HMOX1 Expression Research Articles

Iso-alpha acids from hops (Humulus lupulus) inhibit hepatic steatosis, inflammation, and fibrosis

Non-alcoholic fatty liver disease (NAFLD) is considered to be the hepatic manifestation of the metabolic syndrome. Iso-alpha acids (IAAs), hop-derived bitter compounds in beer, have been shown to beneficially affect different components of the metabolic syndrome such as insulin resistance and dyslipidemia. However, IAAs have not yet been studied in the context of chronic liver disease. Here we analyzed the effect of IAA on the pathogenesis of NAFLD. Once, we applied IAA to mice in combination with a NAFLD-inducing Western-type diet (WTD), and observed that IAA significantly inhibited WTD-induced body weight gain, glucose intolerance, and hepatic steatosis. Fitting to this, IAA dose-dependently inhibited cellular lipid accumulation in primary human hepatocytes (PHH) in vitro. Reduced expression of PPAR-gamma and key enzymes of lipid synthesis as well as increased expression of PPAR-alpha, indicative for increased lipid combustion, were identified as underlying mechanisms of reduced hepatocellular steatosis in vitro and in vivo. Analysis of hepatic HMOX1 expression indicated reduced oxidative stress in IAA-treated mice, which was paralleled by reduced activation of the JNK pathway and pro-inflammatory gene expression and immune cell infiltration. Furthermore, IAA reduced hepatic stellate cell (HSC) activation and pro-fibrogenic gene expression. Similarly, IAA also dose-dependently reduced oxidative stress and JNK activation in steatotic PHH, inhibited HSC activation, and reduced proliferation and pro-fibrogenic gene expression in already activated HSC in vitro. In conclusion, IAAs inhibit different pathophysiological steps of disease progression in NAFLD. Together with previous studies, which demonstrated the safety of even long-term application of IAA in humans, our data suggest IAA as promising therapeutic agent for the prevention and treatment of (non)alcoholic (fatty) liver disease.Iso-alpha-acids (IAAs) are hops-derived compounds. In this study, the authors show that IAAs inhibit development of non-alcoholic fatty liver disease. They found that IAAs reduced expression of PPAR-γ and key enzymes of lipid synthesis as well as increased expression of PPAR-α, indicative of increased lipid metabolism. IAAs also reduced oxidative stress and JNK-activation, inhibited hepatic stellate cell activation and reduced proliferation and pro-fibrogenic gene expression.

Hydroxyurea Exhibits Antioxidant Activity Via the Nrf2 Pathway- Antioxidant/Electrophile Response Element Regulated By p62/ SQSTM1

Introduction: The pathophysiology of sickle cell anemia (HF) is characterized by hemolytic and intermittent vasoconstrictive events with increased redox status in the vascular microenvironment that favors the chronic inflammation. Objectives: To investigate whether hydroxyurea (HU) acts in the inhibition/minimization of reactive oxygen/nitrogen species (ROS/RNS) and in the modulation of in vitro the antioxidant genes expression.Methods: DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay was performed to determine the antioxidant activity of HU using L-ascorbate (L-Asc) and butylated hydroxytoluene (BHT) as antioxidant controls. Human umbilical vein endothelial cell (HUVEC) and peripheral blood mononuclear cells (PBMC) were challenged with HU in the presence or absence of hemin for evaluation the cytotoxicity, inhibition of superoxide anions, nitrate/nitrite production and expression of antioxidant enzymes SOD1 (superoxide dismutase-1); GPx (glutathione peroxidase); GSR (glutathione-disulfide reductase); and HMOX1 (heme oxygenase-1 by RT-qPCR. Microarray analyses were performed on HUVEC stimulated with HU.Results: HU showed scavenging activity (similar to BHT) at concentrations equivalent to that found in the plasma of patients taking the drug (~200 μM). Treatments with HU alone or in combination with hemin did not induce toxicity in PBMC and HUVEC. HU decreases the accumulation of superoxide anions in PBMC in the presence or absence of hemin and the combined treatment of HU with hemin stimulated nitrate/nitrite production in PBMC. HU increases expression of SOD1 and GPx in PBMC and HUVEC. The increase in GSR expression was observed in PBMC and HUVEC submitted to the combined treatment of HU with hemin. HU did not induce HMOX1 expression and did not decrease its expression in combination with hemin in both, PBMC and HUVEC (Figure 1). The microarray assay showed that HU induces the expression of cellular antioxidant components, such as SOD2, GSR, GST1 (glutathione S-transferase-1, GSTM2 (glutathione S-transferase mu 2), MGST1 (microsomal glutathione S-transferase 1), CBR1 (carbonyl reductase 1), protein kinases phosphatidylinositol 3-phosphate C (PRKCB, PRKCZ, PIK3C2B) and p62/SQSTM1 (sequestosome 1) (Table 1). In contrast, a decrease in BACH1 (BTB domain and CNC homolog 1) transcriptional factor expression was observed. Upstream analysis demonstrated prediction of activation for the transcriptional factor JUN and miR-155-5p.Conclusion: The findings indicate that HU can act (i) directly inhibiting on the direct inhibition of ROS/RNS; (ii) inducing nitrite/nitrate production in the presence of hemin; (iii) besides stimulating the antioxidant cellular response by inducing the antioxidant/electrophile response element (ARE/EpRE) mediated by Nrf2 under p62/SQSTM1 regulation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

