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Rolf Graf

Publications and source records attributed to Rolf Graf.

At least 19 recordsLinked to original sources
PubMed2006

Caerulein-induced acute pancreatitis in mice that constitutively overexpress Reg/PAP genes.

BACKGROUND: The cystic fibrosis (CF) mouse pancreas has constitutively elevated expression of the Reg/PAP cell stress genes (60-fold greater Reg3alpha, and 10-fold greater PAP/Reg3beta and Reg3gamma). These genes are suggested to be involved in protection or recovery from pancreatic injury. METHODS: To test this idea the supramaximal caerulein model was used to induce acute pancreatitis in wild type and CF mice. Serum amylase, pancreatic water content (as a measure of edema), pancreatic myeloperoxidase activity, and Reg/PAP expression were quantified. RESULTS: In both wild type and CF mice caerulein induced similar elevations in serum amylase (maximal at 12 h), pancreatic edema (maximal at 7 h), and pancreatic myeloperoxidase activity (MPO, a marker of neutrophil infiltration; maximal at 7 h). By immunohistochemistry, Reg3alpha was strongly expressed in the untreated CF pancreas but not in wild type. During pancreatitis, Reg3alpha was intensely expressed in foci of inflamed tissue in both wild type and CF. CONCLUSION: These data demonstrate that the severity of caerulein-induced pancreatitis is not ameliorated in the CF mouse even though the Reg/PAP stress genes are already highly upregulated. While Reg/PAP may be protective they may also have a negative effect during pancreatitis due to their anti-apoptotic activity, which has been shown to increase the severity of pancreatitis.

Animals↗
PubMed2006

Portal vein ligation and partial hepatectomy differentially influence growth of intrahepatic metastasis and liver regeneration in mice.

BACKGROUND/AIMS: Since modern treatment of liver metastases includes serial hepatectomies and portal vein obstruction, we investigated the effects of portal vein ligation (PVL) and partial hepatectomy (PH) on tumor growth. METHODS: The effects of 70% PH and PVL on liver and lung metastases were evaluated in mice. Tumor growth and liver regeneration were assessed by morphometry and immunohistochemistry for PCNA and BrdU. The effect of growth factors of liver regeneration on CT-26 cells was tested in vitro, and TGF-beta secretion of CT-26 cells was measured by ELISA. RNA synthesis of TGF-beta and activin A was measured by RT-PCR. RESULTS: Liver regeneration after PH and PVL was similar in tumor-free mice. Intrahepatic tumor growth was lower after PH than after PVL (p=0.016). Extrahepatic tumor growth was not different. In contrast to PVL, liver regeneration was delayed after PH in metastatic livers (p=0.001). Tested growth factors of liver regeneration stimulated CT-26 cells in vitro, and CT-26 secreted significant amounts of TGF-beta in vitro and in vivo. CONCLUSIONS: Although similar in tumor-free mice, liver regeneration significantly differed between PVL and PH in metastatic livers. In addition, PH and PVL differently affected intrahepatic tumor growth.

Animals↗
PubMed2006

Platelet-derived serotonin mediates liver regeneration.

The liver can regenerate its volume after major tissue loss. In a mouse model of liver regeneration, thrombocytopenia, or impaired platelet activity resulted in the failure to initiate cellular proliferation in the liver. Platelets are major carriers of serotonin in the blood. In thrombocytopenic mice, a serotonin agonist reconstituted liver proliferation. The expression of 5-HT2A and 2B subtype serotonin receptors in the liver increased after hepatectomy. Antagonists of 5-HT2A and 2B receptors inhibited liver regeneration. Liver regeneration was also blunted in mice lacking tryptophan hydroxylase 1, which is the rate-limiting enzyme for the synthesis of peripheral serotonin. This failure of regeneration was rescued by reloading serotonin-free platelets with a serotonin precursor molecule. These results suggest that platelet-derived serotonin is involved in the initiation of liver regeneration.

5-Hydroxytryptophan↗
PubMed2006

Kupffer cell-dependent TNF-alpha signaling mediates injury in the arterialized small-for-size liver transplantation in the mouse.

