Heme Oxygenase-1 Gene Transfer Research Articles

Heme oxygenase-1 gene delivery for altering high mobility group box-1 protein in pancreatic islet

Pancreatic islet transplantation is a promising strategy for the treatment of type I diabetes. High-mobility group box-1 (HMGB1), highly expressed in islet cells, is a potent immune stimulator in immune rejection. Heme oxygenase-1 (HO1) gene therapy can modulate the release of HMGB1 by altering intracellular molecules for successful cell transplantation. After delivery of the heme oxygenase-1 (HO1) gene to islet cells using an adeno-associated viral vector (AAV), it was evaluated the changes in cytoplasmic Ca2+ ions and calcineurin activity as well as histone acetyltransferase (HAT) and Poly(ADP) ribose polymerase-1 (PARP-1). Inhibition of HMGB1 release was evaluated through altering these intracellular molecules. Then, after transplantation of HO1-transduced islets, the therapeutic effect of them was evaluated through measuring blood glucose level to diabetic mice and through immunohistochemical analysis. The transduced HO1 gene significantly inhibited HMGB1 release in islets that was under the cell damage by hypoxia exposure. It was confirmed that this result was initially due to the decrease in cytoplasmic Ca2+ ion concentration and calcineurin activity. In addition, the delivered HO1 gene simultaneously reduced the activity of HAT and PARP-1, which are involved in the translocation of HMGB1 from the nucleus to the cytoplasm. As a result, when the HO1 gene-transduced islets were transplanted into diabetic mice, the treatment efficiency of diabetes was effectively improved by increasing the survival rate of the islets. Collectively, these results suggest that HO1 gene transfer can be used for successful islet transplantation by altering the activity of intracellular signal molecules and reducing HMGB1 release.

HIF-regulated HO-1 gene transfer improves the post-ischemic limb recovery and diminishes TLR-triggered immune responses — Effects modified by concomitant VEGF overexpression

Heme oxygenase-1 (HO-1) mitigates cellular injury by antioxidant, anti-apoptotic, anti-inflammatory and proangiogenic effects. Vascular endothelial growth factor (VEGF) is a critical regulator of blood vessel growth. Their coordinated action was analyzed in a model of femoral artery ligation (FAL) in mice lacking HO-1 gene (HO-1 KO). Gastrocnemius skeletal muscles of HO-1 KO mice were preemptively injected with plasmids containing hypoxia-response element (HRE) driving the expression of only HO-1 (pHRE-HO1) or both HO-1 and VEGF (pHRE-HO1–VEGF). At day 14th the pHRE-HO1 vector increased an impaired post-ischemic blood flow recovery in HO-1 KO mice to the level observed in wild-type (WT) mice subjected to FAL and pHRE-HO1–VEGF restored it already at day 7. The pHRE-HO1 gene therapy diminished, when compared to control pHRE-empty-treated HO-1 KO mice, the expression of toll-like receptors (TLR4 and TLR9) and inflammatory cytokines (IL-1β, IL-6 and TNFα) at day 3, whereas opposite effects were observed following concomitant HO-1 and VEGF gene transfer. Moreover, HO-1 diminished ischemia-induced expression of MyoD involved in satellite cell differentiation in HO-1 KO mice. Our results confirm the therapeutic potential of HO-1 and VEGF against critical limb ischemia although, their concomitant delivery may have contradictory actions on the resolution of inflammation.

Nucleotide sequencing, cloning, and expression of Capra hircus Heme Oxygenase-1 in caprine islets to promote insulin secretion in vitro.

Transplantation of islets of Langerhans that have been isolated from whole pancreas is an attractive alternative for the reversal of Type 1 diabetes. However, in vitro culture of isolated pancreatic islets has been reported to cause a decrease in glucose response over time. Hence, the improvement in islet culture conditions is an important goal in islet transplantation. Heme Oxygenase-1 (HO-1) is a stress protein that has been described as an inducible protein with the capacity of preventing apoptosis and cytoprotection via radical scavenging. Therefore, this study was aimed to assess the influence of endogenous HO-1 gene transfer on insulin secretion of caprine islets. The full-length cDNA sequence of Capra hircus HO-1 was determined using specific designed primers and rapid amplification of cDNA ends of pancreatic tissue. The HO-1 cDNA was then cloned into the prokaryotic expression vectors and transfected into caprine islets using lipid carriers. Efficiency of lipid carriers to transfect caprine islets was determined by flow cytometry. Insulin secretion assay was carried out by ovine insulin ELISA. The finding demonstrated that endogenous HO-1 gene transfer could improve caprine islet function in in vitro culture. Consequently, strategies using HO-1 gene transfer to islets might lead to better outcome in islet transplantation.

