Heme oxygenase-1 in growth control and its clinical application to vascular disease.

To explore the role of hydrogen sulfide/cystathionine-gamma-lyase (H(2)S/CSE) system in lipopolysaccharide (LPS)- induced acute lung injury (ALI) in rats and the underlying mechanisms. Sixty-four Sprague-Dawley (SD) rats were randomly divided into four groups: control, LPS (instilled intratracheally to induce ALI), sodium hydrosulfide (NaHS), propargylglycine (PPG). Animals were sacrificed at 4 and 8 hours (n=8) after administration of the above agents. Morphological changes in lung tissues were determined, H(2)S, nitrogen monoxide (NO) and carbon monoxide (CO) concentration in plasma were determined. Malondialdehyde (MDA) content, and myeloperoxidase (MPO), CSE, inducible nitric oxide synthase (iNOS), heme oxygenase (HO) activity of the lung were also determined. The level of P-selectin of lung tissue was measured by radioimmunoassay. Immunohistochemistry technique was performed to examine the expression of iNOS and HO-1 protein in lung tissues. Severe injuries of lung tissues and raised MDA content, MPO activity and P-selectin level were observed in rats treated with LPS. LPS also led to a drop in plasma H(2)S concentration and lung CSE activity. The enzyme activity of iNOS and HO, and their protein expression, plasma NO, and CO levels increased after LPS instillation (P<0.05 or P<0.01). Pre-administration of NaHS before LPS could attenuate the changes induced by LPS. Pre-administration of PPG exacerbated the injuries induced by LPS, with increased MDA content, MPO activity, P-selectin level, the plasma NO level, lung iNOS activity and its protein expression, but there was no prominent variation in CO level, HO activity and HO-1 protein expression compared with those of LPS group. Downregulation of H(2)S/CSE is involved in the pathogenesis of ALI induced by LPS. Endogenous and exogenous H(2)S provide protection against ALI, which may be explained by its anti-oxidative effects, attenuation of inflammatory over-reaction in lung induced by polymorphonuclear neutrophils, downregulation of NO/iNOS system and the upregulation of CO/HO-1 system.

Perfusion of hippocampal slices with an inhibitor nitric oxide (NO) synthase blocked induction of long-term potentiation (LTP) produced by a one-train tetanus and significantly reduced LTP by a two-train tetanus, but only slightly reduced LTP by a four-train tetanus. Inhibitors of heme oxygenase, the synthetic enzyme for carbon monoxide (CO), significantly reduced LTP by either a two-train or four-train tetanus. These results suggest that NO and CO are both involved in LTP but may play somewhat different roles. One possibility is that NO serves a phasic, signaling role, whereas CO provides tonic, background stimulation. Another possibility is that NO and CO are phasically activated under somewhat different circumstances, perhaps involving different receptors and second messengers. Because NO is known to be activated by stimulation of NMDA receptors during tetanus, we investigated the possibility that CO might be activated by stimulation of metabotropic glutamate receptors (mGluRs). Consistent with this idea, long-lasting potentiation by the mGluR agonist tACPD was blocked by inhibitors of heme oxygenase but not NO synthase. Potentiation by tACPD was also blocked by inhibitors of soluble guanylyl cyclase (a target of both NO and CO) or cGMP-dependent protein kinase, and guanylyl cyclase was activated by tACPD in hippocampal slices. However, biochemical assays indicate that whereas heme oxygenase is constitutively active in hippocampus, it does not appear to be stimulated by either tetanus or tACPD. These results are most consistent with the possibility that constitutive (tonic) rather than stimulated (phasic) heme oxygenase activity is necessary for potentiation by tetanus or tACPD, and suggest that mGluR activation stimulates guanylyl cyclase phasically through some other pathway.

Powering the powerhouse: Heme oxygenase-1 regulates mitochondrial function in non-alcoholic fatty liver disease (NAFLD).

