR regulate the innate inflammatory response in vitro and in vivo. In this study we aimed to elucidate the role of BVR in vivo in response acetaminophen‐mediated liver injury using newly generated BVR conditional knockout mice.We confirmed lack of BVR in the Cre‐Lyz‐BVR‐fl/fl macrophages in vivo as well as in bone‐marrow derived macrophgaes. We validated decreased bilirubin levels in BVR deficient mice. Importantly after challenge with LPS+D‐Gal Cre‐Lyz‐BVR‐fl/fl mice show greater injury as compare to BVR‐fl/fl control mice. However, Cre‐Lyz‐BVR‐fl/fl mice were resistant to acetaminophen induced liver injury. Lack of BVR in macrophages led to low inflammatory responses to TLR9 ligands (CpG rich region) in vitro and in vivo. We showed that BVR binds specific DNA sequences in the membrane and may be a novel mediator of TLR9 signaling.In summary, BVR in macrophages is critical for acetaminophen mediated liver injury due to its interaction with DNA released from necrotic cells and amplification of inflammation.

Expression of Biliverdin Reductase A in peripheral blood leukocytes is associated with treatment response in HCV-infected patients.

Background and AimsHepatitis C virus (HCV) infection is associated with systemic oxidative stress. Since the heme catabolic pathway plays an important role in antioxidant protection, we attempted to assess the gene expression of key enzymes of heme catabolism, heme oxygenase 1 (HMOX1), heme oxygenase 2 (HMOX2), and biliverdin reductase A (BLVRA) in the liver and peripheral blood leukocytes (PBL) of patients chronically infected with HCV.MethodsGene expressions (HMOX1, HMOX2, BLVRA) and HCV RNA were analyzed in PBL of HCV treatment naïve patients (n = 58) and controls (n = 55), with a subset of HCV patients having data on hepatic gene expression (n = 35). Based upon the therapeutic outcome, HCV patients were classified as either responders (n = 38) or treatment-failure patients (n = 20). Blood samples in HCV patients were collected at day 0, and week 12, 24, 36, and 48 after the initiation of standard antiviral therapy.ResultsCompared to the controls, substantially increased BLVRA expression was detected in PBL (p<0.001) of therapeutically naïve HCV patients. mRNA levels of BLVRA in PBL closely correlated with those in liver tissue (r2 = 0.347,p = 0.03). A marked difference in BLVRA expression in PBL between the sustained responders and patients with treatment failure was detected at week 0 and during the follow-up (p<0.001). Multivariate analysis revealed that BLVRA basal expression in PBL was an independent predictor for sustained virological response (OR 15; 95% CI 1.05–214.2; P = 0.046). HMOX1/2 expression did not have any effect on the treatment outcome.ConclusionOur results suggest that patients with chronic HCV infection significantly upregulate BLVRA expression in PBL. The lack of BLVRA overexpression is associated with non-responsiveness to standard antiviral therapy; whereas, HMOX1/2 does not seem to have any predictive potential.

Mesobiliverdin IXα Enhances Rat Pancreatic Islet Yield and Function

The aims of this study were to produce mesobiliverdin IXα, an analog of anti-inflammatory biliverdin IXα, and to test its ability to enhance rat pancreatic islet yield for allograft transplantation into diabetic recipients. Mesobiliverdin IXα was synthesized from phycocyanobilin derived from cyanobacteria, and its identity and purity were analyzed by chromatographic and spectroscopic methods. Mesobiliverdin IXα was a substrate for human NADPH biliverdin reductase. Excised Lewis rat pancreata infused with mesobiliverdin IXα and biliverdin IXα-HCl (1–100 μM) yielded islet equivalents as high as 86.7 and 36.5%, respectively, above those from non-treated controls, and the islets showed a high degree of viability based on dithizone staining. When transplanted into livers of streptozotocin-induced diabetic rats, islets from pancreata infused with mesobiliverdin IXα lowered non-fasting blood glucose (BG) levels in 55.6% of the recipients and in 22.2% of control recipients. In intravenous glucose tolerance tests, fasting BG levels of 56 post-operative day recipients with islets from mesobiliverdin IXα infused pancreata were lower than those for controls and showed responses that indicate recovery of insulin-dependent function. In conclusion, mesobiliverdin IXα infusion of pancreata enhanced yields of functional islets capable of reversing insulin dysfunction in diabetic recipients. Since its production is scalable, mesobiliverdin IXα has clinical potential as a protectant of pancreatic islets for allograft transplantation.

