Adenovirus-mediated Heme Oxygenase-1 Research Articles

Hesperetin attenuated acetaminophen-induced hepatotoxicity by inhibiting hepatocyte necrosis and apoptosis, oxidative stress and inflammatory response via upregulation of heme oxygenase-1 expression

Acetaminophen (APAP) is a common antipyretic and analgesic drug, but its overdose can induce acute liver failure with lack of effective therapies. Hesperetin, a dihydrogen flavonoid compound, has been revealed to exert multiple pharmacological activities. Here, we explored the protective effects and mechanism of hesperetin on APAP-induced hepatotoxicity. The results showed that pretreatment with hesperetin dose-dependently attenuated APAP-induced acute liver injury in mice, as measured by alleviated serum enzymes activities, hepatic pathological damage and apoptosis. Moreover, hesperetin mitigated APAP-induced oxidative stress and inflammatory response in mice by inhibiting oxidative molecules but increasing antioxidative molecules production, reducing inflammatory cells infiltration and proinflammatory cytokines production, blocking Toll-like receptor (TLR)-4 signal activation. In vitro experiment indicated that hesperetin dose-dependently inhibited APAP-primed cytotoxicity, apoptosis, and reactive oxygen species (ROS) in murine AML12 hepatocytes. Notably, hesperetin up-regulated expression of heme oxygenase-1 (HO-1) mRNA and protein in the liver of mice and AML12 cells exposed to APAP. Furthermore, knockdown of HO-1 by adenovirus-mediated HO-1 siRNA reverted these beneficial effects of hesperetin on APAP-induced hepatocytotoxicity as well as ROS and inflammatory response in vivo and in vitro. These findings demonstrated that hesperetin exerted a protective prophylaxis on APAP-induced acute liver injury by inhibiting hepatocyte necrosis and apoptosis, oxidative stress and inflammatory response via up-regulating HO-1 expression.

Adenovirus-Mediated Heme Oxygenase-1 Gene Transfer to Neonatal Porcine Islet-Like Cluster Cells: The Effects on Gene Expression and Protection from Cell Stress

Adenovirus-Mediated Heme Oxygenase-1 Gene Transfer to Neonatal Porcine Islet-Like Cluster Cells: The Effects on Gene Expression and Protection from Cell Stress

Adenovirus-mediated heme oxygenase-1 gene transfer to neonatal porcine islet-like cluster cells: the effects on gene expression and protection from cell stress

Porcine islet xenotransplantation is a promising strategy for the treatment of diabetes that overcomes donor shortages. However, islet xenografts are susceptible to oxidative stress and apoptosis. Heme oxygenase-1 (HO-1) has been shown to protect cells from oxidative stress, apoptosis and inflammation. Here, we investigated whether introduction of human HO-1 (hHO-1) into neonatal porcine islet-like cell clusters (NPCCs) can induce beneficial transcriptional changes in NPCCs against cellular stress. NPCCs were transduced with either adenovirus-HO-1 (Ad-HO-1) or control adenovirus-GFP (Ad-GFP). After treatment with hydrogen peroxide (H2O2) for 24 hours, nitrite oxide (NO) production assays were performed to detect oxidative stress. Microarray analysis was performed using a pig oligonucleotide 44 K gene chip. We profiled transcriptional changes to apoptosis, oxidant and inflammatory genes, and real-time PCR analysis was also performed to confirm the microarray results. Survival of NPCCs after treatment with H2O2 was significantly higher in the Ad-HO-1 group (p<0.001), and NO production also decreased in the Ad-HO-1 group (p<0.01). The microarray results showed that the expression of pro-apoptosis genes such as CASP3, CASP7, CASP10, CIDE-B and CIDE-C was significantly decreased in the Ad-HO-1 virus group (CASP10; p<0.05, CASP3, CIDE-C; p<0.01, CASP7, CIDE-B; p<0.001). We also found that the expression of oxidative stresses genes including COX1, COX2, CYB5A, SDHD and NOS2 was decreased, and that the anti-oxidant genes Gpx1 and SOD2 were increased in the Ad-HO-1 group (NOS2; p<0.05, COXI, COX2, CYB5A, SDHD, SOD2, GPX1; p<0.001). However, inflammatory gene expression was not significantly changed. Realtime PCR analysis confirmed the results of the microarray analysis. These results shed light on the underlying mechanisms of the protective effects of hHO-1 on porcine islets from cellular stresses and suggest that hHO-1 could be a promising target gene for the production of transgenic pigs that confer improved islet xenograft survival.