Heme-oxygenase-1 (HO-1) is an inducible cytoprotective molecule that displays antioxidant, antiapoptotic, and antiinflammatory effects. In addition, HO-1 appears to have a complex role in angiogenesis. Recent in vivo studies report that vascular endothelial growth factor (VEGF) regulates HO-1 expression and activity in vascular endothelial cells (ECs) and that inhibition of HO-1 abrogates VEGF-induced endothelial activation and subsequent angiogenesis, while promoting VEGF-induced monocyte recruitment and inflammatory angiogenesis. HO-1 may also regulate the synthesis and activity of VEGF, resulting in a positive-feedback loop. In contrast, HO-1 activity has the opposite effect on lipopolysaccharide-driven inflammatory angiogenesis, inhibiting leukocyte invasion and preventing subsequent angiogenesis. In this review, we summarize the current understanding of the role of HO-1 in angiogenesis. We conclude that further investigation, using targeted molecular approaches specifically to alter HO-1 activity, are required to develop our understanding of the role of HO-1 and its products, carbon monoxide, biliverdin, bilirubin, and free iron in angiogenesis. We propose that during chronic inflammation, HO-1 has two roles, first an antiinflammatory action inhibiting leukocyte infiltration, and second, promotion of VEGF-driven noninflammatory angiogenesis, which facilitates tissue repair. Additional studies will help determine whether modulating the activity of HO-1 and/or its products has therapeutic potential in chronic inflammatory dise.

Heme oxygenase in diabetes-induced oxidative stress in the heart

Heme oxygenase-1 (HO-1), the inducible enzyme responsible for the rate-limiting step in the heme catabolism, is expressed in AIDS-Kaposi sarcoma (KS) lesions. Its expression is up-regulated by the Kaposi sarcoma-associated herpesvirus (KSHV) in endothelial cells, but the mechanisms underlying KSHV-induced HO-1 expression are still unknown. In this study we investigated whether the oncogenic G protein-coupled receptor (KSHV-GPCR or vGPCR), one of the key KSHV genes involved in KS development, activated HO-1 expression. Here we show that vGPCR induces HO-1 mRNA and protein levels in fibroblasts and endothelial cells. Moreover, targeted knock-down gene expression of HO-1 by small hairpin RNA and chemical inhibition of HO-1 enzymatic activity by tin protoporphyrin IX (SnPP), impaired vGPCR-induced survival, proliferation, transformation, and vascular endothelial growth factor (VEGF)-A expression. vGPCR-expressing cells implanted in the dorsal flank of nude mice developed tumors with elevated HO-1 expression and activity. Chronic administration of SnPP to the implanted mice, under conditions that effectively blocked HO-1 activity and VEGF-A expression in the transplanted cells, strikingly reduced tumor growth, without apparent side effects. On the contrary, administration of the HO-1 inducer cobalt protoporphyrin (CoPP) further enhanced vGPCR-induced tumor growth. These data postulate HO-1 as an important mediator of vGPCR-induced tumor growth and suggest that inhibition of intratumoral HO-1 activity by SnPP may be a potential therapeutic strategy.

Salt stress induced an increase in endogenous carbon monoxide (CO) production and the activity of the CO synthetic enzyme haem oxygenase (HO) in wheat seedling roots. In addition, a 50% CO aqueous solution, applied daily, not only resulted in the enhancement of CO release, but led to a significant reversal in dry weight (DW) and water loss caused by 150 mm NaCl treatment, which was mimicked by the application of two nitric oxide (NO) donors sodium nitroprusside (SNP) and diethylenetriamine NO adduct (DETA/NO). Further analyses showed that CO, as well as SNP, apparently up-regulated H(+)-pump and antioxidant enzyme activities or related transcripts, thus resulting in the increase of K/Na ratio and the alleviation of oxidative damage. Whereas, the CO/NO scavenger haemoglobin (Hb), NO scavenger or synthetic inhibitor methylene blue (MB) or N(G)-nitro-l-arginine methyl ester hydrochloride (l-NAME) differentially blocked these effects. Furthermore, CO was able to mimic the effect of SNP by strongly increasing NO release in the root tips, whereas the CO-induced NO signal was quenched by the addition of l-NAME or cPTIO, the specific scavenger of NO. The results suggested that CO might confer an increased tolerance to salinity stress by maintaining ion homeostasis and enhancing antioxidant system parameters in wheat seedling roots, both of which were partially mediated by NO signal.

