Higher Levels Of Heme Oxygenase-1 Research Articles

The BRD4 Inhibitor I-BET-762 Reduces HO-1 Expression in Macrophages and the Pancreas of Mice

In pancreatic cancer, the tumor microenvironment (TME) accounts for up to 90% of the tumor mass. Pancreatitis, characterized by the increased infiltration of macrophages into the pancreas, is a known risk factor for pancreatic cancer. The NRF2 (nuclear factor erythroid 2-related factor 2) transcription factor regulates responses to oxidative stress and can promote cancer and chemoresistance. NRF2 also attenuates inflammation through the regulation of macrophage-specific genes. Heme oxygenase 1 (HO-1) is expressed by anti-inflammatory macrophages to degrade heme, and its expression is dependent on NRF2 translocation to the nucleus. In macrophages stimulated with conditioned media from pancreatic cancer cells, HO-1 protein levels increased, which correlated with higher NRF2 expression in the nuclear fraction. Significant differences in macrophage infiltration and HO-1 expression were detected in LSL-KrasG12D/+; Pdx-1-Cre (KC) mice, Nrf2 whole-body knockout (KO) mice and wildtype mice with pancreatitis. Since epigenetic modulation is a mechanism used by tumors to regulate the TME, using small molecules as epigenetic modulators to activate immune recognition is therapeutically desirable. When the bromodomain inhibitor I-BET-762 was used to treat macrophages or mice with pancreatitis, high levels of HO-1 were reduced. This study shows that bromodomain inhibitors can be used to prevent physiological responses to inflammation that promote tumorigenesis.

Serum heme oxygenase-1 level predicts clinical outcome after acute ischemic stroke.

The relationship between heme oxygenase-1 (HO-1) and human ischemic stroke outcome remains unclear, which was investigated in this study. Acute ischemic stroke patients admitted within 24 h were enrolled. Serum HO-1 levels at baseline were measured via ELISA. Poor 3-month functional outcome was defined as modified Rankin Scale (mRS) score 3-6. Multivariable-adjusted binary logistic regression and restricted cubic spline models were employed to examine association between serum HO-1 and functional outcome. HO-1's additive prognostic utility was assessed by net reclassification index (NRI) and integrated discrimination improvement (IDI). Of 194 eligible patients, 79 (40.7%) developed poor functional outcomes at 3-month follow-up. The highest quartile of serum HO-1 was independently associated with a lower risk of poor functional outcome (adjusted OR 0.13, 95% CI 0.04-0.45; p = 0.001) compared with the lowest HO-1 category. The relationship between higher HO-1 levels and reduced risk of poor functional outcome was linear and dose responsive (p = 0.002 for linearity). Incorporating HO-1 into the analysis with conventional factors significantly improved reclassification for poor functional outcomes (NRI = 41.2%, p = 0.004; IDI = 5.0%, p = 0.004). Elevated serum HO-1 levels at baseline were independently associated with improved 3-month functional outcomes post-ischemic stroke. Serum HO-1 measurement may enhance outcome prediction beyond conventional clinical factors.

Implication of noise exposure on hearing with emphasis to hOGG1 and GPx-1 polymorphisms and HO-1 protein among textile workers

Noise exposure is a health hazard in the textile industry. In cochlear hair cells, DNA damage caused by 8-oxoguanine (8-oxo G) can result in noise-induced hearing loss. Human 8-hydroxyguanine glycosylase (hOGG1) is a DNA repair enzyme that excises (8-oxo G) in the DNA and repairs DNA damage. Glutathione peroxidase-1 (GPx) is a crucial antioxidant enzyme that aids in limiting cochlear damages. Heme oxygenase-1 (HO-1) is a stress-inducible protein with a high fold change in the hair cells of the cochlea. The study aimed to investigate the association of either hOGG1 and GPx-1 polymorphisms with audiometric notches and HO-1 protein among textile workers. hOGG1 and GPx genotypes were analyzed by PCR–RFLP, and HO-1 levels were measured by ELISA in 115 male textile workers. Blood pressure and audiogram were performed. Results recorded the relation between audiometric notches and ear complaints among workers. Older age workers showed audiometric notches at > 25 dB with a significant decrease in HO-1 levels and higher levels in workers with normal audiogram. Ser/Cys genotype of hOGG1 gene was associated with age and work duration while CC genotype of GPx is associated with HO-1 levels and diastolic pressure. Ser/Cys genotype of hOGG1 gene was associated with age while Cys/Cys genotype was associated with work duration among workers. CC genotype of GPx gene was associated with higher HO-1 levels and TT genotype was associated with high diastolic pressure. Finally, hearing impairment was dependent on the duration of exposure to noise, older age, and the presence of heterozygote TC genotype of GPx gene among textile workers.

