Nitric oxide-mediated cytoprotection of hepatocytes from glucose deprivation-induced cytotoxicity: involvement of heme oxygenase-1

Heme oxygenase-1 (HO-1) is the rate-limiting enzyme in heme catabolism, which leads to the generation of carbon monoxide (CO), biliverdin, and free iron. One of 3 mammalian HO isoforms, HO-1, is a stress-responsive protein and known to modulate such cellular functions as cytokine production, cell proliferation, and apoptosis to protect organs and tissues from acute injury. Although nitric oxide (NO)-mediated cytoprotective effects against cytotoxicity induced by glucose deprivation have been well recognized, the underlying mechanisms remain to be elucidated. Thus, we investigate the involvement of HO-1 in the cytoprotective effects of NO. Deprivation of glucose markedly reduced the viability of BNL CL.2 cells and primary rat hepatocytes. Pretreatment with NO donor, sodium nitroprusside (SNP), protected hepatocytes from glucose deprivation-induced cytotoxicity; zinc protoporphyrin (ZnPP) IX, an inhibitor of HO, was found to block the SNP-induced cytoprotection. SNP increased the induction of HO-1 protein as well as its activity in hepatocytes. A cytoprotective effect comparable to SNP was observed when the cells were transfected with HO-1 gene or preincubated with another HO-1 inducer, hemin. Additional experiments revealed the involvement of CO in the cytoprotective effect of SNP/HO-1 in BNL CL.2 cells. CO mediated cytoprotective effect through suppression of ERK MAPK activation. In conclusion, our results show that SNP protects hepatocytes from glucose deprivation-induced cytotoxicity through up-regulation of HO-1. Thus, HO-1 might be an important cellular target of NO donor with clinical implications for the prevention of acute liver injury in several pathological conditions.     

Increased vascular heme oxygenase-1 expression contributes to arterial vasodilation in experimental cirrhosis in rats

Vascular heme oxygenase (HO) regulates vascular tone in normal conditions and in some pathologic circumstances (e.g., sepsis). However, its possible role in the pathogenesis of arterial vasodilation in cirrhosis is unknown. To address this question, the expression and activity of HO in arterial vessels was studied in rats at 1, 2, and 4 weeks after bile duct ligation (BDL) or sham operation. A progressively increased expression of HO-1 was found in aorta and mesenteric arteries of BDL rats in a close chronologic relationship with the progression from acute cholestatic liver injury (1 week) to the fully developed cirrhosis with intense systemic arterial vasodilation (4 weeks). No changes were found in the expression of the constitutive isoform HO-2. HO-1 was mainly located in vascular smooth muscle cells of the arterial wall. Aortic HO activity increased in parallel with the expression of HO-1 (up to 600% in rats with cirrhosis compared with sham rats) and correlated with hemodynamic parameters. Increased expression of HO-1 and HO activity were also found in other organs, such as liver and spleen, though to a lesser extent compared with vascular tissue. The acute administration of an inhibitor of HO to cirrhotic rats, at a dose that normalized aortic HO activity, was associated with significantly greater effects on arterial pressure, total peripheral vascular resistance, and cardiac index, compared with effects in sham rats. In conclusion, these findings are consistent with a role for HO in the pathogenesis of arterial vasodilation in cirrhosis.     