1 - Cigarette smoke impairs the anti-oxidative response by reducing heme levels and inducing Bach1

Introduction Cigarette smoke is a major risk factor of cardiovascular diseases. Heme regulates the activity of enzymes and transcription factors including Bach1. Bach1 competes with Nrf2 transcriptional activity and inhibits the expression of anti-oxidative enzymes. However, the molecular mechanism of Bach1 activation and its impact on the anti-oxidative response after exposure to cigarette smoke is not clear. We aim to understand the mechanism through which cigarette smoke impairs the anti-oxidative response. Methods Primary human umbilical vein endothelial cells (HUVEC) were exposed to a cigarette smoke extract (CSE) under static and unidirectional shear stress conditions. Shear stress was induced using the ibidi pump system and cone-and-plate viscometer. Levels of reduced glutathione (GSH) were measured using the fluorescent probe o-phthalaldehyde. Heme levels were measured after oxalic acid extraction. Gene and miR expression was quantified by RT-qPCR. Immunofluorescence imaging was performed on fixed HUVEC. MiR-125b overexpression was achieved by transfecting mimics. Inhibition of Bach1 was done using siRNA transfection. Apoptosis was assessed using caspase 3/7 activity. Human umbilical veins as an ex vivo model were exposed to CSE under static conditions. Results Exposure to CSE induced the expression of BACH1 and reduced total heme levels in HUVEC. It also reduced miR-125b expression in HUVEC and umbilical veins, whereas the expression of miR-125b target AhR repressor (AHRR) increased. Inhibiting BACH1 increased miR-125b expression and reduced AHRR expression. Acute exposure to CSE led to a high oxidative stress load. This observation was supported by a decrease in glutathione levels, an increase in nuclear:cytosolic ratio of the upstream transcription factor Nrf2 and an increase in expression of HMOX1 and NQO1. CSE-induced apoptosis was reversible by preconditioning with N-acetyl cysteine or overexpression of miR-125b. Conclusions cigarette smoke-induced Bach1 impairs the anti-oxidative response through inhibiting miR-125b. Reduced heme levels could be involved in the upregulation of Bach1 activity.

224 - Microglia undergo metabolic adaptation during heme detoxification

Intracerebral hemorrhage (ICH) is a devastating form of stroke that results in significant mortality, and survivors typically suffer from lasting neurological deficits. Despite the severity of ICH and its complications, there are currently no therapies specifically targeted to reduce oxidation-induced damage after ICH. After ICH, damaged erythrocytes release heme into the surrounding environment, where it promotes oxidative damage and inflammation. Microglia, the tissue-resident macrophages of the central nervous system, are responsible for clearing the heme released after ICH. It has been shown that knockdown of heme clearance enzymes, including Heme Oxygenase 1 (HMOX1), in microglia worsens outcome in models of ICH, but the role of metabolic adaptation in microglial heme clearance remains unexplored. Since improving the rate of heme clearance through metabolic manipulation might prove a potential avenue for ICH therapy, we sought to fully understand the interplay between metabolism and heme clearance in heme-exposed microglia. Here we show that heme rapidly alters the metabolic profile of BV2 murine microglia, increasing glycolytic rate at low doses (1-10μM) and decreasing oxygen consumption at higher doses (50μM) using extracellular flux analysis. We also show that subsequent treatment with Ferrostatin-1, an antioxidant shown to prevent iron-dependent cell death (“ferroptosis”), rescues the heme-induced decrease in oxygen consumption. Heme also upregulates expression of both Hmox1 and the Pentose Phosphate Pathway (PPP) genes, Glucose-6-phosphate dehydrogenase (G6pd) and 6-Phosphogluconate dehydrogenase (Pgd), in a dose-dependent manner by Reverse Transcription-Quantitative Polymerase Chain Reaction (RTqPCR). We show that intracellular accumulation of heme in microglia is also dose-dependent and occurs rapidly after heme exposure. Additionally, pharmacologic inhibition of fatty acid and glutamine flux into the tricarboxylic acid (TCA) cycle decreased the rate of heme loading and degradation. Together, these data suggest that metabolic manipulation of microglia is a potential avenue to improve heme clearance and prevent oxidative damage after ICH.