Amelioration of radiation-induced skin injury by adenovirus-mediated heme oxygenase-1 (HO-1) overexpression in rats

ObjectiveRadiation-induced skin injury remains a serious concern for radiation therapy. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme catabolism, has been reported to have potential antioxidant and anti-apoptotic properties. However, the role of HO-1 in radiation-induced skin damage remains unclear. This study aims to elucidate the effects of HO-1 on radiation-induced skin injury in rats.MethodsA control adenovirus (Ad-EGFP) and a recombinant adenovirus (Ad-HO1-EGFP) were constructed. Rats were irradiated to the buttock skin with a single dose of 45 Gy followed by a subcutaneous injection of PBS, 5 × 109 genomic copies of Ad-EGFP or Ad-HO1-EGFP (n = 8). After treatment, the skin MDA concentration, SOD activity and apoptosis were measured. The expression of antioxidant and pro-apoptotic genes was determined by RT-PCR and real-time PCR. Skin reactions were measured at regular intervals using the semi-quantitative skin injury score.ResultsSubcutaneous injection of Ad-HO1-EGFP infected both epidermal and dermal cells and could spread to the surrounding regions. Radiation exposure upregulated the transcription of the antioxidant enzyme genes, including SOD-1, GPx2 and endogenous HO-1. HO-1 overexpression decreased lipid peroxidation and inhibited the induction of ROS scavenging proteins. Moreover, HO-1 exerted an anti-apoptotic effect by suppressing FAS and FASL expression. Subcutaneous injection of Ad-HO1-EGFP demonstrated significant improvement in radiation-induced skin injury.ConclusionsThe present study provides evidences for the protective role of HO-1 in alleviating radiation-induced skin damage in rats, which is helpful for the development of therapy for radiation-induced skin injury.

Heme Oxygenase-1/Carbon Monoxide Induces Vascular Endothelial Growth Factor Expression via p38 Kinase-dependent Activation of Sp1

Heme oxygenase-1 (HO-1) is a stress-inducible enzyme catalyzing the oxidative degradation of heme to free iron, CO, and biliverdin. Previous studies demonstrated that HO-1 overexpression promoted VEGF expression and angiogenesis in the ischemic heart. However, the underlying mechanism remained elusive. Here we show that adenovirus-mediated HO-1 transduction of rat primary cardiomyocytes and H9C2 myocytes resulted in significant induction of VEGF expression, and a similar effect was seen in cells directly exposed to CO gas or a CO-releasing compound, tricarbonyldichlororuthenium (II) dimer. HO-1/CO-induced VEGF expression was significantly suppressed by pharmacological inhibition of p38 kinase, but not of AKT, activation. VEGF promoter-luciferase reporter assays, electrophoretic mobility shift assays, supershift assay, and chromatin immunoprecipitation showed that CO-induced VEGF promoter activation requires the binding of the Sp1 transcriptional factor to a cis-regulatory sequence located at the VEGF promoter. Western blot analysis and immunostaining experiments demonstrated that HO-1/CO induced p38-dependent phosphorylation of Sp1 at Thr-453 and Thr-739 both in vitro and in vivo. Overexpression of Sp1 protein with an alanine mutation at Thr-453 or Thr-739 suppressed CO-induced Sp1 binding to the VEGF promoter and its transcriptional activation. Collectively, these data suggest that p38-dependent phosphorylation of Sp1 at Thr-453/Thr-739 is crucial for HO-1/CO-induced VEGF expression in myocytes.