Iron cycle disruption by heme oxygenase-1 activation leads to a reduced breast cancer cell survival

Heme oxygenase-1 (HO-1), which catalyzes heme degradation releasing iron, regulates several processes related to breast cancer. Iron metabolism deregulation is also connected with several tumor processes. However the regulatory relationship between HO-1 and iron proteins in breast cancer remains unclear. Using human breast cancer biopsies, we found that high HO-1 levels significantly correlated with low DMT1 levels. Contrariwise, high HO-1 levels significantly correlated with high ZIP14 and prohepcidin expression, as well as hemosiderin storage. At mRNA level, we found that high HO-1 expression significantly correlated with low DMT1 expression but high ZIP14, L-ferritin and hepcidin expression. In in vivo experiments in mice with genetic overexpression or pharmacological activation of HO-1, we detected the same expression pattern observed in human biopsies. In in vitro experiments, HO-1 activation induced changes in iron proteins expression leading to an increase of hemosiderin, ROS levels, lipid peroxidation and a decrease of the growth rate. Such low growth rate induced by HO-1 activation was reversed when iron levels or ROS levels were reduced. Our findings demonstrate an important role of HO-1 on iron homeostasis in breast cancer. The changes in iron proteins expression when HO-1 is modulated led to the iron accumulation deregulating the iron cell cycle, and consequently, generating oxidative stress and low viability, all contributing to impair breast cancer progression.

Impact of Oxidative Stress on Risk of Death and Readmission in African Children With Severe Malaria: A Prospective Observational Study.

We hypothesized that oxidative stress in Ugandan children with severe malaria is associated with mortality. We evaluated biomarkers of oxidative stress in children with cerebral malaria (CM, n = 77) or severe malarial anemia (SMA, n = 79), who were enrolled in a randomized clinical trial of immediate vs delayed iron therapy, compared with community children (CC, n = 83). Associations between admission biomarkers and risk of death during hospitalization or risk of readmission within 6 months were analyzed. Nine children with CM and none with SMA died during hospitalization. Children with CM or SMA had higher levels of heme oxygenase-1 (HO-1) (P < .001) and lower superoxide dismutase (SOD) activity than CC (P < .02). Children with CM had a higher risk of death with increasing HO-1 concentration (odds ratio [OR], 6.07 [95% confidence interval {CI}, 1.17-31.31]; P = .03) but a lower risk of death with increasing SOD activity (OR, 0.02 [95% CI, .001-.70]; P = .03). There were no associations between oxidative stress biomarkers on admission and risk of readmission within 6 months of enrollment. Children with CM or SMA develop oxidative stress in response to severe malaria. Oxidative stress is associated with higher mortality in children with CM but not with SMA. NCT01093989.

Recruitment of monocytes primed to express heme oxygenase-1 ameliorates pathological lung inflammation in cystic fibrosis

Overwhelming neutrophilic inflammation is a leading cause of lung damage in many pulmonary diseases, including cystic fibrosis (CF). The heme oxygenase-1 (HO-1)/carbon monoxide (CO) pathway mediates the resolution of inflammation and is defective in CF-affected macrophages (MΦs). Here, we provide evidence that systemic administration of PP-007, a CO releasing/O2 transfer agent, induces the expression of HO-1 in a myeloid differentiation factor 88 (MyD88) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)-dependent manner. It also rescues the reduced HO-1 levels in CF-affected cells induced in response to lipopolysaccharides (LPS) or Pseudomonas aeruginosa (PA). Treatment of CF and muco-obstructive lung disease mouse models with a single clinically relevant dose of PP-007 leads to effective resolution of lung neutrophilia and to decreased levels of proinflammatory cytokines in response to LPS. Using HO-1 conditional knockout mice, we show that the beneficial effect of PP-007 is due to the priming of circulating monocytes trafficking to the lungs in response to infection to express high levels of HO-1. Finally, we show that PP-007 does not compromise the clearance of PA in the setting of chronic airway infection. Overall, we reveal the mechanism of action of PP-007 responsible for the immunomodulatory function observed in clinical trials for a wide range of diseases and demonstrate the potential use of PP-007 in controlling neutrophilic pulmonary inflammation by promoting the expression of HO-1 in monocytes/macrophages.

A Dual Role of Heme Oxygenase-1 in Tuberculosis.