Nitric oxide synthase and heme oxygenase expressions in human liver cirrhosis

AIM:Portal hypertension is a common complication ofliver cirrhosis.Intrahepatic pressure can be elevatedin several ways.Abnormal architecture affectingthe vasculature,an increase in vasoconstrictors andincreased circulation from the splanchnic viscera intothe portal system may all contribute.It follows thatendogenous vasodilators may be able to alleviate thehypertension.We therefore aimed to investigate thelevels of endogenous vasodilators,nitric oxide(NO)andcarbon monoxide(CO)through the expression of nitricoxide synthase(NOS)and heme oxygenase(HO).METHOD:Cirrhotic(n=20)and non-cirrhotic(n=20)livers were obtained from patients whohad undergone surgery.The mRNA and proteinexpressions of the various isoforms of NOS and HOwere examined using competitive PCR,Western Blot andimmunohistochemistry.RESULTS:There was no significant change in eitherinducible NOS(iNOS)or neuronal NOS(nNOS)expressions while endothelial NOS(eNOS)was up-regulated in cirrhotic livers.Concomitantly,caveolin-1,anestablished down-regulator of eNOS,was up-regulated.Inducible HO-1 and constitutive HO-2 were found toshow increased expression in cirrhotic livers albeit indifferent localizations.CONCLUSION:The differences of NOS expressionmight be due to their differing roles in maintaining liverhomeostasis and/or involvement in the pathology ofcirrhosis.Sheer stress within the hypertensive liver mayinduce increased expression of eNOS.In turn,caveolin-1 is also increased.Whether this serves as a defensemechanism against further cirrhosis or is a consequenceof cirrhosis,is yet unknown.The elevated expressionof HO-1 and HO-2 suggest that CO may compensatein its role as a vasodilator albeit weakly.It is possiblethat CO and NO have parallel or coordinated functionswithin the liver and may work antagonistically in thepathophysiology of portal hypertension.     

Altered expression of heme oxygenase-1 in the livers of patients with portal hypertensive diseases

This study was designed to determine changes in expression of heme oxygenase (HO)-1, the stress-inducible and carbon monoxide-producing enzyme, in normotensive and portal hypertensive human livers. GTS-1, a monoclonal antibody against rat HO-1 cross-reacted with the human HO-1 and blocked its enzyme activity, allowing us to examine the activity and localization of HO-1. In controls, approximately 50% of the total HO activity was from HO-1 as judged by the sensitivity to GTS-1, while the rest of activity was from other isozymes such as HO-2. HO-1 was expressed mainly in a subpopulation of Kupffer cells, and the expression in hepatic stellate cells, sinusoidal endothelial cells, and hepatocytes was little, if any. The HO-1 expression exhibited quite different pictures in the livers of portal hypertensive diseases. In cirrhotic livers, which undergo portal hypertension through increases in intrasinusoidal resistance and regenerative changes in the parenchyma, HO-1 occurred in a majority of Kupffer cells and was also observed in hepatocytes. Consequently, the total HO-1 activities became significantly greater in these tissues than those from normal individuals. By contrast, livers of idiopathic portal hypertension that are characterized by an increase in presinusoidal resistance displayed a significant decrease in the HO-1 expression in Kupffer cells, and its hepatocellular expression was not detectable. Although factors involved in altered HO-1 expression in these cells remain unknown, the results suggest that Kupffer cells could alter their expression of HO-1 in response to local hemodynamic changes associated with chronic portal hypertension in humans.     

实验性肝损伤大鼠肝脏HO-1的表达及CO水平变化

目的研究急性肝损伤时大鼠肝脏HO-1的表达情况和CO水平,探讨HO-1和内源性CO在大鼠急性肝损伤中的作用.方法制备急性四氯化碳肝损伤模型,采用RT-PCR和免疫组化法测定不同时间点大鼠肝脏HO-1 mRNA和蛋白的表达情况;测定各时间点肝组织SOD、MDA含量变化,同时测定股静脉血中HbCO水平和ALT、AST肝功能指标.结果HO-1mRNA在正常大鼠有弱表达,染毒3 h后表达显著增强,于24 h时间点表达最强,与对照组相比差异非常显著(P＜0.01);免疫组化结果显示;HO-1蛋白在正常大鼠表达较低或无,染毒3h后即有明显表达,16至48h的时间点内表达均显著增强,主要定位于肝实质细胞、库普细胞的胞浆内.对照组HbCO水平极低,给予四氯化碳3 h后HbCO水平开始升高,此后各时间点均明显高于对照组,差异有显著性,这与HO-1表达情况相一致.此外,染毒后大鼠血清ALT、AST和MDA明显升高,SOD活性则显著降低,和对照组相比差异均十分显著.结论大鼠急性肝损伤后出现HO-1表达持续上调和血中CO水平迅速增高,提示HO/CO系统参与急性肝损伤的病理生理过程,其表达增加可能对机体有重要调节作用.     

