Cobalt Protoporphyrin IX Research Articles

Metal-ligand delocalization of iron and cobalt porphyrin complexes in aqueous solutions probed by soft X-ray absorption spectroscopy.

Metal-ligand delocalization of metal porphyrin complexes in aqueous solutions was investigated by analyzing the electronic structure of both the metal and ligand sides using soft X-ray absorption spectroscopy (XAS) at the metal L2,3-edges and nitrogen K-edge, respectively. In the N K-edge XAS spectra of the ligands, the energies of the CN π* peaks of cobalt protoporphyrin IX (CoPPIX) are higher than iron protoporphyrin IX (FePPIX). The energy difference between the two lowest peaks in the XAS spectrum of CoPPIX is also larger than that of FePPIX. Nitrogen K-edge inner-shell calculations of metalloporphyrins with different central metals indicate that the energy differences between these peaks reflect the electronic configurations and spin multiplicities of metalloporphyrins. We also investigated the hydration structure of CoPPIX in aqueous solution by analyzing the electronic structure of the ligand and revealed that CoPPIX maintains its five-coordination geometry in aqueous solution. The present study shows high performance of N K-edge XAS of ligands for studying the coordination structures of metalloporphyrins in solutions rather than the metal L2,3-edges of central metals.

Limited Heme Oxygenase Contribution to Modulating the Severity of Salmonella enterica serovar Typhimurium Infection.

An important virulence trait of Salmonella enterica serovar Typhimurium (S. Typhimurium) is the ability to avoid the host immune response, generating systemic and persistent infections. Host cells play a crucial role in bacterial clearance by expressing the enzyme heme oxygenase 1 (Hmox1), which catalyzes the degradation of heme groups into Fe2+, biliverdin, and carbon monoxide (CO). The role of Hmox1 activity during S. Typhimurium infection is not clear and previous studies have shown contradictory results. We evaluated the effect of pharmacologic modulation of Hmox1 in a mouse model of acute and persistent S. Typhimurium infection by administering the Hmox1 activity inductor cobalt protoporphyrin-IX (CoPP) or inhibitor tin protoporphyrin-IX (SnPP) before infection. To evaluate the molecular mechanism involved, we measured the colocalization of S. Typhimurium and autophagosome and lysosomal markers in macrophages. Administering CoPP reduced the bacterial burden in organs of mice 5 days post-infection, while SnPP-treated mice showed bacterial loads similar to vehicle-treated mice. Furthermore, CoPP reduced bacterial loads when administered after infection in macrophages in vitro and in a persistent infection model of S. Typhimurium in vivo, while tin protoporphyrin-IX (SnPP) treatment resulted in a bacterial burden similar to vehicle-treated controls. However, we did not observe significant differences in co-localization of green fluorescent protein (GFP)-labeled S. Typhimurium with the autophagic vesicles marker microtubule-associated protein 1A/1B-light chain 3 (LC3) and the lysosomal marker lysosomal-associated membrane protein 1 (LAMP-1) in macrophages treated with CoPP. Our results suggest that CoPP can enhance antimicrobial activity in response to Salmonella infection, reducing bacterial dissemination and persistence in mice, in a CO and autophagy- independent manner.

De novo biosynthesis of a nonnatural cobalt porphyrin cofactor in E. coli and incorporation into hemoproteins

Enzymes that bear a nonnative or artificially introduced metal center can engender novel reactivity and enable new spectroscopic and structural studies. In the case of metal-organic cofactors, such as metalloporphyrins, no general methods exist to build and incorporate new-to-nature cofactor analogs in vivo. We report here that a common laboratory strain, Escherichia coli BL21(DE3), biosynthesizes cobalt protoporphyrin IX (CoPPIX) under iron-limited, cobalt-rich growth conditions. In supplemented minimal media containing CoCl2, the metabolically produced CoPPIX is directly incorporated into multiple hemoproteins in place of native heme b (FePPIX). Five cobalt-substituted proteins were successfully expressed with this new-to-nature cobalt porphyrin cofactor: myoglobin H64V V68A, dye decolorizing peroxidase, aldoxime dehydratase, cytochrome P450 119, and catalase. We show conclusively that these proteins incorporate CoPPIX, with the CoPPIX making up at least 95% of the total porphyrin content. In cases in which the native metal ligand is a sulfur or nitrogen, spectroscopic parameters are consistent with retention of native metal ligands. This method is an improvement on previous approaches with respect to both yield and ease-of-implementation. Significantly, this method overcomes a long-standing challenge to incorporate nonnatural cofactors through de novo biosynthesis. By utilizing a ubiquitous laboratory strain, this process will facilitate spectroscopic studies and the development of enzymes for CoPPIX-mediated biocatalysis.