Iron metabolism is vital for the survival of both humans and microorganisms. Heme oxygenase-1 (HO-1) is an essential stress-response enzyme highly expressed in the lungs, and catabolizes heme into ferrous iron, carbon monoxide (CO), and biliverdin (BV)/bilirubin (BR), especially in pathological conditions which cause oxidative stress and inflammation. Ferrous iron (Fe2+) is an important raw material for the synthesis of hemoglobin in red blood cells, and patients with iron deficiency are often associated with decreased cellular immunity. CO and BR can inhibit oxidative stress and inflammation. Thus, HO-1 is regarded as a cytoprotective molecule during the infection process. However, recent study has unveiled new information regarding HO-1. Being a highly infectious pathogenic bacterium, Mycobacterium tuberculosis (MTB) infection causes acute oxidative stress, and increases the expression of HO-1, which may in turn facilitate MTB survival and growth due to increased iron availability. Moreover, in severe cases of MTB infection, excessive reactive oxygen species (ROS) and free iron (Fe2+) due to high levels of HO-1 can lead to lipid peroxidation and ferroptosis, which may promote further MTB dissemination from cells undergoing ferroptosis. Therefore, it is important to understand and illustrate the dual role of HO-1 in tuberculosis. Herein, we critically review the interplay among HO-1, tuberculosis, and the host, thus paving the way for development of potential strategies for modulating HO-1 and iron metabolism.

Restriction of HIV-1 infection in sickle cell trait

AbstractPatients with sickle cell disease (SCD) have a lower risk for HIV-1 infection. We reported restriction of ex vivo HIV-1 infection in SCD peripheral blood mononuclear cells (PBMCs) that was due, in part, to the upregulation of antiviral, inflammatory, and hemolytic factors, including heme oxygenase-1 (HO-1). Here, we investigated whether individuals with sickle cell trait (SCT), who develop mild hemolysis, also restrict HIV-1 infection. Ex vivo infection of SCT PBMCs exhibited an approximately twofold reduction of HIV-1 replication and lower levels of HIV-1 reverse transcription products, 2-long terminal repeat circle, HIV-1 integration, and gag RNA expression. SCT PBMCs had higher HO-1 messenger RNA (mRNA) and protein levels and reduced ribonucleotide reductase 2 (RNR2) protein levels. HO-1 inhibition by tin porphyrin eliminated ex vivo HIV-1 restriction. Among Howard University clinic recruits, higher levels of HO-1 and RNR2 mRNA and lower HIV-1 env mRNA levels were found in SCT individuals living with HIV-1. To determine the population-level effect of SCT on HIV-1 prevalence, we assessed SCT among women living with HIV (WLH) in the WIHS (Women Interagency HIV-1 Study). Among WIHS African-American participants, the prevalence of SCT was lower among women with HIV compared with uninfected women (8.7% vs 14.2%; odds ratio, 0.57; 95% confidence interval, 0.36-0.92; P = .020). WIHS WLH with SCT had higher levels of CD4+/CD8+ ratios over 20 years of follow-up (P = .003) than matched WLH without SCT. Together, our findings suggest that HIV-1 restriction factors, including HO-1 and RNR2, might restrict HIV-1 infection among individuals with SCT and limit the pathogenicity of HIV.

Clinical Significance of Heme Oxygenase 1 in Tumor Progression.

Heme oxygenase 1 (HO-1) plays a key role in cell adaptation to stressors through the antioxidant, antiapoptotic, and anti-inflammatory properties of its metabolic products. For these reasons, in cancer cells, HO-1 can favor aggressiveness and resistance to therapies, leading to poor prognosis/outcome. Genetic polymorphisms of HO-1 promoter have been associated with an increased risk of cancer progression and a high degree of therapy failure. Moreover, evidence from cancer biopsies highlights the possible correlation between HO-1 expression, pathological features, and clinical outcome. Indeed, high levels of HO-1 in tumor specimens often correlate with reduced survival rates. Furthermore, HO-1 modulation has been proposed in order to improve the efficacy of antitumor therapies. However, contrasting evidence on the role of HO-1 in tumor biology has been reported. This review focuses on the role of HO-1 as a promising biomarker of cancer progression; understanding the correlation between HO-1 and clinical data might guide the therapeutic choice and improve the outcome of patients in terms of prognosis and life quality.

Increased Plasma Heme Oxygenase-1 Levels in Patients With Early-Stage Parkinson's Disease.

