Repression of heme oxygenase-1 by hypoxia in vascular endothelial cells

Heme oxygenase 1 (HO-1), a rate-limiting enzyme in heme catabolism, has been reported to be induced by hypoxia. Unexpectedly, here we show that expression of HO-1 mRNA is repressed by hypoxia in primary cultures of human umbilical vein endothelial cells (HUVECs), but is increased by cobalt chloride (CoCl(2)) that is known to mimic hypoxia. Under the culture conditions used, the DNA-binding and transactivation activities of hypoxia-inducible factor 1 were increased in HUVECs by hypoxia or CoCl(2). Therefore, hypoxia and cobalt showed opposing effects on HO-1 mRNA expression, despite activation of hypoxia-inducible factor 1. The half-life of HO-1 mRNA was not changed by hypoxia, but was significantly prolonged by CoCl(2). Hypoxia also represses HO-1 mRNA expression in human coronary artery endothelial cells and astrocytes. The repression of HO-1 expression may represent the adaptation to hypoxia in certain cell types.     

Disrupted postnatal lung development in heme oxygenase-1 deficient mice

Background

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.

Methods

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.

Results

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.

Conclusions

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.     

Effect of heme oxygenase-1 overexpression in two models of lung inflammation

An increasing number of studies implicate heme oxygenase-1 (HO-1) in the regulation of inflammation. Although the mechanisms involved in this cytoprotection are largely unknown, HO-1 and its enzymatic products, carbon monoxide and bilirubin, downregulate the inflammatory response by either attenuating the expression of adhesion molecules and thus inhibiting leukocyte recruitment or by repressing the induction of cytokines and chemokines. In the present study we used genetically engineered mice that express high levels of a human cDNA HO-1 transgene in lung epithelium to assess the effect of HO-1 on lung inflammation. Two separate models of inflammation were studied: hypoxic exposure and lipopolysaccharide (LPS) challenge. We found that both mRNA and protein levels of specific cytokines and chemokines were significantly elevated in response to hypoxia in the lungs of wild-type mice after 2 and 5 days of exposure but significantly suppressed in the hypoxic lungs of transgenic mice, suggesting that inhibition of these cytokines was caused by overexpression of HO-1. However, LPS treatment resulted in a very pronounced increase in mRNA levels of several cytokines in both wild-type and transgenic mice. Despite the high mRNA levels, significantly lower cytokine protein levels were detected in the bronchoalveolar lavage of HO-1 overexpressing mice compared with wild type, indicating that HO-1 leads to repression of cytokines in the airway. These results demonstrate that HO-1 activity operates through distinct molecular mechanisms to confer cytoprotection in the hypoxic and the LPS models of inflammation.     

内毒素引起的乳鼠心肌细胞血红素加氧酶-1基因的表达

为了探讨在内毒素作用下的乳鼠心肌细胞（neonatal rat cardiomyocytes,NRCMs）血红素加氧酶－1（heme oxygenase-1,HO-1）基因的表达及其在细胞损伤中的作用,分别用10、30及50μg/ml的脂多糖（lipopolysaccharide,LPS）,10μg/ml LPS 10μmol/ml锌原卟啉Ⅸ（Zn-protoporphyrin-Ⅸ,ZnPPⅨ）和单纯10μmol/ml ZnPPⅨ与培养的NRCMs共同孵育6h,以及10μg/ml LPS与NRCMs共同孵育9h和18h。分别观察细胞HO－1 mRNA表达、MDA含量、LDH释放量与台盼蓝摄取率的变化。结果显示,同样与细胞孵育6h,LPS10μg/ml时HO－1 mRNA表达比对照组增加81.2％,30μg/ml时表达量增加126.3％,50μg/ml时表达量增加92.8％;LPS为10μg/ml时,孵育9h后HO－1 mRNA的表达量比对照组增加93.6％,孵育18h后一增加105.8％。LPS30、50μg/ml,10μg/ml LPS＋10μmol/ml ZnPPⅨ与细胞孵育6h及LPS 10μg/ml孵育18h后,细胞MDA含量、LDH释放量与台盼蓝摄取率明显增加（P＜0.01）;单纯10μg/ml LPS与单纯10μmol/ml ZnPPⅨ孵育6h后,上述指标均无明显升高。结果表明,LPS可诱导NRCMs HO－1 mRNA的表达,且在较低LPS剂量范围内具有时间依赖性和浓度依赖性;NRCMs HO-1 mRNA的表达可减低LPS引起的细胞损伤,这可能是细胞产生的一种自身保护性反应。     

