Heme Turnover Research Articles

The impact of heme oxygenase-2 on pharmacological research: A bibliometric analysis and beyond.

Heme oxygenase (HO-2) is an enzyme mainly involved in the physiologic turnover of heme and intracellular gas sensing, and it is very abundant in the brain, testes, kidneys and vessels. Since 1990, when HO-2 was discovered, the scientific community has underestimated the role of this protein in health and disease, as attested by the small amount of articles published and citations received. One of the reason that have contributed to the lack of interest in HO-2 was the difficulty in upregulating or inhibiting this enzyme. However, over the last 10years, novel HO-2 agonists and antagonists have been synthesized, and the availability of these pharmacological tools should increase the appeal of HO-2 as drug target. In particular, these agonists and antagonists could help explain some controversial aspects, such as the neuroprotective versus neurotoxic roles of HO-2 in cerebrovascular diseases. Furthermore, the discovery of HO-2 genetic variants and their involvement in Parkinson's disease, in particular in males, opens new avenues for pharmacogenetic studies in gender medicine.

Ontogeny of Carbon Monoxide-Related Gene Expression in a Deep-Diving Marine Mammal.

Marine mammals such as northern elephant seals (NES) routinely experience hypoxemia and ischemia-reperfusion events to many tissues during deep dives with no apparent adverse effects. Adaptations to diving include increased antioxidants and elevated oxygen storage capacity associated with high hemoprotein content in blood and muscle. The natural turnover of heme by heme oxygenase enzymes (encoded by HMOX1 and HMOX2) produces endogenous carbon monoxide (CO), which is present at high levels in NES blood and has been shown to have cytoprotective effects in laboratory systems exposed to hypoxia. To understand how pathways associated with endogenous CO production and signaling change across ontogeny in diving mammals, we measured muscle CO and baseline expression of 17 CO-related genes in skeletal muscle and whole blood of three age classes of NES. Muscle CO levels approached those of animals exposed to high exogenous CO, increased with age, and were significantly correlated with gene expression levels. Muscle expression of genes associated with CO production and antioxidant defenses (HMOX1, BVR, GPX3, PRDX1) increased with age and was highest in adult females, while that of genes associated with protection from lipid peroxidation (GPX4, PRDX6, PRDX1, SIRT1) was highest in adult males. In contrast, muscle expression of mitochondrial biogenesis regulators (PGC1A, ESRRA, ESRRG) was highest in pups, while genes associated with inflammation (HMOX2, NRF2, IL1B) did not vary with age or sex. Blood expression of genes involved in regulation of inflammation (IL1B, NRF2, BVR, IL10) was highest in pups, while HMOX1, HMOX2 and pro-inflammatory markers (TLR4, CCL4, PRDX1, TNFA) did not vary with age. We propose that ontogenetic upregulation of baseline HMOX1 expression in skeletal muscle of NES may, in part, underlie increases in CO levels and expression of genes encoding antioxidant enzymes. HMOX2, in turn, may play a role in regulating inflammation related to ischemia and reperfusion in muscle and circulating immune cells. Our data suggest putative ontogenetic mechanisms that may enable phocid pups to transition to a deep-diving lifestyle, including high baseline expression of genes associated with mitochondrial biogenesis and immune system activation during postnatal development and increased expression of genes associated with protection from lipid peroxidation in adulthood.