Heme Oxygenase-1 as a Novel Metabolic Player

Heme Oxygenase-1 as a Novel Metabolic Player

Bilirubin participates in protecting of heme oxygenase-1 induction by quercetin against ethanol hepatotoxicity in cultured rat hepatocytes

To attenuate alcohol liver disease (ALD) is extremely urgent since ALD has been emerged as a major liver disease. The aim of the present study is to investigate the hepatoprotective effect against ethanol-induced injury of bilirubin, a product of heme metabolism degradation via HO and biliverdin reductase catalysis. Ethanol-incubated primary rat hepatocytes (100 mmol/L) were treated by quercetin, bilirubin, inflammatory factors, and/or HO-1 inducer/inhibitor for 24 h, and the cellular damage was assayed. Quercetin lowered ethanol-induced glutathione depletion and superoxide dismutase inactivation, inhibited the overproduction of malondialdehyde and reactive oxygen species, and decreased the leakage of cellular aspartate aminotransferase and lactate dehydrogenase, accompanying the normalization of bilirubin level. The effect of quercetin was mimicked by exogenous bilirubin in a dose-dependent manner to some extent (within 25 μmol/L) and pharmacological HO-1 inducer hemin, but abolished by HO-1 inhibitor zinc protoporphyrin-IX. Inflammatory challenge of TNF-α plus IL-6 further aggravated ethanol-induced oxidative damage, which was also attenuated by bilirubin in part. These findings shed a light on the anti-oxidative and anti-inflammatory role of bilirubin released from quercetin/HO-1 and biliverdin reductase pathway against ethanol hepatotoxicity and highlight a prospective strategy of nutritional intervention for ALD by naturally occurring quercetin to induce HO-1 with the release of bioactive end-products.

Scalable production of biliverdin IXα by Escherichia coli

BackgroundBiliverdin 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.ResultsRecombinant 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.ConclusionsMethods 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.

Gene Polymorphisms in the Heme Degradation Pathway and Outcome of Severe Human Sepsis

Heme and its breakdown products CO, Fe, and bilirubin are being recognized as signaling molecules or even therapeutic agents, but also exert adverse effects when released at high concentrations. Manipulating the pathway confers protection in rodent sepsis models via both control of free heme and formation of its first and higher-order products. Thus, regulatory elements present in human heme oxygenase 1 (HMOX1) and biliverdin reductases (BLVRA/B) genes might impact outcome. We tested whether a highly polymorphic (GT)n microsatellite and single-nucleotide polymorphisms in HMOX1 and BLVRA/B genes are associated with outcome of sepsis. Two cohorts (n = 430 and 398 patients) with severe sepsis were screened for single-nucleotide polymorphisms and/or the microsatellite by fragment length analysis and genotyping techniques. Heme oxygenase 1 plasma levels were determined in additional patients with severe sepsis (n = 92) by enzyme-linked immunosorbent assay. Based on mean Sepsis-related Organ Failure Assessment scores, patients homozygous for rs2071746 A allele or medium length (GT)n microsatellites of HMOX1 showed higher 28-day mortality (P = 0.047 and P = 0.033) in one cohort compared with other genotypes, whereas 90-day mortality rates showed no association. The T allele was less frequently observed in both cohorts than would be expected according to Hardy-Weinberg equilibrium. Heme oxygenase 1 plasma levels were elevated in septic patients, independent of the genotype. Single-nucleotide polymorphisms within BLVRA/B showed no association with outcome. Short (GT)n repeats that are in linkage disequilibrium with the T allele of rs2071746 in HMOX1 are associated with favorable outcome, whereas no association with gene variants of BLVRA/B, involved in the generation of higher-order metabolites, was noticed.

