Postischemic cardiac recovery in heme oxygenase‐1 transgenic ischemic/reperfused mouse myocardium

Heme oxygenase-1 (HO-1) transgenic mice (Tg) were created using a rat HO-1 genomic transgene. Transgene expression was detected by RT-PCR and Western blots in the left ventricle (LV), right ventricle (RV) and septum (S) in mouse hearts, and its function was demonstrated by the elevated HO enzyme activity. Tg and non-transgenic (NTg) mouse hearts were isolated and subjected to ischemia/reperfusion. Significant post-ischemic recovery in coronary flow (CF), aortic flow (AF), aortic pressure (AOP) and first derivative of AOP (AOPdp/dt) were detected in the HO-1 Tg group compared to the NTg values. In HO-1 Tg hearts treated with 50 μmol/kg of tin protoporphyrin IX (SnPPIX), an HO enzyme inhibitor, abolished the post-ischemic cardiac recovery. HO-1 related carbon monoxide (CO) production was detected in NTg, HO-1 Tg and HO-1 Tg + SnPPIX treated groups, and a substantial increase in CO production was observed in the HO-1 Tg hearts subjected to ischemia/reperfusion. Moreover, in ischemia/reperfusion-induced tissue Na⁺ and Ca²⁺ gains were reduced in HO-1 Tg group in comparison with the NTg and HO-1 Tg + SnPPIX treated groups; furthermore K⁺ loss was reduced in the HO-1 Tg group. The infarct size was markedly reduced from its NTg control value of 37 ± 4% to 20 ± 6% (P < 0.05) in the HO-1 Tg group, and was increased to 47 ± 5% (P < 0.05) in the HO-1 knockout (KO) hearts. Parallel to the infarct size reduction, the incidence of total and sustained ventricular fibrillation were also reduced from their NTg control values of 92% and 83% to 25% (P < 0.05) and 8% (P < 0.05) in the HO-1 Tg group, and were increased to 100% and 100% in HO-1 KO^−/− hearts. Immunohistochemical staining of HO-1 was intensified in HO-1 Tg compared to the NTg myocardium. Thus, the HO-1 Tg mouse model suggests a valuable therapeutic approach in the treatment of ischemic myocardium.     

Reduction of reperfusion‐induced ventricular fibrillation and infarct size via heme oxygenase‐1 overexpression in isolated mouse hearts

Heme oxygenase‐1 (HO‐1), also known as heat shock protein 32 (hsp‐32) is a stress‐induced cytoprotective protein. The present investigation evaluated the capacity of HO‐1 to reduce the incidence of reperfusion‐induced ventricular fibrillation (VF) and infarct size. HO‐1 transgenic (Tg) mice were generated using a rat HO‐1 genomic transgene. Isolated mouse hearts obtained from Tg and non‐transgenic (NTg) groups were exposed to 20 min. of global ischemia and 120 min. of reperfusion. Epicardial electrocardiogram was recorded to monitor the incidence of reperfusion‐induced VF and at the end of the reperfusion period, detection of HO‐1 by immunohistochemistry and measurement of infarct size using the tetrazolium chloride method were carried out. Results shown here provide additional support for cardioprotective effects of HO‐1 as demonstrated by the reduced infarct size. Moreover, overexpression of the HO‐1 efficiently reduced the incidence of ischemia/reperfusion induced VF in HO‐1 Tg mice.     

Haem oxygenase‐1 inhibits phosphorylation of the Helicobacter pylori oncoprotein CagA in gastric epithelial cells