Expression of HO1 and PER2 can predict the incidence of delirium in trauma patients with concomitant brain injury

Intensive care unit (ICU)-acquired delirium is associated with adverse outcome in trauma patients with concomitant traumatic brain injury (TBI), but diagnosis remains challenging. Quantifying circadian disruption by analyzing expression of the circadian gene period circadian regulator 2 (PER2) and heme oxygenase 1 (HO1), which determines heme turnover, may prove to be potential diagnostic tools. Expression of PER2 and HO1 was quantified using qPCR from blood samples 1 day and 7 days after trauma. Association analysis was performed comparing mRNA expression levels with parameters of trauma (ISS—injury severity score), delirium, acute kidney injury (AKI) and length of ICU stay. 48 polytraumatized patients were included (equal distribution of TBI versus non-TBI) corrected for ISS, age and gender using a matched pairs approach. Expression levels of PER2 and HO1 were independent of age (PER2: P = 0.935; HO1: P = 0.988), while expression levels were significantly correlated with trauma severity (PER2: P = 0.009; HO1: P < 0.001) and longer ICU length of stay (PER2: P = 0.018; HO1: P < 0.001). High expression levels increased the odds of delirium occurrence (PER2: OR = 4.32 [1.14–13.87]; HO1: OR = 4.50 [1.23–14.42]). Patients with TBI showed a trend towards elevated PER2 (OR = 3.00 [0.84–9.33], P = 0.125), but not towards delirium occurrence (P = 0.556). TBI patients were less likely to develop AKI compared to non-TBI (P = 0.022). Expression levels of PER2 and HO1 correlate with the incidence of delirium in an age-independent manner and may potentially improve diagnostic algorithms when used as delirium biomarkers.Trial registration: German Clinical Trials Register (Trial-ID DRKS00008981; Universal Trial Number U1111-1172-6077; Jan. 18, 2018).

A unique case of black choledocholithiasis: a video-based case review in the Pancreato-Biliary Division\u2013Endoscopy Unit of National Liver Institute (NLI)

BackgroundBlack gall stones are consistently described in relation to haem turnover and hemolytic disorders. Black gall stones are also a frequent presentation in cirrhotic patients, principally due to hemolysis-related hypersplenism.Case presentationHerein, we present a case of an average built 83-year-old lady who was suffering from biliary-type abdominal episodes of pain. Neither clinical nor laboratory evidence of overt biliary obstruction, cholangitis, or pancreatitis was detected. Sonographic examination revealed the presence of calcular gall bladder with many small stones, dilated common bile duct (CBD), and biliary radicles, along with homogenous average-sized liver, average-sized spleen with normal echogenicity and no focal lesions. Endoscopic retrograde cholangiography (ERC) was performed with wide papillotomy which was surprisingly followed by a drift of innumerable intensely black small stones.ConclusionIn aged population, absence of the full-blown clinical syndrome in relation to complicated CBD stones should not exclude endoscopic intervention.

Significance of Heme and Heme Degradation in the Pathogenesis of Acute Lung and Inflammatory Disorders

The heme molecule serves as an essential prosthetic group for oxygen transport and storage proteins, as well for cellular metabolic enzyme activities, including those involved in mitochondrial respiration, xenobiotic metabolism, and antioxidant responses. Dysfunction in both heme synthesis and degradation pathways can promote human disease. Heme is a pro-oxidant via iron catalysis that can induce cytotoxicity and injury to the vascular endothelium. Additionally, heme can modulate inflammatory and immune system functions. Thus, the synthesis, utilization and turnover of heme are by necessity tightly regulated. The microsomal heme oxygenase (HO) system degrades heme to carbon monoxide (CO), iron, and biliverdin-IXα, that latter which is converted to bilirubin-IXα by biliverdin reductase. Heme degradation by heme oxygenase-1 (HO-1) is linked to cytoprotection via heme removal, as well as by activity-dependent end-product generation (i.e., bile pigments and CO), and other potential mechanisms. Therapeutic strategies targeting the heme/HO-1 pathway, including therapeutic modulation of heme levels, elevation (or inhibition) of HO-1 protein and activity, and application of CO donor compounds or gas show potential in inflammatory conditions including sepsis and pulmonary diseases.