Bilirubin, the Gold Within

When heme-containing proteins such as hemoglobin and cytochrome P450 are degraded, their prosthetic groups cannot be metabolized and have to be excreted. Elimination of heme could be considered straightforward detoxification, but a large body of evidence shows that this pathway plays a role in various physiological processes as well. Article see p 2556 The first step in the catabolism of heme is cleavage of the porphyrin ring by heme oxygenase (HO); this reaction yields biliverdin, carbon monoxide, and iron. Two HO isoforms mediate this process, constitutively expressed HO-2 and inducible HO-1. Many cell types express HO, with high expression present in cells of the reticuloendothelial system involved in the degradation of erythrocytes.1 The first step in heme catabolism has attracted considerable interest because HO activity is involved in the regulation of processes such as arthrosclerosis, inflammation, and diabetes mellitus.1 This regulation is mediated by the products of HO action on heme, with the potent signaling molecule carbon monoxide likely playing a major role.1 The other product of HO action on heme is the green and water-soluble compound biliverdin. In mammals, levels of biliverdin are low because it is quickly converted to the hydrophobic yellow compound bilirubin by ubiquitously expressed biliverdin reductase. However, other animal species, such as fish,2 do not completely metabolize all biliverdin to bilirubin and excrete significant amounts of the green pigment. Because bilirubin is poorly water soluble, mammals require efficient conjugation with glucuronic acid by UDP glucuronyltransferase before bilirubin glucuronides can be excreted into bile. This aspect of heme metabolism has always puzzled scientists. Why is bilirubin the sole end product of heme degradation in mammals? Bilirubin is toxic, and the absence of bilirubin UDP glucuronyltransferase (UGT1A1) in patients with Crigler-Najjar syndrome causes high unconjugated bilirubin levels and subsequent severe brain damage. In …

Two Structures of a Thiazolinyl Imine Reductase from Yersinia enterocolitica Provide Insight into Catalysis and Binding to the Nonribosomal Peptide Synthetase Module of HMWP1

The thiazolinyl imine reductase from Yersinia enterocolitica (Irp3) catalyzes the NADPH-dependent reduction of a thiazoline ring in an intermediate for the formation of the siderophore yersiniabactin. Two structures of Irp3 were determined in the apo (1.85 Å) and NADP(+)-bound (2.31 Å) forms. Irp3 is structurally homologous to sugar oxidoreductases such as glucose-fructose oxidoreductase and 1,5-anhydro-d-fructose reductase, as well as to biliverdin reductase. A homology model of the thiazolinyl imine reductase from Pseudomonas aeruginosa (PchG) was generated. Extensive loop insertions are observed in the C-terminal domain that are unique to Irp3 and PchG and not found in the structural homologues that recognize small molecular substrates. These loops are hypothesized to be important for binding of the nonribosomal peptide synthetase modules (found in HMWP1 and PchF, respectively) to which the substrate of the reductase is covalently attached. A catalytic mechanism for the donation of a proton from a general acid (either histidine 101 or tyrosine 128) and the donation of a hydride from C4 of nicotinamide of the NADPH cofactor is proposed for reduction of the carbon-nitrogen double bond of the thiazoline.