The cytotoxin‐associated gene A protein (CagA) plays a pivotal role in the aetiology of Helicobacter pylori‐associated gastric diseases. CagA is injected into the cytoplasm of host cells by a type IV secretion system, and is phosphorylated on tyrosine residues by the host enzyme c‐Src. We previously reported that the enzyme haem oxygenase‐1 (HO‐1) inhibits IL‐8 secretion by H. pylori‐infected cells. However, the cellular mechanism by which HO‐1 regulates the innate immune function of infected cells remains unknown. We now show that nitric oxide and haemin, two inducers of HO‐1, decrease the level of phosphorylated CagA (p‐CagA) in H. pylori‐infected gastric epithelial cells and this is blocked by either pharmacological inhibition of HO‐1 or siRNA knockdown of hmox‐1. Moreover, forced expression of HO‐1 by transfection of a plasmid expressing hmox‐1 also results in a strong attenuation of CagA phosphorylation. This occurs through the inhibition of H. pylori‐induced c‐Src phosphorylation/activation by HO‐1.Consequently, H. pylori‐induced cytoskeletal rearrangements and activation of the pro‐inflammatory response mediated by p‐CagA are inhibited in HO‐1‐expressing cells. These data highlight a mechanism by which the innate immune response of the host can restrict the pathogenicity of H. pylori by attenuating CagA phosphorylation in gastric epithelial cells.     

The apolipoprotein A‐I mimetic peptide,D‐4F,restrains neointimal formation through heme oxygenase‐1 up‐regulation

D‐4F, an apolipoprotein A‐I (apoA‐I) mimetic peptide, possesses distinctly anti‐atherogenic effects. However, the biological functions and mechanisms of D‐4F on the hyperplasia of vascular smooth muscle cells (VSMCs) remain unclear. This study aimed to determine its roles in the proliferation and migration of VSMCs. In vitro, D‐4F inhibited VSMC proliferation and migration induced by ox‐LDL in a dose‐dependent manner. D‐4F up‐regulated heme oxygenase‐1 (HO‐1) expression in VSMCs, and the PI3K/Akt/AMP‐activated protein kinase (AMPK) pathway was involved in these processes. HO‐1 down‐regulation with siRNA or inhibition with zinc protoporphyrin (Znpp) impaired the protective effects of D‐4F on the oxidative stress and the proliferation and migration of VSMCs. Moreover, down‐regulation of ATP‐binding cassette transporter A1 (ABCA1) abolished the activation of Akt and AMPK, the up‐regulation of HO‐1 and the anti‐oxidative effects of D‐4F. In vivo, D‐4F restrained neointimal formation and oxidative stress of carotid arteries in balloon‐injured Sprague Dawley rats. And inhibition of HO‐1 with Znpp decreased the inhibitory effects of D‐4F on neointimal formation and ROS production in arteries. In conclusion, D‐4F inhibited VSMC proliferation and migration in vitro and neointimal formation in vivo through HO‐1 up‐regulation, which provided a novel prophylactic and therapeutic strategy for anti‐restenosis of arteries.     

The protection by heme oxygenase‐1 induction against thioacetamide‐induced liver toxicity is associated with changes in arginine and asymmetric dimethylarginine

This study was designed to investigate the role of HO‐1 induction in prevention of thioacetamide (TAA)‐induced oxidative stress, inflammation and liver damage. The changes in hepatic dimethylarginine dimethylaminohydrolase (DDAH) activity as well as plasma arginine and asymmetric dimethylarginine (ADMA) levels were also measured to evaluate nitric oxide (NO) bioavailability. Rats were divided into four groups as control, hemin, TAA and hemin + TAA groups. Hemin (50 mg kg^?1, i.p.) was injected to rats 18 h before TAA treatment to induce HO‐1 enzyme expression. Rats were given TAA (300 mg kg^?1, i.p.) and killed 24 h after treatment. Although TAA treatment produced severe hepatic injury, upregulation of HO‐1 ameliorated TAA‐induced liver damage up to some extent as evidence by decreased serum alanine transaminase, aspartate transaminase and arginase activities and histopathological findings. Induction of HO‐1 stimulated antioxidant system and decreased lipid peroxidation in TAA‐treated rats. Myeloperoxidase activity and inducible NO synthase protein expression were decreased, whereas DDAH activity was increased by hemin injection in TAA‐treated rats. Induction of HO‐1 was associated with increased arginine levels and decreased ADMA levels, being the main determinants of NO production, in plasma of TAA‐treated rats. In conclusion, our results indicate that HO‐1 induction alleviated increased oxidative stress and inflammatory reactions together with deterioration in NO production in TAA‐induced liver damage in rats. Copyright © 2012 John Wiley & Sons, Ltd.     

