흰쥐 관절연골세포에서 NO donor에 의해 유도된 HO-1 발현에서 peroxynitrite의 관련성 연구

In addition to cholesterol-lowering properties, statins exhibit lipid-independent immunomodulatory, anti-inflammatory actions. However, high concentrations are typically required to induce these effects in vitro, raising questions concerning therapeutic relevance. We present evidence that endothelial cell sensitivity to statins depends upon shear stress. Using heme oxygenase-1 expression as a model, we demonstrate differential heme oxygenase-1 induction by atorvastatin in atheroresistant compared with atheroprone sites of the murine aorta. In vitro, exposure of human endothelial cells to laminar shear stress significantly reduced the statin concentration required to induce heme oxygenase-1 and protect against H(2)O(2)-mediated injury. Synergy was observed between laminar shear stress and atorvastatin, resulting in optimal expression of heme oxygenase-1 and resistance to oxidative stress, a response inhibited by heme oxygenase-1 small interfering RNA. Moreover, treatment of laminar shear stress-exposed endothelial cells resulted in a significant fall in intracellular cholesterol. Mechanistically, synergy required Akt phosphorylation, activation of Kruppel-like factor 2, NF-E2-related factor-2 (Nrf2), increased nitric-oxide synthase activity, and enhanced HO-1 mRNA stability. In contrast, heme oxygenase-1 induction by atorvastatin in endothelial cells exposed to oscillatory flow was markedly attenuated. We have identified a novel relationship between laminar shear stress and statins, demonstrating that atorvastatin-mediated heme oxygenase-1-dependent antioxidant effects are laminar shear stress-dependent, proving the principle that biomechanical signaling contributes significantly to endothelial responsiveness to pharmacological agents. Our findings suggest statin pleiotropy may be suboptimal at disturbed flow atherosusceptible sites, emphasizing the need for more specific therapeutic agents, such as those targeting Kruppel-like factor 2 or Nrf2.

1. Haem oxygenase-1 (HO-1) can exert protective effects against oxidative stress and inflammation. Fibroblasts participate in inflammatory responses where they produce high levels of prostaglandins (PGs) and nitric oxide (NO). However, little is known of the presence of HO-1 in these cells and the possible interactions among these pathways. Incubation of cells with NO donors, spermine nonoate (SPNO) and S-nitroso-N-acetylpenicillamine (SNAP), induced a dose- and time-dependent expression of HO-1 protein. 2. NO donors increased basal PGE(2) release although they reduced PGE(2) accumulated in the medium and cyclo-oxygenase (COX) activity when cells were stimulated with lipopolysaccharide (LPS). COX-2 protein was weakly induced by SPNO in basal conditions and in the presence of LPS a synergy for HO-1 and COX-2 protein expression was observed. 3. Our results indicate that reactive oxygen species participate in the inductive effect of NO donors or LPS on HO-1 expression, whereas endogenous NO production may play a role in the mechanism of the synergy exhibited by SPNO and LPS on HO-1 and COX-2 expression. In this system, zinc protoporphyrin IX did not affect nitrite levels but reduced COX activity. 4. The selective COX-2 inhibitors SC58125 and NS398 as well as the non-selective COX inhibitor, indomethacin, strongly reduced PGE(2) synthesis and showed a synergy with NO donors in HO-1 and COX-2 induction. Addition of PGE(2) had no effect, suggesting a mechanism independent of PGs formation. 5. In inflammatory conditions a number of factors could cooperate to induce HO-1 and COX-2, with a positive regulation by COX inhibitors.