Hmox1 Upregulation Is a Mutual Marker in Human Tumor Cells Exposed to Physical Plasma-Derived Oxidants.

Increasing numbers of cancer deaths worldwide demand for new treatment avenues. Cold physical plasma is a partially ionized gas expelling a variety of reactive oxygen and nitrogen species, which can be harnesses therapeutically. Plasmas and plasma-treated liquids have antitumor properties in vitro and in vivo. Yet, global response signatures to plasma treatment have not yet been identified. To this end, we screened eight human cancer cell lines to investigate effects of low-dose, tumor-static plasma-treated medium (PTM) on cellular activity, immune-modulatory properties, and transcriptional levels of 22 redox-related genes. With PTM, a moderate reduction of metabolic activity and modest modulation of chemokine/cytokine pattern and markers of immunogenic cell death was observed. Strikingly, the Nuclear factor (erythroid-derived 2)-like 2 (nrf2) target heme oxygenase 1 (hmox1) was upregulated in all cell lines 4 h post PTM-treatment. nrf2 was not changed, but its baseline expression inversely and significantly correlated with hmox1 expression after exposure to PTM. Besides awarding hmox1 a central role with plasma-derived oxidants, we present a transcriptional redox map of 22 targets and chemokine/cytokine secretion map of 13 targets across eight different human tumor cell lines of four tumor entities at baseline activity that are useful for future studies in this field.

Protective role of down-regulated microRNA-31 on intestinal barrier dysfunction through inhibition of NF-\u03baB/HIF-1\u03b1 pathway by binding to HMOX1 in rats with sepsis

BackgroundIntestinal barrier dysfunction is a significant clinical problem, commonly developing in a variety of acute or chronic pathological conditions. Herein, we evaluate the effect of microRNA-31 (miR-31) on intestinal barrier dysfunction through NF-κB/HIF-1α pathway by targeting HMOX1 in rats with sepsis.MethodsMale Sprague-Dawley rats were collected and divided into the sham group, and the cecum ligation and perforation group which was subdivided after CACO-2 cell transfection of different mimic, inhibitor, or siRNA. Levels of serum D-lactic acid, diamine oxidase and fluorescence isothiocyanate dextran, FITC-DX concentration, and bacterial translocation were detected. Superoxidedismutase (SOD) activity and malondialdehyde (MDA) content were evaluated using the colorimetric method and an automatic microplate reader, respectively. Additionally, the levels of tumor necrosis factor, interleukin (IL)-6, and IL-10 were tested using enzyme-linked immunosorbent assay. The expression of miR-31, HMOX1, NF-κB, HIF-1α, IκB, ZO-1 and Occludin were assessed by reverse transcription quantitative polymerase chain reaction and Western blot analysis.ResultsInhibition of miR-31 decreased intestinal mucosal permeability and intestinal barrier function. The increased levels of miR-31 could cause oxidative damage and affect the expression of inflammatory factors in intestinal tissue of rats. HMOX1 was confirmed as a target gene of miR-31. MiR-31 affected intestinal mucosal permeability and intestinal barrier function, as well as oxidative damage and inflammation level by regulating HMOX1. Down-regulation of miR-31 inhibited NF-κB/HIF-1α pathway related genes by regulating HMOX1 expression. Furthermore, inhibition of miR-31 increased survival rates of rats.ConclusionOverall, the current study found that inhibition of miR-31 protects against intestinal barrier dysfunction through suppression of the NF-κB/HIF-1α pathway by targeting HMOX1 in rats with sepsis.

Environmental Enrichment Improves Cognitive Deficits, AD Hallmarks and Epigenetic Alterations Presented in 5xFAD Mouse Model.