Haem oxygenase protects against thrombocytopaenia and malaria-associated lung injury

BackgroundMalaria-triggered lung injury can occur in both severe and non-severe cases. Platelets may interact with parasitized erythrocytes, leukocytes and endothelium. These interactions can lead to microvessel obstructions and induce release of inflammatory mediators. Induction of the haem oxygenase enzyme is important in the host’s response to free haem and to several other molecules generated by infectious or non-infectious diseases. In addition, an important role for the haem oxygenase-1 isotype has been demonstrated in experimental cerebral malaria and in clinical cases. Therefore, the present work aims to determine the influence of haem oxygenase in thrombocytopaenia and acute pulmonary injury during infection with Plasmodium berghei strain NK65.MethodsC57BL/6 mice were infected with P. berghei and analysed 7-10 days post-infection. For each experiment, Cobalt Protoporphyrin IX/CoPPIX or saline were administered. Bronchoalveolar lavage fluid was used for total and differential leukocyte count and for protein measurement. Lungs were used for histological analyses or for analysis of cytokines and western blotting. The lung permeability was analysed by Evans blue dye concentration. Platelet-leukocyte aggregate formation was assayed using the flow cytometer.ResultsPlasmodium berghei NK65 infection generated an intense lung injury, with increased levels of inflammatory mediators, oedema, and cell migration into the lung. Plasmodium berghei infection was also accompanied by marked thrombocytopaenia and formation of platelet-leukocyte aggregates in peripheral blood. Treatment with the HO-1 inducer cobalt protoporphyrin IX (CoPPIX) modified the inflammatory response but did not affect the evolution of parasitaemia. Animals treated with CoPPIX showed an improvement in lung injury, with decreased inflammatory infiltrate in the lung parenchyma, oedema and reduced thrombocytopaenia.ConclusionData here presented suggest that treatment with CoPPIX inducer leads to less severe pulmonary lung injury and thrombocytopaenia during malaria infection, thus increasing animal survival.

Inhibited autophagy may contribute to heme toxicity in cardiomyoblast cells

Several groups have demonstrated that induction of heme-oxygenase-1 (HO-1) could protect the myocardium against ischemic events; however, heme accumulation could lead to toxicity. The aim of the present study was to investigate the role of autophagy in heme toxicity.H9c2 cardiomyoblast cells were treated with different dose of hemin or cobalt-protoporphyrin IX (CoPPIX) or vehicle. Cell viability was measured by MTT assay. DCF and MitoSOX staining was employed to detect reactive oxygen species. Western blot analysis was performed to analyse the levels of HO-1, certain autophagy related proteins and pro-caspase-3 as an apoptosis marker. To study the autophagic flux, CytoID staining was carried out and cells were analyzed by fluorescence microscope and flow cytometry.Decreased cell viability was detected at high dose of hemin and CoPPIX treated H9c2 cells in a dose-dependent manner. Furthermore, at concentration of the inducers used in the present study a significantly enhanced level of ROS were detected. As it was expected both treatments induced a robust elevation of HO-1 level. In addition, the Beclin-1- independent autophagy was significantly increased, but caused a defective autophagic flux with triggered activation of caspase-3.In conclusion, these results suggest that overexpression of HO-1 by high dose of hemin and CoPPIX can induce cell toxicity in H9c2 cells via enhanced ROS level and impaired autophagy.

Hyperhomocysteinemia Accelerates Acute Kidney Injury to Chronic Kidney Disease Progression by Downregulating Heme Oxygenase-1 Expression.