The heme-regulatory motifs of heme oxygenase-2 contribute to the transfer of heme to the catalytic site for degradation

Heme-regulatory motifs (HRMs) are present in many proteins that are involved in diverse biological functions. The C-terminal tail region of human heme oxygenase-2 (HO2) contains two HRMs whose cysteine residues form a disulfide bond; when reduced, these cysteines are available to bind Fe3+-heme. Heme binding to the HRMs occurs independently of the HO2 catalytic active site in the core of the protein, where heme binds with high affinity and is degraded to biliverdin. Here, we describe the reversible, protein-mediated transfer of heme between the HRMs and the HO2 core. Using hydrogen-deuterium exchange (HDX)-MS to monitor the dynamics of HO2 with and without Fe3+-heme bound to the HRMs and to the core, we detected conformational changes in the catalytic core only in one state of the catalytic cycle-when Fe3+-heme is bound to the HRMs and the core is in the apo state. These conformational changes were consistent with transfer of heme between binding sites. Indeed, we observed that HRM-bound Fe3+-heme is transferred to the apo-core either upon independent expression of the core and of a construct spanning the HRM-containing tail or after a single turnover of heme at the core. Moreover, we observed transfer of heme from the core to the HRMs and equilibration of heme between the core and HRMs. We therefore propose an Fe3+-heme transfer model in which HRM-bound heme is readily transferred to the catalytic site for degradation to facilitate turnover but can also equilibrate between the sites to maintain heme homeostasis.

Active Turnover of Heme in Hibernation Period in Mammals.

Heme oxygenase (HO)-1 plays an important role during hibernation by catalyzing the degradation of heme to biliverdin/bilirubin, ferrous iron, and carbon monoxide, which activates the protective mechanisms against stress. In this context, it was important to analyze the metabolic processes of heme. Nevertheless, to date, no study has approached on biosynthesis of heme. Therefore, our study aims to understand the process of heme biosynthesis, which regulates cell survival in conditions of hypothermia and calorie restriction (CR). During hibernation, the mRNA levels of enzymes responsible for de novo heme biosynthesis were increased in the liver tissue of a Syrian hamster model of hibernation. Moreover, heme trafficking and iron metabolism were found to be more active, as assessed based on the changes in the levels of heme transporter and ferroportin mRNA. The levels of HO-1, a powerful antioxidant, were also upregulated during hibernation. Additionally, increased levels of Sirt-1 mRNA were also observed. These enzymes are known to act as cellular metabolic sensors that activate the cytoprotective mechanisms. These results indicate that HO-1 induction, brought about by the upregulation of heme during the pre-hibernation period, may protect against external stress. Here, we describe heme catabolism during hibernation by analyzing the regulation of the key molecular players involved in heme metabolism. Therefore, this study offers a new strategy for the better regulation of intracellular heme concentrations during hypothermia and other stresses.

Copper and iron metabolism in Ostreococcus tauri - the role of phytotransferrin, plastocyanin and a chloroplast copper-transporting ATPase.

Iron and copper are essential elements for practically all living organisms. Their metabolism is frequently interconnected, and while copper is relatively abundant in the ocean, iron is often a limiting factor for the growth of many marine microorganisms. In the present study, we aimed to elucidate the metabolisms of copper and iron and the connection of both in the marine picoalga Ostreococcus tauri. We show that O. tauri adjusts its copper economy in response to copper deficiency by downregulation of the expression of plastocyanin in favor of cytochrome c oxidase without significant changes in growth and physiology. Copper deprivation leads to increased expression of copper transporting ATPase and proteins involved in tetrapyrrole synthesis, most likely to ensure higher turnover of chlorophyll and/or heme. Elucidation of the effect of copper on the incorporation of iron into O. tauri proteins led us to identify the major iron uptake mediating protein, Ot-Fea1, whose expression and binding of iron is copper dependent. Based on our investigation of the incorporation of iron into Ot-Fea1 and ferritin, we hypothesize that O. tauri possesses another Fea1-independent iron uptake system.