Effect of heme oxygenase‐1 transduced bone marrow mesenchymal stem cells on damaged intestinal epithelial cells in vitro

In this study, we explored the effects of mesenchymal stem cells (MSCs) from bone marrow overexpressing heme oxygenase‐1 (HO‐1) on the damaged human intestinal epithelial barrier in vitro. Rat MSCs were isolated from bone marrow and transduced with rat HO‐1 recombinant adenovirus (HO‐MSCs) for stable expression of HO‐1. Colorectal adenocarinoma 2 (Caco2) cells were treated with tumor necrosis factor‐α (TNF‐α) to establish a damaged colon epithelial model. Damaged Caco2 were cocultured with MSCs, Ad‐MSCs, Ad‐HO + MSCs or HO‐MSCs. mRNA and protein expression of Zona occludens‐1 (ZO‐1) and human HO‐1 and the release of cytokines were measured. ZO‐1 and human HO‐1 in Caco2 were significantly decreased after treatment with TNF‐α; and this effect was reduced when coculture with MSCs from bone marrow. Expression of ZO‐1 was not significantly affected by Caco2 treatment with TNF‐α, Ad‐HO, and MSCs. In contrast, ZO‐1 and human HO‐1 increased significantly when the damaged Caco2 was treated with HO‐MSCs. HO‐MSCs showed the strongest effect on the expression of ZO‐1 in colon epithelial cells. Coculture with HO‐MSCs showed the most significant effects on reducing the expression of IL‐2, IL‐6, IFN‐γ and increasing the expression of IL‐10. HO‐MSCs protected the intestinal epithelial barrier, in which endogenous HO‐1 was involved. HO‐MSCs play an important role in the repair process by reducing the release of inflammatory cytokines and increasing the release of anti‐inflammatory factors. These results suggested that HO‐MSCs from bone marrow were more effective in repairing the damaged intestinal epithelial barrier, and the effectiveness of MSCs was improved by HO‐1 gene transduction, which provides favorable support for the application of stem cell therapy in the intestinal diseases.     

3‐Cl‐AHPC inhibits pro‐HGF maturation by inducing matriptase/HAI‐1 complex formation

Matriptase is an epithelia‐specific membrane‐anchored serine protease, and its dysregulation is highly related to the progression of a variety of cancers. Hepatocyte growth factor activator inhibitor‐1 (HAI‐1) inhibits matriptase activity through forming complex with activated matriptase. The balance of matriptase activation and matriptase/HAI‐1 complex formation determines the intensity and duration of matriptase activity. 3‐Cl‐AHPC, 4‐[3‐(1‐adamantyl)‐4‐hydroxyphenyl]‐3‐chlorocinnamic acid, is an adamantly substituted retinoid‐related molecule and a ligand of retinoic acid receptor γ (RARγ). 3‐Cl‐AHPC is of strong anti‐cancer effect but with elusive mechanisms. In our current study, we show that 3‐Cl‐AHPC time‐ and dose‐ dependently induces matriptase/HAI‐1 complex formation, leading to the suppression of activated matriptase in cancer cells and tissues. Furthermore, 3‐Cl‐AHPC promotes matriptase shedding but without increasing the activity of shed matriptase. Moreover, 3‐Cl‐AHPC inhibits matriptase‐mediated cleavage of pro‐HGF through matriptase/HAI‐1 complex induction, resulting in the suppression of pro‐HGF‐stimulated signalling and cell scattering. Although 3‐Cl‐AHPC binds to RARγ, its induction of matriptase/HAI‐1 complex is not RARγ dependent. Together, our data demonstrates that 3‐Cl‐AHPC down‐regulates matriptase activity through induction of matriptase/HAI‐1 complex formation in a RARγ‐independent manner, providing a mechanism of 3‐Cl‐AHPC anti‐cancer activity and a new strategy to inhibit abnormal matriptase activity via matriptase/HAI‐1 complex induction using small molecules.     

