Close Relationship between Systemic Arterial and Portal Venous Pressure in an Animal Model with Healthy Liver.

It is unclear to what extent systemic arterial blood pressure influences portal pressure. This relationship is clinically important as drugs, which are conventionally used for therapy of portal hypertension, may also influence systemic arterial blood pressure. This study investigated the potential correlation between mean arterial (MAP) and portal venous pressure (PVP) in rats with healthy livers. In a rat model with healthy livers, we investigated the effect of manipulation of MAP on PVP. Interventions consisted of 0.9% NaCl (group 1), 0.1 mg/kg body weight (bw) Sildenafil (low dose), an inhibitor of phosphodiesterase-5 (group 2), and 1.0 mg/kg bw Sildenafil (high dose, group 3) in 600 µL saline injected intravenously. Norepinephrine was used to increase MAP in animals with circulatory failure while PVP was monitored. Injection of the fluids induced a transient drop in MAP and PVP, probably due to a reversible cardiac decompensation. The drop in MAP and drop in PVP are significantly correlated. The time lag between change in MAP and change in PVP by 24 s in all groups suggests a cause-and-effect relationship. Ten minutes after the injection of the fluid, cardiac function was normalized. Thereafter, MAP gradually decreased. In the NaCl group, PVP decreases by 0.485% for a 1% drop of MAP, by 0.550% in the low-dose sildenafil group, and by 0.651% in the high-dose sildenafil group (p < 0.05 for difference group two vs. group one, group three vs. group one, and group three vs. group two). These data suggest that Sildenafil has an inherent effect on portal pressure that exceeds the effect of MAP. Injection of norepinephrine led to a sudden increase in MAP followed by an increase in PVP after a time lag. These data show a close relationship between portal venous pressure and systemic arterial pressure in this animal model with healthy livers. A change in MAP is consequently followed by a change in PVP after a distinct time lag. This study, furthermore, suggests that Sildenafil influences portal pressure. Further studies should be performed in a model with cirrhotic livers, as these may be important in the evaluation of vasoactive drugs (e.g., PDE-5-inhibitors) for therapy of portal hypertension.

Carbon monoxide releasing molecule-2 inhibits pancreatic stellate cell proliferation by activating p38 mitogen-activated protein kinase/heme oxygenase-1 signaling.

Proliferation of pancreatic stellate cells (PSCs) plays a cardinal role during fibrosis development. Therefore, the suppression of PSC growth represents a therapeutic option for the treatment of pancreatic fibrosis. It has been shown that up-regulation of the enzyme heme oxygenase-1 (HO-1) could exert antiproliferative effects on PSCs, but no information is available on the possible role of carbon monoxide (CO), a catalytic byproduct of the HO metabolism, in this process. In the present study, we have examined the effect of CO releasing molecule-2 (CORM-2) liberated CO on PSC proliferation and have elucidated the mechanisms involved. Using primary rat PSCs, we found that CORM-2 inhibited PSC proliferation at nontoxic concentrations by arresting cells at the G(0)/G(1) phase of the cell cycle. This effect was associated with activation of p38 mitogen-activated protein kinase (MAPK) signaling, induction of HO-1 protein, and up-regulation of the cell cycle inhibitor p21(Waf1/Cip1). The p38 MAPK inhibitor 4-(4-flurophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)imidazole (SB203580) abolished the inhibitory effect of CORM-2 on PSC proliferation and prevented both CORM-2-induced HO-1 and p21(Waf1/Cip1) up-regulation. Treatment with tin protoporphyrin IX, an HO inhibitor, or transfection of HO-1 small interfering RNA abolished the inductive effect of CORM-2 on p21(Waf1/Cip1) and reversed the suppressive effect of CORM-2 on PSC growth. The ability of CORM-2 to induce cell cycle arrest was abrogated in p21(Waf1/Cip1)-silenced cells. Taken together, our results suggest that CORM-2 inhibits PSC proliferation by activation of the p38/HO-1 pathway. These findings may indicate a therapeutic potential of CO carriers in the treatment of pancreatic fibrosis.

Heme oxygenase-1 inhibits the proliferation of pancreatic stellate cells by repression of the extracellular signal-regulated kinase1/2 pathway.