Small interfering RNA targeting heme oxygenase-1 enhances ischemia-reperfusion-induced lung apoptosis

Heme oxygenase-1 (HO-1) is emerging as an important cytoprotective enzyme system in a variety of injury models. To optimize future therapeutic applications of HO-1, it is necessary to delineate the precise functions and mechanisms as well as modes of externally regulating HO-1 expression. Investigations have been limited by difficulties with the generation of HO-1 null mice and the lack of specific HO-1 inhibitors. Lung ischemia-reperfusion (I-R) injury is the inciting event in acute lung failure following transplantation, surgery, and shock. To study the function of HO-1 in I-R-induced lung injury, we designed small interfering RNA (siRNA) sequences that effectively suppress HO-1 expression both in vitro and in vivo in an organ-specific manner. In this study we show that there is enhanced apoptosis, via increased Fas expression and caspase 3 activity, in the presence of HO-1 siRNA in endothelial cells and mouse lung during I-R injury, whereas HO-1 overexpression attenuates apoptosis. To the best of our knowledge, we are the first to demonstrate that lung-specific siRNA delivery can be achieved by intranasal administration without the need for viral vectors or transfection agents in vivo, thereby obviating potential concerns for toxicity if siRNA technology is to have clinical application in the future.     

Down-regulation of heme oxygenase-2 is associated with the increased expression of heme oxygenase-1 in human cell lines

Intracellular heme concentrations are maintained in part by heme degradation, which is catalyzed by heme oxygenase. Heme oxygenase consists of two structurally related isozymes, HO-1 and HO-2. Recent studies have identified HO-2 as a potential oxygen sensor. To gain further insights into the regulatory role of HO-2 in heme homeostasis, we analyzed the expression profiles of HO-2 and the biochemical consequences of HO-2 knockdown with specific short interfering RNA (siRNA) in human cells. Both HO-2 mRNA and protein are expressed in the eight human cancer cell lines examined, and HO-1 expression is detectable in five of the cell lines, including HeLa cervical cancer and HepG2 hepatoma. Down-regulation of HO-2 expression with siRNA against HO-2 (siHO-2) caused induction of HO-1 expression at both mRNA and protein levels in HeLa and HepG2 cells. In contrast, knockdown of HO-1 expression did not noticeably influence HO-2 expression. HO-2 knockdown prolonged the half-life of HO-1 mRNA twofold in HeLa cells. Transient transfection assays in HeLa cells revealed that the 4.5-kb human HO-1 gene promoter was activated with selective knockdown of HO-2 in a sequence-dependent manner. Moreover, HO-2 knockdown caused heme accumulation in HeLa and HepG2 cells only when exposed to exogenous hemin. HO-2 knockdown may mimic a certain physiological change that is important in the maintenance of cellular heme homeostasis. These results suggest that HO-2 may down-regulate the expression of HO-1, thereby directing the co-ordinated expression of HO-1 and HO-2.     

Endothelial heme oxygenase-1 induction by hypoxia. Modulation by inducible nitric-oxide synthase and S-nitrosothiols

The stress protein heme oxygenase-1 (HO-1) is induced in endothelial cells exposed to nitric oxide (NO)-releasing agents, and this process is finely modulated by thiols (Foresti, R., Clark, J. E., Green, C. J., and Motterlini R. (1997) J. Biol. Chem. 272, 18411-18417). Here, we report that up-regulation of HO-1 in aortic endothelial cells by severe hypoxic conditions (pO(2) 相似文献   

人体血红素加氧酶-1的研究进展

血红素加氧酶（heme oxygenase,HO)是哺乳动物中血红素代谢的限速酶,HO－1是HO同功酶之一,主要分布在肝、脾、肺等多种脏器,具有调节和保护功能。作者拟从人体HO－1蛋白的晶体结构、HO－1的功能和HO－1表达的诱导因素,以及HO－1基因的表达与调控等研究进展做一综述。     