Experimental infection with Anguillicola crassus alters immune gene expression in both spleen and head kidney of the European eel (Anguilla anguilla)

Invasive parasites have been implicated in the declines of several freshwater species. The swim bladder nematode Anguillicola crassus was introduced into Europe in the 1980s and is considered a threat to the European eel (Anguilla anguilla). Infection affects stress resistance and swimming behaviour. European eels produce an immune response against the parasite during the late stages of infection and after repeated infections. We used RNA-seq to examine the molecular response to infection during the poorly understood early stage and identify expression of genes and associated processes that are modified in two immune organs of European eels 3 days post infection with A. crassus. In the spleen, 67 genes were differentially expressed, 32 of which were annotated. Most of these were involved in immune processes and their regulation. Other differentially expressed genes in the spleen were important for heme metabolism and heme turn-over. In the head kidney, 257 genes (134 annotated) were differentially expressed. Several of these were associated with immune functions. Other differentially expressed genes in the head kidney were related to renal function, in particular osmoregulation and paracellular flow. We conclude that the early response of European eels to A. crassus is complex and involves various processes aside from the immune system. We identified molecular changes occurring early during the infection and identified candidate genes and processes which will facilitate future studies aimed at determining the factors affecting European eel viability in the face of this invasive parasite.

Mitochondrial Dysfunction in Lung Pathogenesis.

Remarkable new roles for mitochondria in calcium handling, apoptosis, heme turnover, inflammation, and oxygen and nutrient sensing have been discovered for organelles that were once thought to be simple energy converters. Although deficits in mitochondrial function are often associated with energy failure and apoptosis, working cells maintain a mitochondrial reserve that affords the organelles distinct homeostatic sensing and regulatory abilities in lung cells. As primary intracellular sources of oxidants, mitochondria serve as critical monitors and modulators of vital oxidation-reduction processes, including mitochondrial biogenesis, mitophagy, inflammasome activation, cell proliferation, and prevention of fibrosis. These processes participate in disease pathogenesis in all lung regions mainly when interference with mitochondrial quality control mechanisms impedes their roles in maintenance of lung health. Sharper identification of mitochondrial-driven signaling mechanisms in specific lung cell types will better refine our understanding of respiratory disease pathogenesis and lead to new diagnostic and therapeutic measures to support mitochondrial quality.

The Oxidant Response Transcription Factor NRF2 Mediates Heme Activation of Placenta Growth Factor Expression in Erythroid Cells, a Contributor to Pulmonary Hypertension in Sickle Cell Disease

