Heme oxygenase-1 prevents superoxide anion-associated endothelial cell sloughing in diabetic rats

Heme oxygenase-1 (HO-1) represents a key defense mechanism against oxidative injury. Hyperglycemia has been linked to increased oxidative stress, leading to endothelial dysfunction, delayed cell replication, and enhanced apoptosis. The effect of streptozotocin (STZ)-induced diabetes on HO activity, HO-1 promoter activity, superoxide anion (O*-2, and the number of circulating endothelial cells was measured. The expression of HO-1/HO-2 protein was unchanged, but HO activity was decreased in aortas of diabetic rats compared with control (p < 0.05). High glucose decreased HO-1 promoter activity (p < 0.05). Hyperglycemia increased O*-2 and this increase was augmented with HO-1 inhibition and diminished with HO-1 upregulation (p < 0.05). Circulating endothelial cells were significantly higher in diabetic rats and were decreased or increased with administration of the HO-1 inducer (CoPP) or inhibitor (SnMP), respectively (p<0.05). In conclusion, HO-1 upregulation in diabetic rats brings about an increase in serum bilirubin, a reduction in O*-2 production, and a decrease in endothelial cell sloughing.     

Cobalt protoporphyrin inhibition of lipopolysaccharide or lipoteichoic acid-induced nitric oxide production via blocking c-Jun N-terminal kinase activation and nitric oxide enzyme activity

In the present study, low doses (0.5, 1, and 2 μM) of cobalt protoporphyrin (CoPP), but not ferric protoporphyrin (FePP) or tin protoporphyrin (SnPP), significantly inhibited lipopolysaccharide (LPS) or lipoteichoic acid (LTA)-induced inducible nitric oxide (iNOS) and nitric oxide (NO) production with an increase in heme oxygenase 1 (HO-1) protein in RAW264.7 macrophages under serum-free conditions. IC₅₀ values of CoPP inhibition of NO and iNOS protein individually induced by LPS and LTA were around 0.25 and 1.7 μM, respectively. This suggests that CoPP is more sensitive at inhibiting NO production than iNOS protein in response to separate LPS and LTA stimulation. NO inhibition and HO-1 induction by CoPP were blocked by the separate addition of fetal bovine serum (FBS) and bovine serum albumin (BSA). Decreasing iNOS/NO production and increasing HO-1 protein by CoPP were observed with CoPP pretreatment, CoPP co-treatment, and CoPP post-treatment with LPS and LTA stimulation. LPS- and LTA-induced NOS/NO productions were significantly suppressed by the JNK inhibitor, SP600125, but not by the ERK inhibitor, PD98059, through a reduction in JNK protein phosphorylation. Transfection of a dominant negative JNK plasmid inhibited LPS- and LTA-induced iNOS/NO production and JNK protein phosphorylation, suggesting that JNK activation is involved in LPS- and LTA-induced iNOS/NO production. Additionally, CoPP inhibition of LPS- and LTA-induced JNK, but not ERK, protein phosphorylation was identified in RAW264.7 cells. Furthermore, CoPP significantly reduced NO production in a cell-mediated, but not cell-free, iNOS enzyme activity assay accompanied by HO-1 induction. However, attenuation of HO-1 protein stimulated by CoPP via transfection of HO-1 siRNA did not affect NO's inhibition of CoPP against LPS stimulation. CoPP effectively suppressing LPS- and LTA-induced iNOS/NO production through blocking JNK activation and iNOS enzyme activity via a HO-1 independent manner is first demonstrated herein.     

Dimethylsulfoxide (DMSO) induces downregulation of heme oxygenase-1 (HO-1) in HL-60 cells: involvement of HO-1 in HL-60 cell differentiation

Heme oxygenase-1 (HO-1), an inducible enzyme with broad tissue expression, is wel1-regulated in response to hematopoietic stress and preserves vascular homeostasis. We investigated the involvement of HO-1 in HL-60 cell differentiation. Dimethyl sulfoxide (DMSO) completely decreased HO-1 expression in a time-dependent manner, but clearly induced HL-60 cell differentiation, as evidenced by a marked increase in CD11b expression. Interestingly, zinc protoporphyrin (ZnPP), a strong inhibitor of HO-1, induced HL-60 cell differentiation. In contrast, treatment with cobalt protoporphyrin (CoPP), an activator of HO-1, decreased CD11b expression. Additionally, ZnPP downregulated HO-1 protein expression in HL-60 cells, whereas CoPP induced upregulation. These results suggest that HO-1 might have a negative function in DMSO-induced HL-60 cell differentiation. This study provides the first evidence that HO-1 plays an important role in DMSO-induced HL-60 cell differentiation.     