Identification of kaempferol‐regulated proteins in rat calvarial osteoblasts during mineralization by proteomics

Kaempferol, a flavonoid, promotes osteoblast mineralization in vitro and bone formation in vivo; however, its mechanism of action is yet unknown. We adopted proteomic approach to identify the differential effect of kaempferol on rat primary calvarial osteoblasts during mineralization. The primary rat calvarial osteoblasts were treated with kaempferol (5.0 μM) for 9 days under mineralizing condition that resulted in significant increase in alkaline phosphatase activity and mineralization of the cells. Further, 2‐D analysis of the kaempferol‐treated osteoblast lysates revealed 18 differentially expressed proteins (nine upregulated and nine downregulated) on the basis of >/<2.0‐fold as cut‐off (p<0.01) that were then identified by MALDI‐TOF MS. These included cytoskeletal proteins, intracellular signaling protein, chaperone, extracellular matrix protein, and proteins involved in glycolysis and cell–matrix interactions. Proteomics data were confirmed by Western blotting and quantitative real‐time PCR by randomly selecting two upregulated and two downregulated proteins. Western blot analysis confirmed upregulation of HSP‐70 and cytokeratin‐14 levels, and downregulation of aldose reductase and caldesmon expression. We further demonstrated that kaempferol treatment inhibits aldose reductase activity in osteoblasts indicating an altered cellular metabolism by decelerating polyol pathway that was associated with the kaempferol‐induced osteoblast mineralization. In conclusion, this is a first comprehensive study on the differential regulation of proteins by kaempferol in primary osteoblast, which would further help to elucidate the role of the identified proteins in the process of osteoblast mineralization.     

Oxygen‐induced transcriptional dynamics in human osteoblasts are most prominent at the onset of mineralization

Oxygen tension plays an important role in the regulation of cellular processes. During hematopoietic stem cell (HSC) differentiation, HSCs migrate from one stem cell niche to the next, each with a different oxygen tension that determines which signaling pathways are on and off, determining the differentiation stage of the cell. Oxygen tension influences osteoblast differentiation and mineralization. Low oxygen levels inhibit matrix formation and mineralization. We were interested in the regulatory mechanisms that underlie this inhibition and wondered whether a switch in oxygen tension could have varying effects depending on the differentiation phase of the osteoblasts. We performed an oxygen tension switch phase study in which we switched osteoblasts from high to low oxygen tension during their 3 week differentiation and mineralization process. We performed microarray expression profiling on samples collected during this 3 week period and analyzed biochemical and histo‐chemical endpoint parameters to determine the effect of a switch in oxygen levels on mineralization. We found that low oxygen tension has the most profound impact on mineralization when administered during the period of matrix maturation. Additionally, a large set of genes was regulated by oxygen, independent of the differentiation phase. These genes were involved in cell metabolisms and matrix formation. Our study demonstrates that variation in oxygen tension strongly affects gene expression in differentiating osteoblasts. The magnitude of this change for either expression levels or the number of regulated probes, depends on the osteoblast differentiation stage, with the phase prior to the onset of mineralization being most sensitive. J. Cell. Physiol. 228: 1863–1872, 2013. © 2013 Wiley Periodicals, Inc.     

Inhibition of bromodomain‐containing protein 4 ameliorates oxidative stress–mediated apoptosis and cartilage matrix degeneration through activation of NF‐E2–related factor 2‐heme oxygenase‐1 signaling in rat chondrocytes