Activation of pancreatic stellate cells (PSCs) is the key process in the development of pancreatic fibrosis, a common feature of chronic pancreatitis and pancreatic cancer. In recent studies, curcumin has been shown to inhibit PSC proliferation via an extracellular signal-regulated kinase (ERK)1/2-dependent mechanism. In addition, curcumin is a potent inducer of the cytoprotective enzyme heme oxygenase-1 (HO-1) in other cell types. Therefore, the aims of this study were to 1) characterize the effect of curcumin on HO-1 gene expression in PSCs, 2) explore whether HO-1 induction contributes to the inhibitory effect of curcumin on PSC proliferation, and 3) clarify the involvement of the mitogen-activated protein kinase (MAPK) family in this context. Cultured rat PSCs were incubated with curcumin and assessed for HO-1 up-regulation by Northern blot analysis, immunoblotting, and activity assays. The effect of HO-1 on platelet-derived growth factor (PDGF)-induced PSC proliferation and MAPK activation was determined by immunoblotting, cell proliferation assays, and cell count analyses. Curcumin induced HO-1 gene expression in PSCs in a time- and dose-dependent manner and inhibited PDGF-mediated ERK1/2 phosphorylation and PSC proliferation. These effects were blocked by treatment of PSCs with tin protoporphyrin IX, an HO inhibitor, or transfection of HO-1 small interfering RNA. Our data provide evidence that HO-1 induction contributes to the inhibitory effect of curcumin on PSC proliferation. Therefore, therapeutic up-regulation of HO-1 could represent a mode for inhibition of PSC proliferation and thus may provide a novel strategy in the prevention of pancreatic fibrosis.

Conformation-specific blockade of the integrin GPIIb/IIIa: a novel antiplatelet strategy that selectively targets activated platelets.

Platelet activation causes conformational changes of integrin GPIIb/IIIa (alpha(IIb)beta3), resulting in the exposure of its ligand-binding pocket. This provides the unique possibility to design agents that specifically block activated platelets only. We used phage display of single-chain antibody (scFv) libraries in combination with several rounds of depletion/selection to obtain human scFvs that bind specifically to the activated conformation of GPIIb/IIIa. Functional evaluation of these scFv clones revealed that fibrinogen binding to human platelets and platelet aggregation can be effectively inhibited by activation-specific scFvs. In contrast to clinically used GPIIb/IIIa blockers, which are all conformation unspecific, activation-specific GPIIb/IIIa blockers do not induce conformational changes in GPIIb/IIIa or outside-in signaling, as evaluated by ligand-induced binding-site (LIBS) exposure in flow cytometry or P-selectin expression in immunofluorescence microscopy, respectively. In contrast to the conformation-unspecific blocker abciximab, activation-specific scFvs permit cell adhesion and spreading on immobilized fibrinogen, which is mediated by nonactivated GPIIb/IIIa. Mutagenesis studies and computer modeling indicate that exclusive binding of activation-specific scFv is mediated by RXD motifs in the heavy-chain complementary-determining region (CDR) 3 of the antibodies, which in comparison with other antibodies forms an exceptionally extended loop. In vivo experiments in a ferric-chloride thrombosis model of the mouse carotid artery demonstrate similar antithrombotic potency of activation-specific scFv, when compared with the conformation-unspecific blockers tirofiban and eptifibatide. However, in contrast to tirofiban and eptifibatide, bleeding times are not prolonged with the activation-specific scFvs, suggesting lower bleeding risks. In conclusion, activation-specific GPIIb/IIIa blockade via human single-chain antibodies represents a promising novel strategy for antiplatelet therapy.

Targeting ligand-induced binding sites on GPIIb/IIIa via single-chain antibody allows effective anticoagulation without bleeding time prolongation.