Superoxide in AVF dysfunction: a new target for intervention

an adequately functioning vascular access is an absolute requisite for patients receiving hemodialysis. Among the three currently available (fistulae, grafts, and tunneled catheters) vascular accesses, the arteriovenous fistula (AVF) is considered the “gold standard” and is the most common mode

The Heme Oxygenase 1 Inducer (CoPP) Protects Human Cardiac Stem Cells against Apoptosis through Activation of the Extracellular Signal-regulated Kinase (ERK)/NRF2 Signaling Pathway and Cytokine Release

Intracoronary delivery of c-kit-positive human cardiac stem cells (hCSCs) is a promising approach to repair the infarcted heart, but it is severely limited by the poor survival of donor cells. Cobalt protoporphyrin (CoPP), a well known heme oxygenase 1 inducer, has been used to promote endogenous CO generation and protect against ischemia/reperfusion injury. Therefore, we determined whether preconditioning hCSCs with CoPP promotes CSC survival. c-kit-positive, lineage-negative hCSCs were isolated from human heart biopsies. Lactate dehydrogenase release assays demonstrated that preconditioning CSCs with CoPP markedly enhanced cell survival after oxidative stress induced by H(2)O(2), concomitant with up-regulation of heme oxygenase 1, COX-2, and anti-apoptotic proteins (BCL2, BCL2-A1, and MCL-1) and increased phosphorylation of NRF2. Apoptotic cytometric assays showed that pretreatment of CSCs with CoPP enhanced the cells' resistance to apoptosis induced by oxidative stress. Conversely, knocking down HO-1, COX-2, or NRF2 by shRNA gene silencing abrogated the cytoprotective effects of CoPP. Further, preconditioning CSCs with CoPP led to a global increase in release of cytokines, such as EGF, FGFs, colony-stimulating factors, and chemokine ligand. Conditioned medium from cells pretreated with CoPP conferred naive CSCs remarkable resistance to apoptosis, demonstrating that cytokines released by preconditioned cells play a key role in the anti-apoptotic effects of CoPP. Preconditioning CSCs with CoPP also induced an increase in the phosphorylation of Erk1/2, which are known to modulate multiple pro-survival genes. These results potentially provide a simple and effective strategy to enhance survival of CSCs after transplantation and, therefore, their efficacy in repairing infarcted myocardium.

The Human Biliverdin Reductase-based Peptide Fragments and Biliverdin Regulate Protein Kinase Cδ Activity: THE PEPTIDES ARE INHIBITORS OR SUBSTRATE FOR THE PROTEIN KINASE C

PKCδ, a Ser/Thr kinase, promotes cell growth, tumorigenesis, and apoptosis. Human biliverdin reductase (hBVR), a Ser/Thr/Tyr kinase, inhibits apoptosis by reducing biliverdin-IX to antioxidant bilirubin. The enzymes are activated by similar stimuli. Reportedly, hBVR is a kinase-independent activator of PKCδ and is transactivated by the PKC (Gibbs, P. E., Miralem, T., Lerner-Marmarosh, N., Tudor, C., and Maines, M. D. (2012) J. Biol. Chem. 287, 1066-1079). Presently, we examined interactions between the two proteins in the context of regulation of their activities and defining targets of hBVR phosphorylation by PKCδ. LC-MS/MS analysis of PKCδ-activated intact hBVR identified phosphorylated serine positions 21, 33, 230, and 237, corresponding to the hBVR Src homology-2 domain motif (Ser(230) and Ser(237)), flanking the ATP-binding motif (Ser(21)) and in PHPS sequence (Ser(33)) as targets of PKCδ. Ser(21) and Ser(230) were also phosphorylated in hBVR-based peptides. The Ser(230)-containing peptide was a high affinity substrate for PKCδ in vitro and in cells; the relative affinity was PKCδ > PKCβII > PKCζ. Two overlapping peptides spanning this substrate, KRNRYLSF and SFHFKSGSL, were effective inhibitors of PKCδ kinase activity and PKCδ-supported activation of transcription factors Elk1 and NF-κB. Only SFHFKSGSL, in PKCδ-transfected phorbol 12-myristate 13-acetate-stimulated cells, caused membrane blebbing and cell loss. Biliverdin noncovalently inhibited PKCδ, whereas PKCδ potentiated hBVR reductase activity and accelerated the rate of bilirubin formation. This study, together with previous findings, reveals an unexpected regulatory interplay between PKCδ and hBVR in modulating cell death/survival in response to various activating stimuli. In addition, this study has identified novel substrates for and inhibitors of PKCδ. We suggest that hBVR-based technology may have utility to modulate PKCδ-mediated functions in the cell.

