Effects of Heme Oxygenase-1 on c-Kit-Positive Cardiac Cells.

Heme oxygenase-1 (HO-1) is one of the most powerful cytoprotective proteins known. The goal of this study was to explore the effects of HO-1 in c-kit-positive cardiac cells (CPCs). LinNEG/c-kitPOS CPCs were isolated and expanded from wild-type (WT), HO-1 transgenic (TG), or HO-1 knockout (KO) mouse hearts. Compared with WT CPCs, cell proliferation was significantly increased in HO-1TG CPCs and decreased in HO-1KO CPCs. HO-1TG CPCs also exhibited a marked increase in new DNA synthesis during the S-phase of cell division, not only under normoxia (21% O2) but after severe hypoxia (1% O2 for 16 h). These properties of HO-1TG CPCs were associated with nuclear translocation (and thus activation) of Nrf2, a key transcription factor that regulates antioxidant genes, and increased protein expression of Ec-SOD, the only extracellular antioxidant enzyme. These data demonstrate that HO-1 upregulates Ec-SOD in CPCs and suggest that this occurs via activation of Nrf2, which thus is potentially involved in the crosstalk between two antioxidants, HO-1 in cytoplasm and Ec-SOD in extracellular matrix. Overexpression of HO-1 in CPCs may improve the survival and reparative ability of CPCs after transplantation and thus may have potential clinical application to increase efficacy of cell therapy.

Heme oxygenase-1/carbon monoxide signaling participates in the accumulation of triterpenoids of Ganoderma lucidum.

Ganoderic triterpenoids (GTs) are the primary bioactive constituents of the Basidiomycotina fungus, Ganoderma lucidum. These compounds exhibit antitumor, anti-hyperlipidemic, and immune-modulatory pharmacological activities. This study focused on GT accumulation in mycelia of G. lucidum mediated by the heme oxygenase-1 (HO-1)/carbon monoxide (CO) signaling. Compared with the control, hemin (10 µmol/L) induced an increase of 60.1% in GT content and 57.1% in HO-1 activity. Moreover, carbon monoxide-releasing molecule-2 (CORM-2), CO donor, increased GT content by 56.0% and HO-1 activity by 18.1%. Zn protoporphyrin IX (ZnPPIX), a specific HO-1 inhibitor, significantly reduced GT content by 26.0% and HO-1 activity by 15.8%, while hemin supplementation reversed these effects. Transcriptome sequencing showed that HO-1/CO could function directly as a regulator involved in promoting GT accumulation by regulating gene expression in the mevalonate pathway, and modulating the reactive oxygen species (ROS) and Ca2+ pathways. The results of this study may help enhance large-scale GT production and support further exploration of GT metabolic networks and relevant signaling cross-talk.

Hemin-induced increase in saponin content contributes to the alleviation of osmotic and cold stress damage to Conyza blinii in a heme oxygenase 1-dependent manner.

Hemin can improve the stress resistance of plants through the heme oxygenase system. Additionally, substances contained in plants, such as secondary metabolites, can improve stress resistance. However, few studies have explored the effects of hemin on secondary metabolite content. Therefore, the effects of hemin on saponin synthesis and the mechanism of plant injury relief by hemin in Conyza blinii were investigated in this study. Hemin treatment promoted plant growth and increased the antioxidant enzyme activity and saponin content of C. blinii under osmotic stress and cold stress. Further study showed that hemin could provide sufficient precursors for saponin synthesis by improving the photosynthetic capacity of C. blinii and increasing the gene expression of key enzymes in the saponin synthesis pathway, thus increasing the saponin content. Moreover, the promotion effect of hemin on saponin synthesis is dependent on heme oxygenase-1 and can be reversed by the inhibitor Zn-protoporphyrin-IX (ZnPPIX). This study revealed that hemin can increase the saponin content of C. blinii and alleviate the damage caused by abiotic stress, and it also broadened the understanding of the relationship between hemin and secondary metabolites in plant abiotic stress relief.

Fluid shear stress regulates placental growth factor expression via heme oxygenase 1 and iron.

