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Iron staining in lungs of neonatal mice transfected with pBI-L/HO-1pTet-Off. Lung sections were incubated with Perl's solution in presence of urea-H 2 O and counterstained with Gill no. 3 hematoxylin. Slides were viewed at 200. A: left (untransfected) lung with hyperoxic exposure. B: right middle lung with hyperoxic exposure. Note increased iron staining (arrow) in alveolar and bronchiolar epithelia of right (transfected) lung.
Source publication
Somatic cell gene transfer is a potentially useful strategy to alter lung function. However, achieving efficient transfer to the alveolar epithelium, especially in smaller animals, has not been demonstrated. In this study, the rat heme oxygenase-1 (HO-1) gene was delivered to the lungs of neonatal mice via transpulmonary injection. A bidirectional...
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Context 1
... oxidative stress has been well documented to induce apoptosis in vivo in lung cells (12), p53 protein was measured by immunocytochemistry as an index of apoptosis (21). Forty-eight hours after gene transfer, p53 protein was increased in the lung transfected with the HO-1 expression plasmid compared with that in the untransfected lung (data not shown). Accumulation of iron is associated with HO-1 overex- pression. Iron deposition was evaluated in the trans- fected lungs because this molecule is a toxic by-product of the HO reaction (30). In hyperoxia, lungs transfected with HO-1 demonstrated increased evidence of iron staining in the alveoli and blood vessels compared with that in nontransfected lungs (Fig. 5), indicating that HO-1 overexpression may be associated with increased iron deposition in the ...
Context 2
... oxidative stress has been well documented to induce apoptosis in vivo in lung cells (12), p53 protein was measured by immunocytochemistry as an index of apoptosis (21). Forty-eight hours after gene transfer, p53 protein was increased in the lung transfected with the HO-1 expression plasmid compared with that in the untransfected lung (data not shown). Accumulation of iron is associated with HO-1 overex- pression. Iron deposition was evaluated in the trans- fected lungs because this molecule is a toxic by-product of the HO reaction (30). In hyperoxia, lungs transfected with HO-1 demonstrated increased evidence of iron staining in the alveoli and blood vessels compared with that in nontransfected lungs (Fig. 5), indicating that HO-1 overexpression may be associated with increased iron deposition in the ...
Citations
... HO is the rating-limiting enzyme to catalyze heme into bilirubin [19]. HO-1, one of HO isoforms, has been regarded as an inducible antioxidant [20]. The size of GT-repeat alleles in the HO-1 promoter can alter the inducibility of HO-1 [21]. ...
Background
Hyperbilirubinemia is a common disorder during neonatal period in Taiwan. Gene variants may play an important role in the development of neonatal hyperbilirubinemia. The current study investigated the association between neonatal hyperbilirubinemia and common gene variants involving the production and metabolism of bilirubin. Methods
This prospective study enrolled 444 healthy infants born in the Chang Gung Memorial Hospital at Taipei from 2013–2015. Hyperbilirubinemia was defined as a total bilirubin ≥ 15 mg/dL. A log-binomial model was used to assess the risk of gene variants. ResultsThe most common genetic variant was short heme oxygenase (HO)-1 promoter GT-allele (<24 repeats) (39.4 %), followed by GA at nt388 in hepatic solute carrier organic anion transporter 1B1 (SLCO1B1) (31.1 %), GA at nt211 in UDP-glucuronosyltransferase 1A1 (UGT1A1) (29.3 %), ABO incompatibility (16.2 %), alpha thalassemia (5.0 %), and G6PD deficiency (3.2 %). The log-binomial analysis demonstrated greater risks of hyperbilirubinemia in infants with GA at nt211 in UGT1A1 (RR = 1.548; 95 % CI = 1.096–2.187), short HO-1 promoter GT-repeat (RR = 2.185; 95 % CI = 1.527–3.125), and G6PD deficiency (RR = 1.985; 95 % CI = 1.010–3.901). The other gene variants – including blood type, alpha thalassemia, and SLCO1B1 – carried no significant risk. ConclusionsG6PD deficiency, short HO-1 promoter GT-repeat and GA at nt211 in UGT1A1 are risk factors of neonatal hyperbilirubinemia. The data provide clinical evidence to explain the high incidence of neonatal hyperbilirubinemia in Taiwan.