Patients with sickle cell disease (SCD) have elevated plasma levels of placenta growth factor (PlGF) which promotes expression of the pulmonary vasoconstrictor endothelin-1 (ET-1) contributing to pulmonary hypertension, an important age-related and life-limiting complication of SCD. In SCD patients, markers of high iron burden are associated with the highest PlGF levels, leading us to hypothesize a mechanistic link between excessive iron and the induction of the PlGF protein. We have published evidence that heme-bound iron stimulates the PlGF promoter up to 400-fold in human K562 cells and in primary human erythroid cells. Gene transfer, knockout and reconstitution experiments have documented the requirement for erythroid Kruppel-like factor (EKLF; KLF1), confirmed by chromatin immunoprecipitation. We are currently investigating the role of other transcription factors known to sense intracellular iron.Using cultured human erythroid cells (K562 cells) treated with heme-bound iron (hemin) and quantitative real-time PCR, we have assessed the time course of heme regulation of the transcripts of several members of the Nrf2-small Maf family of transcription factors. The transcripts for the antioxidant NRF2, NFE2, MafF, MafG and BACH1 are dynamically regulated following exposure of the cells to heme. Gene expression knockdown and small molecule inhibitor and activator experiments have revealed a central role of NRF2 in activating the PlGF promoter in response to heme, further supported by chromatin immunoprecipitation experiments. Specifically, the PlGF promoter is robustly activated by the well characterized non-oxidative NRF2 agonist sulforaphane, and also activated by the nonspecific oxidant hydrogen peroxide. Furthermore, heme activation of the PlGF promoter is inhibited by the NRF2 inhibitor brusatol, by siRNA directed against NRF2, and by the nonspecific antioxidant N-acetyl cysteine. Although the small Maf proteins are often considered to be nonspecific, interchangeable heterodimeric partners of NRF2, our knockdown data suggest a specific role for MafG cooperating with NRF2 in transducing the heme signal on the PlGF promoter. Our knockdown and sulforaphane results also support a role for NRF2 in regulating the transcript levels of all the known NRF2 family members. We have found similarities and differences in the regulation of the PlGF promoter with another well described heme-responsive promoter, heme oxygenase-1. We are currently investigating these regulatory effects on heme on PlGF expression in vivo and in primary erythroid cells from Nrf2 knockout mice.Our results to date support a mechanism in which accelerated heme turnover in sickle cell disease promotes robust expression of PlGF in erythroblasts during erythroid differentiation, through a pathway that involves EKLF, NRF2 and MafG. Our prior published data in collaboration with the Malik lab has documented a causal role for PlGF in promoting expression of the potent vasoconstrictor endothelin-1 accompanied by the development of pulmonary hypertension, corroborated by data from humans with SCD. This mechanism helps to explain the clinical observation that heavily transfused, iron overloaded adults with SCD are more likely to develop pulmonary hypertension, as a potential consequence of excess heme trafficking from turnover of transfused red cells. These results might inspire greater adherence to existing approved therapies to chelate iron in SCD. DisclosuresNo relevant conflicts of interest to declare.

Carbon monoxide: a critical quantitative analysis and review of the extent and limitations of its second messenger function.

Endogenously produced carbon monoxide (CO) is commonly believed to be a ubiquitous second messenger involved in a wide range of physiological and pathological responses. The major evidence supporting this concept is that CO is produced endogenously via heme oxygenase-catalyzed breakdown of heme and that experimental exposure to CO alters tissue function. However, it remains to be conclusively demonstrated that there are specific receptors for CO and that endogenous CO production is sufficient to alter tissue function. Unlike other signaling molecules, CO is not significantly metabolized, and it is removed from cells solely via rapid diffusion into blood, which serves as a near infinite sink. This non-metabolizable nature of CO renders the physiology of this gas uniquely susceptible to quantitative modeling. This review analyzes each of the steps involved in CO signaling: 1) the background CO partial pressure (PCO) and the blood and tissue CO binding; 2) the affinity of the putative CO receptors; 3) the rate of endogenous tissue CO production; and 4) the tissue PCO that results from the balance between this endogenous CO production and diffusion to the blood sink. Because existing data demonstrate that virtually all endogenous CO production results from the routine “housekeeping” turnover of heme, only a small fraction can play a signaling role. The novel aspect of the present report is to demonstrate via physiological modeling that this small fraction of CO production is seemingly insufficient to raise intracellular PCO to the levels required for the conventional, specific messenger receptor activation. It is concluded that the many physiological alterations observed with exogenous CO administration are probably produced by the non-specific CO inhibition of cytochrome C oxidase activity, with release of reactive oxygen species (ROS) and that this ROS signaling pathway is a potential effector mechanism for endogenously produced CO.

End-Alveolar Carbon Monoxide As a Measure of Erythrocyte Survival and Hemolytic Severity in Sickle Cell Disease