Implantation of small liver grafts causes liver injury and defective regeneration leading to graft failure. We investigated whether Kupffer cell-dependent TNF-alpha signaling contributes to this poor outcome. Partial 30% liver transplantation was performed in C57BL/6 wild-type mice (control group), and in three groups with down-regulation of the TNF-alpha pathway: (i) TNF receptor 1 knockout [TNFR-1(-/-)] mice, and mice pretreated with (ii) gadolinium chloride or (iii) pentoxifylline (PTX). Fifty-percent partial liver transplantation, a model associated with full recovery, and transplantation in IL-6 knockout [IL-6(-/-)] mice were performed in some experiments. Graft injury, regeneration, portal flow, liver microcirculation, leukocyte adhesion, and animal survival were assessed. Animal survival rates were 14% in the control group vs. 43% in the gadolinium chloride group, 57% for the TNFR-1(-/-) group, and 86% in the PTX group (P < 0.001). Markers of liver injury were reduced in all treated groups when compared with controls. Each treated group disclosed better portal flow and sinusoid perfusion, decreased leukocyte adherence, particularly in the PTX group. Liver regeneration occurred only in the treated groups. IL-6 and IL-10 levels were dramatically up-regulated (50x) in the PTX group, and at lower levels in other experimental groups. The protective effect of PTX was lost in IL-6(-/-) mice and protection was restored by a single dose of r-IL-6. In conclusion, interruption of TNF-alpha signaling or depletion of Kupffer cells improves survival after 30% liver transplantation, reduces liver injury, and enhances regeneration. The superior effects of PTX are mediated by IL-6.

Animals↗
PubMed2006

Bile salt toxicity aggravates cold ischemic injury of bile ducts after liver transplantation in Mdr2+/- mice.

Intrahepatic bile duct strictures are a serious complication after orthotopic liver transplantation (OLT). We examined the role of endogenous bile salt toxicity in the pathogenesis of bile duct injury after OLT. Livers from wild-type mice and mice heterozygous for disruption of the multidrug resistance 2 Mdr2 gene (Mdr2+/-) were transplanted into wild-type recipient mice. Mdr2+/- mice secrete only 50% of the normal amount of phospholipids into their bile, leading to an abnormally high bile salt/phospholipid ratio. In contrast to homozygous Mdr2-/- mice, the Mdr2+/- mice have normal liver histology and function under normal conditions. Two weeks after OLT, bile duct injury and cholestasis were assessed by light and electron microscopy, as well as through molecular and biochemical markers. There were no signs of bile duct injury or intrahepatic cholestasis in liver grafts from wild-type donors. Liver grafts from Mdr2+/- donors, however, had enlarged portal tracts with cellular damage, ductular proliferation, biliostasis, and a dense inflammatory infiltrate after OLT. Parallel to this observation, recipients of Mdr2+/- livers had significantly higher serum transaminases, alkaline phosphatase, total bilirubin, and bile salt levels, as compared with recipients of wild-type livers. In addition, hepatic bile transporter expression was compatible with the biochemical and histological cholestatic profile found in Mdr2+/- grafts after OLT. In conclusion, toxic bile composition, due to a high biliary bile salt/phospholipid ratio, acted synergistically with cold ischemia in the pathogenesis of bile duct injury after transplantation.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2006

Biochemistry and biology of SPINK-PSTI and monitor peptide.

This article summarizes structural and functional properties of pancreatic secretory trypsin inhibitor (PSTI), which has been identified in many species. Its prominent role is to protect the pancreas from prematurely activated trypsinogen before entry into the duodenum. In the rat there are two isoforms, one of which is PSTI-I, a 61-amino acid peptide involved in the feedback regulation of pancreatic enzymes. Independent investigations in neoplastic diseases led to the discovery of tumor-associated trypsin inhibitor,which is identical to PSTI.

Amino Acid Sequence↗
PubMed2006

Novel short-term hypothermic oxygenated perfusion (HOPE) system prevents injury in rat liver graft from non-heart beating donor.

OBJECTIVE: To assess a machine perfusion system in rescuing liver grafts from non-heart-beating donors (NHBD). SUMMARY BACKGROUND DATA: The introduction of extracorporeal liver perfusion systems in the clinical routine depends on feasibility. Conceivably, perfusion could be performed during recipient preparation. We investigated whether a novel rat liver machine perfusion applied after in situ ischemia and cold storage can rescue NHBD liver grafts. METHODS: We induced cardiac arrest in male Brown Norway rats by phrenotomy and ligation of the subcardial aorta. We studied 2 experimental groups: 45 minutes of warm in situ ischemia + 5 hours cold storage versus 45 minutes of warm in situ ischemia + 5 hours cold storage followed by 1 hour hypothermic oxygenated extracorporeal perfusion (HOPE). In both groups, livers were reperfused in a closed sanguineous isolated liver perfusion device for 3 hours at 37 degrees C. To test the benefit of HOPE on survival, we performed orthotopic liver transplantation in both experimental groups. RESULTS: After cold storage and reperfusion, NHBD livers showed necrosis of hepatocytes, increased release of AST, and decreased bile flow. HOPE improved NHBD livers significantly with a reduction of necrosis, less AST release, and increased bile flow. ATP was severely depleted in cold-stored NHBD livers but restored in livers treated by HOPE. After orthotopic liver transplantation, grafts treated by HOPE demonstrated a significant extension on animal survival. CONCLUSIONS: We demonstrate a beneficial effect of HOPE by preventing reperfusion injury in a clinically relevant NHBD model.