Increased fluid shear stress (FSS) is a key initiating stimulus for arteriogenesis, the outward remodeling of collateral arterioles in response to upstream occlusion. Placental growth factor (PLGF) is an important arteriogenic mediator. We previously showed that elevated FSS increases PLGF in a reactive oxygen species (ROS)-dependent fashion both in vitro and ex vivo. Heme oxygenase 1 (HO-1) is a cytoprotective enzyme that is upregulated by stress and has arteriogenic effects. In the current study, we used isolated murine mesentery arterioles and co-cultures of human coronary artery endothelial cells (EC) and smooth muscle cells (SMC) to test the hypothesis that HO-1 mediates the effects of FSS on PLGF. HO-1 mRNA was increased by conditions of increased flow and shear stress in both co-cultures and vessels. Both inhibition of HO-1 with zinc protoporphyrin and HO-1 knockdown abolished the effect of FSS on PLGF. Conversely, induction of HO-1 activity increased PLGF. To determine which HO-1 product upregulates PLGF, co-cultures were treated with a CO donor (CORM-A1), biliverdin, ferric ammonium citrate (FAC), or iron-nitrilotriacetic acid (iron-NTA). Of these FAC and iron-NTA induced an increase PLGF expression. This study demonstrates that FSS acts through iron to induce pro-arteriogenic PLGF, suggesting iron supplementation as a novel potential treatment for revascularization.

Dasatinib protects against acute respiratory distress syndrome via Nrf2-regulated M2 macrophages polarization.

Dasatinib, a tyrosine kinase inhibitor, has a protective effect on experimental acute respiratory distress syndrome (ARDS). This study investigated the effect and mechanism of dasatinib in ARDS. C57BL/6 mice were administered with dasatinib (1 and 10 mg/kg) after lipopolysaccharide (LPS) treatment to evaluate the effect of dasatinib on white blood cells (WBC), neutrophils, lymphocytes and macrophages in bronchoalveolar lavage fluid (BALF). The levels and mRNA expressions of inflammation-related cytokines in lung tissues and RAW 264.7 cells were detected by enzyme-linked immunosorbent assay and quantitative real-time PCR, respectively. The protein expressions of nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase 1 (HO1) were determined by Western blot. MTT assay was performed to detect the viability of RAW 264.7 cell. Rescue experiments were used to assess the effect of Nrf2 silencing on the LPS- and dasatinib-treated mice. Under LPS treatment, levels of the WBC, neutrophils, lymphocytes and macrophages in BALF and mRNA expressions of IL-6, TNF-α and IL-10 as well as expression of iNOS were increased, but the expression of arginase-1 was inhibited, while no obvious changes of the protein expressions of Nrf2 and HO1 were observed. Dasatinib partially reversed the effects of LPS above, and further promoted the mRNA expression of IL-10 and the protein expressions of Nrf2 and HO1, while Nrf2 silencing counteracted the effect of dasatinib. Dasatinib induced the polarization of M2 subtype of macrophages and alleviated LPS-induced ARDS through activating Nrf2 signaling pathway, which may provide a new strategy for the treatment of ARDS.

Diannexin Can Ameliorate Acute Respiratory Distress Syndrome in Rats by Promoting Heme Oxygenase-1 Expression.