Thromboembolism is still a major problem in almost all fields of clinical medicine. The inducible enzyme heme oxygenase 1 (HO-1) provides endogenous iron, vasodilative carbon monoxide (CO) and anti-oxidant biliverdin by decomposition of heme. The aim of this study was to examine whether the expression of HO-1 increases the anti-thrombotic properties of the endothelium and subsequently modulates the process of microvascular thrombus formation in vivo. In micro-vessels of mouse cremaster muscle preparations ferric chloride-induced thrombus formation was analyzed using intravital fluorescence microscopy. When mice were pretreated with an intraperi-toneal injection of hemin, a HO-1 inducer, Western blot protein analysis of cremaster muscle tissue revealed a marked induction of HO-1. Immunohistochemistry displayed a preferential expression of HO-1 in the vascular endothelium of arterioles and venules. In these animals, superfusion with ferric chloride caused arteriolar and venular thrombus growth, which however was significantly delayed (complete occlusion: 670 ± 96 s and 647 ± 129 s) when compared with thrombus formation in control animals (complete occlusion: 143 ± 18 s and 130 ± 25 s). The delay in thrombus formation in hemin-treated mice was abolished by tin protoporphyrin-IX, a HO-1 inhibitor, but not by copper protoporphyrin-IX, which does not inhibit the enzyme. Application of the vitamin E analogue Trolox in HO-1-blocked animals almost completely restored the delay in thrombus formation, implying that - beside CO - the anti-oxidant biliverdin mainly contributes to the anti-thrombotic effect of HO-1. Animals with HO-1 induction exhibited a reduced P-selectin protein expression in cremaster muscle tissue, most probably presenting the molecular basis for delayed thrombus growth. Local induction of HO-1 activity may therefore be of preventive and therapeutic value for clinical disorders with an increased risk of thrombotic events.

1. Heme oxygenase 1 (HO-1) has been shown to play a pivotal role in the maintenance of cellular homeostasis when the liver undergoes sublethal stress, such as ischaemia-reperfusion (I/R) injury. In the present study, we investigated the protective role of HO-1 in hyperbaric oxygen (HBO) preconditioning against liver injury after I/R. 2. A total hepatic ischaemia (30 min) and reperfusion (60 min) injury model in rats was used in the present study. Preconditioned groups were exposed to HBO 24 h prior to the induction of I/R injury. Other groups were injected with zinc protoporphyrin IX (ZnPP) intraperitoneally 1 h before I/R to inhibit HO-1 activity. At the end of the reperfusion period, blood and liver samples were collected for the analysis of liver injury markers, morphological changes, and HO-1 expression and activity in the liver. 3. In untreated rats, I/R induced an increase in hepatic injury markers, such as plasma transaminases, inflammatory cytokines (tumour necrosis factor-α and interleukin-1β), and tissue malondialdehyde. However, HBO preconditioning attenuated the I/R-induced increases in these hepatic injury markers, and prevented both the necrosis and apoptosis of hepatocytes induced by I/R injury. Furthermore, HBO preconditioning significantly increased HO-1 mRNA and protein levels in the liver. In rats in which HO-1 activity had been inhibited with ZnPP pretreatment, the protective effects of HBO preconditioning against I/R injury were abolished. 4. In conclusion, HBO preconditioning can protect the liver against I/R injury and it appears that this effect might be mediated by the induction of HO-1.

Quercetin suppressed CYP2E1-dependent ethanol hepatotoxicity via depleting heme pool and releasing CO

The enzyme heme oxygenase-1 (HO-1) has cytoprotective effects by catalyzing the degradation of heme to produce carbon monoxide, iron and biliverdin. Furthermore, HO-1 activity has been associated with successful pregnancy. On the other hand, in the context of certain inflammatory conditions, HO-1 can induce iron overload and cell death. To investigate the role of HO-1 in gestational malaria, pregnant BALB/c mice were infected with Plasmodium berghei ANKA in early, mid and late gestation. We found that malaria affected the pregnancy outcome in the three periods evaluated. However, only poor pregnancy outcomes in early pregnancy were related to HO-1 upregulation, iron overload, lipid peroxidation and necrosis of the decidua, which were prevented by HO-1 inhibition. In conclusion, HO-1 expression must be finely tuned in gestational malaria to avoid the deleterious effect of increased enzyme activity.

Heme oxygenase (HO) is the rate limiting enzyme in the catabolism of heme molecules to the bile pigments which have recently been demonstrated to be strong antioxidants. In this study we analyzed the activity of HO in inflammatory cells isolated from a model of carrageenin induced acute inflammation in the rat. HO activity was significantly higher 24 hours after induction of the inflammation, this increase in activity coincided with the appearance of the highly inducible isoform of HO, Heat Shock Protein 32 kDa (HSP32) as detected by Western Blot analysis. Pre-treatment of animals with Tin protoporphyrin, a HO inhibitor, increased cell exudate at 24 hour in this model by 128% as compared to vehicle control. In comparison pre-treatment with a HO inducer, Ferriprotoporphyrin, decreased inflammatory cell number by 50% and cell exudate by 73% at 24 hours compared to control. These results suggest that HO may represent an endogenous protective mechanism against free radicals in acute inflammation and may be involved in the resolution of acute inflammation. The HSP32 isoform of HO may therefore represent a novel therapeutic target for the modulation of the inflammatory response.

