Viktória Jeney

Mechanisms of cell protection by heme Oxygenase-1

Heme oxygenases (HO) catabolize free heme, that is, iron (Fe) protoporphyrin (IX), into equimolar amounts of Fe(2+), carbon monoxide (CO), and biliverdin. The stress-responsive HO-1 isoenzyme affords protection against programmed cell death. The mechanism underlying this cytoprotective effect relies on the ability of HO-1 to catabolize free heme and prevent it from sensitizing cells to undergo programmed cell death. This cytoprotective effect inhibits the pathogenesis of a variety of immune-mediated inflammatory diseases.

Heme oxygenase-1 and carbon monoxide suppress the pathogenesis of experimental cerebral malaria

Cerebral malaria claims more than 1 million lives per year. We report that heme oxygenase-1 (HO-1, encoded by Hmox1) prevents the development of experimental cerebral malaria (ECM). BALB/c mice infected with Plasmodium berghei ANKA upregulated HO-1 expression and activity and did not develop ECM. Deletion of Hmox1 and inhibition of HO activity increased ECM incidence to 83% and 78%, respectively. HO-1 upregulation was lower in infected C57BL/6 compared to BALB/c mice, and all infected C57BL/6 mice developed ECM (100% incidence). Pharmacological induction of HO-1 and exposure to the end-product of HO-1 activity, carbon monoxide (CO), reduced ECM incidence in C57BL/6 mice to 10% and 0%, respectively. Whereas neither HO-1 nor CO affected parasitemia, both prevented blood-brain barrier (BBB) disruption, brain microvasculature congestion and neuroinflammation, including CD8+ T-cell brain sequestration. These effects were mediated by the binding of CO to hemoglobin, preventing hemoglobin oxidation and the generation of free heme, a molecule that triggers ECM pathogenesis.

A Central Role for Free Heme in the Pathogenesis of Severe Sepsis

Heme from red blood cells released in septic shock worsens organ dysfunction and increases the risk of death, but can be overcome by a scavenger of free heme. Casting Heme in a New Light Sepsis, or severe systemic infection, is a deadly disease that has always been difficult to treat. Despite modern-day antibiotics and intensive care management, patients with sepsis still have a high rate of major complications and death. These severe consequences are thought to be a result of simultaneous overwhelming infection and an overexuberant immune response, which together damage tissues and lead to organ dysfunction. One cell type that is injured during sepsis is the erythrocyte. As these red blood cells lyse, hemoglobin is released and oxidized, releasing free heme into the circulation. This heme is not an innocent bystander, however, as Larsen et al. now report. It increases inflammation and cell death, exacerbating the damage to the body and increasing the risk of death. The authors found that mice lacking heme oxygenase 1, the enzyme that breaks down heme into harmless by-products, have more free circulating heme, which makes them more susceptible to death from sepsis than are matching wild-type mice. In addition, giving extra heme to wild-type mice suffering from sepsis greatly increases their risk of organ dysfunction and death without affecting the number of bacteria in their blood. Moreover, hemopexin, a protein produced by the body to scavenge free heme, protects mice and human patients with sepsis from the deleterious effects of heme and decreases the risk of complications and death. Because these authors have shown that heme concentrations are associated with worse prognosis in sepsis patients, we may now have a new way to monitor patients’ health status and, eventually, to treat them. Measurements of heme and hemopexin in patients with sepsis may predict who needs more intensive interventions, potentially allowing for more timely treatment before organ failure ensues. In addition, high-risk patients could be given extra hemopexin or other heme-neutralizing substances to possibly save them from death caused by sepsis, even when all the current treatments fail. Low-grade polymicrobial infection induced by cecal ligation and puncture is lethal in heme oxygenase-1–deficient mice (Hmox1−/−), but not in wild-type (Hmox1+/+) mice. Here we demonstrate that the protective effect of this heme-catabolizing enzyme relies on its ability to prevent tissue damage caused by the circulating free heme released from hemoglobin during infection. Heme administration after low-grade infection in mice promoted tissue damage and severe sepsis. Free heme contributed to the pathogenesis of severe sepsis irrespective of pathogen load, revealing that it compromised host tolerance to infection. Development of lethal forms of severe sepsis after high-grade infection was associated with reduced serum concentrations of the heme sequestering protein hemopexin (HPX), whereas HPX administration after high-grade infection prevented tissue damage and lethality. Finally, the lethal outcome of septic shock in patients was also associated with reduced HPX serum concentrations. We propose that targeting free heme by HPX might be used therapeutically to treat severe sepsis.