BACKGROUND: The recombinant protein diannexin can inhibit platelet-mediated events, which contribute to acute respiratory distress syndrome (ARDS). Here, we investigated the effect of diannexin and its effect on heme oxygenase-1 (HO-1) in ARDS. METHODS: A total of 32 rats were randomized into sham, ARDS, diannexin (D), and diannexin+HO-1 inhibitor (DH) groups. Alveolar-capillary permeability was evaluated by testing the partial pressure of oxygen to fraction of inspired oxygen (PaO2/FiO2) ratio, lung wet/dry weight ratio, and protein levels in the lung. Inflammation was assessed by measuring cytokine levels in the bronchial alveolar lavage fluid (BALF) and serum and nuclear factor-κB (NF-κB) in the lung tissue. Inducible nitric oxide synthase (iNOS), malondialdehyde (MDA), and myeloperoxidase (MPO) were measured to evaluate the oxidative stress response. Lung tissue pathology and apoptosis were also evaluated. We measured HO-1 expression in the lung tissue to investigate the effect of diannexin on HO-1 in ARDS. RESULTS: Compared with the ARDS group, diannexin improved PaO2/FiO2, lung wet/dry weight ratio, and protein levels in the BALF and decreased levels of cytokines and NF-κB in the lung and serum. Diannexin inhibited the oxidative stress response and significantly ameliorated pathological lung injury and apoptosis. The partial reversal of diannexin effects by a HO-1 inhibitor suggests that diannexin may promote HO-1 expression to ameliorate ARDS. CONCLUSIONS: We showed that diannexin can improve alveolar-capillary permeability, inhibit the oxidative stress response and inflammation, and protect against ARDS-induced lung injury and apoptosis.

Extracellular Superoxide Dismutase Prevents Skin Aging by Promoting Collagen Production through the Activation of AMPK and Nrf2/HO-1 Cascades.

With aging, the skin becomes thin and drastically loses collagen. Extracellular superoxide dismutase (EC-SOD), also known as superoxide dismutase (SOD) 3, is the major SOD in the extracellular matrix of the tissues and is well-known to maintain the reduction‒oxidation homeostasis and matrix components of such tissues. However, the role of EC-SOD in aging-associated reductions of skin thickness and collagen production is not well-studied. In this study, we compared the histological differences in the dorsal skin of EC-SOD‒overexpressing transgenic mice (Sod3+/+) of different age groups with that in wild-type mice and also determined the underlying signaling mechanism. Our data showed that the skin thickness in Sod3+/+ mice significantly increased with aging compared with that in wild-type male mice. Furthermore, Sod3+/+ mice had promoted collagen production through the activation of adenosine monophosphate-activated protein kinase and Nrf2/HO-1 pathways in aged mice. Interestingly, subcutaneous injection of adeno-associated virus‒overexpressing EC-SOD exhibited increased skin thickness and collagen expression. Furthermore, combined recombinant EC-SOD and dihydrotestosterone treatment synergistically elevated collagen production through the activation of TGFß in human dermal fibroblasts. Altogether, these results showed that EC-SOD prevents skin aging by promoting collagen production in vivo and in vitro. Therefore, we propose that EC-SOD may be a potential therapeutic target for antiaging in the skin.

Cephalosporin antibiotics specifically and selectively target nasopharyngeal carcinoma through HMOX1-induced ferroptosis.

AIMS: Many antibiotics derived from mold metabolites have been found to possess anticarcinogenic properties. We aimed to investigate whether they may elicit anticancer activity, especially against nasopharyngeal carcinoma. MAIN METHODS: The response of nasopharyngeal and other carcinoma cell lines to cephalosporin antibiotics was evaluated in vitro and in vivo. MTT and clonogenic colony formation assays assessed the viability and proliferation of cultured cells. Flow cytometry was used to assess cell cycle parameters and apoptotic markers. Tumor growth was determined using a xenograft model in vivo. Microarray and RT-qPCR expression analyses investigate differential gene expression. Mechanistic assessment of HMOX1 in cefotaxime-mediated ferroptosis was tested with Protoporphyrin IX zinc. KEY FINDINGS: Cephalosporin antibiotics showed highly specific and selective anticancer activity on nasopharyngeal carcinoma CNE2 cells both in vitro and vivo with minimal toxicity. Cefotaxime sodium significantly regulated 11 anticancer relevant genes in CNE2 cells in a concentration-dependent manner. Pathway analyses indicate apoptotic and the ErbB-MAPK-p53 signaling pathways are significantly enriched. HMOX1 represents the top one ranked upregulated gene by COS and overlaps with 16 of 42 enriched apoptotic signaling pathways. Inhibition of HMOX1 significantly reduced the anticancer efficacy of cefotaxime in CNE2 cells. SIGNIFICANCE: Our discovery is the first to highlight the off-label potential of cephalosporin antibiotics as a specific and selective anticancer drug for nasopharyngeal carcinoma. We mechanistically show that induction of ferroptosis through HMOX1 induction mediates cefotaxime anticancer activity. Our findings provide an alternative treatment for nasopharyngeal carcinoma by showing that existing cephalosporin antibiotics are specific and selective anticancer drugs.