Inhibition of filovirus replication by the zinc finger antiviral protein

The zinc finger antiviral protein (ZAP) was recently shown to inhibit Moloney murine leukemia virus and Sindbis virus replication. We tested whether ZAP also acts against Ebola virus (EBOV) and Marburg virus (MARV). Antiviral effects were observed after infection of cells expressing the N-terminal part of ZAP fused to the product of the zeocin resistance gene (NZAP-Zeo) as well as after infection of cells inducibly expressing full-length ZAP. EBOV was inhibited by up to 4 log units, whereas MARV was inhibited between 1 to 2 log units. The activity of ZAP was dependent on the integrity of the second and fourth zinc finger motif, as tested with cell lines expressing NZAP-Zeo mutants. Heterologous expression of EBOV- and MARV-specific sequences fused to a reporter gene suggest that ZAP specifically targets L gene sequences. The activity of NZAP-Zeo in this assay was also dependent on the integrity of the second and fourth zinc finger motif. Time-course experiments with infectious EBOV showed that ZAP reduces the level of L mRNA before the level of genomic or antigenomic RNA is affected. Transient expression of ZAP decreased the activity of an EBOV replicon system by up to 95%. This inhibitory effect could be partially compensated for by overexpression of L protein. In conclusion, the data demonstrate that ZAP exhibits antiviral activity against filoviruses, presumably by decreasing the level of viral mRNA.     

Down-regulation of heme oxygenase-1 by SVCV infection

Heme oxygenase (HO-1) is a cytoprotective enzyme that plays a critical role in defending the body against oxidant-induced injury during inflammatory processes. In mammalian systems, viral infection or antigen expression can down-regulate the expression of HO-1. In turn, the induction of HO-1 or overexpression of HO-1 results in potent and direct antiviral activity that targets the replication of several mammalian viruses. In this study, the HO-1 gene of Cyprinus carpio was cloned, and the expression profile of HO-1 was investigated during spring viremia of carp virus (SVCV) infection. The results demonstrate that the expression of HO-1 was down-regulated during SVCV infection in the EPC cells and in common carp. These results indicated that SVCV infection could induce host oxidative stress, which may contribute to tissue injury in affect fish.     

Long-Lasting Expression of HO-1 Delays Progression of Type I Diabetes in NOD Mice

Heme oxygenase-1 (HO-1) is crucial in regulating oxidative injury. The present study was designed to assess whether HO-1 upregulation by cobalt protoporphyrin IX (CoPP) moderates or prevents the diabetic state in non-obese diabetic (NOD) mice, an animal model for Type 1 diabetes (T1D). HO-1 expression and HO activity were upregulated in the pancreas by the intermittent administration of CoPP. This was associated with decreases in blood glucose and pancreatic O2-, but increased pAKT and BcL-XL and cell survival. A considerable number of beta cells were preserved in the islets of CoPP-treated NOD mice, while none were found in untreated diabetic mice. The number of CD11c+ dendritic cells was decreased in the pancreas of CoPP-treated NOD mice (p     

Differential effect of cobalt protoporphyrin on distributions of heme oxygenase in renal structure and on blood pressure in SHR.

Heme oxygenase (HO) is a microsomal enzyme that oxidatively cleaves heme to form biliverdin, releasing iron and carbon monoxide (CO). Thus, HO not only controls the availability of heme for the synthesis of hemeproteins but also generates CO, which binds to the heme moiety of hemoproteins, thereby affecting their enzymatic activity. The present study was undertaken to explore changes in the relative expression of renal HO-1 and HO-2 in response to modulators and the effect on blood pressure regulation in spontaneously hypertensive rats (SHR). Immunohistochemistry confirmed a cobalt protoporphyrin (CoPP)-mediated increase in HO-1 protein. After a single injection of CoPP (5 mg/100 gram body weight) in 7-week-old SHR, blood pressure significantly decreased (p<0.01) while renal HO activity increased 6-fold over controls. CoPP pretreatment deceased the levels of the renal cytochrome P450-derived arachidonic acid metabolite, 20-HETE, a powerful vasoconstrictor, by 65% in renal tissue. Western blot analysis demonstrated that CoPP significantly increased HO-1 protein expression in the cortex and outer medulla and, to a lesser degree, in the inner medulla of the rat kidney. HO-2 was constitutively expressed in all parts of the kidney, and did not significantly change after treatment with CoPP. These results indicate that selective induction of cortical and outer medullary HO-1 is associated with a decrease in 20-HETE and blood pressure, suggesting an important role for HO-1 activity in the regulation of urine volume, electrolyte excretion and blood pressure.     