Kupffer-cell specific induction of heme oxygenase 1 (hsp32) by the atrial natriuretic peptide--role of cGMP

BACKGROUND/AIMS: Pretreatment with atrial natriuretic peptide (ANP) attenuates ischemia-reperfusion injury of livers via cGMP. Heme oxygenase-1 (HO-1) is known as a protective mediator in ischemia-reperfusion injury. The aim of this study was to investigate whether ANP affects the expression of HO-1. METHODS: Rat livers were perfused with KH-buffer with/without ANP or 8-Br-cGMP, kept in UW solution (4 degrees C, 24 h), and reperfused. HO-1 mRNA and protein was determined by Northern and Western blot, in situ hybridization, and immunohistochemistry in livers or isolated liver cells. RESULTS: ANP significantly elevated HO-1 mRNA expression at the end of the preconditioning period and was without effects at the end of ischemia and during reperfusion. 8-Br-cGMP did not affect HO-1 mRNA expression. In situ hybridization as well as immunohistological double-staining revealed that Kupffer cells but not hepatocytes showed HO-1 mRNA and protein expression. Hepatocytes revealed no changes in HO-1 protein whereas Kupffer cells showed a marked increase in HO-1 protein after ANP treatment. Inhibition of HO-1 did not abrogate hepatoprotection conveyed by ANP. CONCLUSION: Our data show the potency of ANP to specifically induce HO-1 in Kupffer cells independently of cGMP. This increased expression of HO-1 is not involved in hepatoprotection conferred by ANP being in line with the knowledge that ANP mediates hepatoprotection via cGMP.     

Identification of heme oxygenase in human endometrium

The aim of the present study was to investigate the presence of heme oxygenase (HO)-1 and HO-2 in human endometrium at various stages of the menstrual cycle using RT-PCR, Western blotting, and immunohistochemistry. RT-PCR detected mRNA for HO-1 and HO-2 in human endometrium at all stages of the menstrual cycle. Western blotting also revealed the expression of the two distinct HO proteins throughout the menstrual cycle. HO-1 was constitutively expressed, whereas HO-2 expression was apparently greater in the secretory phase than in the menstrual and proliferative phases. Immunohistochemistry showed that distribution of the two HO isoforms had distinct topographic patterns: HO-1 was observed in endometrial epithelial cells and macrophages, whereas HO-2 was found in endothelial cells and smooth muscle cells of blood vessels in the endometrium. The detection of mRNA and protein for HO-1 and HO-2 in normal human endometrium suggests that the carbon monoxide/HO system may play a role in the local control of endometrial function.     

Induction of anaphylatoxin C5a receptors in rat hepatocytes by lipopolysaccharide in vivo: mediation by interleukin-6 from Kupffer cells

BACKGROUND & AIMS: In normal rat liver, anaphylatoxin C5a induces glucose output from hepatocytes indirectly via prostanoids released from Kupffer cells. Correspondingly, it was found that hepatocytes, in contrast to Kupffer cells, did not express C5a receptors. Lipopolysaccharide (LPS) has been reported to enhance C5a receptor expression in murine livers. This might be the result of de novo expression in hepatocytes. METHODS: C5a receptor expression was investigated in hepatocytes after in vivo treatment of rats with LPS and in vitro stimulation of isolated cells with LPS and proinflammatory cytokines on messenger RNA (mRNA) and protein level, and functionally in isolated hepatocytes and perfused liver. RESULTS: In vivo treatment of rats with LPS induced C5a receptor mRNA and protein in hepatocytes with a maximum after 8-10 hours. At this time-point, C5a directly activated glycogen phosphorylase in isolated hepatocytes and enhanced glucose output in perfused livers without the involvement of prostanoids. LPS failed to induce C5a receptors in cultured hepatocytes in vitro, whereas interleukin (IL) 6 and IL-1beta, which are known to be released from Kupffer cells on stimulation with LPS, did so. In cocultures of hepatocytes with Kupffer cells, LPS induced C5a receptors in hepatocytes in an IL-6-dependent manner. CONCLUSIONS: Thus, IL-6 from Kupffer cells appears to be the main mediator of LPS-induced de novo expression of C5a receptors in hepatocytes.     