Animals↗
PubMed2006

Cationic long-chain ceramide LCL-30 induces cell death by mitochondrial targeting in SW403 cells.

Ceramides are sphingolipid second messengers that are involved in the mediation of cell death. There is accumulating evidence that mitochondria play a central role in ceramide-derived toxicity. We designed a novel cationic long-chain ceramide [omega-pyridinium bromide D-erythro-C16-ceramide (LCL-30)] targeting negatively charged mitochondria. Our results show that LCL-30 is highly cytotoxic to SW403 cells (and other cancer cell lines) and preferentially accumulates in mitochondria, resulting in a decrease of the mitochondrial membrane potential, release of mitochondrial cytochrome c, and activation of caspase-3 and caspase-9. Ultrastructural analyses support the concept of mitochondrial selectivity. Interestingly, levels of endogenous mitochondrial C16-ceramide decreased by more than half, whereas levels of sphingosine-1-phosphate increased dramatically and selectively in mitochondria after administration of LCL-30, suggesting the presence of a mitochondrial sphingosine kinase. Of note, intracellular long-chain ceramide levels and sphingosine-1-phosphate remained unaffected in the cytosolic and extramitochondrial (nuclei/cellular membranes) cellular fractions. Furthermore, a synergistic effect of cotreatment of LCL-30 and doxorubicin was observed, which was not related to alterations in endogenous ceramide levels. Cationic long-chain pyridinium ceramides might be promising new drugs for cancer therapy through their mitochondrial preference.

Antibiotics, Antineoplastic↗
PubMed2005

Exocrine meets endocrine: pancreatic stone protein and regenerating protein--two sides of the same coin.

BACKGROUND: Regenerating protein (reg) and pancreatic stone protein (PSP) have been discovered independently in the fields of diabetes and pancreatitis. MATERIALS AND METHODS: These proteins are identical; however, because of the gap between the endocrine and exocrine field, there was never a consensus and the nomenclature has not been rectified. Since the time of the initial discovery, more isoforms have been unified. Historically, PSP was discovered long before reg, yet, in many areas outside of the pancreatitis research field, reg is being used. RESULTS: For PSP/reg, a role in proliferation and regeneration of islet cells has been postulated. A hitherto insufficiently understood phenomenon is the massive up-regulation of PSP/reg in pancreatic tissue and juice under conditions of stress. Similarly, PAP (pancreatitis-associated protein)/reg III has been attributed various functional roles. Structurally, the ability to form fibrils after tryptic cleavage is a striking common features of both proteins. However, this biochemical transformation is in itself not enough to gain functional insight. Thus, physiological and genetic approaches are required to further characterize the role of these proteins in the pancreas. Recently, more evidence has been presented in support of the theory that PSP/reg plays a key role in islet neogenesis/regeneration. CONCLUSIONS: In this review we discuss the debate on the localization and functional roles of PSP/reg and PAP/regIII. Therefore, we have summarized hypotheses and experimental results supporting such hypotheses.

Animals↗
PubMed2005

Mouse livers with macrosteatosis are more susceptible to normothermic ischemic injury than those with microsteatosis.

BACKGROUND/AIMS: Fatty livers are increasingly used for transplantation due to the dramatic organ shortage. While steatosis is an established risk factor for post-operative complications, the impact of macro- vs. microvesicular steatosis on ischemic injury is unclear. METHODS: The effects of ischemia and reperfusion were tested in two different models of steatotic mice: ob/ob as a model disclosing predominantly macrovesicular steatosis and choline deficient diet having mainly microvesicular steatosis. Steatotic and lean livers were subjected to 45 min of ischemia. Serial markers of hepatocellular injury, animal survival were measured. Hepatic tissue blood flow and portal vein perfusion were assessed. RESULTS: Ob/ob mice had a significantly lower tolerance to hepatic ischemia. with increased AST release and decreased survival in comparison to the choline deficient mice. No difference in ATP content was found between both steatosis models, but hepatic perfusion and portal vein flow were significantly lower after reperfusion in the ob/ob mice when compared to the choline deficient animals. Ischemic pre-conditioning significantly improved liver reperfusion and injury in both models of steatosis. CONCLUSIONS: Livers with macrovesicular steatosis have a lower tolerance to ischemic injury than those with microvesicular steatosis. Low intrahepatic and portal vein perfusion in macrovesicular fatty livers is, at least partially, responsible for the poorer outcome.