Introduction: Heme oxygenase-1 (HO-1) is a 32 kDa stress-response protein implicated in the pathogenesis of Parkinson’s disease (PD). Biliverdin is derived from heme through a reaction mediated by HO-1 and protects cells from oxidative stress. However, iron and carbon monoxide produced by the catabolism of HO-1 exert detrimental effects on patients with PD. The purpose of this study was to determine whether plasma HO-1 levels represent a biomarker of PD and to further explore the underlying mechanism of increased HO-1 levels by applying voxel-based morphometry (VBM).Methods: We measured plasma HO-1 levels using an enzyme-linked immunosorbent assay (ELISA) in 156 subjects, including 81 patients with early- and advanced-stage PD and 75 subjects without PD. The analyses were adjusted to control for confounders such as age, sex, and medication. We analyzed T1-weighted magnetic resonance imaging (MRI) data from 74 patients with PD using VBM to elucidate the association between altered brain volumes and HO-1 levels. Then, we compared performance on MMSE sub-items between PD patients with low and high levels of HO-1 using Mann-Whitney U tests.Results: Plasma HO-1 levels were significantly elevated in PD patients, predominantly those with early-stage PD, compared with controls (p < 0.05). The optimal cutoff value for patients with early PD was 2.245 ng/ml HO-1 [area under the curve (AUC) = 0.654]. Plasma HO-1 levels were unaffected by sex, age, and medications (p > 0.05). The right hippocampal volume was decreased in the subset of PD patients with high HO-1 levels (p < 0.05). A weak correlation was observed between right hippocampal volume and plasma HO-1 levels (r = −0.273, p = 0.018). There was no difference in total MMSE scores between the low- and high-HO-1 groups (p > 0.05), but the high-HO-1 group had higher language scores than the low-HO-1 group (p < 0.05).Conclusions: Plasma HO-1 levels may be a promising biomarker of early PD. Moreover, a high plasma concentration of the HO-1 protein is associated with a reduction in right hippocampal volume.

SIAH2-mediated and organ-specific restriction of HO-1 expression by a dual mechanism

The intracellular levels of the cytoprotective enzyme heme oxygenase-1 (HO-1) are tightly controlled. Here, we reveal a novel mechanism preventing the exaggerated expression of HO-1. The analysis of mice with a knock-out in the ubiquitin E3 ligase seven in absentia homolog 2 (SIAH2) showed elevated HO-1 protein levels in specific organs such as heart, kidney and skeletal muscle. Increased HO-1 protein amounts were also seen in human cells deleted for the SIAH2 gene. The higher HO-1 levels are not only due to an increased protein stability but also to elevated expression of the HO-1 encoding HMOX1 gene, which depends on the transcription factor nuclear factor E2-related factor 2 (NRF2), a known SIAH2 target. Dependent on its RING (really interesting new gene) domain, expression of SIAH2 mediates proteasome-dependent degradation of its interaction partner HO-1. Additionally SIAH2-deficient cells are also characterized by reduced expression levels of glutathione peroxidase 4 (GPX4), rendering the knock-out cells more sensitive to ferroptosis.

Activation of Nrf2/HO-1 Pathway and Human Atherosclerotic Plaque Vulnerability:an In Vitro and In Vivo Study.

Reactive oxygen species (ROS) induce nuclear factor erythroid 2–related factor 2 (Nrf2) activation as an adaptive defense mechanism, determining the synthesis of antioxidant molecules, including heme-oxygenase-1 (HO-1). HO-1 protects cells against oxidative injury, degrading free heme and inhibiting ROS production. HO-1 is highly expressed in macrophages during plaque growth. Macrophages are morpho-functionally heterogeneous, and the prevalence of a specific phenotype may influence the plaque fate. This heterogeneity has also been observed in monocyte-derived macrophages (MDMs), a model of macrophages infiltrating tissue. The study aims to assess oxidative stress status and Nrf2/HO-1 axis in MDM morphotypes obtained from healthy subjects and coronary artery disease (CAD) patients, in relation to coronary plaque features evaluated in vivo by optical coherence tomography (OCT). We found that MDMs of healthy subjects exhibited a lower oxidative stress status, lower Nrf2 and HO-1 levels as compared to CAD patients. High HO-1 levels in MDMs were associated with the presence of a higher macrophage content, a thinner fibrous cap, and a ruptured plaque with thrombus formation, detected by OCT analysis. These findings suggest the presence of a relationship between in vivo plaque characteristics and in vitro MDM profile, and may help to identify patients with rupture-prone coronary plaque.