Cumulative evidence shows that modifications in lifestyle factors constitute an effective strategy to modulate molecular events related to neurodegenerative diseases, confirming the relevant role of epigenetics. Accordingly, Environmental Enrichment (EE) represents an approach to ameliorate cognitive decline and neuroprotection in Alzheimer’s disease (AD). AD is characterized by specific neuropathological hallmarks, such as β-amyloid plaques and Neurofibrillary Tangles, which severely affect the areas of the brain responsible for learning and memory. We evaluated EE neuroprotective influence on 5xFAD mice. We found a better cognitive performance on EE vs. Control (Ct) 5xFAD mice, until being similar to Wild-Type (Wt) mice group. Neurodegenerative markers as β-CTF and tau hyperphosphorylation, reduced protein levels whiles APPα, postsynaptic density 95 (PSD95) and synaptophysin (SYN) protein levels increased protein levels in the hippocampus of 5xFAD-EE mice group. Furthermore, a reduction in gene expression of Il-6, Gfap, Hmox1 and Aox1 was determined. However, no changes were found in the gene expression of neurotrophins, such as Brain-derived neurotrophic factor (Bdnf), Nerve growth factor (Ngf), Tumor growth factor (Tgf) and Nerve growth factor inducible (Vgf) in mice with EE. Specifically, we found a reduced DNA-methylation level (5-mC) and an increased hydroxymethylation level (5-hmC), as well as an increased histone H3 and H4 acetylation level. Likewise, we found changes in the hippocampal gene expression of some chromatin-modifying enzyme, such as Dnmt3a/b, Hdac1, and Tet2. Extensive molecular analysis revealed a correlation between neuronal function and changes in epigenetic marks after EE that explain the cognitive improvement in 5xFAD.

Abstract B097: NRF2 antioxidant signaling mediates the ability of PIM protein kinases to induce resistance of prostate cancer to PI3K inhibitor therapy

Abstract Aberrant activation of the PI3K pathway leading to increases in AKT activity is seen in many prostate cancer patients refractory to androgen blockade therapy. A number of small-molecule inhibitors of PI3K, including BKM120 (Novartis), have entered clinical trials. As with PI3K-AKT activation, highly expressed Pim kinase has been shown to drive the growth of prostate cancer. Recent studies have demonstrated that Pim kinase plays a critical role in promoting resistance of breast cancer to PI3K inhibition. Similarly, when the Pim-1 protein kinase is overexpressed, prostate cancer cell lines become resistant to PI3K inhibitor. In both tissue culture and animal models, the combination of a Pim inhibitor, PIM447, and BKM120 acted synergistically in blocking the survival and growth of prostate cancer cells. We therefore investigated the biochemical mechanism by which Pim-1 mediates cell resistance to the PI3K inhibitor. Previously, we have demonstrated that knockout of Pim kinases led to accumulation of reactive oxygen species (ROS) accompanied by a lower level of expression of ROS scavengers. To explore whether Pim-1 overexpression reverses this phenotype in prostate epithelial cancer, using lentiviral transduction, we expressed Pim-1 in mouse prostate epithelial cancer cells derived from spontaneous prostate adenocarcinomas that arose in a mouse model in which Tp53 and Rb1 are deleted in prostate epithelium. Pim-1 overexpression promoted anchorage-independent growth and formation of mouse prostate epithelial tumors in vivo. Gene expression arrays demonstrated that Pim-1 induced an Nrf2 antioxidant response element gene signature, including increases in Heme oxygenase-1 (Hmox1), NAD(P)H quinone oxidoreductase 1 (Nqo1), and glutathione S-transferase kappa1 (Gstk1) genes. Similarly, overexpression of Pim-1 in human prostate cancer LNCaP cells results in increased expression of Nrf2 and Hmox1 protein. As expected, depletion of Nrf2 with shRNA prevented Pim-1-mediated increases in Hmox1 expression, indicating that Nrf2 activation is required for the Pim-1 induction of ROS scavengers. In line with this result, in Pim-1 overexpressing cell lines knockdown of either Hmox1 or Nrf2 restored sensitivity to BKM120 treatment. A vital role of ROS scavengers in mediating the resistance to BKM120 was further confirmed by the treatment of cells and xenografted tumors with buthionine sulfoximine (inhibitor of glutathione synthesis), which resensitized the Pim-1 overexpressing cells to BKM120 via ROS induction. Our data further demonstrated that Pim-1 overexpression decreases the ubiquitination of Nrf2 protein, thus preventing its degradation, and increasing its half-life. Conversely, inhibition of Pim kinase promoted Nrf2 ubiquitination and proteasomal degradation. Taken together, our results show that the increased expression of Pim kinases in prostate cancer blocks therapeutic efficacy of PI3K inhibitor by inducing Nrf2 and antioxidant scavengers that inhibit the induction of ROS. Citation Format: Jin H. Song, Libia A. Leuvano, Neha Singh, Andrew S. Kraft. NRF2 antioxidant signaling mediates the ability of PIM protein kinases to induce resistance of prostate cancer to PI3K inhibitor therapy [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr B097.