The risk factors promoting acute kidney injury (AKI) to chronic kidney disease (CKD) progression remain largely unknown. The aim of the present study was to investigate whether hyperhomocysteinemia (Hhcy) accelerates the development of renal fibrosis after AKI. Hhcy aggravated ischemia-reperfusion-induced AKI and the subsequent development of renal fibrotic lesions characterized by excessive extracellular matrix deposition. Mechanistically, the RNA binding protein human antigen R (HuR) bound to the 3'-untranslated region (3'-UTR) of heme oxygenase-1 (HO-1) messenger RNA (mRNA). Homocysteine (Hcy) downregulated HuR expression, reduced the binding of HuR to the 3'-UTR of HO-1, and thereafter decreased HO-1 expression. Administration of the HO-1 inducer cobalt protoporphyrin-IX significantly hindered Hhcy-augmented reactive oxygen species production and renal fibrotic lesions. Innovation and Conclusion: These data indicate that Hhcy might be a novel risk factor that promotes AKI to CKD progression. Lowering Hcy level or HO-1 induction might be a potential therapeutic strategy to improve the outcome of AKI.

Various roles of heme oxygenase-1 in response of bone marrow macrophages to RANKL and in the early stage of osteoclastogenesis

Heme oxygenase-1 (HO-1; encoded by Hmox1), a downstream target of the Nrf2 transcription factor, has been postulated to be a negative regulator of osteoclasts (OCLs) differentiation. Here, we further explored such a hypothesis by examining HO-1 effects in different stages of osteoclastogenesis. We confirmed the inhibition of the expression of OCLs markers by Nrf2. In contrast, both the lack of the active Hmox1 gene or HO-1 silencing in OCLs precursor cells, bone marrow macrophages (BMMs), decreased their differentiation towards OCLs, as indicated by the analysis of OCLs markers such as TRAP. However, no effect of HO-1 deficiency was observed when HO-1 expression was silenced in BMMs or RAW264.7 macrophage cell line pre-stimulated with RANKL (considered as early-stage OCLs). Moreover, cobalt protoporphyrin IX (CoPPIX) or hemin, the known HO-1 inducers, inhibited OCLs markers both in RANKL-stimulated RAW264.7 cells and BMMs. Strikingly, a similar effect occurred in HO-1−/− cells, indicating HO-1-independent activity of CoPPIX and hemin. Interestingly, plasma of HO-1−/− mice contained higher TRAP levels, which suggests an increased number of bone-resorbing OCLs in the absence of HO-1 in vivo. In conclusion, our data indicate that HO-1 is involved in the response of bone marrow macrophages to RANKL and the induction of OCLs markers, but it is dispensable in early-stage OCLs. However, in vivo HO-1 appears to inhibit OCLs formation.

Heme-oxygenase-1 Production by Intestinal CX3CR1+ Macrophages Helps to Resolve Inflammation and Prevents Carcinogenesis.

CX3CR1+ macrophages in the intestinal lamina propria contribute to gut homeostasis through the immunomodulatory interleukin IL10, but there is little knowledge on how these cells or the CX3CR1 receptor may affect colorectal carcinogenesis. In this study, we show that CX3CR1-deficient mice fail to resolve gut inflammation despite high production of IL10 and have increased colitis and adenomatous polyps in chemical and genetic models of colon carcinogenesis. Mechanistically, CX3CL1-mediated engagement of the CX3CR1 receptor induced upregulation of heme-oxygenase-1 (HMOX-1), an antioxidant and anti-inflammatory enzyme. CX3CR1-deficient mice exhibited significantly lower expression of HMOX-1 in their adenomatous colon tissues. Combining LPS and CX3CL1 displayed a strong synergistic effect in vitro, but HMOX-1 levels were significantly lower in KO macrophages. Cohousing of wild-type and CX3CR1-/- mice during the AOM/DSS treatment attenuated disease severity in CX3CR1-/- mice, indicating the importance of the microbiome, but did not fully reinstate HMOX-1 levels and did not abolish polyp formation. In contrast, pharmacologic induction of HMOX-1 in vivo by cobalt protoporphyrin-IX treatment eradicated intestinal inflammation and fully protected KO mice from carcinogenesis. Taken together, our results establish an essential role for the receptor CX3CR1 in gut macrophages in resolving inflammation in the intestine, where it helps protects against colitis-associated cancer by regulating HMOX-1 expression. Cancer Res; 77(16); 4472-85. ©2017 AACR.

Haem oxygenase-1 polymorphisms can affect HCV replication and treatment responses with different efficacy in humanized mice.

