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BACKGROUND: Mesenchymal stem cells (MSCs) are used for the treatment of osteoarthritis (OA), and MSC genetic engineering is expected to enhance cartilage repair. Here, we aimed to investigate the effect of MSCs overexpressing platelet-derived growth factor (PDGF) or heme oxygenase-1 (HO-1) in chondrocytes and synovial cells with an OA phenotype and assess the in vivo efficacy of intra-articular injections of these MSCs in canine OA models. METHODS: Canine adipose-derived MSCs were transfected with canine PDGF (PDGF-MSCs) or HO-1 (HO-1-MSCs) using lentiviral vectors. Canine chondrocytes or synovial cells were stimulated with lipopolysaccharide (LPS) to mimic the inflammatory OA model and then co-cultured with MSCs, PDGF-MSCs, or HO-1-MSCs for 24 h and 72 h. The mRNA levels of pro-inflammatory, extracellular matrix-degradative/synthetic, or pain-related factors were measured after co-culture by real-time PCR. Furthermore, a surgery-induced canine OA model was established and the dogs were randomized into four groups: normal saline (n = 4), MSCs (n = 4), PDGF-MSCs (n = 4), and HO-1-MSCs (n = 4). The OA symptoms, radiographic OA severity, and serum matrix metallopeptidase (MMP)-13 levels were assessed before and 10 weeks after treatment, to evaluate the safety and efficacy of the modified MSCs. RESULTS: PDGF or HO-1 overexpression significantly reduced the expression of pro-inflammatory factors, MMP-13, and nerve growth factor elicited by LPS and increased that of aggrecan and collagen type 2 in chondrocytes (P < 0.05). In addition, the expression of aggrecanases was significantly downregulated in synovial cells, whereas that of tissue inhibitor of metalloproteinases was upregulated (P < 0.05). Furthermore, the co-cultured MSCs highly expressed genes that contributed to the maintenance of joint homeostasis (P < 0.05). In vivo studies showed that OA symptoms improved after administration of all MSCs. Also, PDGF-MSCs significantly improved limb function and reduced pain (P < 0.05). The results of the radiographic assessment and serum MMP-13 levels did not vary significantly compared to those of the control. CONCLUSIONS: Genetically modifying PDGF and HO-1 in MSCs is an effective strategy for treating OA, suggesting that PDGF-MSCs can be novel therapeutic agents for improving OA symptoms.

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The blood-spinal cord barrier (BSCB) plays a key role in maintaining the microenvironment and is primarily composed of tight junction proteins and nonfenestrated capillary endothelial cells. After injury, BSCB damage results in increasing capillary permeability and release of inflammatory factors. Recent studies have reported that haem oxygenase-1 (HO-1) fragments lacking 23 amino acids at the C-terminus (HO-1C[INCREMENT]23) exert novel anti-inflammatory and antioxidative effects in vitro. However, no study has identified the role of HO-1C[INCREMENT]23 in vivo. We aimed to investigate the protective effects of HO-1C[INCREMENT]23 on the BSCB after spinal cord injury (SCI) in a rat model. Here, adenoviral HO-1C[INCREMENT]23 (Ad-GFP-HO-1C[INCREMENT]23) was intrathecally injected into the 10th thoracic spinal cord segment (T10) 7 days before SCI. In addition, nuclear and cytoplasmic extraction and immunofluorescence staining of HO-1 were used to examine the effect of Ad-GFP-HO-1C[INCREMENT]23 on HO-1 nuclear translocation. Evan's blue staining served as an index of capillary permeability and was detected by fluorescence microscopy at 633 nm. Western blotting was also performed to detect tight junction protein expression. The Basso, Beattie and Bresnahan score was used to evaluate kinematic functional recovery through the 28th day after SCI. In this study, the Ad-GFP-HO-1C[INCREMENT]23 group showed better kinematic functional recovery after SCI than the Ad-GFP and Vehicle groups, as well as smaller reductions in TJ proteins and capillary permeability compared with those in the Ad-GFP and Vehicle groups. These findings indicated that Ad-GFP-HO-1C[INCREMENT]23 might have a potential therapeutic effect that is mediated by its protection of BSCB integrity.