Role of heme oxygenase-1 in human placenta on iron supply to fetus.

INTRODUCTION: To date, details on how iron is supplied from the mother to the fetus through the placenta have remained unclear. Recently, increasing evidence has shown that heme oxygenase (HO)-1, which is an inducible isoform of the rate-limiting enzyme in the heme degradation pathway, may be involved in the effective reutilization of iron. In this study, we examined the distribution and gene expression of HO-1 in the villous tissue of human placenta at various periods of pregnancy. METHODS: Using the placenta of 38 samples for which consent was obtained, chronological changes in the localization of HO-1 protein were examined by histological examination. RT-PCR was also performed to examine the expression of HO-1, transferrin receptor-1, and ferroportin 1. Ferric iron in the tissues was analyzed by Prussian blue staining. RESULTS: Immunohistochemical studies showed that HO-1 protein was exclusively expressed in trophoblastic cells throughout gestation. In the miscarriage placenta in the first trimester, ho-1 mRNA levels were significantly higher than normal. Placenta with fetal death (miscarriage) in the first and second trimester indicate significantly higher ratio of ho-1 gene for iron production to the fpn-1 gene for iron excretion than normal. These suggest that the role of HO-1 with various physiological functions is changing throughout pregnancy. DISCUSSION: These findings suggest that HO-1 in placenta plays an important role in iron supplying system in the second trimester to support fetal development.

Myeloid tumor necrosis factor and heme oxygenase-1 regulate the progression of colorectal liver metastases during hepatic ischemia-reperfusion.

The liver ischemia-reperfusion (IR) injury that occurs consequently to hepatic resection performed in patients with metastases can lead to tumor relapse for not fully understood reasons. We assessed the effects of liver IR on tumor growth and the innate immune response in a mouse model of colorectal (CR) liver metastasis. Mice subjected to liver ischemia 2 days after intrasplenic injection of CR carcinoma cells displayed a higher metastatic load in the liver, correlating with Kupffer cells (KC) death through the activation of receptor-interating protein 3 kinase (RIPK3) and caspase-1 and a recruitment of monocytes. Interestingly, the immunoregulatory mediators, tumor necrosis factor-α (TNF-α) and heme oxygenase-1 (HO-1) were strongly upregulated in recruited monocytes and were also expressed in the surviving KC following IR. Using TNFflox/flox LysMcre/wt mice, we showed that TNF deficiency in macrophages and monocytes favors tumor progression after IR. The antitumor effect of myeloid cell-derived TNF involved direct tumor cell apoptosis and a reduced expression of immunosuppressive molecules such as transforming growth factor-ß, interleukin (IL)-10, inducible nitric oxyde synthase (iNOS), IL-33 and HO-1. Conversely, a monocyte/macrophage-specific deficiency in HO-1 (HO-1flox/flox LysMcre/wt ) or the blockade of HO-1 function led to the control of tumor progression post-liver IR. Importantly, host cell RIPK3 deficiency maintains the KC number upon IR, inhibits the IR-induced innate cell recruitment, increases the TNF level, decreases the HO-1 level and suppresses the tumor outgrowth. In conclusion, tumor recurrence in host undergoing liver IR is associated with the death of antitumoral KC and the recruitment of monocytes endowed with immunosuppressive properties. In both of which HO-1 inhibition would reinforce their antitumoral activity.

Anti-aging Klotho Protects SH-SY5Y Cells Against Amyloid ß1-42 Neurotoxicity: Involvement of Wnt1/pCREB/Nrf2/HO-1 Signaling.