Enhancement of host anti-oxidant enzymes, such as haemoxygenase-1, may attenuate virus-mediated hepatocyte injury, while the induction of HO-1 by cobalt-protoporphyrin-IX (CoPP) administration, as the application of its haem degradation product biliverdin (BV), was shown to hinder HCV replication in vitro. In addition, (GT)n -repeats length in the polymorphic region of the HO-1 promoter may affect HO-1 expression and responsiveness to infection and disease severity. Aim of this study was to investigate the antiviral and hepatoprotective effects of CoPP-mediated HO-1 induction, alone or in combination with interferon alpha (peg-IFNα), in HCV-infected mice harbouring hepatocytes from donors with different HO-1-promoter polymorphisms. Upon establishment of HCV infection, CoPP, BV and peg-IFNα were given alone or in combination. Viraemia changes and intrahepatic human gene expression were determined by qRT-PCR and immunohistochemistry. CoPP administration increased human HO-1 expression and significantly reduced viraemia, although changes correlated with promoter length (Δ0.5log and Δ2log reduction with medium- and short-polymorphism respectively). Polymorphisms did not influence BV-mediated antiviral effects (Δ1log). Notably, HO-1 induction attenuated basal HCV-driven enhancement of interferon genes and pro-inflammatory cytokines, both in cells with short- or medium-polymorphisms. Moreover, simultaneous administration of CoPP and peg-IFNα reduced viraemia even stronger (median 3log), whereas 1log viraemia reduction was determined in mice receiving peg-IFNα monotherapy. Although the protective function of HO-1 could be elicited in vivo with both host polymorphisms, the strength of HO-1 induction and suppression of HCV occurred in a polymorphism-dependent manner, indicating that host-genetic determinants may affect disease progression and infection outcome.

Differences in vulnerability of neurons and astrocytes to heme oxygenase-1 modulation: Implications for mitochondrial ferritin.

Induction of the antioxidant enzyme heme oxygenase-1 (HO-1) was observed in both astrocytes and neurons in the substantia nigra of patients with Parkinson’s disease (PD). In the current study, we investigated whether HO-1 behaves differently between neurons and astrocytes under the condition of neurotoxicity related to PD. The results showed a time-dependent HO-1 upregulation in primary cultured ventral mesencephalon neurons and astrocytes treated with the mitochondria complex I inhibitor 1-methyl-4-phenylpyridinium (MPP+) or recombinant α-synuclein. However, HO-1 upregulation appeared much later in neurons than in astrocytes. The HO-1 inhibitor zinc protoporphyrin (ZnPP) aggravated MPP+- or α-synuclein-induced oxidative damage in both astrocytes and neurons, indicating that this HO-1 response was cytoprotective. For neurons, the HO-1 activator cobalt protoporphyrin IX (CoPPIX) exerted protective effects against MPP+ or α-synuclein during moderate HO-1 upregulation, but it aggravated damage at the peak of the HO-1 response. For astrocytes, CoPPIXalways showed protective effects. Higher basal and CoPPIX-induced mitochondrial ferritin (MtFt) levels were detected in astrocytes. Lentivirus-mediated MtFt overexpression rescued the neuronal damage induced by CoPPIX, indicating that large MtFt buffering capacity contributes to pronounced HO-1 tolerance in astrocytes. Such findings suggest that astrocyte-targeted HO-1 interventions and MtFt modulations have potential as novel pharmacological strategies in PD.

Preferential Heme Oxygenase-1 Activation in Striatal Astrocytes Antagonizes Dopaminergic Neuron Degeneration in MPTP-Intoxicated Mice.

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) accompanied by increased oxidative damage. Astrocytes, which are the most abundant glial cell types in the brain, possess higher antioxidant potential partially due to preferentially activated nuclear factor E2-related factor 2 genes. Heme oxygenase isoform 1 (HO-1) is crucial for the response to oxidative stress via the catabolism of heme to carbon monoxide, bilirubin, and iron. In the present study, we aimed to investigate astroglial expression of HO-1 in the SNpc, especially in the striatum of a subacute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mouse model of PD, and to investigate the neuroprotective effects of intraventricularly administrated HO-1 activator cobalt protoporphyrin IX (CoPPIX). The results showed that HO-1 was faintly distributed in neurons but not astrocytes in the normal SNpc and striatum. MPTP triggered a robust HO-1 response in the astrocytes of the striatum after 1-day treatment, but the HO-1 levels declined dramatically at day 3 and were completely undetectable at day 5. Intraventricular administration of CoPPIX for 8days could preferentially activate HO-1 in astrocytes in the striatum but not SNpc. The content of striatal dopamine and its derivatives was restored in the subacute MPTP models. CoPPIX also increased the number of dopaminergic neurons and the tyrosine hydroxylase levels in the SNpc. These results suggest that inadequate HO-1 in striatal astrocytes might contribute to the limited antioxidant defense and dopaminergic neuron degeneration in PD, and preferential HO-1 activation in striatal astrocytes might be neuroprotective. The study thus sheds light on the targeting of HO-1 in striatal astrocytes for PD therapeutics.