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Liver ischemia-reperfusion injury (IRI) represents a major risk factor of early graft dysfunction and a key obstacle to expanding the donor pool in orthotopic liver transplantation (OLT). Although graft autophagy is essential for resistance against hepatic IRI, its significance in clinical OLT remains unknown. Despite recent data identifying heme oxygenase-1 (HO-1) as a putative autophagy inducer, its role in OLT and interactions with sirtuin-1 (SIRT1), a key autophagy regulator, have not been studied. We aimed to examine HO-1-mediated autophagy induction in human OLT and in a murine OLT model with extended (20 hours) cold storage, as well as to analyze the requirement for SIRT1 in autophagy regulation by HO-1. Fifty-one hepatic biopsy specimens from OLT patients were collected under an institutional review board protocol 2 hours after portal reperfusion, followed by Western blot analyses. High HO-1 levels correlated with well-preserved hepatocellular function and enhanced SIRT1/LC3B expression. In mice, HO-1 overexpression by genetically modified HO-1 macrophage therapy was accompanied by decreased OLT damage and increased SIRT1/LC3B expression, whereas adjunctive inhibition of SIRT1 signaling diminished HO-1-mediated hepatoprotection and autophagy induction. Our translational study confirms the clinical relevance of HO-1 cytoprotection and identifies SIRT1-mediated autophagy pathway as a new essential regulator of HO-1 function in IR-stressed OLT.

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An efficient gene carrier to the brain is required for successful gene therapy of ischemic stroke. In this study, deoxycholic acid-conjugated polyethylenimine (DA-PEI) was synthesized and evaluated as a heme oxygenase-1 (HO-1) gene carrier for ischemic stroke gene therapy. Gel retardation assay and heparin competition assay showed that DA-PEI formed a stable complex with plasmid DNA. In vitro transfection assays with the luciferase gene showed that DA-PEI had higher transfection efficiency than polyethylenimine (25 kDa, PEI25k) and lipofectamine in Neuro2A cells. Furthermore, DA-PEI had less toxicity than lipofectamine. To evaluate the therapeutic effects of the pß-HO-1/DA-PEI complex, the complex was injected locally in the brain of the transient middle cerebral artery occlusion animal model. In in vivo studies, DA-PEI was more effective than PEI25k in delivering pß-HO-1 to the ischemic brain and achieved higher HO-1 expression. As a result, the pß-HO-1/DA-PEI complexes more effectively reduced infarct volume and the number of apoptotic cells compared with the pß-HO-1/PEI25k complex. The results suggest that DA-PEI will be useful for HO-1 gene therapy of ischemic stroke.

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BACKGROUND/PURPOSE: Liver transplantation is the treatment of choice in patients with end-stage liver disease. During liver transplantation, ischemia-reperfusion injury (IRI) occurs, which is an inevitable consequence of the transplantation process. To reduce the extent of cellular injury, one of the proteins that have been extensively investigated is heme oxygenase 1 (HO-1), which plays an important role in protecting the organs against IRI. The aim of this study was to introduce an active and functional HO-1 protein conjugated to a cell-penetrating peptide (CPP) in vitro and ex vivo into liver cells in hypothermic and anoxic conditions and to assert its cytoprotective effects. METHODS: We generated an enzymatically active soluble (s)HO-1-CPP recombinant protein. The ability of the sHO-1-CPP protein to penetrate McA-RH7777, Clone 9, and Hep G2 cells, primary hepatocytes, and Kupffer and human umbilical vein endothelial cells in vitro, as well as its ability to penetrate a whole liver ex vivo under hypothermic and anoxic conditions, was assessed. An in vitro hypoxia-reoxygenation (HR) model was used to determine the cytoprotective effect of the sHO-1-CPP protein. RESULTS: We showed that our recombinant protein sHO-1-CPP can cross cell membranes into rodent and human liver cells in vitro, and the results were further validated ex vivo, where rodent livers were perfused with an organ preservation solution supplemented with sHO-1-CPP under anoxic and hypothermic conditions. Immunohistochemistry revealed an intracellular localization of sHO-1-CPP in zones 1-3 of the perfused livers. The CPP did not exert any significant toxicity on the cells. Treating cells with sHO-1-CPP showed significant cytoprotection in the in vitro HR model. CONCLUSIONS: Our findings show that the recombinant protein sHO-1-CPP can be successfully delivered to cells of a whole organ in an ex vivo hypothermic and anoxic perfusion model and that it provides cytoprotection to hepatocytes in an in vitro HR model. These results hold great potential for future repair and protection of donor organs. Future experiments are planned to confirm these data in in vivo models of IRI.