Introduction: In sickle cell disease, polymerization of sickle hemoglobin gives rise to changes in the integrity and viability of the erythrocyte, leading to both extravascular and intravascular hemolysis and shortened life-span. Current gold standard techniques to estimate RBC survival require that a label be placed on the cells that can be followed while the RBCs age in the circulation. These studies are infrequently employed in practice, due to the need for multiple blood draws and an extended period of observation (6 weeks) in order to obtain the necessary time points. Endogenous CO is a technique that has been used in multiple studies to assess RBC survival, based upon the principal that virtually all CO produced in human beings results from cleavage of the α-methene bond of heme and is completely excreted via the lungs. Because RBC destruction accounts for approximately 80% of heme turnover in the body, endogenous CO production can be used as a quantitative indicator of RBC life span. Furne has reported on the development of a simple, rapid, and noninvasive method for determining RBC life span based on gas chromatography measurement of exhaled alveolar CO (EACO) concentration immediately upon awakening corrected for atmospheric CO, as determined with a device that simulates the body’s equilibration with CO.We set up to investigate the use of early morning end-alveolar CO (EACO) concentration as a quantitative measure of RBC life span and correlate it to indirect measurements of hemolytic rate in subjects with sickle cell disease.Material and methods: EACO was measured within 24 hours of breath collection in 67 SCD adult patients (67 HbSS, 4 HbSC and 1 HbSb-Thalassemia) and 14 HbAA race matched controls on an Ametrek TA 7000 Gas Purity Monitor. Subjects were at steady state, at least 30 days from transfusion or acute exacerbations. Breath samples were collected immediately upon awakening the same day as blood sampling for additional tests. EACO groups were compared using the Mann-Whitney test. EACO values were used to calculate the red blood cell lifespan by the method of Furne (PMID 12878986), using the calculation: RBC survival (days) = 1,380 · [Hb]/EACO. Correlation analyses are reported using Pearson’s correlation coefficient. The statistical significance was set at a two-side p < .05. Analyses were performed with R version 2.13.1 (2011-07-08).Results: EACO was nearly four-fold higher in patients than controls (median 2.25 vs. 0.58 ppm, p<.0001, figure). In patients with SCD, unadjusted EACO values were correlated with %HbS (p<.01), indirect markers of hemolysis: absolute reticulocyte count (p<.02), and percentage of reticulocytes (p<.01), indirect bilirubin (p<.001), and negatively with plasma nitrite (p<.02). EACO-derived red blood cell lifespan correlated negatively with LDH (p<.04), hematocrit (p<.01), hemoglobin (p<.06) indirect bilirubin (p<.01), absolute and percent reticulocyte counts (p<.01), AST (p<.01), and %HbS (p<.01).Conclusions: Our results confirm the usefulness of exhaled CO measurement as a marker of hemolysis and red cell survival and in substantial, prospective cohort of adult patients with SCD, confirming and considerably extending results of other investigators. EACO-derived RBC survival can be used to update estimates from Crosby in 1955 regarding the proportion of overall hemolysis falling into the extravascular and intravascular categories, the latter related to oxidative stress and scavenging of nitric oxide. This assay may be a useful biomarker in clinical trials of therapeutic interventions aimed at improving red cell life-span in SCD and other hemolytic anemias. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Heme-iron utilization by Leptospira interrogans requires a heme oxygenase and a plastidic-type ferredoxin-NADP+ reductase

BackgroundHeme oxygenase catalyzes the conversion of heme to iron, carbon monoxide and biliverdin employing oxygen and reducing equivalents. This enzyme is essential for heme-iron utilization and contributes to virulence in Leptospira interrogans. MethodsA phylogenetic analysis was performed using heme oxygenases sequences from different organisms including saprophytic and pathogenic Leptospira species. L. interrogans heme oxygenase (LepHO) was cloned, overexpressed and purified. The structural and enzymatic properties of LepHO were analyzed by UV–vis spectrophotometry and 1H NMR. Heme-degrading activity, ferrous iron release and biliverdin production were studied with different redox partners. ResultsA plastidic type, high efficiently ferredoxin-NADP+ reductase (LepFNR) provides the electrons for heme turnover by heme oxygenase in L. interrogans. This catalytic reaction does not require a ferredoxin. Moreover, LepFNR drives the heme degradation to completeness producing free iron and α-biliverdin as the final products. The phylogenetic divergence between heme oxygenases from saprophytic and pathogenic species supports the functional role of this enzyme in L. interrogans pathogenesis. ConclusionsHeme-iron scavenging by LepHO in L. interrogans requires only LepFNR as redox partner. Thus, we report a new substrate of ferredoxin-NADP+ reductases different to ferredoxin and flavodoxin, the only recognized protein substrates of this flavoenzyme to date. The results presented here uncover a fundamental step of heme degradation in L. interrogans. General significanceOur findings contribute to understand the heme-iron utilization pathway in Leptospira. Since iron is required for pathogen survival and infectivity, heme degradation pathway may be relevant for therapeutic applications.