Heme oxygenase 1 (HO-1) is a representative mediator of antioxidants and cytoprotectants against various stress stimuli including oxidants in vascular cells. Intensive insulin treatment can delay the onset and progression of diabetic retinopathy and other vascularopathies, yet little is known about insulin regulation of anti-apoptotic and antioxidant molecules such as HO-1 in vascular cells. Intravitreous injection or in vitro addition of insulin increased HO-1 protein expression in rat retina and in cultured bovine retinal pericytes, retinal endothelial cells, and retinal pigment epithelial cells. In bovine retinal pericytes, insulin induced mRNA and protein expression of HO-1 in a time- and concentration-dependent manner. Using HO-1 promoter analysis, the luciferase reporter assay showed that induction of HO-1 expression by insulin is mediated by additional response elements in the ho-1 promoter gene, which was not responsive to antioxidants. Insulin-induced HO-1 mRNA expression through activation of PI3-kinase/Akt pathway without affecting ERK and p38 MAPK. Overexpression of an adenoviral vector of native IRS1, IRS2, and Akt dominant negative or small interfering RNA transfection of Akt1 and Akt2 targeted gene demonstrated that insulin regulated HO-1 expression via IRS1 and Akt2 pathway, selectively. Further, insulin treatment prevented H(2)O(2)-induced NF-kappaB and caspase-8 activation and apoptosis via the IRS1/PI3K/Akt2/HO-1 pathway in the pericytes. In conclusion, we suggest that the anti-apoptotic properties of insulin are mediated partly by increasing HO-1 expression at transcriptional level via IRS1/PI3K/Akt2 activation, a potential explanation for how insulin is retarding the progression of microvascular complications induced by diabetes.

Nitric oxide induces heme oxygenase-1 gene expression in mesangial cells

Heme oxygenase-1 (HO-1) catabolizes the degradation of heme into bilirubin, carbon monoxide, and iron ions. The HO-1 products provide antioxidant cytoprotection in addition to having potent antiinflammatory and immunomodulatory functions. HO-1 is induced by its substrate heme and environmental factors including oxidative and heat stresses. Although previous studies reported that lipopolysaccharide (LPS) induced the expression of both the HO-1 gene and its protein in macrophages, the major regulators of HO-1 expression remain unknown. To identify these regulators, we used two types of cell, the murine macrophage-like cell line J774.1/JA-4 and its LPS-resistant mutant, LPS1916. Based on a comparison of the results obtained with these cells, we found that nitric oxide (NO) was closely linked to the induction of HO-1. Real-time polymerase chain reaction (PCR) showed that the time course for inducible HO-1 mRNA by LPS or LPS+interferon (IFN)-γ was similar to that for inducible NO synthase (iNOS) mRNA. Furthermore, the expression of iNOS mRNA and protein increased earlier than that of HO-1 mRNA and protein. N-Nitro-L-arginine methyl ester, an NO synthase inhibitor, reduced both HO-1 expression and NO production in LPS+IFN-γ-treated JA-4 cells. Furthermore, NOC-12, an NO donor, significantly induced HO-1 expression not only in JA-4 but also in LPS1916 cells. Reactive oxygen species (ROS) scavengers, such as superoxide dismutase and catalase, did not affect HO-1 protein expression in LPS+IFN-γ-treated JA-4 cells. These results suggest that, among ROS, NO plays an important role in HO-1 induction in activated macrophages treated with LPS+IFN-γ.

Expression of Heme Oxygenase-1 in Response to Myocardial Infarction in Rats

Expression of Heme Oxygenase-1 in Response to Myocardial Infarction in Rats

Nitric oxide (NO) and NO donors were among the first reported inducers of the tissue-protective protein heme oxygenase-1 (HO-1) with a potential for eventual use in humans. Besides other clinically established NO releasing drugs, sodium nitroprusside (SNP) has frequently been employed as an experimental tool to explore effects of NO on HO-1 and other biological targets. In this issue of Molecular Pharmacology, Kim et al. (p. 1633) demonstrate that the effects of SNP on expression of HO-1 are mainly due to free iron released from SNP in aqueous solution, whereas NO plays a negligible role, if any, as the mediator of response to SNP. Downstream effects of iron, after being dissociated from SNP, include increases in intracellular cAMP that are causally linked to subsequent phosphorylation of specific MAPK targets and enhanced HO-1 protein levels. Based on the data reported by Kim et al. (2006), the use of SNP as an experimental tool to mimic intracellular effects of NO should be avoided in the future. This work not only helps revise concepts in NO and HO-1 research but also may direct future efforts to the role of iron and reactive oxygen species in the regulation of adenylyl cyclase.