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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-1ß, 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.

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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 (0.1 mL/100 g). Normal saline (1 mL/animal) or adenoviral HO-1 gene (Adv-HO-1, 2.0 × 10 PFU/mL/animal) or adenoviral empty vectors (Adv-0, 2.0 × 10 PFU/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.

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Cardioprotection can be evoked through extracardiac approaches. This prompted us to investigate whether remote transplantation of stem cells confers protection of the heart against ischemic injury. The cardioprotective effect of subcutaneous transplantation of naïve versus heme oxygenase-1 (HMOX-1)-overexpressing mouse mesenchymal stem cells (MSC) to mice was investigated in hearts subjected to ischemia-reperfusion in a Langendorff perfusion system. Mice were transplanted into the interscapular region with naïve or HMOX-1 transfected MSC isolated from transgenic luciferase reporter mice and compared to sham-treated animals. The fate of transplanted cells was followed by in vivo bioluminescence imaging, revealing that MSC proliferated, but did not migrate detectably from the injection site. Ex vivo analysis of the hearts showed that remote transplantation of mouse adipose-derived MSC (mASC) resulted in smaller infarcts and improved cardiac function after ischemia-reperfusion compared to sham-treated mice. Although HMOX-1 overexpression conferred cytoprotective effects on mASC against oxidative stress in vitro, no additive beneficial effect of HMOX-1 transfection was noted on the ischemic heart. Subcutaneous transplantation of MSC also improved left ventricular function when transplanted in vivo after myocardial infarction. Plasma analysis and gene expression profile of naïve- and HMOX-1-mASC after transplantation pointed toward pentraxin 3 as a possible factor involved in the remote cardioprotective effect of mASC. These results have significant implications for understanding the behavior of stem cells after transplantation and development of safe and noninvasive cellular therapies with clinical applications. Remote transplantation of MSC can be considered as an alternative procedure to induce cardioprotection.