Carbon monoxide reduces neuropathic pain and spinal microglial activation by inhibiting nitric oxide synthesis in mice

Background

Carbon monoxide (CO) synthesized by heme oxygenase 1 (HO-1) exerts antinociceptive effects during inflammation but its role during neuropathic pain remains unknown. Our objective is to investigate the exact contribution of CO derived from HO-1 in the modulation of neuropathic pain and the mechanisms implicated.

Methodology/Principal Findings

We evaluated the antiallodynic and antihyperalgesic effects of CO following sciatic nerve injury in wild type (WT) or inducible nitric oxide synthase knockout (NOS2-KO) mice using two carbon monoxide-releasing molecules (CORM-2 and CORM-3) and an HO-1 inducer (cobalt protoporphyrin IX, CoPP) daily administered from days 10 to 20 after injury. The effects of CORM-2 and CoPP on the expression of HO-1, heme oxygenase 2 (HO-2), neuronal nitric oxide synthase (NOS1) and NOS2 as well as a microglial marker (CD11b/c) were also assessed at day 20 after surgery in WT and NOS2-KO mice. In WT mice, the main neuropathic pain symptoms induced by nerve injury were significantly reduced in a time-dependent manner by treatment with CO-RMs or CoPP. Both CORM-2 and CoPP treatments increased HO-1 expression in WT mice, but only CoPP stimulated HO-1 in NOS2-KO animals. The increased expression of HO-2 induced by nerve injury in WT, but not in NOS2-KO mice, remains unaltered by CORM-2 or CoPP treatments. In contrast, the over-expression of CD11b/c, NOS1 and NOS2 induced by nerve injury in WT, but not in NOS2-KO mice, were significantly decreased by both CORM-2 and CoPP treatments. These data indicate that CO alleviates neuropathic pain through the reduction of spinal microglial activation and NOS1/NOS2 over-expression.

Conclusions/Significance

This study reports that an interaction between the CO and nitric oxide (NO) systems is taking place following sciatic nerve injury and reveals that increasing the exogenous (CO-RMs) or endogenous (CoPP) production of CO may represent a novel strategy for the treatment of neuropathic pain.     

Inhibition of heme oxygenase-1 interferes with the transforming activity of the Kaposi sarcoma herpesvirus-encoded G protein-coupled receptor

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.     

Heme oxygenase-1 induction by cobalt protoporphyrin enhances fever and inhibits pyrogenic tolerance to lipopolysaccharide

Heme oxygenase-1 (HO-1) is an enzyme that catalyzes degradation of the heme and regulates its availability for newly synthetized hemeproteins such as cyclooxygenases, NO synthases and cytochrome P450. Moreover, HO-1 activity modulates synthesis of cytokines and prostaglandins. All of these factors are well-defined components of fever and pyrogenic tolerance mechanisms. We examine the effect of HO-1 induction and activation using cobalt protoporphyrin (CoPP) on changes in body temperature (T_b), plasma levels of interleukin-6 (IL-6), prostaglandin E₂ (PGE₂) and HO-1 protein in the course of these processes. Intraperitoneally (i.p.) pre-treatment of rats with CoPP (5 mg kg⁻¹) significantly accelerated and enhanced the early stage of lipopolysaccharide (LPS)-induced fever and shortened a post-fever recovery to normal temperature. Pre-treatment with CoPP significantly potentiated the increase in plasma IL-6, PGE₂ and HO-1 levels measured 4 h after the LPS administration. Furthermore, induction of HO-1 attenuated the development of pyrogenic tolerance to repeated injections of LPS. Based on these data we conclude that heme oxygenase-1 may act as a physiological regulator of the febrile response intensity to bacterial infections.     