OBJECTIVE: Therapeutic anticoagulation is widely used, but limitations in efficacy and bleeding complications cause an ongoing search for new agents. However, with new agents developed it seems to be an inherent problem that increased efficiency is accompanied by an increase in bleeding complications. We investigate whether targeting of anticoagulants to activated platelets provides a means to overcome this association of potency and bleeding. METHODS AND RESULTS: Ligand-induced binding sites (LIBS) on fibrinogen/fibrin-binding GPIIb/IIIa represent an abundant clot-specific target. We cloned an anti-LIBS single-chain antibody (scFv(anti-LIBS)) and genetically fused it with a potent, direct factor Xa (fXa) inhibitor, tick anticoagulant peptide (TAP). Specific antibody binding of fusion molecule scFv(anti-LIBS)-TAP was proven in flow cytometry; anti-fXa activity was demonstrated in chromogenic assays. In vivo anticoagulative efficiency was determined by Doppler-flow in a ferric chloride-induced carotid artery thrombosis model in mice. ScFv(anti-LIBS)-TAP prolonged occlusion time comparable to enoxaparine, recombinant TAP, and nontargeted mutant-scFv-TAP. ScFv(anti-LIBS)-TAP revealed antithrombotic effects at low doses at which the nontargeted mutant-scFv-TAP failed. In contrast to the other anticoagulants tested, bleeding times were not prolonged by scFv(anti-LIBS)-TAP. CONCLUSIONS: The novel clot-targeting approach of anticoagulants via single-chain antibody directed against a LIBS-epitope on GPIIb/IIIa promises effective anticoagulation with reduced bleeding risk.

Analysis of the nitric oxide-cyclic guanosine monophosphate pathway in experimental liver cirrhosis suggests phosphodiesterase-5 as potential target to treat portal hypertension.

AIM: To investigate the potential effect of inhibitors of phosphodiesterase-5 (PDE-5) for therapy of portal hypertension in liver cirrhosis. METHODS: In the rat model of thioacetamide-induced liver fibrosis/cirrhosis the nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathway was investigated. Expression and localization of PDE-5, the enzyme that converts vasodilating cGMP into inactive 5'-GMP, was in the focus of the study. Hepatic gene expression of key components of the NO-cGMP pathway was determined by qRT-PCR: Endothelial NO synthase (eNOS), inducible NO synthase (iNOS), soluble guanylate cyclase subunits α1 and ß1 (sGCa1, sGCb1), and PDE-5. Hepatic PDE-5 protein expression and localization were detected by immunohistochemistry. Serum cGMP concentrations were measured using ELISA. Acute effects of the PDE-5 inhibitor Sildenafil (0.1 mg/kg or 1.0 mg/kg) on portal and systemic hemodynamics were investigated using pressure transducers. RESULTS: Hepatic gene expression of eNOS (2.2-fold; P = 0.003), sGCa1 (1.7-fold; P = 0.003), sGCb1 (3.0-fold; P = 0.003), and PDE-5 (11-fold; P = 0.003) was increased in cirrhotic livers compared to healthy livers. Overexpression of PDE-5 (7.7-fold; P = 0.006) was less pronounced in fibrotic livers. iNOS expression was only detected in fibrotic and cirrhotic livers. In healthy liver, PDE-5 protein was localized primarily in zone 3 hepatocytes and to a lesser extent in perisinusoidal cells. This zonation was disturbed in cirrhosis: PDE-5 protein expression in perisinusoidal cells was induced approximately 8-fold. In addition, PDE-5-expressing cells were also found in fibrous septa. Serum cGMP concentrations were reduced in rats with cirrhotic livers by approximately 40%. Inhibition of PDE-5 by Sildenafil caused a significant increase in serum cGMP concentrations [+ 64% in healthy rats (P = 0.024), + 85% in cirrhotic rats (P = 0.018)]. Concomitantly, the portal venous pressure was reduced by 19% in rats with liver cirrhosis. CONCLUSION: Overexpression and abrogated zonation of PDE-5 likely contribute to the pathogenesis of cirrhotic portal hypertension. PDE-5 inhibition may therefore be a reasonable therapeutic approach for portal hypertension.

Carbon monoxide releasing molecule-2 CORM-2 represses global protein synthesis by inhibition of eukaryotic elongation factor eEF2.