... Furthermore, our previous study has demonstrated an induction of heme oxygenase (HO)-1 among G6PD deficiency [32]. HO-1 is an inducible antioxidant to protect against a wide variety of immune-mediated infections [33][34][35][36]. Taken together, we suggest that the up-regulation of HO-1 in G6PD deficiency may be associated with the compensation of the susceptibility to the oxidative injury of infection. ...
G6PD-deficient adults are reported to be susceptible to severe infection, and decreased cytokine responses have been postulated as the underlying mechanism. However, investigating the association of G6PD deficiency and cytokine responses during infancy is lacking. The current study aims to determine whether cytokine responses of tumor necrosis factor ()-α, interleukins (IL)-6, and IL-10 are impaired in the G6PD-deficient infants. Upon agreements with informed consents, peripheral blood mononuclear cells (PBMCs) of enrolled infants were collected twice at 1 month and 1 year of age. PBMCs were then stimulated with toll-like receptor (TLR) agonists-including PAM3csk4 for TLR1-2, poly (I:C) for TLR3, and lipopolysaccharide for TLR4-to analyze the expression of TNF-α, IL-6, and IL-10. Males (P = .004) and phototherapy during neonatal period (P = .008) were more common among G6PD-deficient infants than G6PD-normal subjects. After the stimulation of TLR agonists, there was no significant difference in the expression of TNF-α, IL-6, and IL-10 between PBMCs of G6PD-deficient and -normal infants at both 1 month and 1 year of age. In conclusion, the clinical characteristics of G6PD-deficient infants are different from those of G6PD-normal subjects. The data suggest that the innate immune responses to TLR agonists in G6PD-deficienct infants are not different from those of G6PD-normal infants.
... LA pretreatment can downmodulate the response mediated by Nrf2 and provide protection to As 3+ exposed HepG2 cells [49]. Interestingly, LA induced a small increase in HO-1, this finding was consistent with several reports linking HO-1 cytoprotection with low (less than fivefold) induction of the enzyme, while high levels of HMOX1 expression (greater than 15-fold) were associated with significant oxygen cytotoxicity [50]. HO-1 is a cytoprotective molecule, which has potent anti-inflammatory cardioprotective properties, [51] but additionally, its essential metabolite, carbon monoxide (CO), appears toxic when CO binds to hemoglobin (Hb). ...
Diabetes has emerged as a major threat to worldwide health. The exact mechanisms underlying the disease are unknown; however, there is growing evidence that the excess generation of reactive oxygen species (ROS) associated with hyperglycemia, causes oxidative stress in a variety of tissues. In this context, various natural compounds with pleiotropic actions like α-lipoic acid (LA) are of interest, especially in metabolic diseases such as diabetes. LA, either as a dietary supplement or a therapeutic agent, modulates redox potential because of its ability to match the redox status between different subcellular compartments as well as extracellularly. Both the oxidized (disulfide) and reduced (di-thiol: dihydro-lipoic acid, DHLA) forms of LA show antioxidant properties. LA exerts antioxidant effects in biological systems through ROS quenching but also via an action on transition metal chelation. Dietary supplementation with LA has been successfully employed in a variety of in vivo models of disease associated with an imbalance of redox status: diabetes and cardiovascular diseases. The complex and intimate association between increased oxidative stress and increased inflammation in related disorders such as diabetes, makes it difficult to establish the temporal sequence of the relationship.
... LA pretreatment can downmodulate the response mediated by Nrf2 and provide protection to As 3+ exposed HepG2 cells [49]. Interestingly, LA induced a small increase in HO-1, this finding was consistent with several reports linking HO-1 cytoprotection with low (less than fivefold) induction of the enzyme, while high levels of HMOX1 expression (greater than 15-fold) were associated with significant oxygen cytotoxicity [50]. HO-1 is a cytoprotective molecule, which has potent anti-inflammatory cardioprotective properties, [51] but additionally, its essential metabolite, carbon monoxide (CO), appears toxic when CO binds to hemoglobin (Hb). ...
... Importantly, ferrous iron accumulated in high HO-1 expressing cells, and the addition of iron chelators or specific HO-1 inhibitors significantly reduced all measures of oxidative tissue injury [27]. The notion that high levels of HO-1 activity may potentiate, rather than attenuate ROS toxicity, and that this is related to the increased availability of ferrous iron is further supported by several in vitro studies [80,81], as well as studies in animals [82,83]. Thus, we hypothesize that, up to a certain level, induction of HO-1 is protective, while excessive upregulation of HO-1 in response to an inflammatory stimulus is deleterious. ...