Sickle Hemoglobin Confers Tolerance to Plasmodium Infection

Sickle human hemoglobin (Hb) confers a survival advantage to individuals living in endemic areas of malaria, the disease caused by Plasmodium infection. As demonstrated hereby, mice expressing sickle Hb do not succumb to experimental cerebral malaria (ECM). This protective effect is exerted irrespectively of parasite load, revealing that sickle Hb confers host tolerance to Plasmodium infection. Sickle Hb induces the expression of heme oxygenase-1 (HO-1) in hematopoietic cells, via a mechanism involving the transcription factor NF-E2-related factor 2 (Nrf2). Carbon monoxide (CO), a byproduct of heme catabolism by HO-1, prevents further accumulation of circulating free heme after Plasmodium infection, suppressing the pathogenesis of ECM. Moreover, sickle Hb inhibits activation and/or expansion of pathogenic CD8(+) T cells recognizing antigens expressed by Plasmodium, an immunoregulatory effect that does not involve Nrf2 and/or HO-1. Our findings provide insight into molecular mechanisms via which sickle Hb confers host tolerance to severe forms of malaria.

Peroxisome Proliferator-activated Receptor γ-regulated ABCG2 Expression Confers Cytoprotection to Human Dendritic Cells

ABCG2, a member of the ATP-binding cassette transporters has been identified as a protective pump against endogenous and exogenous toxic agents. ABCG2 was shown to be expressed at high levels in stem cells and variably regulated during cell differentiation. Here we demonstrate that functional ABCG2 is expressed in human monocyte-derived dendritic cells by the activation of a nuclear hormone receptor, PPARγ. We identified and characterized a 150-base pair long conserved enhancer region, containing three functional PPAR response elements (PPARE), upstream of the human ABCG2 gene. We confirmed the binding of the PPARγ·RXR heterodimer to this enhancer region, suggesting that PPARγ directly regulates the transcription of ABCG2. Consistent with these results, elevated expression of ABCG2 mRNA was coupled to enhanced protein production, resulting in increased xenobiotic extrusion capacity via ABCG2 in PPARγ-activated cells. Furthermore PPARγ instructed dendritic cells showed increased Hoechst dye extrusion and resistance to mitoxantrone. Collectively, these results uncovered a mechanism by which up-regulation of functional ABCG2 expression can be achieved via exogenous or endogenous activation of the lipid-activated transcription factor, PPARγ. The increased expression of the promiscuous ABCG2 transporter can significantly modify the xenobiotic and drug resistance of human myeloid dendritic cells.

Red Cells, Hemoglobin, Heme, Iron, and Atherogenesis

Objective—We investigated whether red cell infiltration of atheromatous lesions promotes the later stages of atherosclerosis. Methods and Results—We find that oxidation of ferro (FeII) hemoglobin in ruptured advanced lesions occurs generating ferri (FeIII) hemoglobin and via more extensive oxidation ferrylhemoglobin (FeIII/FeIV=O). The protein oxidation marker dityrosine accumulates in complicated lesions, accompanied by the formation of cross-linked hemoglobin, a hallmark of ferrylhemoglobin. Exposure of normal red cells to lipids derived from atheromatous lesions causes hemolysis and oxidation of liberated hemoglobin. In the interactions between hemoglobin and atheroma lipids, hemoglobin and heme promote further lipid oxidation and subsequently endothelial reactions such as upregulation of heme oxygenase-1 and cytotoxicity to endothelium. Oxidative scission of heme leads to release of iron and a feed-forward process of iron-driven plaque lipid oxidation. The inhibition of heme release from globin by haptoglobin and sequestration of heme by hemopexin suppress hemoglobin-mediated oxidation of lipids of atheromatous lesions and attenuate endothelial cytotoxicity. Conclusion—The interior of advanced atheromatous lesions is a prooxidant environment in which erythrocytes lyse, hemoglobin is oxidized to ferri- and ferrylhemoglobin, and released heme and iron promote further oxidation of lipids. These events amplify the endothelial cell cytotoxicity of plaque components.

A central role for free heme in the pathogenesis of severe malaria: The missing link?

Malaria, the disease caused by Plasmodium infection, is endemic to poverty in so-called underdeveloped countries. Plasmodium falciparum, the main infectious Plasmodium species in sub-Saharan countries, can trigger the development of severe malaria, including cerebral malaria, a neurological syndrome that claims the lives of more than one million children (<5 years old) per year. Attempts to eradicate Plasmodium infection, and in particular its lethal outcomes, have so far been unsuccessful. Using well-established rodent models of malaria infection, we found that survival of a Plasmodium-infected host is strictly dependent on the host’s ability to up-regulate the expression of heme oxygenase-1 (HO-1 encoded by the gene Hmox1). HO-1 is a stress-responsive enzyme that catabolizes free heme into biliverdin, via a reaction that releases Fe and generates the gas carbon monoxide (CO). Generation of CO through heme catabolism by HO-1 prevents the onset of cerebral malaria. The protective effect of CO is mediated via its binding to cell-free hemoglobin (Hb) released from infected red blood cells during the blood stage of Plasmodium infection. Binding of CO to cell-free Hb prevents heme release and thus generation of free heme, which we found to play a central role in the pathogenesis of cerebral malaria. We will address hereby how defense mechanisms that prevent the deleterious effects of free heme, including the expression of HO-1, impact on the pathologic outcome of Plasmodium infection and how these may be used therapeutically to suppress its lethal outcomes.