Heat shock protein-mediated protection against Cisplatin-induced hair cell death.

Cisplatin is a highly successful and widely used chemotherapy for the treatment of various solid malignancies in both adult and pediatric patients. Side effects of cisplatin treatment include nephrotoxicity and ototoxicity. Cisplatin ototoxicity results from damage to and death of cells in the inner ear, including sensory hair cells. We showed previously that heat shock inhibits cisplatin-induced hair cell death in whole-organ cultures of utricles from adult mice. Since heat shock protein 70 (HSP70) is the most upregulated HSP in response to heat shock, we investigated the role of HSP70 as a potential protectant against cisplatin-induced hair cell death. Our data using utricles from HSP70 (-/-) mice indicate that HSP70 is necessary for the protective effect of heat shock against cisplatin-induced hair cell death. In addition, constitutive expression of inducible HSP70 offered modest protection against cisplatin-induced hair cell death. We also examined a second heat-inducible protein, heme oxygenase-1 (HO-1, also called HSP32). HO-1 is an enzyme responsible for the catabolism of free heme. We previously showed that induction of HO-1 using cobalt protoporphyrin IX (CoPPIX) inhibits aminoglycoside-induced hair cell death. Here, we show that HO-1 also offers significant protection against cisplatin-induced hair cell death. HO-1 induction occurred primarily in resident macrophages, with no detectable expression in hair cells or supporting cells. Depletion of macrophages from utricles abolished the protective effect of HO-1 induction. Together, our data indicate that HSP induction protects against cisplatin-induced hair cell death, and they suggest that resident macrophages mediate the protective effect of HO-1 induction.

Small molecule activators of the Nrf2-HO-1 antioxidant axis modulate heme metabolism and inflammation in BV2 microglia cells

The nuclear factor erythroid derived 2-related factor 2 (Nrf2) and the antioxidant protein heme oxygenase-1 (HO-1) are crucial components of the cellular stress response. These two systems work together to combat oxidative stress and inflammation and are attractive drug targets for counteracting different pathologies, including neuroinflammation. We aimed to identify the most effective Nrf2/HO-1 activators that modulate the inflammatory response in microglia cells. In the present study, we searched the literature and selected 56 compounds reported to activate Nrf2 or HO-1 and analyzed them for HO-1 induction at 6 and 24h and cytotoxicity in BV2 microglial cells in vitro. Approximately 20 compounds up-regulated HO-1 at the concentrations tested (5–20μM) with carnosol, supercurcumin, cobalt protoporphyrin-IX and dimethyl fumarate exhibiting the best induction/low cytotoxicity profile. Up-regulation of HO-1 by some compounds resulted in increased cellular bilirubin levels but did not augment the expression of proteins involved in heme synthesis (ALAS 1) or biliverdin reductase. Bilirubin production by HO-1 inducers correlated with their potency in inhibiting nitrite production after challenge with interferon-γ (INF-γ) or lipopolysaccharide (LPS). The compounds down-regulated the inflammatory response (TNF-α, PGE2 and nitrite) more strongly in cells challenged with INF-γ than LPS, and silencing HO-1 or Nrf2 with shRNA differentially affected the levels of inflammatory markers. These findings indicate that some small activators of Nrf2/HO-1 are effective modulators of microglia inflammation and highlight the chemical scaffolds that can serve for the synthesis of potent new derivatives to counteract neuroinflammation and neurodegeneration.