PP131. Regulation of heme oxygenase/carbon monoxide (ho/co) and l-arginine/nitric oxide (no) pathways by human chorionic gonadotropin (hcg) in human fetal endothelium

HO/CO and l-arginine/NO pathways are critical regulators of vascular function in early human pregnancy. HO is the rate-limiting enzyme in heme catabolism leading to the formation of biliverdin, which is subsequently reduced to bilirubin by biliverdin reductase, carbon monoxide, and free iron. HO-1, the inducible isoform of the enzyme, is essential to promote placental function and fetal development (1). HO-1 is also required to maintain nitric oxide synthesis (2). Human chorionic gonadotropin (hCG) is the first blastocyst-derived hormonal signal playing a significant role in implantation and placentation (3). We hypothesized that hCG upregulates HO-1 and thus positively regulates early vascular events in human pregnancy. In this study we used human umbilical vein endothelial cells (HUVECs) to demonstrate that hCG up-regulates mRNA and protein abundance of HO-1. The effect of hCG on l-arginine transport and the expression of hCAT1, the main cationic amino acid transporter involved in endothelial nitric oxide synthesis were evaluated. HUVEC were isolated from normal pregnancies (approval from the Institutional Review Board) by collagenase digestion and grown (37°C, 5% CO2) in medium 199 supplemented with 20% newborn and fetal calf sera. Cells were treated with hCG (0.5-100IU/mL, 0-24hours and CoCl2 (150μM, 6hours) as positive control. Total proteins were separated by SDS-PAGE electrophoresis, transferred to nitrocellulose membranes and incubated with monoclonal anti human HO-1 or anti β-actin and respective secondary antibody conjugated with horseradish peroxidase. Protein bands were detected by enhanced chemiluminiscence. Total RNA was isolated and cDNA was obtained by reverse transcription. PCR assays were performed in a reaction mix containing 0.5μM primers, dNTPs, Taq DNA polymerase, 2 mM MgCl2. Relative abundance of HO-1, hCAT-1 and 28S were determined. l-[(3)H]Arginine transport (100μM, 2 μCi ml(-1), 37°C, 1min) were determined in HUVECs exposed (30min) to hCG (0.5-100IU/mL) and CORM-2 (CO donor). The highest level of HO-1 protein were obtained at 5IU/mL of hCG and declined at higher concentrations. hCG increased HO-1 protein abundance and maximal response was observed at 6 hours (4-fold), decreasing at 24hours exposure. These effects of hCG on HO-1 protein abundance are well correlated with changes in HO-1 mRNA regarding time of exposure and concentration of hCG. L-arginine transport and hCAT-1 mRNA were also increased (2.8-fold and 4.5-fold, respectively) by hCG. Upregulation of HO-1 as well as the increase in l-arginine transport and hCAT-1 mRNA in HUVECs are novel effects of hCG, which occur at physiological plasma concentrations of the hormone, as those found in the first weeks of pregnancy. These changes could be relevant for the maintenance of the NO synthesis in fetal and maternal vasculature, ensuring the predominance of the vasodilator tone. These effects could be also critical for vascular remodelling of uterine circulation and placental vascular development in early stages of pregnancy, the lack of which could result in abnormal placentation as described in preeclampsia and fetal growth restriction.