Acute intermittent porphyria with psychiatric manifestations: A case report

The porphyrias are a group of eight genetic disorders of heme biosynthesis that result from the deficient activity of a certain enzyme in the biosynthesis pathway. These disorders are separated by symptom manifestation and classified as acute porphyrias and cutaneous porphyrias. One of the acute porphyrias, acute intermittent porphyria (AIP), is caused by a prophobilinogen deaminase deficiency and usually presents as abdominal pain, nausea, constipation, and confusion.

Intracellular accumulation of bilirubin as a defense mechanism against increased oxidative stress

Antioxidant, anti-inflammatory and anti-atherogenic effects have been associated with elevations of unconjugated bilirubin (UCB) in serum and with the induction of heme oxygenase-1 (HO-1), the rate-limiting enzyme in UCB synthesis. The aim of this study was to investigate the intracellular metabolism and antioxidant properties of UCB in human hepatoblastoma HepG2 cells and tissues of Wistar rats exposed to oxidative stressors and lipopolysaccharide (LPS), respectively. Intracellular UCB concentrations in HepG2 cells correlated with its levels in culture media (p<0.001) and diminished lipid peroxidation in a dose-dependent manner (p<0.001). Moreover, induction of HO-1 with sodium arsenite led to 2.4-fold (p=0.01) accumulation of intracellular UCB over basal level while sodium azide-derived oxidative stress resulted in a 60% drop (p<0.001). This decrease was ameliorated by UCB elevation in media or by simultaneous induction of HO-1. In addition, hyperbilirubinemia and liver HO-1 induction in LPS-treated rats resulted in a 2-fold accumulation of tissue UCB (p=0.01) associated with enhanced protection against lipid peroxidation (p=0.02). In conclusion, hyperbilirubinemia and HO-1 induction associated with inflammation and oxidative stress increase intracellular concentrations of UCB, thus enhancing the protection of cellular lipids against peroxidation. Therefore, the previously reported protective effects of hyperbilirubinemia and HO-1 induction are at least in part due to intracellular accumulation of UCB.

Mechanism of Heme-Heme Oxygenase System Impairment of Endothelium Contraction in the Spontaneously Hypertensive Rat

See related article, pp 926–934 Heme oxygenase (HO) 1 and HO-2 gene expressions are known to increase cellular antioxidant and anti-inflammatory properties through activation of a battery of cytoprotective systems, including decreases in cyclooxygenase 1 and NADPH oxidase (NOX-2) and elevated levels of extracellular superoxide dismutase and NO. The heme-HO system (HO-1 and HO-2) acts as an endogenous anti-inflammatory and protective circuit critical for a self-resolving inflammatory-reparative process. A study by Li et al,1 which appears in this issue of Hypertension , shows that upregulation of heme oxygenase (HO) 1 prevents endothelium contraction in aorta of spontaneously hypertensive rats (SHRs) via a decrease in cyclooxygenase (COX) 1, not COX-2. Induction of HO-1 was observed in cultured endothelial cells and in vivo after treatment with both heme and hemoglobin. In the present article, Li et al1 showed that, in aorta isolated from either wild-type or SHR animals, levels of HO-1 are low. HO-1 under physiological conditions is expressed at low levels and does not seem to affect the basal levels of heme-dependent enzyme activity. On the other hand, HO-2, the constitutive enzyme, is the main determinant for the regulation of the basal physiological levels of various hemeproteins, including COX, NOX2, inducible NO synthase, and CYP450. COX-1/-2 are hemeproteins that catalyze the conversion of arachidonic acid (AA) to prostaglandin H2, the precursor of prostanoids that participate in the regulation of vascular function. Heme binds to the COX …