Lentivirus Mediated HO-1 Gene Transfer Enhances Myogenic Precursor Cell Survival After Autologous Transplantation in Pig

Effects of heme oxygenase‐1 on bacterial antigen‐induced articular chondrocyte catabolism in vitro

Nitric oxide (NO) has been shown to exert antiproliferative and antiapoptotic effects on human T cells. Heme oxygenase-1 (HO-1), which degrades heme into biliverdin, free iron (Fe(2+)), and carbon monoxide (CO), has also been known to have antiproliferative and antiapoptotic effects. Recent evidence suggests that HO-1 is an important cellular target of NO; whether HO-1 expression contributes to the antiproliferative and/or antiapoptotic effects mediated by NO remains to be investigated. In the present study, we examined the effects of NO on HO-1 expression and possible roles of HO-1 in T cell proliferation and apoptosis. Using human Jurkat T cells, we found that the NO donor sodium nitroprusside (SNP) induced HO-1 expression and that preincubation with SNP suppressed T cell proliferation induced by concanavalin A and apoptosis triggered by anti-Fas antibody. Suppressions of T cell proliferation and apoptosis comparable with SNP were also observed when the T cells were preincubated with the HO-1 inducer cobalt protoporphyrin. A phosphorothioate-linked HO-1 antisense oligonucleotide blocked HO-1 expression, and subsequently abrogated the antiproliferative and antiapoptotic effects of SNP. Overexpression of the HO-1 gene after transfection into Jurkat T cells resulted in significant decreases in T cell proliferation and apoptosis. The CO donor tricarbonyldichlororuthenium (II) dimer mimicked the antiproliferative effect of SNP, and the Fe(2+) donor FeSO(4) blocked anti-Fas-induced apoptosis. Taken together, our results suggest that NO induces HO-1 expression in T cells and that suppressions of T cell proliferation and apoptosis afforded by NO are associated with an increased expression of HO-1 by NO.

Expression of Heme Oxygenase and Inducible Nitric Oxide Synthase mRNA in Human Brain Tumors

Comparison of the mechanisms underlying the relaxation induced by two nitric oxide donors: Sodium nitroprusside and a new ruthenium complex

Effect of nitric oxide on shock-induced hepatic heme oxygenase-1 expression in the rat

Heme is a strong inducer and substrate of the stress protein heme oxygenase-1 (HO-1), which produces carbon monoxide, iron, and bilirubin. We have reported recently that nitric oxide (NO) augments the incorporation of free hemin in endothelial cells, resulting in amplified HO-1 expression and production of bilirubin. Here, we extend our studies by showing that both NO+ and NO- donors interacted with reduced (HbA0) or oxidized (metHb) hemoglobin, as well as hemoglobin from sickle cell disease (HbS), to strongly magnify HO-1, with a pattern of induction dependent on the oxidation state of the hemoglobin used. A corresponding enhancement of endothelial heme uptake was observed following exposure of HbA0 or HbS to the NO donors, which also increased the uptake of free hemin. We postulated that this effect may be caused by formation of heme-nitrosyl (H-NO) complexes, and indeed endothelial cells exposed to preformed H-NO showed greater heme incorporation than free hemin. Furthermore, NO donors directly affected the permeability of membranes to free hemin. In conclusion, our data indicate a novel role for NO in the modulation of heme transport and HO-1 induction in endothelial cells, which may be relevant for hematological disorders characterized by disruption of the heme-NO equilibrium.