Alzheimer's disease (AD) is considered a prevalent neurological disorder with a neurodegenerative nature in elderly people. Oxidative stress and neuroinflammation due to amyloid ß (Aß) peptides are strongly involved in AD pathogenesis. Klotho is an anti-aging protein with multiple protective effects that its deficiency is involved in development of age-related disorders. In this study, we investigated the beneficial effect of Klotho pretreatment at different concentrations of 0.5, 1, and 2 nM against Aß1-42 toxicity at a concentration of 20 µM in human SH-SY5Y neuroblastoma cells. Our findings showed that Klotho could significantly and partially restore cell viability and decrease reactive oxygen species (known as ROS) and improve superoxide dismutase activity (SOD) in addition to reduction of caspase 3 activity and DNA fragmentation following Aß1-42 challenge. In addition, exogenous Klotho also reduced inflammatory biomarkers consisting of nuclear factor-kB (NF-kB), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) in Aß-exposed cells. Besides, Klotho caused downregulation of Wnt1 level, upregulation of phosphorylated cyclic AMP response element binding (pCREB), and mRNA levels of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) with no significant alteration of epsilon isoform of protein kinase C (PKCε) after Aß toxicity. In summary, Klotho could alleviate apoptosis, oxidative stress, and inflammation in human neuroblastoma cells after Aß challenge and its beneficial effect is partially exerted through appropriate modulation of Wnt1/pCREB/Nrf2/HO-1 signaling.

Cannabinoid-Induced Autophagy and Heme Oxygenase-1 Determine the Fate of Adipose Tissue-Derived Mesenchymal Stem Cells under Stressful Conditions.

The administration of adipose tissue-derived mesenchymal stem cells (ADMSCs) represents a promising therapeutic option after myocardial ischemia or myocardial infarction. However, their potential is reduced due to the high post-transplant cell mortality probably caused by oxidative stress and mitogen-deficient microenvironments. To identify protection strategies for ADMSCs, this study investigated the influence of the non-psychoactive phytocannabinoid cannabidiol (CBD) and the endocannabinoid analogue R(+)-methanandamide (MA) on the induction of heme oxygenase-1 (HO-1) and autophagy under serum-free conditions. At a concentration of 3 µM, CBD induced an upregulation of HO-1 mRNA and protein within 6 h, whereas for MA only a late and comparatively lower increase in the HO-1 protein could be detected after 48 h. In addition, both cannabinoids induced time- and concentration-dependent increases in LC3A/B-II protein, a marker of autophagy, and in metabolic activity. A participation of several cannabinoid-binding receptors in the effect on metabolic activity and HO-1 was excluded. Similarly, knockdown of HO-1 by siRNA or inhibition of HO-1 activity by tin protoporphyrin IX (SnPPIX) had no effect on CBD-induced autophagy and metabolic activity. On the other hand, the inhibition of autophagy by bafilomycin A1 led to a significant decrease in cannabinoid-induced metabolic activity and to an increase in apoptosis. Under these circumstances, a significant induction of HO-1 expression after 24 h could also be demonstrated for MA. Remarkably, inhibition of HO-1 by SnPPIX under conditions of autophagy deficit led to a significant reversal of apoptosis in cannabinoid-treated cells. In conclusion, the investigated cannabinoids increase metabolic viability of ADMSCs under serum-free conditions by inducing HO-1-independent autophagy but contribute to apoptosis under conditions of additional autophagy deficit via an HO-1-dependent pathway.

Significance of heme oxygenase-1(HMOX1) gene on fetal hemoglobin induction in sickle cell anemia patients.