Characterization of the haem oxygenase protein family in Arabidopsis thaliana reveals a diversity of functions

HOs (haem oxygenases) catalyse the oxidative cleavage of haem to BV (biliverdin), iron and carbon monoxide. In plants, the product of the reaction is BV IXalpha, the precursor of the PHY (phytochrome) chromophore and is thus essential for proper photomorphogenesis. Arabidopsis thaliana contains one major biochemically characterized HO (HY1) and three additional putative HOs (HO2, HO3 and HO4). All four proteins are encoded in the nucleus but contain chloroplast translocation sequences at their N-termini. The transit peptides of all four proteins are sufficient for chloroplast translocalization as shown by GFP (green fluorescent protein) reporter gene fusions. Overall, all four proteins can be divided into two subfamilies: HO1 and HO2. Here we show that all members of the HO1 subfamily (HY1, HO3 and HO4) are active monomeric HOs and can convert haem to BV IXalpha using spinach Fd (ferredoxin) as an electron donor. Addition of a second electron donor, such as ascorbate, led to a 10-fold increase in the haem conversion rate. Furthermore, haem turnover is also promoted by light when spinach thylakoids are present. All HO1 family members displayed similar kinetic parameters indicating they all have a possible involvement in PHY chromophore biosynthesis. HO2 did not yield sufficient amounts of soluble protein and therefore required the construction of a synthetic gene adapted to the codon usage of Escherichia coli. HO2 is unable to bind or degrade haem and therefore it is not a haem oxygenase. However, HO2 shows strong binding of proto IX (protoporphyrin IX), a precursor for both haem and chlorophyll biosynthesis. A possible function of HO2 in the regulation of tetrapyrrole metabolism is discussed.

Cell Surface Biliverdin Reductase Mediates Biliverdin-induced Anti-inflammatory Effects via Phosphatidylinositol 3-Kinase and Akt

Biliverdin reductase A (BVR) catalyzes the reduction of biliverdin (BV) to bilirubin (BR) in all cells. Others and we have shown that biliverdin is a potent anti-inflammatory molecule, however, the mechanism by which BV exerts its protective effects is unclear. We describe and elucidate a novel finding demonstrating that BVR is expressed on the external plasma membrane of macrophages (and other cells) where it quickly converts BV to BR. The enzymatic conversion of BV to BR on the surface by BVR initiates a signaling cascade through tyrosine phosphorylation of BVR on the cytoplasmic tail. Phosphorylated BVR in turn binds to the p85alpha subunit of phosphatidylinositol 3-kinase and activates downstream signaling to Akt. Using bacterial endotoxin (lipopolysaccharide) to initiate an inflammatory response in macrophages, we find a rapid increase in BVR surface expression. One of the mechanisms by which BV mediates its protective effects in response to lipopolysaccharide is through enhanced production of interleukin-10 (IL-10) the prototypical anti-inflammatory cytokine. IL-10 regulation is dependent in part on the activation of Akt. The effects of BV on IL-10 expression are lost with blockade of Akt. Inhibition of surface BVR with RNA interference attenuates BV-induced Akt signaling and IL-10 expression and in vivo negates the cytoprotective effects of BV in models of shock and acute hepatitis. Collectively, our findings elucidate a potentially important new molecular mechanism by which BV, through the enzymatic activity and phosphorylation of surface BVR (BVR)(surf) modulates the inflammatory response.

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-IXalpha, 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.