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OBJECTIVE: To explore the effects of heme oxygenase-1 (HO-1) on the apoptosis, expression of caspase-3 and cytochrome-C (Cyt-C) on hydrogen peroxide (H(2)O(2)) oxidative damage in primary cultured rat's type II alveolar epithelium cell (AECII). METHODS: AECII from male Sprague-Dawley (SD) rats were separated and cultured. The cells were randomly divided into four groups: control group (A group, the cells were cultured with normal saline), H(2)O(2) group (B group, cultured with 0.5 mmol/L H(2)O(2)), HO-1 pretreatment group (C group, pretreatment with 0.2 µmol/L HO-1 for 2 hours followed by 0.5 mmol/L H(2)O(2)), HO-1 inhibition group [D group, pretreatment with 10 µmol/L zinc protoporphyrin IX (ZnppIX) for 2 hours followed by 0.5 mmol/L H(2)O(2)]. The cells in each group were cultured after different treatment. The cell apoptosis rate was determined by flow cytometry at 2, 6, 12 hours after the intervention. The protein expression of caspase-3 and Cyt-C were determined by Western Blot. RESULTS: The cell apoptosis rate in each group was gradually increased with prolonged time of H(2)O(2) treatment. The apoptosis rate at different time points after treatment in B group was significantly higher than that in A group, while the rate in C group was significantly lower than that in B group [2 hours: (11.46 ± 1.47)% vs. (20.83 ± 1.55)%, 6 hours: (12.30 ± 1.37)% vs. (27.14 ± 1.53)%, 12 hours: (12.62 ± 1.39)% vs. (35.66 ± 0.74)%, all P<0.05]. There were no significant differences in apoptotic rate at 2, 6, 12 hours between D group [(24.33 ± 1.36)%, (31.67 ± 1.24)%, (36.93 ± 2.40)%] and B group. The protein expression of caspase-3 and Cyt-C was gradually increased with prolonged time of H(2)O(2) treatment. The protein expression of caspase-3 and Cyt-C at different time points in B group was significantly higher than that in A group. The protein expression of caspase-3 (gray scale) in C group was significantly lower than that in B group (2 hours: 0.250 ± 0.039 vs. 0.650 ± 0.072, 6 hours: 0.470 ± 0.080 vs. 0.960 ± 0.118, 12 hours: 0.680 ± 0.099 vs. 1.830 ± 0.220, all P<0.05), and the Cyt-C protein expression (gray scale) was also significantly lowered in C group (2 hours: 0.250 ± 0.074 vs. 0.390 ± 0.069, 6 hours: 0.340 ± 0.043 vs. 0.670 ± 0.120, 12 hours: 0.470 ± 0.072 vs. 1.360 ± 0.112, all P<0.05). There were no significant differences in protein expression of caspase-3 and Cyt-C between D group (caspase-3: 0.720 ± 0.052, 1.060 ± 0.109, 1.880 ± 0.159; Cyt-C: 0.500 ± 0.110, 0.860 ± 0.050, 1.480 ± 0.140) and B group. CONCLUSIONS: HO-1 preconditioning reduced the apoptotic rate of the AECII oxidative damaged by H(2)O(2) at different time points (2-12 hours) and decreased the expression of caspase-3 and Cyt-C. The mitochondrial apoptosis pathway participated in the protection mechanism of HO-1 in oxidative damage AECII.

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Recent studies have uncovered that overexpression of heme oxygenase-1 (HO-1) by induction or gene transfer provides myocardial protection. In the present study, we investigated whether HO-1 protein mediated by cell-penetrating peptide PEP-1 could confer cardioprotection in a rat model of myocardial ischemia/reperfusion (I/R) injury. Male Sprague-Dawley rats were subjected to 30 minutes of ischemia by occluding the left anterior descending coronary artery and to 120 minutes of reperfusion to prepare the model of I/R. Animals were randomized to receive PEP-1-HO-1 fusion protein or saline 30 minutes before a 30-minute occlusion. I/R increased myocardial infarct size and levels of malondialdehyde, serum tumor necrosis factor alpha, and interleukin 6 and reduced myocardial superoxide dismutase activity. Administration of PEP-1-HO-1 reduced myocardial infarct size and levels of malondialdehyde, serum tumor necrosis factor alpha, and interleukin 6 and increased myocardial superoxide dismutase and HO-1 activities. His-probe protein was only detected in PEP-1-HO-1-transduced hearts. In addition, transduction of PEP-1-HO-1 markedly reduced elevated myocardial tissue nuclear factor-κB induced by I/R. The results suggested that transduction of PEP-1-HO-1 fusion protein decreased myocardial reperfusion injury, probably by attenuating the production of oxidants and proinflammatory cytokines regulated by nuclear factor-κB.

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OBJECTIVE: Radiation-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. METHODS: A 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. RESULTS: Subcutaneous 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. CONCLUSIONS: The 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.