Patrolling monocytes in sickle cell hemolytic conditions

Patients with sickle cell disease (SCD) suffer from intravascular hemolysis associated with vascular injury and dysfunction. Painful vaso-occlusive crisis (VOC) involving increased attachment of sickle erythrocytes and activated leukocytes to damaged vascular endothelium is a hallmark of SCD. Patrolling monocytes, which normally scavenge damaged cells and debris from the vasculature, express higher levels of anti-inflammatory heme oxygenase 1 (HO-1), a heme degrading enzyme with anti-cytotoxic and anti-inflammatory properties. Recent data show that patients with SCD have a novel subset of patrolling monocytes expressing very high levels of HO-1 (HO-1hi) which are decreased in numbers in patients who had a recent VOC episode. This patrolling monocyte subset was responsible for protection of endothelium against sickle RBC stasis in an experimental model. This raises the possibility that patrolling monocytes may also offer protection against vascular endothelium damage in hyperhemolytic conditions in SCD.

Patrolling Monocytes Scavenge Endothelial Adherent Sickle Red Blood Cells in Sickle Cell Disease

Sickle cell disease (SCD) is characterized by hemolytic anemia and increased entrapment of sickle red blood cells (RBCs) via attachment to the underlying activated vascular endothelium, resulting in vaso-occlusive crisis (VOC), marked by severe pain. The endothelial scavenging patrolling monocytes (PMos) expressing high levels of the heme oxygenase 1 (HO-1), a heme degrading enzyme, were recently shown to protect against vaso-occlusion in SCD, although their ability to scavenge endothelial-attached sickle RBCs was not tested. Here, we found that circulating PMos from SCD patients showed roughly 5% ± 0.5% engulfed GPA+ or Band3+ RBC specific material as compared to 0.7% ± 0.04% in healthy donor (HD) PMos or 0.85% ± 0.07% in SCD classical monocytes (CMos) as detected by flow as well as imagining flow cytometry, suggesting that PMos uptake RBCs in SCD. To further investigate this, RBCs purified from HDs (to mimic transfused cells) or SCD patients were labelled with CFSE, and co-cultured with purified monocytes without or with human microvascular endothelial cells (HMVEC). We found 11% ± 0.5% CFSE+ PMos in co-cultures with SCD RBCs in presence of HMVEC as compared to 2.7% ± 0.4% when cultured with HD RBCs, indicating that PMos engulf sickle RBCs, but not HD RBCs. Low levels of CFSE+ PMos (2-3%) were detected in cocultures with either sickle or control RBCs in the absence of HMVEC, implicating that PMos preferentially uptake endothelial cell (EC)-attached sickle RBCs. In contrast to PMos, CMos always had minimal CFSE+ reactivity (2-3%) when cultured with sickle RBCs or HD RBCs with or without HMVEC, supporting a role for PMos, but not CMos, as scavengers of sickle RBCs in the vasculature. Further analysis revealed significantly increased (two-fold) levels of annexin V+ apoptotic marker on sickle RBC engulfed PMos (CFSE+PMos), but also higher levels of HO-1 ((50% ± 3%) as compared to non-engulfed (CFSE-) PMos (1.3% ± 0.4%), suggesting that induction of HO-1 upon uptake of sickle RBCs may counteract the cytotoxic effects of engulfed RBC breakdown products in PMos. To test this hypothesis, monocytes were pre-treated with tin protoporphyrin IX (SnPPIX) to inhibit HO-1 activity prior to coculture with sickle RBCs and HMVEC. We found increased apoptosis in PMos from SnPPIX-treated (18% ± 0.8%) as compared to untreated cultures (6.6% ± 0.6%), consistent with a cytoprotective role of HO-1 induction in sickle RBC-engulfed PMos. Antibody blocking studies identified ICAM-1, VCAM-1, CD11a, CD18 as well as CD16 as key molecules involved in PMo-HMVEC-sickle RBC interactions, but not CD11b, CD11c, CD31, PSGL-1, CD32, CD64, Fc a/m receptor and phosphatidylserine. Interestingly, HMVEC activation induced by heme treatment resulted in significantly higher CFSE+ PMos (17.8% ± 0.9%) when cultured with sickle RBCs, but not HD RBCs (3.5% ± 0.6%), indicating that PMos preferentially uptake sickle RBCs bound to heme-treated ECs. To formally test this, DiI labelled mouse sickle or control RBCs were transfused to heme-treated Nr4a1-GFP mice which express GFP on their circulating PMos, followed by perfusion to remove non-EC attached cells. Using confocal microscopy, we detected DiI+ sickle but not control non-sickle RBCs, engulfed by GFP+ PMos in the vasculature of perfused recipients, confirming uptake of EC-attached sickle RBCs by PMos. Consistent with increased EC-attached sickle RBCs during VOC, SCD patients experiencing acute VOC (n=12) showed increased frequency of GPA+ PMos (13% ± 0.1% vs 5% ± 0.2%, p<0.01) but still only 1.8% ± 0.3% GPA+ CMos. However, total numbers of PMos including HO-1hi PMos were more than two-fold lower in these patients as compared to patients at steady state. Altogether, these data demonstrate for the first time that in SCD, PMos scavenge EC-attached sickle RBCs, but not transfused HD RBCs, through interactions involving manly integrins, resulting in HO-1 upregulation to counteract the cytotoxic effects of engulfed RBC breakdown products. With increased adherence of sickle RBCs to vasculature as a result of damage to ECs such as during VOC, PMos scavenge more RBCs, but their numbers become limiting with implications for beneficial effects of transfusions and potential for PMos as therapeutic targets against VOC in SCD. DisclosuresNo relevant conflicts of interest to declare.