Though the patients with sickle cell anemia (SCA) inherit same genetic mutation, they show considerable phenotypic heterogeneity. It has been observed that patients with elevated fetal hemoglobin (HbF) levels have a relatively mild clinical course. There is sparse literature on the association of higher HbF levels leading to reduction in the oxidative stress in SCA patients. Hence in this study, the significance between the HMOX1 gene polymorphisms and the HbF levels has been studied. Preliminary screening was carried out. Genotyping of 3 variants in the HMOX1 gene was performed in 90 SCA patients and 50 healthy controls by PCR-RFLP, GeneScan and direct DNA sequencing. It was observed that SCA patients with higher HbF levels, showed improved hematological indices with an inverse effect on HbS levels. The TT genotype of rs2071746 (A→T) polymorphism was found to be associated with elevated HbF levels (P: 0.012). Also, the long form (> 25 GT repeats) of rs3074372 (GT)n repeats was found to be linked with increased HbF levels. We could not find any association of rs2071749 (A→G) polymorphism with the HbF levels. As, the sickle cell anemia patients show significant oxidative stress due to hemolysis, the study of polymorphisms in the HMOX1 gene may act as a potential independent marker for elevated HbF levels.

[The Regulation of AMPKα1/Nrf2/ HO-1 Pathway Mediated by Galantamine Hydrobromide Lycoremine in Myocardial Ischemia Reperfusion Rats].

OBJECTIVE: To investigate the effects of AMPKα1/Nrf2/heme oxygenase-1 (HO-1) pathway mediated by galantamine hydrobromide lycoremine (Gal) on endoplasmic reticulum stress apoptosis, myocardial apoptosis and fibrosis in rats with myocardial ischemia reperfusion (I/R). METHODS: A myocardial ischemia reperfusion injury rat model was established, and the rats were randomly divided into 5 groups: Control group, I/R model group, Gal 1 mg/kg group, Gal 2 mg/kg group and Gal 4 mg/kg group. Left ventricular ejection fraction (LVEF), left ventricular end-diastolic volume (LVEDV), left ventricular end-systolic volume (LVESV), left ventricular wall thickness (LVWT), and left ventricular short-axis shortening rate (FS) were detected by doppler ultrasound. Hematoxylin eosin staining was used to detect the pathological damage of myocardial tissue. The expression of Caspase-3 was detected by immunohistochemistry. Protein expression levels of CCAAT/enhancer-binding protein homologous protein (CHOP), cleaved Caspase-12, growth arrest and DNA damageinducible protein 34 (GADD34), immunoglobulin heavy-chain-binding protein (BiP), α-smooth muscle actin (α-SMA), Collagen Ⅰ, AMPKα1, Nrf2, and HO-1 were measured by western blot, and AMPK inhibitor Compound C was added for verification. RESULTS: Compared with the I/R model group, the grade of pathological damage of myocardial tissue in each group of Gal was improved, and cleaved Caspase-3 positive expression rate and Caspase-3 mRNA level were significantly reduced ( P<0.05) as well. The results showed that LVWT, FS and LVEF in Gal 2 mg/kg and Gal 4 mg/kg groups were significantly increased ( P<0.05), LVEDV and LVESV were significantly reduced ( P<0.05) compared with I/R model group. CHOP, cleaved Caspase-12, α-SMA, Collagen Ⅰ, AMPKα1, Nrf2, HO-1 protein levels were significantly reduced ( P<0.05), and GADD34 and BiP protein levels were significantly increased ( P<0.05) in Gal 2 mg/kg and Gal 4 mg/kg groups. CONCLUSION: The regulation of AMPKα1/Nrf2/HO-1 pathway mediated by Gal on endoplasmic reticulum stress apoptosis, myocardial apoptosis and fibrosis in myocardial ischemia reperfusion rats.

Involvement of the p62/Nrf2/HO-1 pathway in the browning effect of irisin in 3T3-L1 adipocytes.

Irisin has gained attention because of its potential applications in the treatment of metabolic diseases. Accumulating evidence indicates that irisin attenuates obesity via the browning of white adipose tissue; however, the underlying mechanisms are unclear. Here, we evaluated the effects of irisin on adipocyte browning and the underlying mechanisms. The western blotting and immunofluorescence analyses demonstrated that irisin significantly induced the up-regulation of brown fat-specific proteins (PGC1α, PRDM16, and UCP-1) and HO-1 in 3T3-L1 adipocytes. Moreover, irisin significantly increased the levels of cytosolic p62 and nuclear Nrf2. These effects of irisin in the adipocytes were attenuated by treatment with SnPP or p62 siRNA. In addition, the browning effect of irisin was observed in BAT-WT-1 cells. These findings suggest that irisin induced browning effect via the p62/Nrf2/HO-1 signalling pathway and that it may be a potential candidate for preventing or treating obesity.