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The R3V6 peptides, which are composed of a 3-arginine block and a 6-valine block, formed self-assembled micelles in aqueous solution. Dye quenching assays showed that a hydrophobic fluorescent dye, 5-dodecanoylaminofluorescein (DAF), interacted with and was loaded into the hydrophobic core of the micelles. In this study, dexamethasone-loaded R3V6 peptide micelles (R3V6-Dexa) were evaluated as a gene carrier. R3V6-Dexa had higher gene delivery efficiency in human embryonic kidney 293 cells compared to those of the R3V6 peptides and poly-L-lysine (PLL). Dexamethasone might stabilize the micelle structure of the R3V6 peptides by forming strong hydrophobic cores and enhanced the transfection efficiency. Furthermore, R3V6-Dexa reduced the expression of an inflammatory cytokine, interleukin-6 (IL-6), more efficiently in lipopolysaccharide (LPS)-induced Raw264.7 cells than did dexamethasone, suggesting that R3V6-Dexa is also a useful carrier for dexamethasone delivery. A focal brain ischemia-reperfusion model was produced by middle cerebral artery occlusion (MCAO). A heme oxygenase-1 (HO-1) expression plasmid DNA, pSV-HO-1, was delivered into the brain using R3V6-Dexa as a carrier. The pSV-HO-1/R3V6-Dexa complex was injected into the brain 1hr prior to MCAO. Twenty-four hours later, the HO-1 expression of the pSV-HO-1/R3V6-Dexa injection group was higher than those of the MCAO control, pß-Luc/R3V6-Dexa, and pSV-HO-1/PEI25k injection groups. In addition, the infarct size was reduced due to the delivery of pSV-HO-1/R3V6-Dexa complex. Therefore, R3V6-Dexa may be a useful carrier for HO-1 gene delivery and stroke gene therapy.

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BACKGROUND: Heme oxygenase-1 (HO-1), a heme degradation enzyme with multiple vasoprotective functions, is systemically induced in pathophysiological states associated with oxidative stress. OBJECTIVES: To evaluate the impact of systemic HO-1 expression on circulating endothelial progenitor cells (EPCs) and re-endothelialization after vascular injury in an animal model. METHODS: Mice received an intravenous (i.v.) injection of the adenovirus-bearing HO-1 gene (Adv-HO-1). The serum levels of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) were determined by ELISA and gene expression examined by quantitative real-time PCR. Circulating EPCs were characterized by flow cytometry and in vitro culture. EPC recruitment and re-endothelialization in injured arteries were assessed in mice receiving GFP+-bone marrow transplantation and guide wire-induced carotid injury. The effect of carbon monoxide (CO), a byproduct from heme degradation by HO-1, was assessed by exposing mice to 250 p.p.m. CO for 2 h day(-1). RESULTS: Systemic HO-1 induction led to elevated serum levels of VEGF and SDF-1 and an increase in circulating EPCs. The re-endothelialization of denuded vessels was accelerated in mice with systemic HO-1 overexpression. A further experiment demonstrated that both EPC mobilization and re-endothelialization were significantly attenuated in mice with HO-1 deficiency. The increase in EPC mobilization and enhanced re-endothelialization was also observed in mice exposed to CO prior to carotid injury. The CO-mediated effect was associated with an increase in circulating SDF-1 but not VEGF. CONCLUSION: These findings support a vital role of HO-1 and its reaction byproduct, CO, in vascular repair through enhancing EPC mobilization.

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Heme oxygenase-1 (HO-1) is regarded as a sensitive and reliable indicator of cellular oxidative stress. Studies on carbon monoxide (CO) and bilirubin, two of the three (iron is the third) end products of heme degradation have improved the understanding of the protective role of HO against oxidative injury. CO is a vasoactive molecule and bilirubin is an antioxidant, and an increase in their production through an increase in HO activity assists other antioxidant systems in attenuating the overall production of reactive oxygen species (ROS), thus facilitating cellular resistance to oxidative injury. Gene transfer is used to insert specific genes into cells that are either otherwise deficient in or that underexpress the gene. Successful HO gene transfer requires two essential elements to produce functional HO activity. Firstly, the HO gene must be delivered in a safe vector, e.g., adenoviral, retroviral or leptosome based vectors, currently being used in clinical trials. Secondly, with the exception of HO gene delivery to either ocular or cardiovascular tissue via catheter-based delivery systems, HO delivery must be site and organ specific. This has been achieved in rabbit ocular tissues, rat liver, kidney and vasculature, SHR kidney, and endothelial cells [Abraham et al., 1995a; Abraham et al., 1995b; Abraham et al., 2002c; Quan et al., 2004; Sabaawy et al., 2000; Sabaawy et al., 2001; Yang et al., 2004]. In this review, we discuss the functional significance of the HO system in various pathophysiological conditions and the beneficial therapeutic applications of human HO gene transfer and gene therapy in a variety of clinical circumstances.

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