Interaction between endothelial heme oxygenase-2 and endothelin-1 in altered aortic reactivity after hypoxia in rats

The aim of this study was to determine whether increased expression of heme oxygenase (HO) contributes to impairment of aortic contractile responses after hypoxia through effects on reactivity to endothelin-1 (ET-1). Thoracic aortas from normoxic rats and rats exposed to hypoxia (10% O2) for 16 or 48 h were mounted in organ bath myographs for contractile studies, fixed in paraformaldehyde, or frozen in liquid nitrogen for protein extraction. In rings from normoxic rats, the HO inhibitor tin protoporphyrin IX (SnPP IX, 10 microM) did not alter the response to phenylephrine or ET-1. In rings from rats exposed to 16-h hypoxia, maximum tension generated in response to these agonists was higher in endothelium-intact but not -denuded rings in the presence of SnPP IX. In rings from rats exposed to 48-h hypoxia SnPP IX increased contraction in endothelium-intact but not -denuded rings. In endothelium-intact aortic rings from rats exposed to 16-h hypoxia incubated with endothelin A receptor-specific antagonist BQ-123 (10(-7) M), SnPP IX did not alter phenylephrine-induced contraction. Aortic ET-1 protein levels, measured by radioimmunoassay, were increased in rats exposed to hypoxia for 16 and 48 h. Western blotting showed that HO-1 and HO-2 protein were increased after 16 h of hypoxia and returned to near-control levels after 48 h. Increase in HO-1 protein was detected in endothelium-intact and -denuded rings. Removal of endothelium abolished the increase in HO-2 immunoreactivity. Immunohistochemistry localized expression of HO-1 protein to vascular smooth muscle, whereas HO-2 was only detected in endothelium. HO-2 is expressed by aortic endothelial cells early during hypoxic exposure and impairs ET-1-mediated potentiation of contraction to alpha-adrenoceptor stimulation.     

Improvement of heme oxygenase-1-based heme sensor for quantifying free heme in biological samples

We recently reported a novel heme sensor using fluorescently labeled heme oxygenase-1; however, its inherent enzyme activity would be a potential obstacle in quantifying heme in biological samples. Here, we found that mutation of the catalytically important residue, Asp140, with histidine in the sensor not only diminished the heme degradation activity but also increased heme binding affinity. The sensor with a visible fluorophore was also found to be beneficial to avoid background emission from endogenous substance in biological samples. By using the improved heme sensor, we succeeded in quantifying free heme in rat hepatic samples for the first time.     

Crystal structure of human heme oxygenase-1.

Heme oxygenase catalyzes the first step in the oxidative degradation of heme. The crystal structure of heme oxygenase-1 (HO-1) reported here reveals a novel helical fold with the heme sandwiched between two helices. The proximal helix provides a heme iron ligand, His 25. Conserved glycines in the distal helix near the oxygen binding site allow close contact between the helix backbone and heme in addition to providing flexibility for substrate binding and product release. Regioselective oxygenation of the alpha-meso heme carbon is due primarily to steric influence of the distal helix.     

Down-regulation of heme oxygenase-1 by SVCV infection

Heme oxygenase (HO-1) is a cytoprotective enzyme that plays a critical role in defending the body against oxidant-induced injury during inflammatory processes. In mammalian systems, viral infection or antigen expression can down-regulate the expression of HO-1. In turn, the induction of HO-1 or overexpression of HO-1 results in potent and direct antiviral activity that targets the replication of several mammalian viruses. In this study, the HO-1 gene of Cyprinus carpio was cloned, and the expression profile of HO-1 was investigated during spring viremia of carp virus (SVCV) infection. The results demonstrate that the expression of HO-1 was down-regulated during SVCV infection in the EPC cells and in common carp. These results indicated that SVCV infection could induce host oxidative stress, which may contribute to tissue injury in affect fish.     