Role of statin as inducer of Hmox-1 system in treatment of preeclampsia

Preeclampsia is the major cause of both maternal and neonatal morbidity and mortality. Its insidence remains high and the management has not been established yet because its etiology and pathophysiological are still poorly understood. Theories regarding etiopathogenesis and management of preeclampsia have been postulated yet it remains controversial. Placental ischemic and angiogenic imbalance are suggested to be predisposing factors of preeclampsia. It is thereby targeted in prevention of preeclampsia. Unfortunately, both primary and secondary prevention using various supplements and drugs fails to exhibit good outcome. Overall, these efforts are considered useless. In recent years, researchers have been using statin derivative in management of preclampsia. It has been reported that statin provides protective effect in endothelial cells by inducing expression of Hmox-1 and inihibiting release of sFlt-1 as well as potent antioxidant properties. Thus, statin has been proposed as promising agent to significantly reduce anti-angiogenic in preeclamptic patients which is overviewed in this review based on recent studies.

Abstract 2218: Trim29 regulates hemangiomas neovascularization through targeting HMOX1

Abstract Infantile hemangiomas is the most common benign vascular tumors of childhood, characterized by rapid proliferation after birth and slow involution thereafter. Although several signaling pathways have been implicated in hemangiomas neovascularization, the mechanism is still unrevealed especially the initiative factor. Here we used gene microarray to profile proliferating and involuting phases of infantile hemangiomas and bioinformatic methods to find the distinguished genes. Log2FC>0.585 or <-0.585 and a nominal P value of 0.05 were used as the filtering criteria. A total of 354 significant genes were identified including 262 upregulated genes. 20 genes were selected for screening according to the microarray results. Trim29 was certified to be responsible for the proliferation of IH using Celigo cytometer method, which were reinforced in patient lesions. By overexpression of Trim29, proteomics was used to detect the downstream proteins.After comparing with the microarray results, HMOX1 was identified and selected for further study after reidentification in patient lesions. Through overexpression and knockdown assay in vitro, we found Trim29 could regulate the expression of HMOX1 and affect hemangiomas neovascularization. Propranolol could block this potential pathway to inhibit proliferation of IH in vivo. These results indicated Trim29 could regulate hemangiomas neovascularization through targeting HMOX1. This innovative pathway could be blocked by propranolol and gave a potential target gene for further investigation to find new drug treatment for IH. Citation Format: Qiang Huang, Wei Li, Xin Ge, Peng Li, Ya Gao. Trim29 regulates hemangiomas neovascularization through targeting HMOX1 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2218.

Heme Oxygenase-1 Influences Satellite Cells and Progression of Duchenne Muscular Dystrophy in Mice.

Muscle damage in Duchenne muscular dystrophy (DMD) caused by the lack of dystrophin is strongly linked to inflammation. Heme oxygenase-1 (HO-1; Hmox1) is an anti-inflammatory and cytoprotective enzyme affecting myoblast differentiation by inhibiting myomiRs. The role of HO-1 has not been so far well addressed in DMD. In dystrophin-deficient mdx mice, expression of Hmox1 in limb skeletal muscles and diaphragm is higher than in wild-type animals, being consistently elevated from 8 up to 52 weeks, both in myofibers and inflammatory leukocytes. Accordingly, HO-1 expression is induced in muscles of DMD patients. Pharmacological inhibition of HO-1 activity or genetic ablation of Hmox1 aggravates muscle damage and inflammation in mdx mice. Double knockout animals (Hmox1-/-mdx) demonstrate impaired exercise capacity in comparison with mdx mice. Interestingly, in contrast to the effect observed in muscle fibers, in dystrophin-deficient muscle satellite cells (SCs) expression of Hmox1 is decreased, while MyoD, myogenin, and miR-206 are upregulated compared with wild-type counterparts. Mdx SCs demonstrate disturbed and enhanced differentiation, which is further intensified by Hmox1 deficiency. RNA sequencing revealed downregulation of Atf3, MafK, Foxo1, and Klf2 transcription factors, known to activate Hmox1 expression, as well as attenuation of nitric oxide-mediated cGMP-dependent signaling in mdx SCs. Accordingly, treatment with NO-donor induces Hmox1 expression and inhibits differentiation. Finally, differentiation of mdx SCs was normalized by CO, a product of HO-1 activity. Innovation and Conclusions: HO-1 is induced in DMD, and HO-1 inhibition aggravates DMD pathology. Therefore, HO-1 can be considered a therapeutic target to alleviate this disease. Antioxid. Redox Signal. 00, 000-000.

Hmox1 promotes osteogenic differentiation at the expense of reduced adipogenic differentiation induced by BMP9 in C3H10T1/2 cells.