Protective Effect of Adenoviral Transfer of Heme Oxygenase-1 Gene on Rats With Severe Acute Pancreatitis

It is widely accepted that the pathophysiological mechanism of severe acute pancreatitis (SAP) is characterized by the systemic inflammatory response, which eventually causes systemic inflammatory response syndrome (siRs), multiple organ dysfunction syndrome (MoDs), and even death. Heme oxygenase (HO)-1 has been proved to exert anti-inflammatory benefit in a variety of inflammatory diseases, and a variety of methods by which HO-1 overexpression can be induced have been reported. In this study, we hypothesized that transfer of HO-1 gene by adenoviral vector could inhibit the systemic inflammation and the development of MODs. Sprague–Dawley (sD) rats were used as subjects in this study. Each was made into sAP model by retrograded injection through pancreatic duct with 5% sodium taurocholate (0.1mL/100g). Normal saline (1mL/animal) or adenoviral HO-1 gene (Adv-HO-1, 2.0×109 PFU/mL/animal) or adenoviral empty vectors (Adv-0, 2.0×109PFU/mL/animal) were injected intraperitoneally. HO-1 expression in serum and tissues including pancreas, liver and kidney was measured and observed to be upregulated in the rats treated with Adv-HO-1. The administration of Adv-HO-1 also inhibited the expression of tumor necrosis factor (TNF)-α and boosted the expression of interleukin-10 (IL-10). The decreasing serum concentration of ALT, AST, BUN and CREA, the amelioration of histopathologic damage in pancreas, liver and kidney tissues, the improvement of survival rate were all observed in rats treated with Adv-HO-1 comparing to others. As a result, this study showed that Adv-HO-1 could exert protective effect including anti-inflammation and organ protection through enhancing the expression of HO-1 in SAP.

Heme oxygenase-1 gene transfer mediated by adeno-associated virus induces immunological tolerance in liver allograft in rats

Objective To investigate the efficacy and molecular mechanisms of AAV-HO-1 inducing immuno-telorance in rats.Methods An orthotopic liver transplantation model by DA to Lewis was set up using Kamada' s two cuff technique.PBS,empty AAV or purified AAV-HO-1 was injected into the portal vein and incubated for 2 h at the donor liver cold preserved stage,and then OLT was done.The media survival time (MST),serum levels of interleukin (IL)-2 and tumor necrosis factor (TNF)-α,infiltration of CD4 +,CD8 + and Treg (CD4 + CD25 + Foxp3 +) cells into donor livers and percent of Treg in the spleen were examined.A mixed lymphocyte reaction (MLR) was performed.Results Recipients transplanted with AAV-HO-1-perfused liver allografts had a median survival time of 30 days,which was significantly longer than that in the PBS group (11 days) and empty AAV group (12 days) (both P ＜0.01).Overexpression of HO-1 reduced the production of IL-2 and TNF-α,inhibited infiltration of CD4+ and CD8 + cells,and increased infiltration of Treg ceils into donor livers.Splenocytes from the tolerant recipients had higher percentages of Treg cells,and responded poorly to the allogeneic donor splenocytes.Conclusion Persistent expression of HO-1 in the donor livers by intraportal delivery of AAV-HO-1 can prolong the survival by expanding Treg cells,suggesting that AAV-HO-1 can induce immuno-tolerance in liver allograft in rats. Key words: Liver transplantation ; Immunological tolerance ; Gene therapy ; Adeno-associated virus; Heme oxygenase-1

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.