Cobalt Protoporphyrin Accelerates TFEB Activation and Lysosome Reformation during LPS-Induced Septic Insults in the Rat Heart

Lipopolysaccharide (LPS)-induced myocardial dysfunction is caused, at least in part, by mitochondrial dysfunction. Mitochondrial dysfunction and the oxidative damage associated with it are scavenged through various cellular defense systems such as autophagy to prevent harmful effects. Our recent study has demonstrated that cobalt protoporphyrin IX (CoPPIX), a potent inducer of heme oxygenase-1 (HO-1), ameliorates septic liver injuries by enhancing mitochondrial autophagy in rats. In our current study, we show that CoPPIX (5 mg/kg s.c.) not only accelerates the autophagic response but also promotes lysosome reformation in the rat heart treated with LPS (15 mg/kg i.p.). Lysosomal membrane-associated protein-2 (LAMP2), which is essential to the maintenance of lysosomal functions in the heart, is depleted transiently but restored rapidly during LPS administration in the rat. Activation of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy, was also observed, indicating a hyper consumption and subsequent reformation of the lysosome to meet the increased demand for autophagosome cleaning. CoPPIX was found to promote these processes and tended to restore the LPS-induced suppression of cardiac performances whilst chloroquine (CQ; 20 mg/kg i.p.), an inhibitor of lysosomes and autophagic protein degradation, abrogates these beneficial effects. The cardioprotective effect of CoPPIX against LPS toxicity was also observed via decreased levels of cardiac releasing enzymes in the plasma. Taken together, our current data indicate that lysosome reformation mediated by TFEB may be involved in cardioprotection against LPS-induced septic insults, and serve as a novel mechanism by which CoPPIX protects the heart against oxidative stress.

Effects of interlukin-10 on lipopolysaccharide-induced liver injury in rats

Objective To investigate the effects of interlukin-10 (IL-10) on lipopolysaccharide (LPS)-induced liver injury in rats.Methods One hundred male Wistar rats,aged 10-14 weeks,weighing 250-300 g,were randomly divided into 5 groups (n =20 each):control group (C group),LPS group,IL-10 group,HO-1 inducer cobalt protoporphyrin-Ⅸ group (Co group) and HO-1 inhibitor zinc protoporphyrin-Ⅸ group (Zn group).The animals in LPS,IL-10,Co and Zn groups received intraperitoneal LPS 20 mg/kg.IL-10,Co and Zn groups received recombinant human IL-10 1 μg at 3 h before LPS injection.Co and Zn groups received cobalt protoporphyrin-Ⅸ and zinc protoporphyrin-Ⅸ 25 mg/kg at 2 h before administration of recombinant human IL-10.Ten rats in each group were chosen at 24 h after LPS injection and blood samples were collected from the heart for determination of the levels of serum alanine aminotransferase (ALT),aspartate transaminase (AST),tumor necrosis factor-α (TNF-α) and interlukin-1β (IL-1 β).The animals were then sacrificed and lungs removed for determination of glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) activities,malonodialdehyde (MDA) content and expression of HO-1 mRNA in lung tissues.The left 10 rats in each group were chosen and the survival rates within 72 h after LPS administration were recorded.Results Compared with C group,the levels of serum ALT,AST,TNF-αand IL-1 β and MDA content in lung tissues were significantly increased,and GSH-Px and SOD activities in lung tissues and survival rates were decreased in LPS,IL-10,Co and Zn groups (P ＜ 0.05).Compared with LPS group,the levels of serum ALT,AST,TNF-α,IL-1β and MDA content in lung tissues were significantly decreased,GSH-Px and SOD activities in lung tissues and survival rates were increased,and the expression of HO-1 mRNA was up-regulated (P ＜ 0.05).Compared with IL-10 group,the levels of serum ALT,AST,TNF-α and IL-1β and MDA content in lung tissues were significantly increased,and GSH-Px and SOD activities in lung tissues and survival rates were decreased in group Zn (P ＜ 0.05),and no significant change in the parameters mentioned above was found in Co group (P ＞ 0.05).Conclusion IL-10 can attenuate LPS-induced liver injury in rats by inducing the expression of HO-1. Key words: Interlukin-10 ; Endotoxemia; Liver

Abstract 648: Heme-oxygenase-1 Gene Expression Ameliorates Cardiac Dysfunction Following Myocardial Infarction In C57 Mice By Recruitment Of Angiogenic Factors And Angiogenesis In C57 Mice