Mesenchymal stem cells (MSCs) are multipotent progenitors that can differentiate into a variety of cell types under proper stimuli. Bone morphogenetic protein 9 (BMP9) is able to simultaneously induce both adipogenic and osteogenic differentiation of MSCs although the regulatory molecules involved remain to be fully identified and characterized. Heme oxygenase 1 (Hmox1) plays an essential role not only in fat metabolism, but also in bone development. In the present study, we investigated the functional role of Hmox1 in BMP9-induced osteogenic/adipogenic differentiation in MSCs line C3H10T1/2 and probed the possible mechanism involved. We found that BMP9 promoted the endogenous expression of Hmox1 in C3H10T1/2 cells. Overexpression of Hmox1 or cobalt protoporphyrin (CoPP), an inducer of Hmox1, increased BMP9-induced osteogenic differentiation in vitro. Subcutaneous stem cell implantation in nude mice further confirmed that Hmox1 potentiated BMP9-induced ectopic bone formation in vivo. In contrast, Hmox1 reduced BMP9-induced adipogenic differentiation in C3H10T1/2 cells. Although had no obvious effect on BMP9-induced Smad1/5/8 phosphorylation, Hmox1 enhanced phosphorylation of p38, and AKT, while decreased phosphorylation of ERK1/2. Furthermore, Hmox1 increased total β-catenin protein level, and promoted the nuclear translocation of β-catenin in C3H10T1/2 cells. Taken together, our study strongly suggests that Hmox1 is likely to potentiate osteogenic differentiation and yet decrease adipogenic differentiation induced by BMP9 possibly through regulation of multiple signaling pathways.

Effect of diesel exhaust generated by a city bus engine on stress responses and innate immunity in primary bronchial epithelial cell cultures

Harmful effects of diesel emissions can be investigated via exposures of human epithelial cells, but most of previous studies have largely focused on the use of diesel particles or emission sources that are poorly representative of engines used in current traffic.We studied the cellular response of primary bronchial epithelial cells (PBECs) at the air-liquid interface (ALI) to the exposure to whole diesel exhaust (DE) generated by a Euro V bus engine, followed by treatment with UV-inactivated non-typeable Haemophilus influenzae (NTHi) bacteria to mimic microbial exposure. The effect of prolonged exposures was investigated, as well as the difference in the responses of cells from COPD and control donors and the effect of emissions generated during a cold start. HMOX1 and NQO1 expression was transiently induced after DE exposure. DE inhibited the NTHi-induced expression of human beta-defensin-2 (DEFB4A) and of the chaperone HSPA5/BiP. In contrast, expression of the stress-induced PPP1R15A/GADD34 and the chemokine CXCL8 was increased in cells exposed to DE and NTHi. HMOX1 induction was significant in both COPD and controls, while inhibition of DEFB4A expression by DE was significant only in COPD cells. No significant differences were observed when comparing cellular responses to cold engine start and prewarmed engine emissions.

HSF4 transcriptional regulates HMOX-1 expression in HLECs

The major causes for cataract formation are free radicals, which are neutralized by the endogenous antioxidants. However, how the human lens clean these harmful free radicals is still unclear. Transcriptional factor heat shock factor 4 (HSF4) is a cataract-causing gene and plays important roles during lens development. Here we show that HMOX-1, an anti-oxidase, is a bona fide transcriptional target gene of HSF4 in HLECs (human lens epithelial cells). HSF4 directly binds to the HSE element in HMOX-1 promoter to mediate its mRNA transcription and protein accumulation. The HSE element located at the region of −389 bp to −362 bp upstream from the TSS (transcription start site), which is critical for HMOX-1 transcriptional activation. Furthermore, knockdown of HSF4 by siRNA inhibited HMOX-1 expression. Thus, these data revealed a novel transcription target of HSF4 and provided new insights into anti-oxidation regulation in lens and age-related cataract.

Deregulation of linc-PINT in acute lymphoblastic leukemia is implicated in abnormal proliferation of leukemic cells.

Long Non-Coding RNAs (lncRNAs) are functional RNAs longer than 200 nucleotides in length. Several lncRNAs are involved in cell proliferation and are deregulated in several human tumors. Few lncRNAs have been described to play a role in Acute Lymphoblastic Leukemia (ALL). In this study, we carried out a genome wide lncRNA expression profiling in ALL samples and peripheral blood samples obtained from healthy donors. We detected 43 lncRNAs that were aberrantly expressed in ALL. Interestingly, among them, linc-PINT showed a significant downregulation in T and B-ALL. Re-expression of linc-PINT in ALL cells induced inhibition of leukemic cell growth that was associated with apoptosis induction and cell cycle arrest in G2/M phase. linc-PINT induced the transcription of HMOX1 which reduced the viability of ALL cells. Intriguingly, we observed that treatment with anti-tumoral epigenetic drugs like LBH-589 (Panobinostat) and Curcumin induced the expression of linc-PINT and HMOX1 in ALL. These results indicate that the downregulation of linc-PINT plays a relevant role in the pathogenesis of ALL, and linc-PINT re-expression may be one of the mechanisms exerted by epigenetic drugs to reduce cell proliferation in ALL.