Carbon monoxide (CO) is an endogenous gaseous transmitter that exerts antiproliferative effects in many cell types, but effects of CO on the translational machinery are not described. We examined the effects of the carbon monoxide releasing molecule-2 (CORM-2) on critical steps in translational signaling and global protein synthesis in pancreatic stellate cells (PSCs), the most prominent collagen-producing cells in the pancreas, whose activation is associated with pancreatic fibrosis. PSCs were isolated from rat pancreatic tissue and incubated with CORM-2. CORM-2 prevented the decrease in the phosphorylation of eukaryotic elongation factor 2 (eEF2) caused by serum. By contrast, the activation dependent phosphorylation of initiation factor 4E-binding protein 1 (4E-BP1) was inhibited by CORM-2 treatment. The phosphorylation of eukaryotic initiation factor 2α (eIF2α) and eukaryotic initiation factor 4E (eIF4E) were not affected by CORM-2 treatment. In consequence, CORM-2 mediated eEF2 phosphorylation and inactivation of 4E-BP1 suppressed global protein synthesis. These observations were associated with inhibition of phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin (PI3K-Akt-mTOR) signaling and increased intracellular calcium and cAMP levels. The CORM-2 mediated inhibition of protein synthesis resulted in downregulation of cyclin D1 and cyclin E expression, a subsequent decline in the phosphorylation of the retinoblastoma tumor suppressor protein (Rb) and cell growth arrest at the G(0)/G(1) phase checkpoint of the cell cycle. Our results suggest the therapeutic application of CO releasing molecules such as CORM-2 for the treatment of fibrosis, inflammation, cancer, or other pathologic states associated with excessive protein synthesis or hyperproliferation. However, prolonged exogenous application of CO might also have negative effects on cellular protein homeostasis.

Effects of hydrogen sulfide on rat pancreatic stellate cells.

OBJECTIVES: Pancreatic stellate cells (PSCs) play a crucial role during pancreatic fibrosis development. Hydrogen sulfide (H2S) is a recently discovered gaseous transmitter, whose role in PSCs has not been explored yet. In the present study, we examined the effects of sodium hydrosulfide (NaHS), an H2S donor, on rat PSCs and elucidated the mechanisms involved. METHODS: Primary PSCs were isolated from rat pancreatic tissue. Lactate dehydrogenase and caspase assays were performed to detect cell death. Pancreatic stellate cell proliferation was determined by cell count analyses, bromodeoxyuridine incorporation, and flow cytometry. The role of heme oxygenase-1 (HO-1) was assessed by pharmacological HO inhibition and transfection of HO-1 small interfering RNA. Pancreatic stellate cell migration was determined by a wound healing assay, and PSC contraction was assessed by a gel contraction assay. α-Smooth muscle actin, collagen type I, and fibronectin messenger RNAs were analyzed by real-time polymerase chain reaction. RESULTS: NaHS inhibited PSC proliferation at nontoxic concentrations. This was associated with HO-1-mediated repression of extracellular signal-regulated kinase 1/2 signaling. NaHS suppressed PSC migration and activation as well as extracellular matrix synthesis. CONCLUSIONS: The results of the present study indicate that NaHS inhibits key cell functions of PSCs. Administration of H(2)S-releasing compounds might represent a novel strategy in the treatment of pancreatic fibrosis.

Hepatic steatosis prevents heme oxygenase-1 induction by isoflurane in the rat liver.

AIM: To characterize the inductive effects of isoflurane (ISO) on hepatic heme oxygenase-1 (HO-1) in an animal model of hepatic steatosis. METHODS: Lean (LEAN) and obese (FAT) Zucker rats were randomized into 4 groups: 1: LEAN + pentobarbital sodium (PEN); 2: LEAN + ISO; 3: FAT + PEN; 4: FAT + ISO. The animals were mechanically ventilated for 6 h. In vitro analyses of liver tissue included determination of HO-1 mRNA and protein expression as well as measurement of HO enzyme activity and immunohistochemical analyses. RESULTS: Compared to PEN treatment, ISO administration profoundly induced hepatic HO-1 mRNA and protein expression and significantly increased HO enzyme activity in lean Zucker rats. In contrast, no difference in HO-1 gene expression was observed after ISO or PEN anesthesia in obese Zucker rats. CONCLUSION: The present study demonstrates that ISO is an inducer of hepatic HO-1 gene expression in non-steatotic organs but failed to upregulate HO-1 in steatotic livers.

p38ß-regulated induction of the heat shock response by carbon monoxide releasing molecule CORM-2 mediates cytoprotection in lung cells in vitro.