Heme oxygenase-1 enhances renal mitochondrial transport carriers and cytochrome C oxidase activity in experimental diabetes

Up-regulation of heme oxygenase (HO-1) by either cobalt protoporphyrin (CoPP) or human gene transfer improves vascular and renal function by several mechanisms, including increases in antioxidant levels and decreases in reactive oxygen species (ROS) in vascular and renal tissue. The purpose of the present study was to determine the effect of HO-1 overexpression on mitochondrial transporters, cytochrome c oxidase, and anti-apoptotic proteins in diabetic rats (streptozotocin, (STZ)-induced type 1 diabetes). Renal mitochondrial carnitine, deoxynucleotide, and ADP/ATP carriers were significantly reduced in diabetic compared with nondiabetic rats (p < 0.05). The citrate carrier was not significantly decreased in diabetic tissue. CoPP administration produced a robust increase in carnitine, citrate, deoxynucleotide, dicarboxylate, and ADP/ATP carriers and no significant change in oxoglutarate and aspartate/glutamate carriers. The increase in mitochondrial carriers (MCs) was associated with a significant increase in cytochrome c oxidase activity. The administration of tin mesoporphyrin (SnMP), an inhibitor of HO-1 activity, prevented the restoration of MCs in diabetic rats. Human HO-1 cDNA transfer into diabetic rats increased both HO-1 protein and activity, and restored mitochondrial ADP/ATP and deoxynucleotide carriers. The increase in HO-1 by CoPP administration was associated with a significant increase in the phosphorylation of AKT and levels of BcL-XL proteins. These observations in experimental diabetes suggest that the cytoprotective mechanism of HO-1 against oxidative stress involves an increase in the levels of MCs and anti-apoptotic proteins as well as in cytochrome c oxidase activity.     

Heme oxygenase-1 modulates brain inflammation and apoptosis in mice with angiostrongyliasis

The rat nematode lungworm Angiostrongylus cantonensis undergoes obligatory intracerebral migration in its hosts and causes eosinophilic meningitis or meningoencephalitis. Heme oxygenase 1 (HO-1) has several cytoprotective properties such as anti-oxidative, anti-inflammatory, and anti-apoptotic effects. HO-1 in brain tissues was induced in A. cantonensis-infected group and showed positive modulation in cobalt protoporphyrin (CoPP)-treated groups. Assay methods for the therapeutic effect include western blot analysis, enzyme-linked immunosorbent assay, gelatin zymography, blood–brain barrier permeability evaluation and eosinophil count in cerebrospinal fluid. The combination of albendazole (ABZ) and CoPP significantly decreased pro-inflammatory cytokines, tumor necrosis factor-α, interleukin (IL)-1β, IL-5, and IL-33 but significantly increased anti-inflammatory cytokines IL-10 and transforming growth factor-β. In addition, worm recovery, matrix metalloproteinase-9, BBB permeability, and eosinophil counts were decreased in the ABZ and CoPP co-treated groups. Induction of HO-1 with CoPP strongly inhibited the protein levels of caspase-3 and increased the induction of annexin-V and B-cell leukemia 2. Thus, co-treatment with ABZ and CoPP prevented A. cantonensis-induced eosinophilic meningoencephalitis and its anti-apoptotic effect by promoting HO-1 signaling prior to BBB dysfunction. HO-1 induction might be a therapeutic modality for eosinophilic meningoencephalitis.     

Induction of hemeoxygenase-1 reduces glomerular injury and apoptosis in diabetic spontaneously hypertensive rats