Rational: Angiogenesis is essential in order to increase blood circulation in infarcted tissue of MI (Myocardial infarction). Increased Heme-Oxygenase (HO)-1 gene expression increases angiogenic proteins, e.g. VEGF, bFGF, EGF, angiopoietin and adiponectin. Objective: To investigate whether increased levels of HO-1, after the occurrence of a MI, improves angiogenesis and capillary formation in ischemic myocardium, thereby improving cardiac function. METHODS: Experimental MI was induced by LAD (Left anterior descending artery) ligation. C57BL6 mice were divided into 4 groups: Sham; MI; 5 days after MI treated with the HO-1 inducer, cobalt protoporphyrinIX (CoPP); and, CoPP in the presence of the HO activity inhibitor, Stannous Mesoporphyrin (SnMP). HO-1 downstream signaling proteins were determined including VEGF, CD31 and adiponectin. Echocardiography was performed weekly for 4 weeks after surgery. Results: 5 days after MI, CoPP treatment significantly increased VEGF (p<0.05 vs.MI), CD31 (p<0.05 vs.MI), and adiponectin levels (p<0.05 vs.MI). These findings were associated with a significant increase in capillary formation and blood flow in CoPP-treated animals (p<0.05 vs.MI). Echocardiography showed that left ventricle dilatation, measured as end diastolic area (EDA), was significantly reduced in CoPP- treated animals compared to MI groups (EDA: MI: 0.216±0.02cm2; MI+CoPP: 0.172±0.03 cm2; (-13%) p<0.01). This was associated with a significant decrease in apoptosis and fibrosis (P<0.05). These beneficial effects were reversed by SnMP administration. Conclusion: HO-1 improved cardiac function and enhanced angiogenesis via the recruitment of pro-angiogenic factors.

Abstract 220: Induction of Heme Oxygenase-1 Enhances Matrix Metalloproteinase-9 Secretion and Myocardial Fibrosis

Background: Heme oxygenase (HO)-1 is a stress-inducible rate-limiting enzyme in heme catabolism into carbon monoxide, biliverdin and free iron. Whereas HO-1 exerts cardioprotective effect, such as anti-hypertrophic and anti-apoptotic effect in cardiomyocytes, the influence of HO-1 on cardiac fibroblasts has not been clarified. We examined the effect of HO-1 induced by cobalt protoporphyrin IX (CoPPIX) on matrix metalloproteinase (MMP)-9 secretion, a biomarker of cardiac remodeling, in cardiac fibroblasts. Methods and Results: Cardiac fibroblasts were isolated from male Wistar rats. Secreted MMP-9 protein into culture medium and HO-1 expression in cell lysates were measured by Western blotting. CoPPIX (0.1-1 μ M) increased HO-1 expression and enhanced interleukin (IL)-1β (4 ng/ml, 24 h)-induced MMP-9 secretion (n=4). Mn 2 (CO) 10 (0.3-10 μ M), a carbon monoxide donor, increased IL-1β-induced MMP-9 secretion (n=4), although bilirubin (0.3-10 μ M), a metabolite of biliverdin, and FeSO 4 (0.3-10 μ M), a free iron donor, had no effect. Mn 2 (CO) 10 increased reactive oxygen species (ROS) generation and the enhanced effect of Mn 2 (CO) 10 on IL-1β-induced MMP-9 secretion was abolished by an antioxidant EUK-134 (10 μ M, n=4). We also examined the effect of CoPPIX on isoproterenol (5 mg/kg/day, subcutaneous injection, for 1 or 7 days)-induced cardiac remodeling in male Wistar rats. CoPPIX (1 mg/kg) was administered intraperitoneally every 2 days and this treatment increased HO-1 expression. Hearts were excised at the day 2 or day 8. Myocardial fibrosis was evaluated by Azan staining. At the day 8, CoPPIX worsened isoproterenol-induced myocardial fibrosis significantly (n=6), but had no effect on the increase of heart weight. CoPPIX also enhanced isoproterenol-induced MMP-9 expression at the day 2 (n=10). Conclusion: Induction of HO-1 by CoPPIX enhances IL-1β-induced MMP-9 secretion via ROS generation through heme-derived carbon monoxide in cardiac fibroblasts and isoproterenol-induced myocardial fibrosis and MMP-9 expression. These findings suggest that HO-1 induction does not necessarily exert cardioprotective effects.