Gene transfer as a strategy to achieve permanent cardioprotection II: rAAV-mediated gene therapy with heme oxygenase-1 limits infarct size 1 year later without adverse functional consequences

Extensive evidence indicates that heme oxygenase-1 (HO-1) exerts potent cytoprotective effects in response to stress. Previous studies have shown that gene therapy with HO-1 protects against myocardial ischemia/reperfusion injury for up to 8weeks after gene transfer. However, the long-term effects of HO-1 gene therapy on myocardial ischemic injury and function are unknown. To address this issue, we created a recombinant adeno-associated viral vector carrying the HO-1 gene (rAAV/HO-1) that enables long-lasting transgene expression. Mice received injections in the anterior LV wall of rAAV/LacZ (LacZ group) or rAAV/HO-1 (HO-1 group); 1year later, they were subjected to a 30-min coronary occlusion (O) and 4h of reperfusion (R). Cardiac HO-1 gene expression was confirmed at 1month and 1year after gene transfer by immunoblotting and immunohistochemistry analyses. In the HO-1 group, infarct size (% of risk region) was dramatically reduced at 1year after gene transfer (11.2±2.1%, n=12, vs. 44.7±3.6%, n=8, in the LacZ group; P<0.05). The infarct-sparing effects of HO-1 gene therapy at 1year were as powerful as those observed 24h after ischemic PC (six 4-min O/4-min R cycles) (15.0±1.7%, n=10). There were no appreciable changes in LV fractional shortening, LV ejection fraction, or LV end-diastolic or end-systolic diameter at 1year after HO-1 gene transfer as compared to the age-matched controls or with the LacZ group. Histology showed no inflammation in the myocardium 1year after rAAV/HO-1-mediated gene transfer. These results demonstrate, for the first time, that rAAV-mediated HO-1 gene transfer confers long-term (1year), possibly permanent, cardioprotection without adverse functional consequences, providing proof of principle for the concept of achieving prophylactic cardioprotection (i.e., "immunization against infarction").

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.

Effects of recombinant adeno-associated virus-mediated heme oxygenase-1 gene transfection on inflammatory cytokine in isolated rat hearts with myocardial ischemia-reperfusion injury

Objective To investigate the effects of recombinant adeno-associated virus (rAAV)-mediated heme oxygenase-1 (HO-1) gene transfer on inflammatory cytokine in isolated rat hearts with myocardial ischemia-reperfusion (I/R) injury. Methods Thirty male SD rats weighing 220-280 kg were randomly divided into 3 groups: group Ⅰ normal control (n=6); group Ⅱ normal saline + I/R (n=12) and group Ⅲ rAAV-HO-I +I/R (n=12). In group Ⅲ rAAV-mediated HO-1 was injected intramuscularly at 4 points on the anterior and posterior wall of left ventricle along the left anterior descending branch of coronary artery. In group Ⅱ normal saline was injected instead of rAAV- HO-1 gene. Six animals in each group were sacrificed 3 months after the gene delivery in group Ⅱ and Ⅲ The expression of HO-1 in the injected myocardium was determined by immuno-histnchemical technique. The isolated rat hearts were perfased with an oxygenated (95% O_2-5% CO_2) K-H solution at 37℃ in a Langendorff apparatus and subjected to 40 min of global ischemia followed by 45 min of reperfusion in group Ⅱ and Ⅲ. The left ventricular diastolic pressure (LVDP), left ventricular end-diastolic pressure (LVEDP) anti ±dp/dt_(max) were measured recorded at 15 min of post-preparation equilibration and at 15, 30 and 45 min of repcrfusion. TNF-α and IL-6 contents in myocardium were measured Results The HO-1 expression was significantly higher in group Ⅲ than in group Ⅱ (P < 0.05 or 0.01). The left ventricular function was significantly better and TNF-α and IL-6 contents in myocardium were significantly lower in group Ⅲ than in group Ⅱ (P <0.05 or 0.01). Conclusion rAAV-mediatcd HO-1 gene transfer can protect myocardium against I/R injury in isolated rat hearts through inhibition of inflammatory cytokine production. Key words: Heme oxygenase (decyclizing); Myocardial reperfusion injury; Transfection; Adenoviridae