Kinetics of ROS generation induced by polycyclic aromatic hydrocarbons and organic extracts from ambient air particulate matter in model human lung cell lines

Polycyclic aromatic hydrocarbons (PAHs) associated with particulate matter (PM) may induce oxidative damage via reactive oxygen species (ROS) generation. However, the kinetics of ROS production and the link with antioxidant response induction has not been well studied. To elucidate the differences in oxidative potential of individual PAH compounds and extractable organic matter (EOM) from PM containing various PAH mixtures, we studied ROS formation and antioxidant response [total antioxidant capacity (TAC) and expression of HMOX1 and TXNRD1] in human alveolar basal epithelial cells (A549 cells) and human embryonic lung fibroblasts (HEL12469 cells). We treated the cells with three concentrations of model PAHs (benzo[a]pyrene, B[a]P; 3-nitrobenzanthrone, 3-NBA) and EOM from PM <2.5 μm (PM2.5). ROS levels were evaluated at 8 time intervals (30 min–24 h). In both cell lines, B[a]P treatment was associated with a time-dependent decrease of ROS levels. This trend was more pronounced in HEL12469 cells and was accompanied by increased TAC. A similar response was observed upon 3-NBA treatment in HEL12469 cells. In A549 cells, however, this compound significantly increased superoxide levels. This response was accompanied by the decrease of TAC as well as HMOX1 and TXNRD1 expression. In both cell lines, a short-time exposure to EOMs tended to increase ROS levels, while a marked decrease was observed after longer treatment periods. This was accompanied by the induction of HMOX1 and TXNRD1 expression in HEL12469 cells and increased TAC in A549 cells. In summary, our data indicate that in the studied cell lines B[a]P and EOMs caused a time-dependent decrease of intracellular ROS levels, probably due to the activation of the antioxidant response. This response was not detected in A549 cells following 3-NBA treatment, which acted as a strong superoxide inducer. Pro-oxidant properties of EOMs are limited to short-time exposure periods.

CYP2J2 Expression in Adult Ventricular Myocytes Protects Against Reactive Oxygen Species Toxicity.

Cytochrome P450 2J2 isoform (CYP2J2) is a drug-metabolizing enzyme that is highly expressed in adult ventricular myocytes. It is responsible for the bioactivation of arachidonic acid (AA) into epoxyeicosatrienoic acids (EETs). EETs are biologically active signaling compounds that protect against disease progression, particularly in cardiovascular diseases. As a drug-metabolizing enzyme, CYP2J2 is susceptible to drug interactions that could lead to cardiotoxicity. CYP2J2 has been shown to be resistant to induction by canonical CYP inducers such as phenytoin and rifampin. It is, however, unknown how cellular stresses augment CYP2J2 expression. Here, we determine the effects of oxidative stress on gene expression in adult ventricular myocytes. Further, we assess the consequences of CYP2J2 inhibition and CYP2J2 silencing on cells when levels of reactive oxygen species (ROS) are elevated. Findings indicate that CYP2J2 expression increases in response to external ROS or when internal ROS levels are elevated. In addition, cell survival decreases with ROS exposure when CYP2J2 is chemically inhibited or when CYP2J2 expression is reduced using small interfering RNA. These effects are mitigated with external addition of EETs to the cells. Finally, we determined the results of external EETs on gene expression and show that only two of the four regioisomers cause an increase in HMOX1 expression. This work is the first to determine the consequence of cellular stress, specifically high ROS levels, on CYP2J2 expression in human ventricular myocytes and discusses how this enzyme may play an important role in response to cardiac oxidative stress.

Assessment of lung cell toxicity of various gasoline engine exhausts using a versatile invitro exposure system.