Rosuvastatin upregulates the antioxidant defense protein heme oxygenase-1

Cholesterol-independent, pleiotropic actions of HMG-CoA reductase inhibitors (statins) lead to anti-inflammatory and antioxidant actions by as yet unidentified mechanisms. This study explores the role of heme oxygenase-1 (HO-1) as target and potential mediator of rosuvastatin. In cultured human endothelial cells (ECV 304), rosuvastatin increased HO-1 mRNA and protein levels in a concentration-dependent fashion. HO-1 induction by rosuvastatin remained unaffected by mevalonate and N-nitro-L-arginine-methylester, showing that isoprenoid- and NO-dependent pathways were not involved. Pretreatment of endothelial cells with rosuvastatin reduced NADPH-dependent production of oxygen radicals. The HO-1 metabolite bilirubin, when added exogenously to the cells, virtually abolished NADPH-dependent oxidative stress. Rosuvastatin-induced inhibition of free radical formation was rescued in the presence of the HO inhibitor, tin protoporphyrin-IX. Our results demonstrate that HO-1 is a target site and antioxidant mediator of rosuvastatin in endothelial cells. This novel pathway may contribute to and partially explain the pleiotropic antiatherogenic actions of rosuvastatin.     

Maintenance of lung myeloperoxidase activity in proestrus females after trauma-hemorrhage: upregulation of heme oxygenase-1

Previous studies showed that females in the proestrus stage of the reproductive cycle maintain organ functions after trauma-hemorrhage. However, it remains unknown whether the female reproductive cycle is an important variable in the regulation of lung injury after trauma-hemorrhage and, if so, whether the effect is mediated via upregulation of heme oxygenase (HO)-1. To examine this, female Sprague-Dawley rats during diestrus, proestrus, estrus, and metestrus phases of the reproductive cycle or 14 days after ovariectomy underwent soft tissue trauma and then hemorrhage (mean blood pressure 40 mmHg for 90 min followed by fluid resuscitation). At 2 h after trauma-hemorrhage or sham operation, lung myeloperoxidase (MPO) activity and intercellular adhesion molecule (ICAM)-1, cytokine-induced neutrophil chemoattractant (CINC)-1, CINC-3, and HO-1 protein levels were measured. Plasma 17beta-estradiol concentration was also determined. The results indicated that trauma-hemorrhage increased lung MPO activity and ICAM-1, CINC-1, and CINC-3 levels in ovariectomized females. These parameters were found to be similar to sham-operated animals in proestrus female rats subjected to trauma-hemorrhage. Lung HO-1 protein level in proestrus females was increased significantly compared with female rats subjected to trauma-hemorrhage during diestrus, estrus, and metestrus phases of the reproductive cycle and ovariectomized rats. Furthermore, plasma 17beta-estradiol level was highest in proestrus females. Administration of the HO inhibitor chromium mesoporphyrin prevented the attenuation of shock-induced lung damage in proestrus females. Thus these findings suggest that the female reproductive cycle is an important variable in the regulation of lung injury following trauma-hemorrhage and that the protective effect in proestrus females is likely mediated via upregulation of HO-1.     

Anti-inflammatory effects of mangiferin on sepsis-induced lung injury in mice via up-regulation of heme oxygenase-1

Sepsis, a serious unbalanced hyperinflammatory condition, is a tremendous burden for healthcare systems, with a high mortality and limited treatment. Increasing evidences indicated that some active components derived from natural foods have potent anti-inflammatory properties. Here we show that mangiferin (MF), a natural glucosyl xanthone found in both mango and papaya, attenuates cecal ligation and puncture-induced mortality and acute lung injury (ALI), as indicated by reduced systemic and pulmonary inflammatory responses. Moreover, pretreatment with MF inhibits sepsis-activated mitogen-activated protein kinases and nuclear factor kappa-light-chain-enhancer of activated B cells signaling, resulting in inhibiting production of proinflammatory mediators. Notably, MF dose-dependently up-regulates the expression and activity of heme oxygenase (HO)-1 in the lung of septic mice. Further, these beneficial effects of MF on the septic lung injury were eliminated by ZnPP IX, a specific HO-1 inhibitor. Our results suggest that MF attenuates sepsis by up-regulation of HO-1 that protects against sepsis-induced ALI through inhibiting inflammatory signaling and proinflammatory mediators. Thereby, MF may be effective in treating sepsis with ALI.