Could Heme Oxygenase-1 Be a New Target for Therapeutic Intervention in Malaria-Associated Acute Lung Injury/Acute Respiratory Distress Syndrome?

Malaria is a serious disease and was responsible for 429,000 deaths in 2015. Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is one of the main clinical complications of severe malaria; it is characterized by a high mortality rate and can even occur after antimalarial treatment when parasitemia is not detected. Rodent models of ALI/ARDS show similar clinical signs as in humans when the rodents are infected with murine Plasmodium. In these models, it was shown that the induction of the enzyme heme oxygenase 1 (HO-1) is protective against severe malaria complications, including cerebral malaria and ALI/ARDS. Increased lung endothelial permeability and upregulation of VEGF and other pro-inflammatory cytokines were found to be associated with malaria-associated ALI/ARDS (MA-ALI/ARDS), and both were reduced after HO-1 induction. Additionally, mice were protected against MA-ALI/ARDS after treatment with carbon monoxide- releasing molecules or with carbon monoxide, which is also released by the HO-1 activity. However, high HO-1 levels in inflammatory cells were associated with the respiratory burst of neutrophils and with an intensification of inflammation during episodes of severe malaria in humans. Here, we review the main aspects of HO-1 in malaria and ALI/ARDS, presenting the dual role of HO-1 and possibilities for therapeutic intervention by modulating this important enzyme.

HO-1hi patrolling monocytes protect against vaso-occlusion in sickle cell disease

AbstractPatients with sickle cell disease (SCD) suffer from intravascular hemolysis associated with vascular injury and dysfunction in mouse models, and painful vaso-occlusive crisis (VOC) involving increased attachment of sickle erythrocytes and activated leukocytes to damaged vascular endothelium. Patrolling monocytes, which normally scavenge damaged cells and debris from the vasculature, express higher levels of anti-inflammatory heme oxygenase 1 (HO-1), a heme degrading enzyme. Here, we show that HO-1–expressing patrolling monocytes protect SCD vasculature from ongoing hemolytic insult and vaso-occlusion. We found that a mean 37% of patrolling monocytes from SCD patients express very high levels of HO-1 (HO-1hi) vs 6% in healthy controls and demonstrated that HO-1hi expression was dependent on uptake of heme-exposed endothelium. SCD patients with a recent VOC episode had lower numbers of HO-1hi patrolling monocytes. Heme-mediated vaso-occlusion by mouse SCD red blood cells was exacerbated in mice lacking patrolling monocytes, and reversed following transfer of patrolling monocytes. Altogether, these data indicate that SCD patrolling monocytes remove hemolysis-damaged endothelial cells, resulting in HO-1 upregulation and dampening of VOC, and that perturbation in patrolling monocyte numbers resulting in lower numbers of HO-1hi patrolling monocyte may predispose SCD patients to VOC. These data suggest that HO-1hi patrolling monocytes are key players in VOC pathophysiology and have potential as therapeutic targets for VOC.