COVID-19 and heme oxygenase: novel insight into the disease and potential therapies.

The COVID-19 pandemic needs therapies that are presently available and safe. We propose that subjects with metabolic syndrome, old age, and male gender have the greatest morbidity and mortality and have low stress proteins, in particular, low intracellular heme oxygenase (HO-1), making them particularly vulnerable to the disease. Additionally, COVID-19's heme reduction may contribute to even lower HO-1. Low-grade inflammation associated with these risk factors contributes to triggering a cytokine storm that spreads to multi-organ failure and near death. The high mortality of those treated with ventilator assistance may partially be explained by ventilator-induced inflammation. The cytoprotective and anti-inflammatory properties of HO-1 can limit the infection's damage. A paradox of COVID-19 hospital admissions data suggests that fewer cigarette-smokers are admitted compared with non-smokers in the general population. This unexpected observation may result from smoke induction of HO-1. Therapies with anti-viral properties that raise HO-1 include certain anesthetics (sevoflurane or isoflurane), hemin, estrogen, statins, curcumin, resveratrol, and melatonin. Controlled trials of these HO-1 inducers should be done in order to prevent or treat COVID-19 disease.

Tolerance to FVIII: Role of the Immune Metabolic Enzymes Indoleamine 2,3 Dyoxigenase-1 and Heme Oxygenase-1.

The occurrence of neutralizing anti-FVIII antibodies is a major complication in the treatment of patients affected by hemophilia A. The immune response to FVIII is a complex, multi-factorial process that has been extensively studied for the past two decades. The reasons why only a proportion of hemophilic patients treated with FVIII concentrates develop a clinically significant immune response is incompletely understood. The "danger theory" has been proposed as a possible explanation to interpret the findings of some observational clinical studies highlighting the possible detrimental impact of inflammatory stimuli at the time of replacement therapy on inhibitor development. The host immune system is often challenged to react to FVIII under steady state or inflammatory conditions (e.g., bleeding, infections) although fine tuning of mechanisms of immune tolerance can control this reactivity and promote long-term unresponsiveness to the therapeutically administered factor. Recent studies have provided evidence that multiple interactions involving central and peripheral mechanisms of tolerance are integrated by the host immune system with the environmental conditions at the time of FVIII exposure and influence the balance between immunity and tolerance to FVIII. Here we review evidences showing the involvement of two key immunoregulatory oxygenase enzymes (IDO1, HO-1) that have been studied in hemophilia patients and pre-clinical models, showing that the ability of the host immune system to induce such regulatory proteins under inflammatory conditions can play important roles in the balance between immunity and tolerance to exogenous FVIII.

Heme Oxygenase-1 and Carbon Monoxide Regulate Growth and Progression in Glioblastoma Cells.

In human glioma tumours, heme oxygenase-1 (HO-1) is overexpressed when compared with normal brain tissues and during oligodendroglioma progression. However, the molecular mechanisms mediated by HO-1 to promote glioblastoma remain unknown. We therefore aimed at investigating the effect of HO-1 expression and its selective enzymatic inhibition in two different cell lines (i.e. A172 and U87-MG). HO-1 was induced by hemin treatment (10 µM), and VP13/47 (100 µM) was used as a specific non-competitive inhibitor of HO-1 activity. Cell proliferation was measured by cell index measurement (xCelligence technology) and clonogenic assay, whereas cell migration was assessed by wound healing assay. Carbon monoxide-releasing molecules (CORMs) (i.e. CORM-3 and CORM-A1) were also used in a separate set of experiments to confirm the effect of HO-1 by-product in glioblastoma progression further. Our results were further validated using GSE4412 microarray dataset analysis and comparing biopsies overexpressing HO-1 with the rest of the cases. Our results showed that hemin was able to induce both HO-1 gene and protein expression in a cell-dependent manner being A172 more responsive to pharmacological upregulation of HO-1. Hemin, but not CORMs treatment, resulted in a significant increase of cell proliferation following 24 h of treatment as measured by increased cell index and colony formation capacity and such effect was abolished by VP13/47. Interestingly, both hemin and CORMs showed a significant effect on the wound healing assay also exhibiting cell specificity. Finally, our dataset analysis showed a positive correlation of HO-1 gene expression with ITGBI and ITGBII which are membrane receptors involved in cell adhesion, embryogenesis, tissue repair, immune response and metastatic diffusion of tumour cells. In conclusion, our data suggest that HO-1 and its by-product CO exhibit a cell-specific effect on various aspects of disease progression and are associated with a complex series of molecular mechanisms driving cell proliferation, survival and metastasis.