Heme oxygenase 1 (HO-1) is an essential enzyme induced by heme and multiple stimuli associated with critical illness. In humans, polymorphisms in the HMOX1 gene promoter may influence the magnitude of HO-1 expression. In many diseases including murine malaria, HO-1 induction produces protective anti-inflammatory effects, but observations from patients suggest these may be limited to a narrow range of HO-1 induction, prompting us to investigate the role of HO-1 in malaria infection. In 307 Gambian children with either severe or uncomplicated P. falciparum malaria, we characterized the associations of HMOX1 promoter polymorphisms, HMOX1 mRNA inducibility, HO-1 protein levels in leucocytes (flow cytometry), and plasma (ELISA) with disease severity. The (GT)(n) repeat polymorphism in the HMOX1 promoter was associated with HMOX1 mRNA expression in white blood cells in vitro, and with severe disease and death, while high HO-1 levels were associated with severe disease. Neutrophils were the main HO-1-expressing cells in peripheral blood, and HMOX1 mRNA expression was upregulated by heme-moieties of lysed erythrocytes. We provide mechanistic evidence that induction of HMOX1 expression in neutrophils potentiates the respiratory burst, and propose this may be part of the causal pathway explaining the association between short (GT)(n) repeats and increased disease severity in malaria and other critical illnesses. Our findings suggest a genetic predisposition to higher levels of HO-1 is associated with severe illness, and enhances the neutrophil burst leading to oxidative damage of endothelial cells. These add important information to the discussion about possible therapeutic manipulation of HO-1 in critically ill patients.
... To increase the efficiency of delivery, aliquots of the C/EBP␣ siRNA, control siRNA, or siGLO were dissolved in saline and mixed with Lipofectamine 2000 at room temperature for 1 h. A 30-l (3 mg/kg body wt) aliquot of the mixture was injected into the left axilla of the neonatal mouse at the third intercostal space via a 1-ml insulin syringe, as described previously, resulting in intrapulmonary delivery (20). The mice were returned to their mothers and kept in room air for 16 h prior to hyperoxic exposure. ...
... Because of the small size of the newborn mouse pups, we encountered significant difficulties when administering small volumes of siRNA via intranasal inhalation, and in preliminary experiments, we could not demonstrate inhibition of C/EBP␣ in the neonatal mouse lung with this method (data not shown). We therefore chose an intrapulmonary injection method to deliver the C/EBP␣ siRNA (20). Stability of the injected siRNA was evaluated by injection of a Cy3-labeled control siRNA (siGLO), and fluorescent signals were monitored at 24 and 72 h after injections. ...
Postnatal lung development requires proliferation and differentiation of specific cell types at precise times to promote proper alveolar formation. Hyperoxic exposure can disrupt alveolarization by inhibiting cell growth; however, it is not fully understood how this is mediated. The transcription factor CCAAT/enhancer binding protein-α (C/EBPα) is highly expressed in the lung and plays a role in cell proliferation and differentiation in many tissues. After 72 h of hyperoxia, C/EBPα expression was significantly enhanced in the lungs of newborn mice. The increased C/EBPα protein was predominantly located in alveolar type II cells. Silencing of C/EBPα with a transpulmonary injection of C/EBPα small interfering RNA (siRNA) prior to hyperoxic exposure reduced expression of markers of type I cell and differentiation typically observed after hyperoxia but did not rescue the altered lung morphology at 72 h. Nevertheless, when C/EBPα hyperoxia-exposed siRNA-injected mice were allowed to recover for 2 wk in room air, lung epithelial cell proliferation was increased and lung morphology was restored compared with hyperoxia-exposed control siRNA-injected mice. These data suggest that C/EBPα is an important regulator of postnatal alveolar epithelial cell proliferation and differentiation during injury and repair.
... In the lung, expression of the inducible HO-1 isoform peaks in the perinatal period, a critical phase for alveolar development , then decreases to adult levels [14,15]. At the cellular level, HO-1 is expressed in multiple lung cell types including alveolar type II epithelial cells, macrophages, vascular smooth muscle and endothelial cells161718. HO-1 gene expression can be dramatically induced by hyperoxia, hypoxia, heavy metals, oxidized LDL and inflammation amongst other injuries [13,19]. ...
Heme oxygenase (HO) degrades cellular heme to carbon monoxide, iron and biliverdin. The HO-1 isoform is both inducible and cyto-protective during oxidative stress, inflammation and lung injury. However, little is known about its precise role and function in lung development. We hypothesized that HO-1 is required for mouse postnatal lung alveolar development and that vascular expression of HO-1 is essential and protective during postnatal alveolar development.