Heme Oxygenase-1 Accelerates Cutaneous Wound Healing in Mice

Heme oxygenase-1 (HO-1), a cytoprotective, pro-angiogenic and anti-inflammatory enzyme, is strongly induced in injured tissues. Our aim was to clarify its role in cutaneous wound healing. In wild type mice, maximal expression of HO-1 in the skin was observed on the 2nd and 3rd days after wounding. Inhibition of HO-1 by tin protoporphyrin-IX resulted in retardation of wound closure. Healing was also delayed in HO-1 deficient mice, where lack of HO-1 could lead to complete suppression of reepithelialization and to formation of extensive skin lesions, accompanied by impaired neovascularization. Experiments performed in transgenic mice bearing HO-1 under control of keratin 14 promoter showed that increased level of HO-1 in keratinocytes is enough to improve the neovascularization and hasten the closure of wounds. Importantly, induction of HO-1 in wounded skin was relatively weak and delayed in diabetic (db/db) mice, in which also angiogenesis and wound closure were impaired. In such animals local delivery of HO-1 transgene using adenoviral vectors accelerated the wound healing and increased the vascularization. In summary, induction of HO-1 is necessary for efficient wound closure and neovascularization. Impaired wound healing in diabetic mice may be associated with delayed HO-1 upregulation and can be improved by HO-1 gene transfer.

Heme oxygenase-1 promotes neovascularization in ischemic heart by coinduction of VEGF and SDF-1

Heme oxygenase-1 (HO-1) is a stress-inducible enzyme with multiple protective functions in cardiovascular systems. Studies have shown that the timely cardiac HO-1 overexpression at acute phase of ischemic infarction (MI) provides protection via its anti-apoptotic and anti-inflammatory effects. Here we demonstrate that a delayed HO-1 transduction mediated by a recombinant adeno-associated virus in ischemic hearts of mice with permanent coronary artery ligation significantly attenuated left ventricular fibrosis and cardiac dysfunctions examined at 4 weeks post MI. HO-1-mediated protection was correlated with enhanced vascularization in the ischemic myocardium. HO-1 gene transfer resulted in a notable increase in the number of c-kit +- stem cells recruited to the infarcted area at 10 days after ligation. HO-1-mediated stem cell recruitment was also demonstrated in the heart of non-ischemic mice receiving intravenous infusion of green fluorescent protein-bearing bone marrow stem cells. Additional experiments revealed that vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) were highly induced in HO-1 transduced myocardium. Mononuclear cell infiltration was evident and colocalized with angiogenic factors in the same region. Flow cytometry analysis of the mononuclear cells isolated from HO-1-transduced left ventricles revealed that over 50% of cells expressed CD34, a marker of hematopoietic stem cells and endothelial progenitor cells. VEGF and SDF-1 blockade by neutralizing antibodies significantly attenuated HO-1-mediated neovascularization and protection in infarcted mice. These data suggest that cardiac HO-1 gene transfer post MI provides protection at least in part by promoting neovascularization through inducing angiogenic factors and the recruitment of circulating progenitor/stem cells.

Photoreceptor Protection against Light Damage by AAV-Mediated Overexpression of Heme Oxygenase-1

To investigate whether overexpression of the cytoprotective gene heme oxygenase-1 (HO-1) in photoreceptors by gene delivery attenuates cellular injury caused by intense light damage and to document the possible mechanisms of protection. Recombinant adeno-associated virus type 5 (rAAV5) expressing the mouse HO-1 gene (mHO-1) was delivered to cyclic-light reared Sprague-Dawley (SD) rats by subretinal injection. Three weeks after transfer of HO-1 gene, animals were subjected to 2-hour intense light exposure then were returned to darkness. Expression of HO-1, p53, p38, and cellular FLICE inhibitory protein (c-FLIP) at different times after intense light damage was evaluated by Western blot analysis. HO-1 transgene expression, along with expression of c-fos and bcl-2, was analyzed by immunohistochemistry. In addition, the protective effects of HO-1 were evaluated by determining the morphology of the retina. Finally, apoptosis in photoreceptors was measured using TdT-dUTP terminal nick-end labeling (TUNEL) 24 hours after photic injury. Exogenous administration of HO-1 by gene transfer led to HO-1 transgene expression in photoreceptors. Protection of retina by HO-1 overexpression is evident from the partially preserved retina structure and attenuated apoptosis in photoreceptors after photic injury. Concurrently, overexpression of HO-1 was associated with a decrease in the expression of c-fos and p53, an increase in the activation of p38 and bcl-2, and preserved the expression of c-FLIP. Overexpression of HO-1 in photoreceptors protected themselves from subsequent cellular damage caused by intense light exposure. The anti-apoptotic mechanisms of HO-1 may be related to the induction of p38, bcl-2, and c-FLIP and to the suppression of c-fos and p53.