Adenosine Triphosphate↗
PubMed2005

Pancreatic response to endotoxin after chronic alcohol exposure: switch from apoptosis to necrosis?

Chronic alcohol consumption is known to increase the susceptibility to acute and chronic pancreatitis, and it is likely that a cofactor is required to initiate the progression to alcoholic pancreatitis. The severity and complications of alcoholic and nonalcoholic acute pancreatitis may be influenced by a number of cofactors, including endotoxemia. To explore the effect of a possible cofactor, we used endotoxin [lipopolysaccharide (LPS)] as a tool to induce cellular injury in the alcoholic pancreas. Single, increasing doses of endotoxin were injected in rats fed an alcohol or control diet and killed 24 h after the injection. We examined the mechanism by which LPS exacerbates pancreatic injury in alcohol-fed rats and whether the injury is associated with apoptosis or necrosis. We showed that chronic alcohol exposure alone inhibits apoptosis through the intrinsic pathway and the downstream apoptosis executor caspase-3 compared with the controls. Pancreatic necrosis and inflammation increased after LPS injection in control and alcohol-fed rats in a dose-dependent fashion but with a significantly greater response in the alcohol-fed animals. Caspase activities and TdT-mediated dUTP nick-end labeling positivity were lower in the alcoholic pancreas injected with LPS, whereas the histopathology and inflammation were more severe compared with the control-fed animals. Assessment of a putative indicator of necrosis, the ratio of ADP to ATP, indicated that alcohol exposure accelerates pancreatic necrosis in response to endotoxin. These findings suggest that the pancreas exposed to alcohol is more sensitive to LPS-induced damage because of increased sensitivity to necrotic cell death rather than apoptotic cell death. Similar to the liver, the pancreas is capable of responding to LPS with a more severe response in alcohol-fed animals, favoring pancreatic necrosis rather than apoptosis. We speculate that this mechanism may occur in acute alcoholic pancreatitis patients.

Adenosine Triphosphate↗
PubMed2004

Coordinate regulation of PSP/reg and PAP isoforms as a family of secretory stress proteins in an animal model of chronic pancreatitis.

BACKGROUND: PSP/reg and PAP are secretory stress proteins (SSP) and may be part of a protective mechanism. They share structural homologies and form insoluble fibrils after tryptic activation. To further explore the regulation of these proteins, we investigated the male WBN/Kob rat, a model of pancreatic inflammatory and fibrotic disease similar to chronic pancreatitis. MATERIALS AND METHODS: Expression of PSP/reg and PAP I, II and III in the WBN/Kob rat pancreas was evaluated on the mRNA and protein level, by immunohistochemistry and by highly sensitive isoform specific ELISAs. RESULTS: The SSPs are constitutively secreted, PAP in nanomolar, PSP/reg in micromolar concentrations. Before conventional morphological changes are detectable in the WBN/Kob rat, focally increased expression of secretory stress protein is visible. SSP levels in pancreatic juice of WBN/Kob rats reach peak values 10- to 50-fold higher than in Wistar control rats. The highest expression was localized to acini with inflammatory infiltration. CONCLUSIONS: There is a tight spatial and temporal association between pre-inflammatory changes or inflammation and SSP-expression. These results support our concept that PSP/reg and PAP are coordinately regulated SSP.

Acute-Phase Proteins↗
PubMed2004

Ischemic preconditioning and intermittent clamping improve murine hepatic microcirculation and Kupffer cell function after ischemic injury.

The aim of this study was to evaluate whether the protective effect of intermittent clamping and ischemic preconditioning is related to an improved hepatic microcirculation after ischemia/reperfusion injury. Male C57BL/6 mice were subjected to 75 or 120 min of hepatic ischemia and 1 or 3 hours of reperfusion. The effects of continuous ischemia, intermittent clamping, and ischemic preconditioning before prolonged ischemia on sinusoidal perfusion, leukocyte-endothelial interactions, and Kupffer cell phagocytic activity were analyzed by intravital fluorescence microscopy. Kupffer cell activation was measured by tissue levels of tumor necrosis factor (TNF)-alpha, and the integrity of sinusoidal endothelial cells and Kupffer cells were evaluated by electron microscopy. Continuous ischemia resulted in decreased sinusoidal perfusion rate and phagocytic activity of Kupffer cell, increased leukocyte-endothelial interactions and TNF-alpha levels. Both protective strategies improved sinusoidal perfusion, leukocyte-endothelial interactions and phagocytic activity of Kupffer cells after 75-minutes of ischemia, and intermittent clamping also after 120 minutes ischemia. TNF-alpha release was significantly reduced and sinusoidal wall integrity was preserved by both protective procedures. In conclusion, both strategies are protective against ischemia/reperfusion injury by maintaining hepatic microcirculation and decreasing Kupffer cell activation for clinically relevant ischemic periods, and intermittent clamping appears superior for prolonged ischemia.