Adiponectin-Mediated Heme Oxygenase-1 Induction Protects Against Iron-Induced Liver Injury via a PPARα-Dependent Mechanism

Protective effects of adiponectin (APN; an adipocytokine) were shown against various oxidative challenges; however, its therapeutic implications and the mechanisms underlying hepatic iron overload remain unclear. Herein, we show that the deleterious effects of iron dextran on liver function and iron deposition were significantly reversed by adiponectin gene therapy, which was accompanied by AMP-activated protein kinase (AMPK) phosphorylation and heme oxygenase (HO)-1 induction. Furthermore, AMPK-mediated peroxisome proliferator-activated receptor-α (PPARα) activation by APN was ascribable to HO-1 induction. Additionally, we revealed direct transcriptional regulation of HO-1 by the binding of PPARα to a PPAR-responsive element (PPRE) by various experimental assessments. Interestingly, overexpression of HO-1 in hepatocytes mimicked the protective effect of APN in attenuating iron-mediated injury, whereas it was abolished by SnPP and small interfering HO-1. Furthermore, bilirubin, the end-product of the HO-1 reaction, but not CO, protected hepatocytes from iron dextran-mediated caspase activation. Herein, we demonstrate a novel functional PPRE in the promoter regions of HO-1, and APN-mediated HO-1 induction elicited an antiapoptotic effect and a decrease in iron deposition in hepatocytes subjected to iron challenge.

Adenovirus-Mediated Heme Oxygenase Transfer Inhibits Graft Arteriosclerosis in Rat Aortic Transplants

Purpose Heme oxygenase-1 (HO-1) has been implicated in graft protection because its induction is associated with long-surviving allografts. The aim of this study was to analyze the efficacy of adenovirus-mediated HO-1 overexpression to inhibit graft arteriosclerosis and estimate whether the protective role correlated with nuclear factor κB (NF = κB) expression and grafts apoptosis. Methods Aortic transplantation was performed using inbred Brown-Norway (BN) rats (RT 1n male) as donors and Lewis rats (RT 1l male) as recipients. The experiments were divided into four groups: group A, Lewis–Lewis ( n = 6), no treatment; group B, BN–Lewis ( n = 6), no treatment; group C, BN–Lewis ( n = 6), donor aorta perfused with buffer containing 4 × 10 10 pfu Ad-Null (the noncoding adenoviral vector); and group D, BN–Lewis ( n = 6), donor aorta perfused with buffer containing 4 × 10 10 pfu Ad-HO-1 (adenoviral vector coding for HO-1). All grafts were harvested at 60 days after transplantation. Reverse transcriptase polymerase chain reaction was performed to detect the expression of HO-1 gene. Hematoxylin and eosin staining was used to detect the morphology and cytology of the transplanted vessels. Intimal thickening was detected by Masson staining. Immunohistochemistry was used to detect the localization and expression of HO-1 and NF-κB and TUNEL assays to detect the apoptosis of the grafts. Results Gene transfer of HO-1 grafts using an adenoviral vector resulted in the expression of HO-1 protein in endothelium and adventitia. We observed that the intimal thickness in Ad-HO-1–treated aortas (11.11 ± 0.92 μm) was significantly thinner compared with untreated (85.20 ± 6.90 μm) or Ad-null treated (87.20 ± 6.20 μm) aortas ( P < .01). Immunohistology showed that treatment with Ad-HO-1 resulted in a significant reduction in leukocyte infiltration and a decreased number of vascular smooth muscle cells in the intima, compared with Ad-null–treated aortas. The levels of NF-κB and the number of apoptotic cells in Ad-HO-1–treated aortas showed significantly lower compared with Ad-null–treated arotas ( P < .05). Conclusion HO-1 prevented the development of graft arteriosclerosis in the rat aortic transplant model. The protective role of HO-1 in chronic rejection lesions seemed to correlate with downregulation of the expression of NF-κB and inhibition of apoptosis in the grafts.