Influence of heme oxygenase-1 gene transfer on the viability and function of rat islets inin vitroculture

To investigate the influence of heme oxygenase-1 (HO-1) gene transfer on the viability and function of cultured rat islets in vitro. Islets were isolated from the pancreata of Sprague-Dawley rats by intraductal collagenase digestion, and purified by discontinuous Ficoll density gradient centrifugation. Purified rat islets were transfected with adenoviral vectors containing human HO-1 gene (Ad-HO-1) or enhanced green fluorescent protein gene (Ad-EGFP), and then cultured for seven days. Transfection was confirmed by fluorescence microscopy and Western blot. Islet viability was evaluated by acridine orange/propidium iodide fluorescent staining. Glucose-stimulated insulin release was detected using insulin radioimmunoassay kits and was used to assess the function of islets. Stimulation index (SI) was calculated by dividing the insulin release upon high glucose stimulation by the insulin release upon low glucose stimulation. After seven days culture, the viability of cultured rat islets decreased significantly (92% +/- 6% vs 52% +/- 13%, P < 0.05), and glucose-stimulated insulin release also decreased significantly (6.47 +/- 0.55 mIU/L/30IEQ vs 4.57 +/- 0.40 mIU/L/30IEQ, 14.93 +/- 1.17 mIU/L/30IEQ vs 9.63 +/- 0.71 mIU/L/30IEQ, P < 0.05). Transfection of rat islets with adenoviral vectors at an MOI of 20 was efficient, and did not impair islet function. At 7 d post-transfection, the viability of Ad-HO-1 transfected islets was higher than that of control islets (71% +/- 15% vs 52% +/- 13%, P < 0.05). There was no significant difference in insulin release upon low glucose stimulation (2.8 mmol/L) among Ad-HO-1 transfected group, Ad-EGFP transfected group, and control group (P > 0.05), while when stimulated by high glucose (16.7 mmol/L) solution, insulin release in Ad-HO-1 transfected group was significantly higher than that in Ad-EGFP transfected group and control group, respectively (12.50 +/- 2.17 mIU/L/30IEQ vs 8.87 +/- 0.65 mIU/L/30IEQ; 12.50 +/- 2.17 mIU/L/30IEQ vs 9.63 +/- 0.71 mIU/L/30IEQ, P < 0.05). The SI of Ad-HO-1 transfected group was also significantly higher than that of Ad-EGFP transfected group and control group, respectively (2.21 +/- 0.02 vs 2.08 +/- 0.05; 2.21 +/- 0.02 vs 2.11 +/- 0.03, P < 0.05). The viability and function of rat islets decrease over time in in vitro culture, and heme oxygenase-1 gene transfer could improve the viability and function of cultured rat islets.

Adeno-associated virus-mediated heme oxygenase-1 gene transfer suppresses the progression of micronodular cirrhosis in rats

Aims: Liver cirrhosis is a worldwide health problem. To test the hypothesis that enhancement of the activity of heme oxygenase can interfere with processes of fibrogenesis associated with recurrent liver injury, we investigated the therapeutic potential of over-expression of heme oxygense-1 in a CCl4-induced micronodular cirrhosis model.