During the progression of osteoarthritis, dysregulation of extracellular matrix (ECM) anabolism, abnormal generation of reactive oxygen species, and proteolytic enzymes have been shown to accelerate the degradation process of cartilage. The purpose of the current study was to investigate the functional role of bromodomain‐containing protein 4 (BRD4) in hydrogen peroxide (H₂O₂)–stimulated chondrocyte injury and delineate the underlying molecular mechanisms. We observed that the expression BRD4 was markedly elevated in rat chondrocytes after H₂O₂ stimulation. Additionally, inhibition of BRD4 using small interfering RNA or JQ1 (a selective potent chemical inhibitor) led to repression of H₂O₂‐induced oxidative stress, as revealed by a decrease in the reactive oxygen species production accompanied by a decreased malondialdehyde content, along with increased activities of antioxidant markers superoxide dismutase, catalase, and glutathione peroxidase on exposure of chondrocytes to H₂O₂. Meanwhile, depletion of BRD4 led to repress the oxidative stress–induced apoptosis of chondrocytes triggered by H₂O₂ accompanied by an increase in the expression of anti‐apoptotic Bcl‐2 and a decrease in the expression of pro‐apoptotic Bax and caspase 3 as well as attenuated caspase 3 activity. Moreover, knockdown of BRD4 or treatment with JQ1 markedly attenuated ECM deposition, reflected in a marked upregulation of proteoglycans collagen type II and aggrecan as well as downregulation of ECM–degrading enzymes matrix metalloproteinase 13 and A disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS‐5). More importantly, inhibition of BRD4‐activated NF‐E2–related factor 2 (Nrf2)–heme oxygenase‐1 signaling. Mechanistically, the protective effect of BRD4 inhibition on H₂O₂‐stimulated apoptosis and cartilage matrix degeneration was markedly abrogated by Nrf2 depletion. Altogether, we concluded that the protective effect of BRD4 inhibition against oxidative stress–mediated apoptosis and cartilage matrix degeneration occurred through Nrf2–heme oxygenase‐1 signaling, implying that BRD4 inhibition may be a more effective therapeutic strategy against osteoarthritis.     

Pro‐apoptotic effect of haem oxygenase‐1 in human colorectal carcinoma cells via endoplasmic reticular stress

Several biological effects of haem oxygenase (HO)‐1, including anti‐inflammatory, antiapoptotic and antioxidative properties were reported; however, the role of HO‐1 in apoptosis is still unclear. In the presence of stimulation by cobalt protoporphyrin (CoPP), an HO‐1 inducer, apoptotic characteristics were observed, including DNA laddering, hypodiploid cells, and cleavages of caspase (Casp)‐3 and poly(ADP) ribose polymerase (PARP) proteins in human colon carcinoma COLO205, HCT‐15, LOVO and HT‐29 cells in serum‐free (SF) conditions with increased HO‐1, but not heat shock protein 70 (HSP70) or HSP90. The addition of 10% foetal bovine serum (FBS) or 1% bovine serum albumin accordingly inhibited CoPP‐induced apoptosis and HO‐1 protein expression in human colon cancer cells. CoPP‐induced apoptosis of colon cancer cells was prevented by the addition of the pan‐caspase inhibitor, Z‐VAD‐FMK (VAD), and the Casp‐3 inhibitor, Z‐DEVD‐FMK (DEVD). N‐Acetyl cysteine inhibited reactive oxygen species‐generated H₂O₂‐induced cell death with reduced intracellular peroxide production, but did not affect CoPP‐induced apoptosis in human colorectal carcinoma (CRC) cells. Two CoPP analogs, ferric protoporphyrin and tin protoporphyrin, did not affect the viability of human CRC cells or HO‐1 expression by those cells, and knockdown of HO‐1 protein expression by HO‐1 small interfering (si)RNA reversed the cytotoxic effect elicited by CoPP. Furthermore, the carbon monoxide (CO) donor, CORM, but not FeSO₄ or biliverdin, induced DNA ladders, and cleavage of Casp‐3 and PARP proteins in human CRC cells. Increased phosphorylated levels of the endoplasmic reticular (ER) stress proteins, protein kinase R‐like ER kinase (PERK), and eukaryotic initiation factor 2α (eIF2α) by CORM and CoPP were identified, and the addition of the PERK inhibitor, GSK2606414, inhibited CORM‐ and CoPP‐induced apoptosis. Increased GRP78 level and formation of the HO‐1/GRP78 complex were detected in CORM‐ and CoPP‐treated human CRC cells. A pro‐apoptotic role of HO‐1 against the viability of human CRC cells via induction of CO and ER stress was firstly demonstrated herein.