Heme Oxygenase-1 Attenuates Interleukin-1β-Induced Nitric Oxide Synthase Expression in Vascular Smooth Muscle Cells

Heme oxygenase-1 (HO-1) has been implicated in antioxidant and anti-inflammatory actions. To characterize the role of HO-1 in the vascular inflammatory response, we examined the effect of HO-1 on the expression of inducible nitric oxide synthase (iNOS) induced by interleukin-1β (IL-1β) in rat vascular smooth muscle cells (VSMCs). Western blot analysis demonstrated that IL-1β-induced iNOS expression was significantly reduced by hemin cotreatment or adenovirus-mediated HO-1 gene transfer. Scavenging carbon monoxide (CO), one of the by-products of heme degradation by HO-1, significantly attenuated HO-1-mediated suppression of iNOS gene induction as revealed by Northern blot analysis. Exposure of cells to CO or a CO donor, the tricarbonyldichlororuthenium(II) dimer, also markedly inhibited IL-1β-induced iNOS expression. Transient transfection experiments with a reporter gene construct carrying the rat iNOS gene promoter demonstrated that IL-1β-induced promoter activity was substantially reduced by cotreatment with CO or a CO donor. Furthermore, the effects of CO on iNOS gene promoter activity and protein expression were diminished by cotreatment with the specific guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo-(4,3-a)quinoxalin-1-one. These data support the finding that HO-1 attenuates IL-1β-induced iNOS gene expression in VSMCs. CO appears to mediate the suppressive effect of HO-1, at least in part, through downregulating transcriptional activation of the iNOS gene via a cGMP-dependent pathway.

Heme oxygenase-1 attenuates interleukin-1β-induced nitric oxide synthase expression in vascular smooth muscle cells

Heme oxygenase-1 (HO-1) has been implicated in antioxidant and anti-inflammatory actions. To characterize the role of HO-1 in the vascular inflammatory response, we examined the effect of HO-1 on the expression of inducible nitric oxide synthase (iNOS) induced by interleukin-1beta (IL-1beta) in rat vascular smooth muscle cells (VSMCs). Western blot analysis demonstrated that IL-1beta-induced iNOS expression was significantly reduced by hemin cotreatment or adenovirus-mediated HO-1 gene transfer. Scavenging carbon monoxide (CO), one of the by-products of heme degradation by HO-1, significantly attenuated HO-1-mediated suppression of iNOS gene induction as revealed by Northern blot analysis. Exposure of cells to CO or a CO donor, the tricarbonyldichlororuthenium(II) dimer, also markedly inhibited IL-1beta-induced iNOS expression. Transient transfection experiments with a reporter gene construct carrying the rat iNOS gene promoter demonstrated that IL-1beta-induced promoter activity was substantially reduced by cotreatment with CO or a CO donor. Furthermore, the effects of CO on iNOS gene promoter activity and protein expression were diminished by cotreatment with the specific guanylate cyclase inhibitor, 1H-[1,2,4]oxadiazolo-(4,3-a)quinoxalin-1-one. These data support the finding that HO-1 attenuates IL-1beta-induced iNOS gene expression in VSMCs. CO appears to mediate the suppressive effect of HO-1, at least in part, through downregulating transcriptional activation of the iNOS gene via a cGMP-dependent pathway.

Adenovirus-mediated heme oxygenase-1 gene expression stimulates apoptosis in vascular smooth muscle cells.