Early-Onset Carotid Atherosclerosis Is Associated With Increased Intima-Media Thickness and Elevated Serum Levels of Inflammatory Markers

Background and Purpose— Several factors have been held responsible for the development of atherosclerosis. To avoid the masking effect of age, we evaluated correlates of carotid atherosclerosis in patients <55 years of age. Methods— Plasma lipids, oxidative resistance of low-density lipoprotein, homocysteine, inflammatory markers, plasma viscosity, and red cell deformability were measured in fasting blood samples of 100 subjects: 45 patients with >30% stenosis of the internal carotid artery, 20 patients with carotid occlusion, and 35 control subjects. Stenosis and intima-media thickness (IMT) of the carotid artery were evaluated by duplex ultrasound. Results— White blood cell (WBC) count, plasma fibrinogen, C-reactive protein (CRP), and lipoprotein(a) levels were significantly higher in patients than in control subjects, and patients had increased IMT (P <0.01 for all comparisons). There was a tendency for higher homocysteine levels in patients. Smokers had higher WBC, fibrinogen, and CRP levels. After the effect of smoking was controlled for, WBC count, natural logarithmic transform of homocysteine, and online-measured IMT remained significantly higher in patients than in control subjects. WBC, fibrinogen, and CRP levels were highest in the highest IMT quartile (P =0.012, P =0.007, and P =0.036, respectively). Conclusions— Inflammatory markers and homocysteine have a more important role than lipid factors in early-onset carotid atherosclerosis. We cannot recommend the measurement of low-density lipoprotein peroxidation as a routine screening test to identify high-risk patients for early-onset carotid atherosclerosis. The confounding effect of smoking on inflammatory markers should be considered in studies on atherosclerosis.

Heme Oxygenase 1 Determines Atherosclerotic Lesion Progression Into a Vulnerable Plaque

Background— The molecular regulation for the transition from stable to vulnerable plaque remains to be elucidated. Heme oxygenase 1 (HO-1) and its metabolites have been implicated in the cytoprotective defense against oxidative injury in atherogenesis. In this study, we sought to assess the role of HO-1 in the progression toward plaque instability in carotid artery disease in patients and in a murine model of vulnerable plaque development. Methods and Results— Atherectomy biopsy from 112 patients with clinical carotid artery disease was collected and stratified according to characteristics of plaque vulnerability. HO-1 expression correlated closely with features of vulnerable human atheromatous plaque (P<0.005), including macrophage and lipid accumulation, and was inversely correlated with intraplaque vascular smooth muscle cells and collagen deposition. HO-1 expression levels correlated with the plaque destabilizing factors matrix metalloproteinase-9, interleukin-8, and interleukin-6. Likewise, in a vulnerable plaque model using apolipoprotein E−/− mice, HO-1 expression was upregulated in vulnerable versus stable lesions. HO-1 induction by cobalt protoporphyrin impeded lesion progression into vulnerable plaques, indicated by a reduction in necrotic core size and intraplaque lipid accumulation, whereas cap thickness and vascular smooth muscle cells were increased. In contrast, inhibition of HO-1 by zinc protoporphyrin augmented plaque vulnerability. Plaque stabilizing was prominent after adenoviral transduction of HO-1 compared with sham virus–treated animals, providing proof that the observed effects on plaque vulnerability were HO-1 specific. Conclusions— Here we demonstrate in a well-defined patient group and a murine vulnerable plaque model that HO-1 induction reverses plaque progression from a vulnerable plaque to a more stable phenotype as part of a compensatory atheroprotective response.

Fibulin-5 Is a Novel Binding Protein for Extracellular Superoxide Dismutase

The extracellular superoxide dismutase (ecSOD) plays an important role in atherosclerosis and endothelial function by modulating levels of the superoxide anion (O2·−) in the extracellular space. Although heparan sulfate proteoglycan is an important ligand for ecSOD, little is known about other biological binding partners of ecSOD. The goal of this study was to identify novel proteins that interact with ecSOD. A yeast two-hybrid screening of a human aorta cDNA library using ecSOD as bait identified fibulin-5 as a predominant binding protein for ecSOD. Further analysis showed that the binding domain of ecSOD within fibulin-5 mapped to its C-terminal domain. In vitro pulldown assays and coimmunoprecipitation analysis further confirmed that ecSOD interacts with fibulin-5 in vitro and in vivo. Studies using fibulin-5−/− mice indicated that fibulin-5 is required for binding of ecSOD to vascular tissue. Importantly, the decrease in tissue-bound ecSOD levels in aortas from fibulin-5−/− mice was associated with an increase in vascular O2·− levels. Furthermore, immunohistochemical analysis using ApoE−/− mice suggested a codistribution of ecSOD and fibulin-5 in atherosclerotic vessels. In summary, we provide in this study the first evidence that the ecSOD-fibulin-5 interaction is required for ecSOD binding to vascular tissues, thereby regulating vascular O2·− levels. This interaction may represent a novel mechanism for controlling vascular redox state in the extracellular space in various cardiovascular diseases such as atherosclerosis and hypertension in which oxidative stress is increased.