Adrenocorticotropin induces heme oxygenase-1 expression in adrenal cells

Heme oxygenase (HO) catalyzes the first and rate-controlling step of heme catabolism into biliverdin, iron and carbon monoxide. Three isoforms of HO have been identified so far: the inducible HO-1 and the constitutive HO-2 and HO-3. Both HO-1 and HO-2 were expressed in zona fasciculata (ZF) adrenal cells and in a mouse adrenocortical cell line (Y1). HO-1 but not HO-2 expression was upregulated by adrenocorticotropic hormone (ACTH) and accumulation of HO-1 protein correlated with an increase in HO activity in Y1 cells. ACTH induced HO-1 expression in a time- and dose-dependent manner with a maximum after 5 h of treatment and a threshold concentration of 0.1 mIU/ml. Actinomycin D and cycloheximide completely blocked the effect of ACTH on HO-1 mRNA expression whereas mRNA stability was not affected by ACTH. Permeable analogs of cAMP mimicked the effect of ACTH on HO-1 expression and ACTH induction was prevented by the protein kinase A (PKA) inhibitor H89. Steroid production was significantly increased when both HO-1 and HO-2 activities were inhibited by Sn-protoporphyrin IX (SnPPIX). The lipid peroxidation and increase in carbonyl content triggered by hydrogen peroxide was prevented by treatment of Y1 cells with bilirubin and ACTH.     

Analysis of heme oxygenase isomers in rat

AIM: To purify and identify heme oxygenase (HO) isomers which exist in rat liver, spleen and brain treated with hematin and phenylhydrazine and in untreated rat liver and to investigate the characteristics of HO isomers, to isolate and confirm the rat HO-1 cDNA that actually encodes HO-1 by expressing cDNA in monkey kidney cells (COS-1 cells), to prepare the rat heme oxygenase-1 (HO-1) mutant and to detect inhibition of HO-1 mutated enzyme. METHODS: First, rat liver, spleen and brain microsomal fractions were purified by DEAE-Sephacel and hydroxylapatite. The characteristics including activity, immunity and inducibility of two isomers (HO-1 and HO-2), and their apparent molecular weight were measured by detecting enzymatic activities, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis, respectively. Second, plasmid pcDNA3HO1 containing native rat HO-1 cDNA and pcDNA3HO1D25 carrying mutated rat HO-1 cDNA (His25Ala) were constructed by site-directed mutagenesis. COS-1 cells transfected with pcDNA3HO1 and pcDNA3HO1D25 were collected and disrupted by sonication, the microsomes were prepared by ultracentrifugation. Third, the inhibition of rat HO-1 mutant was analyzed. RESULTS: Two isomers were purified and identified in treated rat liver, spleen, brain and untreated rat liver. HO-1 was the predominant form with a ratio of 2.0:1 and 3.2:1 of HO-1 and HO-2 in liver and spleen, respectively, but only the activity of HO-2 in the brain and untreated liver could be detected. The apparent molecular weights of HO-1 and HO-2 were about M(r)30 000 and M(r) 36 000 under reducing conditions, respectively. The antiserum against liver HO-2 was employed in Western blotting analysis, the reactivity of HO-1 in the liver was not observed. The plasmid pcDNA3HO1 was highly expressed in endoplasmic reticulum of transfected COS-1 cells. The specific activity was -5-fold higher than that of the control. However, the enzyme activity of mutated HO-1 declined. While an equal amount of mutant was added to the enzyme reaction system, the levels of bilirubin decreased 42 %. CONCLUSION: The studies suggest that HO-1 and HO-2 exist in the hematin and phenylhydrazine treated rat liver and spleen, but only HO-2 in the brain and untreated liver. Two constitutive forms are different in molecular weight, inducibility and immunochemical properties. The activity of expressed HO-1 in COS-1 cells is higher than that of purified enzyme from rat spleen tissue. It suggests that this clone has an insert of 1030 base-pairs encodes HO-1. His25Ala mutant reduced the formation of bilirubin and it suggests that the mutant could completely bind the heme with native enzyme.     