Objective: To explore the effect of heme oxygenase-1 (HO-1) on level of reactive oxygen species (ROS) induced by zinc oxide nanoparticles (ZnO-NPs) in Human umbilical vein endothelial cells line EA.hy926. Methods: The EA.hy926 cells in logarithmic growth phase were incubated with 0.0, 2.5, 5.0, 10.0 and 15.0 mg/L ZnO-NPs respectively. The ROS level, reflected by mean fluorescence intensity (MFI), was examined by flow cytometer after 4 hours exposure, the protein expression of HO-1 which was determined by Western Blot after exposed to ZnO-NPs for 24 hours. Cells incubated with 15.0 mg/L were set as the ZnO-NPs group; a blank control group was set at the same time. Cells were pretreated with HO-1 inhibitor zinc protoporphyrin (ZnPPIx) and HO-1 activator cobalt protoporphyrin (CoPPIx), they were classified as ZnPPIx group and CoPPIx group. 15 mg/L ZnO-NPs was chosen to conduct the experiment of HO-1 activation and inhibition. Cells were classified as ZnPPIX+ ZnO-NPs group and CoPPIx+ ZnO-NPs group after pretreated with 10 μmol/L ZnPPIx or CoPPIx for 1 h, added 15 mg/L ZnO-NPs to cell culture medium. In all groups ROS levels were detected after exposed to ZnO-NPs for 4 hours, the protein expression of HO-1 was detected after exposed to ZnO-NPs for 24 hours. Results: With the increased dose of ZnO-NPs, levels of ROS and HO-1 in EA.hy926 cells were clearly elevated (the MFI of 0.0, 2.5, 5.0, 10.0 and 15.0 mg/L ZnO-NPs incubated groups was 22 627.22±718.27, 24 726.47±568.52, 31 141.75±1 312.24, 39 824.82±4 774.74, 50 569.03±1 497.63 respectively, and HO-1 relative expression were 0.16±0.01, 0.19±0.02, 0.16±0.01, 0.23±0.02, 0.92±0.06 respectively). HO-1 expression in ZnPPIx pretreatment group decreased compared with ZnO-NPs group (1.05±0.05 vs. 1.12±0.01, P<0.05), meanwhile ROS level enhanced (62 683.95±2 589.59 vs. 53 654.53±2 229.01, P<0.05). However, CoPPIx pretreatment had higher HO-1 level and lower level of ROS compared with ZnO-NPs group (HO-1: 1.74±0.11 vs. 0.22±0.03, P<0.05; ROS: 32 845.04±993.48 vs. 53 654.53±2 229.01, P<0.05). Conclusions: Exposure to ZnO-NPs significantly induced ROS generation in EA.hy926 cells in a dose-dependent manner. HO-1 regulated ZnO-NPs-induced oxidative stress.

Neurodegenerative disorders and brain damage are initiated by excessive production of reactive oxygen species (ROS), which leads to tissue injury, cellular death and inflammation. In cellular anti-oxidant systems, heme oxygenase-1 (HO-1) is an oxidative-sensor protein induced by ROS generation or carbon monoxide (CO) release. CO releasing molecules (CORMs), including CORM-3, exert anti-oxidant and anti-inflammatory effects. However, the molecular mechanisms of CORM-3-induced HO-1 expression and protection against interleukin (IL)-1β-induced inflammatory responses have not been fully elucidated in rat brain astrocytes (RBA-1). To study the regulation of CORM-3-induced HO-1 expression, signaling pathways, promoter activity, mRNA and protein expression were assessed following treatment with pharmacological inhibitors and gene-specific siRNA knockdown. We found that CORM-3 mediated HO-1 induction via transcritional and translational processes. Furthermore, CORM-3-induced HO-1 expression was mediated by phosphorylation of several protein kinases, such as c-Src, Pyk2, protein kinase Cα (PKCα) and p42/p44 mitogen-activated protein kinase (MAPK), which were inhibited by respective pharmacological inhibitors or by gene-specific knockdown with siRNA transfections. Next, we found that CORM-3 sequentially activated the c-Src/Pyk2/PKCα/p42/p44 MAPK pathway, thereby up-regulating mRNA for the activator protein (AP)-1 components c-Jun and c-Fos; these effects were attenuated by an AP-1 inhibitor (Tanshinone IIA; TSIIA) and other relevant inhibitors. Moreover, CORM-3-induced upregulation of HO-1 attenuated the IL-1β-induced cell migration and matrix metallopeptidase-9 mRNA expression in RBA-1 cells. These effects were reversed by an matrix metalloproteinase (MMP)2/9 inhibitor or by transfection with HO-1 siRNA.

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