Heme oxygenase‐1 gene transfer inhibits angiotensin II‐mediated rat cardiac myocyte apoptosis but not hypertrophy

Cardiac myocyte apoptosis underlies the pathophysiology of cardiomyopathy, and plays a critical role in the transition from myocardial hypertrophy to heart failure. Angiotensin II (Ang II) induces cardiac myocyte apoptosis and hypertrophy which contribute to heart failure possibly through enhanced oxidative stress; however, the mechanisms underlying the activation of both pathways and their interactions remain unclear. In the present study, we have investigated whether overexpression of the antioxidant protein heme oxygenase-1 (HO-1) protects against apoptosis and hypertrophy in cultured rat cardiac myocytes treated with Ang II. Our findings demonstrate that Ang II (100 nM, 24 h) alone upregulates HO-1 expression and induces both myocyte hypertrophy and apoptosis, assessed by measuring terminal deoxynucleotidyltransferase dUTP nick-end labelling (TUNEL) staining, caspase-3 activity and mitochondrial membrane potential. Ang II elicited apoptosis was augmented in the presence of tin protoporphyrin, an inhibitor of HO activity, while HO-1 gene transfer to myocytes attenuated Ang II-mediated apoptosis but not hypertrophy. Adenoviral overexpression of HO-1 was accompanied by a significant increase in Ang II induced phosphorylation of Akt, however, Ang II-mediated p38 mitogen activated protein kinase (MAPK) phosphorylation was attenuated. Inhibition of phosphotidylinositol-3-kinase enhanced myocyte apoptosis elicited by Ang II, however, p38MAPK inhibition had no effect, suggesting that overexpression of HO-1 protects myocytes via augmented Akt activation and not through modulation of p38MAPK activation. Our findings identify the signalling pathways by which HO-1 gene transfer protects against apoptosis and suggest that overexpression of HO-1 in cardiomyopathies may delay the transition from myocyte hypertrophy to heart failure.

Anomalous Renal Effects of Tin Protoporphyrin in a Murine Model of Sickle Cell Disease

In human and murine models of sickle cell disease (SCD), heme oxygenase-1 (HO-1) is induced in the kidney, an organ commonly involved in SCD. The present study assessed the role of HO-1 by using a competitive inhibitor of HO activity, tin protoporphyrin (SnPP), in protocols affording a composite, clinically relevant analysis of the kidney in SCD under unstressed and stressed conditions. Whereas short-term administration of SnPP exerted comparable renal hemodynamic effects in wild-type and sickle mice, chronic administration of SnPP exerted divergent effects: SnPP provoked tubulointerstitial inflammation and up-regulation of injury-related genes in wild-type mice, whereas in sickle mice SnPP reduced expression of injury-related genes and vascular congestion without provoking tubulointerstitial inflammation. SnPP also protected against the heightened sensitivity to renal ischemia observed in sickle mice, preventing ischemia-induced worsening of renal injury in sickle mice above that observed in wild-type mice. Effective and comparable inhibition of HO activity by SnPP in wild-type and sickle mice was confirmed. These findings suggest that induction of HO-1, at least as assessed by this approach, may contribute to renal injury in this murine model of SCD and uncover an experimental maneuver that protects the kidney in murine SCD.

Adeno-associated virus-mediated heme oxygenase-1 gene transfer suppresses the progression of micronodular cirrhosis in rats

To test the hypothesis that enhancement of the activity of heme oxygenase can interfere with processes of fibrogenesis associated with recurrent liver injury, we investigated the therapeutic potential of over-expression of heme oxygense-1 in a CCl(4)-induced micronodular cirrhosis model. Recombinant adeno-associated viruses carrying rat HO-1 or GFP gene were generated. 1x 10(12) vg of adeno-associated viruses were administered through portal injection at the time of the induction of liver fibrosis. Conditioning the rat liver with over-expression of HO-1 by rAAV/HO-1 significantly increased the HO enzymatic activities in a stable manner. The development of micronodular cirrhosis was significantly inhibited in rAAV/HO-1-transduced animals as compared to controls. Portal hypertension was markedly diminished in rAAV/HO-1-transduced animals as compared to controls, whereas there are no significant changes in systolic blood pressure. This finding was accompanied with improved liver biochemistry, less infiltrating macrophages and less activated hepatic stellate cells (HSCs) in rAAV/HO-1-transduced livers. Enhancement of HO activity in the livers suppresses the development of cirrhosis.

Mo-P3:221 Heme oxygenase-1 gene transfer via a hypoxia-responsive adeno-associated viral vector suppresses progressive heart failure after myocardial infarction

Mo-P3:221 Heme oxygenase-1 gene transfer via a hypoxia-responsive adeno-associated viral vector suppresses progressive heart failure after myocardial infarction

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.