Neonatal lung development in wildtype and HO-1 mutant mice was evaluated by histological and molecular methods. Furthermore, these newborn mice were treated with postnatal dexamethasone (Dex) till postnatal 14 days, and evaluated for lung development.
Compared to wildtype littermates, HO-1 mutant mice exhibited disrupted lung alveolar structure including simplification, disorganization and reduced secondary crest formation. These defects in alveolar development were more pronounced when these mice were challenged with Dex treatment. Expression levels of both vascular endothelial and alveolar epithelial markers were also further decreased in HO-1 mutants after Dex treatment.
These experiments demonstrate that HO-1 is required in normal lung development and that HO-1 disruption and dexamethasone exposure are additive in the disruption of postnatal lung growth. We speculate that HO-1 is involved in postnatal lung development through modulation of pulmonary vascular development.
... In our study, we detected a low increase on HMOX1 response by ALA (8 h, 2.2 times versus control) that decreased afterwards to 1.3-times at the end of the study. This finding was consistent with several reports linking the HMOX1 cytoprotection with low (less than 5-fold) induction of the enzyme (Cheng et al., 2006;Suttner and Dennery, 1999;Yeh et al., 2008), while high levels of HMOX1 expression (greater than 15fold) were associated with significant oxygen cytotoxicity (Suttner and Dennery, 1999;Weng et al., 2000) such as that found in the As 3+ groups (Figs. 3A and 4A). ...
Oxidative stress is a known mechanism induced, among other things, by arsenic toxicity. As a response, the cell triggers the synthesis of antioxidant and stress response elements like glutathione and heme oxygenase. Alpha-lipoic acid (ALA) is a well-known antioxidant that confers protection to oxidative stress conditions. We analyzed the effect of ALA pretreatment on Nrf2-responsive gene expression of HepG2 cells exposed to As(3+). Cells were treated with 5mM ALA and 8h later exposed to 50μM As(3+) for 24h, analyzing MTT-activity, glutathione content, Nrf2 induction and antioxidant gene expression. As(3+) increased glutathione (154%), heme oxygenase, glutamate cystein ligase, modifier subunit and metallothionein (35-fold, 10-fold and 9-fold, respectively). ALA prevented the strong expression of heme oxygenase by As(3+) exposure (from 35- to 5-times of control cells), which correlated with the reduction of Nrf2 observed in As(3+) group. ALA pretreatment can down-modulate the response mediated by Nrf2 and provide protection to As(3+) exposed HepG2 cells.
... Twenty micrograms of cell homogenates were separated by electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes using semi-dry electrophoretic transfer cell (Bio-Rad, Hercules, CA, USA). These membranes were incubated with eNOS antibody (Transduction Laboratories, Lexington, KY, USA) as previously described (26). ...
Our purpose was to investigate the gene expression of endothelin-1 (ET-1) and endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells (HUVECs) under various concentrations and periods of exogenous nitric oxide (NO) gas exposure. Cultured HUVECs were exposed to 0, 20, and 80 ppm NO for 0, 24, and 48 h. With NO exposure for 24 h, ET-1 peptide levels decreased in both the 20 and 80 ppm groups. Thereafter, at 48 h, ET-1 peptide levels persistently decreased in the 20 ppm group, but significantly increased in the 80 ppm group. Furthermore, there was a significant decrease in the expression levels of eNOS protein and mRNA in the 80 ppm 48 h group. The data suggest a time- and dose-dependent effect of NO gas exposure on the gene expression of ET-1 and eNOS in HUVECs. A high concentration and long period of NO exposure induces an inhibition of eNOS and enhancement of ET-1, which could lead to vasoconstriction. These findings may have clinical implications in NO therapy regarding the optimal dose and period.
... In our study, we detected a low increase on HMOX1 response by ALA (8 h, 2.2 times versus control) that decreased afterwards to 1.3-times at the end of the study. This finding was consistent with several reports linking the HMOX1 cytoprotection with low (less than 5-fold) induction of the enzyme (Cheng et al., 2006;Suttner and Dennery, 1999;Yeh et al., 2008), while high levels of HMOX1 expression (greater than 15fold) were associated with significant oxygen cytotoxicity (Suttner and Dennery, 1999;Weng et al., 2000) such as that found in the As 3+ groups (Figs. 3A and 4A). ...