Heme oxygenase-1 protects donor livers from ischemia/reperfusion injury:The role of Kupffer cells

AIM:To examine whether heme oxygenase (HO)-1 overexpression would exert direct or indirect effects on Kupffer cells activation, which lead to aggravation of reperfusion injury.METHODS: Donors were pretreated with cobalt protoporphyrin (CoPP) or zinc protoporphyrin (ZnPP), HO-1 inducer and antagonist, respectively. Livers were stored at 4℃ for 24 h before transplantation. Kupffer cells were isolated and cultured for 6 h after liver reperfusion.RESULTS: Postoperatively, serum transaminases were significantly ...     

The mechanisms of hepatic sinusoidal endothelial cell regeneration: A possible communication system associated with vascular endothelial growth factor in liver cells

Vascular endothelial growth factor (VEGF) has been shown to induce proliferation of sinusoidal endothelial cells in primary culture. To elucidate the mechanisms of sinusoidal endothelial cell regeneration in vivo, mRNA expression of VEGF and its receptors, flt-1 and KDR/flk-1, were studied in rat livers. Northern blot analysis revealed that VEGF-mRNA was expressed in hepatocytes immediately after isolation from normal rats. In contrast, non-parenchymal cells, including sinusoidal endothelial cells, expressed VEGF receptor-mRNA. Vascular endothelial growth factor-mRNA expression in hepatocytes was decreased during primary culture, but increased following a peak of DNA synthesis, induced by addition of epidermal growth factor or hepatocyte growth factor to the culture medium at 24 h of plating. In a 70% resected rat liver, VEGF-mRNA expression increased with a peak at 72 h after the operation, and mRNA expression of VEGF receptors between 72 and 168 h. In such a liver, mitosis was maximal in hepatocytes at 36 h and in sinusoidal endothelial cells at 96 h. Also, mRNA expression of both VEGF and its receptors was significantly increased in carbon tetrachloride-intoxicated rat liver compared with normal rat liver. Vascular endothelial growth factor expression was minimal in Kupffer cells isolated from normal rats, but marked in activated Kupffer cells and hepatic macrophages from the intoxicated rats. Vascular endothelial growth factor-mRNA expression was also increased in activated stellate cells from these rats and in the cells activated during primary culture compared with quiescent cells. We conclude that increased levels of VEGF expression in regenerating hepatocytes may contribute to the proliferation of sinusoidal endothelial cells in partially resected rat liver, probably through VEGF receptors up-regulated on the cells. Also, VEGF derived from activated Kupffer cells, hepatic macrophages and stellate cells may be involved in this proliferation in injured rat liver.     