Heme oxygenase-1 (HO-1) catalyzes the degradation of heme into biliverdin, iron, and carbon monoxide (CO). Although HO-1 is induced in vascular smooth muscle cells (SMCs), the biological role of HO-1 in these cells has not been completely characterized. In the present study, we overexpressed HO-1 in rat aortic SMCs by generating a recombinant defective adenovirus containing the rat HO-1 gene (AdHO-1) and examined the effect on SMC proliferation. Infection of SMCs with AdHO-1 resulted in a dose-dependent increase in the expression of HO-1 mRNA, protein, and activity. Infection of SMCs with AdHO-1 inhibited serum-stimulated SMC proliferation in a dose-dependent manner. In contrast, the control adenovirus expressing the green fluorescent protein failed to induce HO-1 expression and had minimal effects on SMC growth. Infection with AdHO-1 stimulated SMC apoptosis in a dose-dependent fashion, as demonstrated by DNA fragmentation, positive annexin V labeling, and caspase-3 activation. HO-1-mediated apoptosis was associated with a marked increase in the expression of the proapoptotic protein p53. Finally, the exogenous administration of biliverdin and bilirubin stimulated SMC apoptosis. In contrast, the administration of CO or iron failed to induce cell death. These results demonstrate that overexpression of HO-1 or the exogenous administration of biliverdin or bilirubin stimulates SMC apoptosis. Adenovirus-mediated transfer of the HO-1 gene may provide a novel therapeutic approach in treating occlusive vascular disease.

Adenovirus-mediated heme oxygenase-1 gene delivery inhibits injury-induced vascular neointima formation.

Recent studies have demonstrated that systemic pharmacological induction of heme oxygenase-1 (HO-1), the inducible isoform of the initial and rate-limiting enzyme for heme catabolism, attenuates neointima formation after experimental vascular injury. We have now investigated the ability of localized adenovirus-mediated HO-1 (Ad-HO-1) gene delivery to modify arterial remodeling after balloon angioplasty. Two weeks after balloon angioplasty in the rat carotid artery, elevated HO-1 protein was observed in the Ad-HO-1 arteries compared with those exposed to empty adenovirus (Ad-E) or to PBS. The arteries exposed to Ad-HO-1 exhibited significantly reduced neointimal area, medial wall area, neointimal area/medial wall area ratio, and neointimal thickness compared with arteries exposed to Ad-E. The Ad-E vessels showed subtle reductions in each morphometric parameter compared with PBS vessels. In a separate group of animals, concomitant treatment of Ad-HO-1 with the HO-1 inhibitor tin protoporphyrin completely restored each morphometric parameter to control levels. Arteries exposed to Ad-HO-1 demonstrated significantly increased TUNEL labeling of apoptotic nuclei and significantly decreased PCNA labeling of DNA synthesis in the medial wall 48 hours after injury. These results indicate that HO-1 represents an important in vivo vasoprotective mediator that is capable of attenuating the pathophysiological remodeling response to endovascular injury and suggest that HO-1 may be a novel target for the treatment of vascular disease.

Adenovirus-mediated heme oxygenase-1 gene transfection prevents hemoglobin-induced contraction of rat basilar artery.

Vasospasm is a consequence of erythrocyte lysis in the subarachnoid space that releases hemoglobin and possibly other spasmogenic substances. Removing the blood clot early after SAH, either surgically or by intracisternal fibrinolysis with tissue plasminogen activator or urokinase, may prevent vasospasm [1]. In view of the critical role that hemoglobin appears to play in the pathogenesis of vasospasm, modification of the metabolism of hemoglobin and heme may be im- portant to preventing vasospasm. Oxidation of ferrous iron in oxy- or deoxyhemoglobin produces meth- emoglobin that more readily releases its heme groups from the globin chains. Globin chains probably are degraded by intra- and extracellular proteases. The heme group is metabolized to biliverdin, carbon mon- oxide (CO) and iron by heme oxygenases (HO) that consist of at least 3 isozymes including the oxidative stress-inducible protein HO-1 [2].