Low expression of GFI-1 Gene is associated with Panobinostat-resistance in acute myeloid leukemia through influencing the level of HO-1

To improve the treatment outcomes of acute myeloid leukemia (AML), epigenetic modification has been widely tested and used in recent years. However, drug-resistance is still a choke point to cure the malignancy. The growth factor independent 1 transcriptional repressor (GFI-1), as a zinc-finger transcriptional repressor, can bind histone deacetylases to allow the transcriptional repression. According to the finding of our study, AML patients with low level of GFI-1 not only implicated poor prognosis but also caused Panobinostat-resistance. In our prevent study revealed that heme oxygenase-1(HO-1) was one of the main factors leading to chemotherapy sensitivity to AML. Thus, this study tried to test the correlation between GFI-1 and HO-1. Our study discovered that AML patients with lower expression of GFI-1 had higher level of HO-1, HDAC1, HDAC2 and HDAC3, which resulted in poor prognosis in AML. The results of the in vitro study were the same. Panobinostat is a promising new class of anti-cancer drugs in AML. However, knocking down GFI-1 by siRNA could eliminate the Panobinostat-induced cell apoptosis. Subsequently, we utilized ZnPP to down regulate the level of HO-1, finding that the Panobinostat-resistance between the low level of GFI-1 and empty vector had eased. After further exploring the mechanism, it could be found that with knock down GFI-1, the phosphorylation of Akt and PI3K could be activated. Subsequently, Akt pathway and HO-1 inhibitor were utilized respectively and the resistance was reversed. It suggested that the resistance of Panobinostat to AML cells at low level of GFI-1 was mainly due to up-regulated level of HO-1 through the PI3K-Akt pathway.

Heme oxygenase-1 regulates sirtuin-1-autophagy pathway in liver transplantation: From mouse to human.

Liver ischemia-reperfusion injury (IRI) represents a major risk factor of early graft dysfunction and a key obstacle to expanding the donor pool in orthotopic liver transplantation (OLT). Although graft autophagy is essential for resistance against hepatic IRI, its significance in clinical OLT remains unknown. Despite recent data identifying heme oxygenase-1 (HO-1) as a putative autophagy inducer, its role in OLT and interactions with sirtuin-1 (SIRT1), a key autophagy regulator, have not been studied. We aimed to examine HO-1-mediated autophagy induction in human OLT and in a murine OLT model with extended (20hours) cold storage, as well as to analyze the requirement for SIRT1 in autophagy regulation by HO-1. Fifty-one hepatic biopsy specimens from OLT patients were collected under an institutional review board protocol 2 hours after portal reperfusion, followed by Western blot analyses. High HO-1 levels correlated with well-preserved hepatocellular function and enhanced SIRT1/LC3B expression. In mice, HO-1 overexpression by genetically modified HO-1 macrophage therapy was accompanied by decreased OLT damage and increased SIRT1/LC3B expression, whereas adjunctive inhibition of SIRT1 signaling diminished HO-1-mediated hepatoprotection and autophagy induction. Our translational study confirms the clinical relevance of HO-1 cytoprotection and identifies SIRT1-mediated autophagy pathway as a new essential regulator of HO-1 function in IR-stressed OLT.

Protective Role of HO-1 Expressing CD16+ Patrolling Monocytes Against Hemolysis-Induced Endothelial Damage and Vaso-Occlusive Crisis in Sickle Cell Disease