Animals↗
PubMed2003

Secretory apparatus assessed by analysis of pancreatic secretory stress protein expression in a rat model of chronic pancreatitis.

Secretory stress proteins (SSP) are a family of proteins including isoforms of pancreatitis-associated protein (PAP) and pancreatic stone protein (PSP/reg). In vitro exposure to trypsin results in the formation of insoluble fibrillar structures. SSP are constitutively secreted into pancreatic juice at low levels. The WBN/Kob rat is a model for chronic pancreatitis, displaying focal inflammation, destruction of the parenchyma and changes in the architecture of the acinar cell; the synthesis and secretion of SSP are also increased. We have investigated the secretory apparatus by SSP immunohistochemistry at the light- and electron-microscopical (EM) levels. Immunocytochemistry of PSP/reg in Wistar control rats reveals low levels, with individual acinar cells exhibiting high immunoreactivity in zymogen granules. PAP is not detectable. In the WBN/Kob rat, PSP/reg and PAP immunoreactivity is markedly increased. Double immunofluorescence for PSP/reg and PAP I or II demonstrates that these proteins colocalize to the same cell. Acinar cells change their secretory architecture by fusion of zymogen granules and elongation of the fused organelles. The immunogold technique has demonstrated an increase of SSP in zymogen granules in WBN/Kob rats. PSP/reg-positive zymogen granules fuse to form elongated structures with fibrillar contents. An extensive PSP/reg-positive fibrillar network is established in the cytosol. Extracellular fibrils have been observed in several ductules. Thus, SSP-derived fibrils form concomitantly with acinar damage in the WBN/Kob rat. Based on the known tryptic cleavage site of SSP, the in vivo generation of fibrils is presumably the result of premature trypsin activation.

Animals↗
PubMed2003

Protective strategies against ischemic injury of the liver.

This article summarizes strategies to protect the liver from injuries caused by ischemia and reperfusion. Three different sections (i.e., surgical and pharmacologic strategies and gene therapy) present approaches to enhance the survival and viability of the liver in various surgical procedures including liver transplantation. The first section reviews approaches using surgical interventions such as ischemic preconditioning and intermittent clamping. Their protective effects are discussed with respect to the mechanism of injury. In the second section, pharmacologic agents targeting microcirculation, oxidative stress, proteases, and inflammation are described. Mechanisms of injury and their suppression by a wide variety of drugs are discussed. The third section focuses on gene therapy. Potential target genes have been identified (e.g., superoxide dismutase or heme oxygenase). Animal experiments in which the liver injury is reduced successfully may pave the way to novel strategies applied to different liver diseases in humans.

Animals↗
PubMed2003

Sub-lethal oxidative stress triggers the protective effects of ischemic preconditioning in the mouse liver.

BACKGROUND/AIMS: While ischemic preconditioning confers significant protection against subsequent prolonged periods of ischemia, the mechanisms triggering protection remain speculative. We hypothesize that a sub-lethal oxidative stress during ischemic preconditioning induces defense mechanisms preventing subsequent lethal injury. METHODS: We used mouse models of partial and total hepatic ischemia for 75 min. Ischemic preconditioning consisted of 10-min ischemia and 15-min reperfusion prior to the prolonged ischemic insult. RESULTS: Tissue levels of peroxides increased about three times after 10 min of ischemia and normalized within 15 min of reperfusion. This limited oxidative stress during ischemic preconditioning prevented the negative effects of subsequent prolonged ischemia as assessed by AST-levels, TUNEL-staining of hepatocytes and animal survival. N-Acetylcysteine inhibited the mild oxidative burst of ischemic preconditioning, and fully reversed the protective effects of preconditioning. The protective role of a sub-lethal oxidative stress was supported by the benefit of delivery of an H2O2-analog through the portal vein prior to a long ischemic insult. This challenge conferred similar protection as ischemic preconditioning. CONCLUSIONS: We conclude that the mild burst of oxidative stress generated during ischemic preconditioning triggers protective mechanisms against subsequent, otherwise lethal, ischemic injury. The pathway possibly includes enhancement of natural anti-oxidative stress mechanisms.

Animals↗