The carbon monoxide releasing molecule tricarbonyldichlororuthenium (CORM-2) displays protective actions like carbon monoxide. The molecular mechanism underlying this effect remains controversial. We hypothesized that CORM-2 mediates cytoprotection via induction of heat shock proteins through activation of p38 mitogen-activated kinase. Embryonic bovine lung cells were incubated with CORM-2. Apoptosis was induced by staurosporine and analyzed by flow cytometry following annexin-V staining, caspase-3 activity assay, and by Western Blot for caspase-3 cleavage. Heat shock response was assessed by DNA-binding activity of heat shock factor 1 and by reporter gene activity. Cells were transfected with siRNA targeting p38 isoforms. Data were analyzed with ANOVA and post-hoc Holm-Sidak test. CORM-2 inhibited staurosporine-induced apoptosis (% annexin-V positive cells: staurosporine = 60 ± 4% vs. CORM-2 10 µM = 48 ± 4%, CORM-2 25 µM=42 ± 5%, CORM-2 50 µM = 40 ± 4% and CORM-2 100 µM = 38 ± 2%, mean ± S.D., P<0.001; caspase-3 activity: staurosporine=92 ± 15 RFUs vs. CORM-2 50 µM=60 ± 14 RFUs, mean ± S.D. P<0.001). CORM-2 induced phosphorylation of p38 MAPK, but not of JNK and ERK1/2. CORM-2 induced DNA-binding of heat shock factor 1 and elicited a 4-fold induction of gene activity (P<0.05). Incubation with the Hsp inhibitors KNK437 attenuated and 17-AAG abolished the anti-apoptotic effect of CORM-2 (P<0.001). p38 inhibition and silencing of p38ß attenuated the anti-apoptotic effect of CORM-2 (P<0.05), most likely by abolishing CORM-2-induced HSF-1 binding activity. These findings suggest that CORM-2-mediated cytoprotection is caused by induction of the heat shock response and by p38 activation. Furthermore, the p38ß isoform activation may represent an upstream mechanism of heat shock response induction.

Up-regulation of heme oxygenase-1 by sevoflurane is not dependent on Kupffer cells and associates with ERK1/2 and AP-1 activation in the rat liver.

Sevoflurane is a potent non-toxic inducer of the hepatoprotective enzyme heme oxygenase-1 (HO-1). So far, little is known about the underlying molecular mechanism. Therefore the aim of this study was to characterize the respective signal transduction pathway and in particular to elucidate the role of Kupffer cells in this context. Rats were treated with or without sevoflurane. The effects on hepatic HO-1 gene expression, mitogen-activated protein kinases and transcription factors were studied by Northern and Western blot analyses, immunostaining, electrophoretic mobility shift assays, and enzymatic activity assays. Kupffer cells were depleted by administration of clodronate liposomes in vivo to characterize their role in HO-1 signal transduction. In additional in vitro experiments, HO-1 mRNA expression in primary rat hepatocytes and HepG2 cells was assessed. Sevoflurane up-regulated HO-1 gene expression in pericentral hepatocytes and increased HO enzyme activity in vivo. This was associated with activation of ERK1/2 and activator protein-1. We identified c-jun/AP-1, JunD, c-fos, and Fra-1 as active subunits of the activator protein-1 complex. Administration of clodronate liposomes to rats led to depletion of Kupffer cells without affecting sevoflurane induced HO-1 expression. Moreover, sevoflurane up-regulated HO-1 mRNA in primary rat hepatocytes but not in HepG2 cells. Our results suggest that sevoflurane induced HO-1 gene expression in pericentral hepatocytes does not depend on Kupffer cells and is associated with activation of ERK1/2 and activator protein-1. Since we could recently demonstrate significant hepatoprotective effects of HO-1 induced by isoflurane, the present results may help to establish new concepts in hepatic organ protection.