Age alters expression and inducibility of heme oxygenase isozymes in mice

Heme oxygenase (HO) performs the rate limiting step in heme degradation and is induced by cell injury or stress. We wished to determine if dietary fatty acid composition, increased age and/or an induced oxidative stress would alter the expression of HO-1 (constitutive and inducible isozyme) or of HO-2 (constitutive isozyme), in mouse liver, spleen and brain. Six-and 24-month-old male B6C3F1 mice were fed AIN-76A diets containing either 5% corn oil (CO, moderately unsaturated, n=5 per age group) or 19% menhaden fish oil plus 1% corn oil (FO, highly polyunsaturated, n=20 per age group). After 2 weeks, 5 CO and 5 FO fed mice in each age group were sacrificed. The remaining FO diet mice (n=15 per age group) were then challenged with a systemic oxidative stress by intraperitoneal injection of 125 mg iron/kg body weight as iron dextran. Five stressed mice from each age group were sacrificed 1, 5, and 24 hours post injection; liver, spleen and brain were removed. Part of each tissue was fixed in formalin, and microsomal protein isolated from the remaining tissue. HO-1 and HO-2 were detected by immunoblot of microsomal protein and by immunohistochemical staining of fixed tissue in the liver and spleen, but only HO-2 was detected in the brain. There was no significant difference in HO-1 or HO-2 expression due to diet. The liver of old unstressed mice had significantly more HO-1 than young mice. However, HO-1 was significantly induced in the livers of young mice, but not of old mice, following oxidative stress. Spleen HO-1 expression was not significantly altered by age or oxidative stress. HO-2 expression was not significantly altered by age or induced oxidative stress in any tissue examined. Age-related alterations in liver HO-1 isozyme expression and inducibility may contribute to increased susceptibility to exogenous stress and disease.     

Expression of nitric oxide synthase protein and gene in the splanchnic organsof liver cirrhosis and portal hypertensive rats

AIM To investigate the expression of endothelial NO synthase (eNOS), inducible NO synthase (iNOS)protein and eNOS mRNA gene in the splanchnic organs of liver cirrhosis and portal hypertensive rats.METHODS In control and CCl4-induced liver cirrhotic rats, the expression of eNOS and iNOS proteins wasdetected by immunohistochemical method, and eNOS mRNA was detected by in situ hybridization.RESULTS The expression of eNOS protein and eNOS mRNA increased in most organs of the cirrhotic rats,including bronchial and alveolar epithelial cells, renal tubular epithelial cells and mesenchyma, endothelialand adventitial cells of aorta and superior mesenteric artery, whereas no significant increase of iNOS proteinwas found. In the hepatic tissue, NOS protein and eNOS mRNA were present in mesenchymal cells and vesseladventitial cells, no difference was observed in the expression between control and cirrhotic rats.CONCLUSION The expression of NOS varied in region. In splanchnic organs and vasculars there was anincreased expression of eNOS which induced aplanchnic vasodilation and increased the inflow of portal vein,while in the liver tissue and blood vessel showed no increased expression, which may be associated withincreased intrahepatic vascular resistance.     

Carbon Monoxide as a Guardian against Hepatobiliary Dysfunction

Abstract : Carbon monoxide (CO) generated through the reaction of heme oxygenase (HO) has attracted great interest in regulation of hepatobiliary homeostasis. The gas generated by HO‐2 in the hepatic parenchyma can modestly activate soluble guanylate cyclase (sGC) expressed in hepatic stellate cells in a paracrine manner and thereby constitutively relax sinusoids. Kupffer cells express HO‐1, the inducible isozyme, even under normal unstimulated conditions and constitutes approximately 30% of the total HO activity in this organ. Upon exposure to a variety of stressors such as cytokines, endotoxin, hypoxia and oxidative stress, the liver induces HO‐1 and overproduces CO. The stress‐inducible CO has been shown to guarantee ample blood supply during detoxification of heme and thus to play a protective role in the liver. However, molecular mechanisms by which CO serves as a protectant for hepatocytes, the cells expressing little sGC, remain to be solved. Previous observation suggested that CO modulates intracellular calcium mobilization through inhibiting cytochrome P‐450 activities and thereby maintain stroke volume of bile canalicular contraction in cultured hepatocytes. CO also stimulates mrp2‐dependent excretion of bilirubin‐IXα and helps heme catabolism. Although a direct molecular target responsible for the latter event remains unknown, such properties of CO could support xenobiotic metabolism through its actions on sinusoidal hemodynamics and hepatobiliary systems.