Nicotine Improves Survivability, Hypotension, and Impaired Adenosinergic Renal Vasodilations in Endotoxic Rats: Role of α7-nAChRs/HO-1 Pathway.

The nicotinic/cholinergic antiinflammatory pathway protects against acute kidney injury and other end-organ damages induced by endotoxemia. In this study, we tested the hypothesis that functional α7-nAChRs/heme oxygenase-1 (HO-1) pathway is imperative for the nicotine counteraction of hemodynamic and renovascular dysfunction caused by acute endotoxemia in rats. Renal vasodilations were induced by cumulative bolus injections of acetylcholine (ACh, 0.01 nmol-7.29 nmol) or ethylcarboxamidoadenosine (NECA, adenosine receptor agonist, 1.6 nmol-100 nmol) in isolated phenylephrine-preconstricted perfused kidneys. The data showed that 6-h treatment with lipopolysaccharide (LPS, 5 mg/kg i.p.) decreased systolic blood pressure and renal vasodilations caused by NECA but not Ach. The endotoxic insult also increased the mortality rate and elevated serum urea and creatinine. These LPS effects were sex-unrelated, except hypotension, and enhanced mortality which were more evident in male rodents, and abrogated after co-administration of nicotine (0.5, 1 mg/kg and 2 mg/kg) in a dose-dependent fashion. The advantageous effects of nicotine on NECA vasodilations, survivability, and kidney biomarkers in endotoxic male rats disappeared upon concurrent exposure to methyllycaconitine citrate (α7-nAChR blocker) or zinc protoporphyrin (HO-1 inhibitor) and were reproduced after treatment with bilirubin, but not hemin (HO-1 inducer) or tricarbonyldichlororuthenium (II) dimer (carbon monoxide-releasing molecule). Together, current biochemical and pharmacological evidence suggests key roles for α7-nAChRs and the bilirubin byproduct of the HO-1 signaling in the nicotine counteraction of renal dysfunction and reduced adenosinergic renal vasodilator capacity in endotoxic rats.

Novel Interplay between p53 and HO-1 in Embryonic Stem Cells.

Stem cells genome safeguarding requires strict oxidative stress control. Heme oxygenase-1 (HO-1) and p53 are relevant components of the cellular defense system. p53 controls cellular response to multiple types of harmful stimulus, including oxidative stress. Otherwise, besides having a protective role, HO-1 is also involved in embryo development and in embryonic stem (ES) cells differentiation. Although both proteins have been extensively studied, little is known about their relationship in stem cells. The aim of this work is to explore HO-1-p53 interplay in ES cells. We studied HO-1 expression in p53 knockout (KO) ES cells and we found that they have higher HO-1 protein levels but similar HO-1 mRNA levels than the wild type (WT) ES cell line. Furthermore, cycloheximide treatment increased HO-1 abundance in p53 KO cells suggesting that p53 modulates HO-1 protein stability. Notably, H2O2 treatment did not induce HO-1 expression in p53 KO ES cells. Finally, SOD2 protein levels are also increased while Sod2 transcripts are not in KO cells, further suggesting that the p53 null phenotype is associated with a reinforcement of the antioxidant machinery. Our results demonstrate the existence of a connection between p53 and HO-1 in ES cells, highlighting the relationship between these stress defense pathways.