Causing leukocyte activation and upregulation of adhesion molecules on endothelial cells. CD16+ monocytes, also known as endothelial patrolling monocytes, normally scavenge the damaged cells and debris from the vasculature. As compared to other monocyte subsets or immune cell types, the CD16+monocyte subset expresses higher levels of the anti-inflammatory heme oxygenase 1 (HO-1), a heme degrading enzyme. Given the role of CD16+ monocytes as scavengers of debris on endothelial cells, we tested the hypothesis that this subset may protect SCD vasculature from the ongoing hemolytic insult through expression of high levels of HO-1. We found roughly 35% of circulating CD16+ monocytes from SCD patients expressed very high levels of HO-1 as compared to 5% in healthy controls. The HO-1hi SCD monocytes expressed significantly (30%) less TNF-a compared to HO-1lo monocytes following stimulation, consistent with anti-inflammatory effects of HO-1. We hypothesized that uptake of free hemoglobin/heme was responsible for high HO-1 expression levels in SCD CD16+ monocytes. To test this, healthy donors (HDs) or SCD patient monocytes were treated with different doses of free heme or hemolysed RBCs. We found dose-dependent HO-1 induction (five-fold at 20mM heme) in purified CD16- monocytes, but surprisingly none in CD16+ subset. However, upon co-culture with human umbilical vein endothelial cells (HUVEC), continuous or prior exposure to heme induced HO-1hi expression exclusively in CD16+ monocytes (5 fold in HD and further two fold in SCD compared to non-heme treated cocultures, p<0.001). Using imagining flow cytometric analysis, we found marked increase in uptake of heme-exposed endothelial cell-derived material by CD16+ monocytes (HD: 2% to 13% ± 3%; in SCD: 20% ± 3% to 30% ± 4%, p< 0.001) but none by CD16- monocytes. Our transwell studies demonstrated that cell-cell contact between CD16+ monocytes and heme-exposed HUVEC was required for HO-1hi expression. We found roughly 4-fold increase in expression of phosphatidylserine (PS, annexin V+), ICAM-1 and vCAM-1 on heme-treated HUVEC cells. Antibody blocking studies identified PS moieties as well as ICAM-1 as key molecules involved in monocyte-HUVEC interactions that mediated HO-1hi induction, suggesting that high levels of HO-1 expression in SCD CD16+ monocytes is in part the result of attachment to and engulfment of apoptotic, activated endothelial cells damaged by heme. SCD patients suffer from vaso-occlusive crisis (VOC), resulting from increased attachment of SCD RBCs to damaged and activated endothelium. We hypothesized that inadequate numbers or lower HO-1hi levels in CD16+ monocyte will predispose SCD patients to episodes of VOC due to decreased removal by CD16+ monocytes of damaged endothelial and sickle RBCs. Amongst SCD patients receiving chronic transfusions, we found a two-fold lower frequency of circulating CD16+ monocytes and half the numbers of CD16+HO-1hi monocytes in patients with a recent history of VOC episode as compared to those without VOC (p< 0.01); the former group also expressed higher levels of circulating sVCAM-1 (997 ± 210 vs 765 ± 236 ng/m, p=0.02), a marker of endothelial activation. To formally test the role of patrolling monocytes in endothelial damage induced by SCD RBCs and heme, we injected RBCs from Townes SCD mice alone or after 24hrs with heme into Nr4a1-knockout mice which have a selective loss of patrolling monocytes. Immunofluorescence analysis of liver vasculature showed a 3 fold increase in the activated endothelial marker, ICAM-1 within 24hrs following injection of sickle RBCs and two-fold increase in circulating sVCAM-1 levels in mice treated with sickle RBC plus heme (p <0.001). Transfer of HO-1+ patrolling monocytes (LY6Clo), but not a classical monocyte subset (LY6C+) reversed activated endothelial phenotype, indicating that patrolling monocytes can inhibit SCD-induced endothelial activation. Altogether, these data suggest that SCD patrolling monocytes remove hemolysis-damaged endothelial cells, resulting in HO-1 upregulation and dampening of vascular inflammation. Perturbations in CD16+ monocyte numbers resulting in lower local HO-1 levels can predispose SCD patients to VOC, thus identifying HO-1+ patrolling monocytes as key players in VOC pathophysiology and as therapeutic targets. DisclosuresNo relevant conflicts of interest to declare.

Heme-Oxygenase-1 Expression Contributes to the Immunoregulation Induced by Fasciola hepatica and Promotes Infection.

Fasciola hepatica, also known as the liver fluke, is a trematode that infects livestock and humans causing fasciolosis, a zoonotic disease of increasing importance due to its worldwide distribution and high economic losses. This parasite immunoregulates the host immune system by inducing a strong Th2 and regulatory T immune response by immunomodulating dendritic cell (DC) maturation and alternative activation of macrophages. In this paper, we show that F. hepatica infection in mice induces the upregulation of heme-oxygenase-1 (HO-1), the rate-limiting enzyme in the catabolism of free heme that regulates the host inflammatory response. We show and characterize two different populations of antigen presenting cells that express HO-1 during infection in the peritoneum of infected animals. Cells that expressed high levels of HO-1 expressed intermediate levels of F4/80 but high expression of CD11c, CD38, TGFβ, and IL-10 suggesting that they correspond to regulatory DCs. On the other hand, cells expressing intermediate levels of HO-1 expressed high levels of F4/80, CD68, Ly6C, and FIZZ-1, indicating that they might correspond to alternatively activated macrophages. Furthermore, the pharmacological induction of HO-1 with the synthetic metalloporphyrin CoPP promoted F. hepatica infection increasing the clinical signs associated with the disease. In contrast, treatment with the HO-1 inhibitor SnPP protected mice from parasite infection, indicating that HO-1 plays an essential role during F. hepatica infection. Finally, HO-1 expression during F. hepatica infection was associated with TGFβ and IL-10 levels in liver and peritoneum, suggesting that HO-1 controls the expression of these immunoregulatory cytokines during infection favoring parasite survival in the host. These results contribute to the elucidation of the immunoregulatory mechanisms induced by F. hepatica in the host and provide alternative checkpoints to control fasciolosis.