Microarray genomic profile of mitochondrial and oxidant response in manganese chloride treated PC12 cells

Environmental or occupational exposure to high levels of manganese (Mn) can lead to manganism, a symptomatic neuro-degenerative disorder similar to idiopathic Parkinson's disease. The underlying mechanism of Mn neurotoxicity remains unclear. In this study, we evaluate the primary toxicological events associated with MnCl2 toxicity in rat PC12 cells using whole genome cDNA microarray, RT-PCR, Western blot and functional studies. The results show that a sub-lethal dose range (38–300μM MnCl2) initiated slight metabolic stress evidenced by heightened glycolytic rate and induction of enolase/aldolase – gene expression. The largest shift observed in the transcriptome was MnCl2 induction of heme-oxygenase 1 (HO-1) [7.7 fold, p<0.001], which was further corroborated by RT-PCR and Western blot studies. Concentrations in excess of 300μM corresponded to dose dependent loss of cell viability which was associated with enhanced production of H2O2 concomitant to elevation of gene expression for diverse antioxidant enzymes; biliverdin reductase, arsenite inducible RNA associated protein, dithiolethione-inducible gene-1 (DIG-1) and thioredoxin reductase 1. Moreover, Mn initiated significant reduction of gene expression of mitochondrial glutaryl-coenzyme A dehydrogenase (GCDH), an enzyme involved with glutaric acidemia, oxidative stress, lipid peroxidation and striatal degeneration observed in association with severe dystonic-dyskinetic movement disorder. Future research will be required to elucidate a defined role for HO-1 and GCDH in Mn toxicity.

Go Green: The Anti-Inflammatory Effects of Biliverdin Reductase

Biliverdin (BV) has emerged as a cytoprotective and important anti-inflammatory molecule. Conversion of BV to bilirubin (BR) is catalyzed by biliverdin reductase (BVR) and is required for the downstream signaling and nuclear localization of BVR. Recent data by others and us make clear that BVR is a critical regulator of innate immune responses resulting from acute insult and injury and moreover, that a lack of BVR results in an enhanced proinflammatory phenotype. In macrophages, BVR is regulated by its substrate BV which leads to activation of the PI3K–Akt-IL-10 axis and inhibition of TLR4 expression via direct binding of BVR to the TLR4 promoter. In this review, we will summarize recent findings on the role of BVR and the bile pigments in inflammation in context with its activity as an enzyme, receptor, and transcriptional regulator.

Direct Antioxidant Properties of Bilirubin and Biliverdin. Is there a Role for Biliverdin Reductase?

Reactive oxygen species (ROS) and signaling events are involved in the pathogenesis of endothelial dysfunction and represent a major contribution to vascular regulation. Molecular signaling is highly dependent on ROS. But depending on the amount of ROS production it might have toxic or protective effects. Despite a large number of negative outcomes in large clinical trials (e.g., HOPE, HOPE-TOO), antioxidant molecules and agents are important players to influence the critical balance between production and elimination of reactive oxygen and nitrogen species. However, chronic systemic antioxidant therapy lacks clinical efficacy, probably by interfering with important physiological redox signaling pathways. Therefore, it may be a much more promising attempt to induce intrinsic antioxidant pathways in order to increase the antioxidants not systemically but at the place of oxidative stress and complications. Among others, heme oxygenase (HO) has been shown to be important for attenuating the overall production of ROS in a broad range of disease states through its ability to degrade heme and to produce carbon monoxide and biliverdin/bilirubin. With the present review we would like to highlight the important antioxidant role of the HO system and especially discuss the contribution of the biliverdin, bilirubin, and biliverdin reductase (BVR) to these beneficial effects. The BVR was reported to confer an antioxidant redox amplification cycle by which low, physiological bilirubin concentrations confer potent antioxidant protection via recycling of biliverdin from oxidized bilirubin by the BVR, linking this sink for oxidants to the NADPH pool. To date the existence and role of this antioxidant redox cycle is still under debate and we present and discuss the pros and cons as well as our own findings on this topic.