Induction of hemeoxygenase-1 (HO-1) lowers blood pressure and reduces organ damage in hypertensive animal models; however, a potential protective role for HO-1 induction against diabetic-induced glomerular injury remains unclear. We hypothesize that HO-1 induction will protect against diabetes-induced glomerular injury by maintaining glomerular integrity and inhibiting renal apoptosis, inflammation, and oxidative stress. Diabetes was induced with streptozotocin in spontaneously hypertensive rats (SHR) as a model where the coexistence of hypertension and diabetes aggravates the progression of diabetic renal injury. Control and diabetic SHR were randomized to receive vehicle or the HO-1 inducer cobalt protoporphyrin (CoPP). Glomerular albumin permeability was significantly greater in diabetic SHR compared with control, consistent with an increase in apoptosis and decreased glomerular nephrin and α(3)β(1)-integrin protein expression in diabetic SHR. CoPP significantly reduced albumin permeability and apoptosis and restored nephrin and α(3)β(1)-integrin protein expression levels in diabetic SHR. Glomerular injury in diabetic SHR was also associated with increases in NF-κB-induced inflammation and oxidative stress relative to vehicle-treated SHR, and CoPP significantly blunted diabetes-induced increases in glomerular inflammation and oxidative stress in diabetic SHR. These effects were specific to exogenous stimulation of HO-1, since incubation with the HO inhibitor stannous mesoporphyrin alone did not alter glomerular inflammatory markers or oxidative stress yet was able to prevent CoPP-mediated decreases in these parameters. These data suggest that induction of HO-1 reduces diabetic induced-glomerular injury and apoptosis and these effects are associated with decreased NF-κB-induced inflammation and oxidative stress.     

AMP-Activated Protein Kinase Is Required for the Macropinocytic Internalization of Ebolavirus

Identification of host factors that are needed for Zaire Ebolavirus (EBOV) entry provides insights into the mechanism(s) of filovirus uptake, and these factors may serve as potential antiviral targets. In order to identify novel host genes and pathways involved in EBOV entry, gene array findings in the National Cancer Institute''s NCI-60 panel of human tumor cell lines were correlated with permissivity for EBOV glycoprotein (GP)-mediated entry. We found that the gene encoding the γ2 subunit of AMP-activated protein kinase (AMPK) strongly correlated with EBOV transduction in the tumor panel. The AMPK inhibitor compound C inhibited infectious EBOV replication in Vero cells and diminished EBOV GP-dependent, but not Lassa fever virus GPC-dependent, entry into a variety of cell lines in a dose-dependent manner. Compound C also prevented EBOV GP-mediated infection of primary human macrophages, a major target of filoviral replication in vivo. Consistent with a role for AMPK in filovirus entry, time-of-addition studies demonstrated that compound C abrogated infection when it was added at early time points but became progressively less effective when added later. Compound C prevented EBOV pseudovirion internalization at 37°C as cell-bound particles remained susceptible to trypsin digestion in the presence of the inhibitor but not in its absence. Mouse embryonic fibroblasts lacking the AMPKα1 and AMPKα2 catalytic subunits were significantly less permissive to EBOV GP-mediated infection than their wild-type counterparts, likely due to decreased macropinocytic uptake. In total, these findings implicate AMPK in macropinocytic events needed for EBOV GP-dependent entry and identify a novel cellular target for new filoviral antivirals.     

Cathepsin B & L Are Not Required for Ebola Virus Replication

Ebola virus (EBOV), family Filoviridae, emerged in 1976 on the African continent. Since then it caused several outbreaks of viral hemorrhagic fever in humans with case fatality rates up to 90% and remains a serious Public Health concern and biothreat pathogen. The most pathogenic and best-studied species is Zaire ebolavirus (ZEBOV). EBOV encodes one viral surface glycoprotein (GP), which is essential for replication, a determinant of pathogenicity and an important immunogen. GP mediates viral entry through interaction with cellular surface molecules, which results in the uptake of virus particles via macropinocytosis. Later in this pathway endosomal acidification activates the cysteine proteases Cathepsin B and L (CatB, CatL), which have been shown to cleave ZEBOV-GP leading to subsequent exposure of the putative receptor-binding and fusion domain and productive infection. We studied the effect of CatB and CatL on in vitro and in vivo replication of EBOV. Similar to previous findings, our results show an effect of CatB, but not CatL, on ZEBOV entry into cultured cells. Interestingly, cell entry by other EBOV species (Bundibugyo, Côte d''Ivoire, Reston and Sudan ebolavirus) was independent of CatB or CatL as was EBOV replication in general. To investigate whether CatB and CatL have a role in vivo during infection, we utilized the mouse model for ZEBOV. Wild-type (control), catB^−/− and catL^−/− mice were equally susceptible to lethal challenge with mouse-adapted ZEBOV with no difference in virus replication and time to death. In conclusion, our results show that CatB and CatL activity is not required for EBOV replication. Furthermore, EBOV glycoprotein cleavage seems to be mediated by an array of proteases making targeted therapeutic approaches difficult.