Expression and function of heme oxygenase-1 in human gastric cancer

Heme oxygenase-1 (HO-1) potently influences tumor growth and metastasis. To date, no study has been performed on HO-1 expression pattern and its clinicopathological significance in human gastric cancer (GC) cases. In this study, the expression of HO-1 in human GC tissues (n = 74) and matched non-tumoral adjacent parenchyma (n = 46) was investigated by immunohistochemistry. The correlation of HO-1 with the clinicopathological characteristics was analyzed. Results showed that HO-1 was expressed in 62 GC tissues from 74 cases (83.8%), which is significantly higher than non-tumoral adjacent parenchyma (20/46, 43.8%, P < 0.05). A high HO-1 expression rate showed a close association with well/moderate histological differentiation and negative lymph node metastasis (P < 0.05). The expression of matrix metallopeptidase 9 (MMP9) and vascular endothelial growth factor A (VEGF-A) as well as chemosensitivity to cisplatin of MKN-45 cell lines with genetically altered HO-1 status were then determined by realtime polymerase chain reaction and 3-(4,5 dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), respectively. Whether the induction or inhibition of HO-1 by cobalt-protoporphyrin-IX (CoPP) or zinc-protoporphyrin-IX (ZnPP) could affect the sensitivity of MKN-45 cells to cisplatin was also studied. Results showed that the expression of MMP9 and VEGF-A were up-regulated in MKN-45 cells overexpressing HO-1, and down-regulated in HO-1 interfered cells. HO-1 overexpression could lead to an increased resistance to cisplatin, whereas down-regulation of HO-1 expression by siRNA or chemical inhibition of HO-1 could lead to increased chemosensitivity to cisplatin in MKN-45 cells. HO-1 may have multiple effects on protection against carcinogenesis and progression in GC.

Up-regulation of heme oxygenase-1 as a resistance mechanism against MPP+ induced cytotoxicity in Raw 264.7 macrophages

To elucidate the effects of heme oxygennase-1 (HO-1) on a neurotoxic substance, 1-methyl-4-phenylpyridinium (MPP+), induced cytotoxicity in immune cells, cell death, and the production of reactive oxygen species (ROS) were studied using Raw 264.7 macrophages. HO-1 is induced by harmful stimuli including oxidative stress, and it exerts a protective effect against cytotoxicity in immune cells. Using Western blotting analysis, we have evaluated the expression of HO-1 in Raw 264.7 macrophages treated with MPP+. Via a WST assay and flow cytometric analysis, we have also evaluated the cytoprotective effect of HO-1 induction against the cytotoxicity induced by exposure to MPP+. In Raw 264.7 macrophage exposed MPP+ cells, HO-1 expression evidenced a rapid increase, peaking at 12 h. The increment of cell death and ROS production due to MPP+ exposure were aggravated further by treatment with Tin protoporphyrin-IX (SnPP-IX, HO-1 inhibitor). The increased cell death and ROS production induced by MPP+ exposure were recovered to a significant degree by treatment with Cobalt protoporphyrin-IX (CoPP-IX, HO-inducer). According to these results, HO-1 induction exerts a cytoprotective effect on MPP+-induced cytotoxicity, which is related to ROS production.

Effect of cobalt on Escherichia coli metabolism and metalloporphyrin formation

Toxicity in Escherichia coli resulting from high concentrations of cobalt has been explained by competition of cobalt with iron in various metabolic processes including Fe-S cluster assembly, sulfur assimilation, production of free radicals and reduction of free thiol pool. Here we present another aspect of increased cobalt concentrations in the culture medium resulting in the production of cobalt protoporphyrin IX (CoPPIX), which was incorporated into heme proteins including membrane-bound cytochromes and an expressed human cystathionine beta-synthase (CBS). The presence of CoPPIX in cytochromes inhibited their electron transport capacity and resulted in a substantially decreased respiration. Bacterial cells adapted to the increased cobalt concentration by inducing a modified mixed acid fermentative pathway under aerobiosis. We capitalized on the ability of E. coli to insert cobalt into PPIX to carry out an expression of CoPPIX-substituted heme proteins. The level of CoPPIX-substitution increased with the number of passages of cells in a cobalt-containing medium. This approach is an inexpensive method to prepare cobalt-substituted heme proteins compared to in vitro enzyme reconstitution or in vivo replacement using metalloporphyrin heme analogs and seems to be especially suitable for complex heme proteins with an additional coenzyme, such as human CBS.