Adverse effect studies of gasoline exhaust are scarce, even though gasoline direct injection (GDI) vehicles can emit a high number of particles. The aim of this study was to conduct an invitro hazard assessment of different GDI exhausts using two different cell culture models mimicking the human airway. In addition to gasoline particle filters (GPF), the effects of two lubrication oils with low and high ash content were assessed, since it is known that oils are important contributors to exhaust emissions. Complete exhausts from two gasoline driven cars (GDI1 and GDI2) were applied for 6 h (acute exposure) to a multi-cellular human lung model (16HBE14o-cell line, macrophages, and dendritic cells) and a primary human airway model (MucilAir™). GDI1 vehicle was driven unfiltered and filtered with an uncoated and a coated GPF. GDI2 vehicle was driven under four settings with different fuels: normal unleaded gasoline, 2% high and low ash oil in gasoline, and 2% high ash oil in gasoline with a GPF. GDI1 unfiltered was also used for a repeated exposure (3 times 6 h) to assess possible adverse effects. After 6 h exposure, no genes or proteins for oxidative stress or pro-inflammation were upregulated compared to the filtered air control in both cell systems, neither in GDI1 with GPFs nor in GDI2 with the different fuels. However, the repeated exposure led to a significant increase in HMOX1 and TNFa gene expression in the multi-cellular model, showing the responsiveness of the system towards gasoline engine exhaust upon prolonged exposure. The reduction of particles by GPFs is significant and no adverse effects were observed invitro during a short-term exposure. On the other hand, more data comparing different lubrication oils and their possible adverse effects are needed. Future experiments also should, as shown here, focus on repeated exposures.

Murine Bone Marrow Mesenchymal Stromal Cells Respond Efficiently to Oxidative Stress Despite the Low Level of Heme Oxygenases 1 and 2.

Mesenchymal stromal cells (MSCs) are heterogeneous cells from adult tissues that are able to differentiate in vitro into adipocytes, osteoblasts, or chondrocytes. Such cells are widely studied in regenerative medicine. However, the success of cellular therapy depends on the cell survival. Heme oxygenase-1 (HO-1, encoded by the Hmox1 gene), an enzyme converting heme to biliverdin, carbon monoxide, and Fe2+, is cytoprotective and can affect stem cell performance. Therefore, our study aimed at assessing whether Hmox1 is critical for survival and functions of murine bone marrow MSCs. Both MSC Hmox1+/+ and Hmox1-/- showed similar phenotype, differentiation capacities, and production of cytokines or growth factors. Hmox1+/+ and Hmox1-/- cells showed similar survival in response to 50 μmol/L hemin even in increased glucose concentration, conditions that were unfavorable for Hmox1-/- bone marrow-derived proangiogenic cells (BDMC). Hmox1+/+ MSCs but not fibroblasts retained low ROS levels even after prolonged incubation with 50 μmol/L hemin, although both cell types have a comparable Hmox1 expression and similarly increase its levels in response to hemin. MSCs Hmox1-/- treated with hemin efficiently induced expression of a vast panel of antioxidant genes, especially enzymes of the glutathione pathway. Innovation and Conclusion: Hmox1 overexpression is a popular strategy to enhance viability and performance of MSCs after the transplantation. However, murine MSCs Hmox1-/- do not differ from wild-type MSCs in phenotype and functions. MSC Hmox1-/- show better resistance to hemin than fibroblasts and BDMCs and rapidly react to the stress by upregulation of quintessential genes in antioxidant response. Antioxid. Redox Signal. 00, 000-000.

Simvastatin Treatment Upregulates HO-1 in Patients with Abdominal Aortic Aneurysm but Independently of Nrf2.

Heme oxygenase-1 (HO-1), encoded by HMOX1 gene and regulated by Nrf2 transcription factor, is a cytoprotective enzyme. Its deficiency may exacerbate abdominal aortic aneurysm (AAA) development, which is also often associated with hyperlipidemia. Beneficial effects of statins, the broadly used antilipidemic drugs, were attributed to modulation of Nrf2/HO-1 axis. However, the effect of statins on Nrf2/HO-1 pathway in patients with AAA has not been studied yet. We analyzed AAA tissue from patients treated with simvastatin (N = 28) or without statins (N = 14). Simvastatin treatment increased HO-1 protein level in AAA, both in endothelial cells (ECs) and in smooth muscle cells (SMCs), but increased Nrf2 localization was restricted only to vasa vasorum. Nrf2 target genes HMOX1, NQO1, and GCLM expression remained unchanged in AAA. In vitro studies showed that simvastatin raises HO-1 protein level slightly in ECs and to much higher extent in SMCs, which is not related to Nrf2/ARE activation, although HMOX1 expression is upregulated by simvastatin in both cell types. In conclusion, simvastatin-induced modulation of HO-1 level in ECs and SMCs in vitro is not related to Nrf2/ARE activity. Likewise, divergent HO-1 and Nrf2 localization together with stable expression of Nrf2 target genes, including HMOX1, in AAA tissue denotes Nrf2 independency.