Sodium thiosulfate refuels the hepatic antioxidant pool reducing ischemia-reperfusion-induced liver injury.

Ischemia-reperfusion injury is a critical liver condition during hepatic transplantation, trauma, or shock. An ischemic deprivation of antioxidants and energy characterizes liver injury in such cases. In the face of increased reactive oxygen production, hepatocytes are vulnerable to the reperfusion driving ROS generation and multiple cell-death mechanisms. In this study, we investigate the importance of hydrogen sulfide as part of the liver's antioxidant pool and the therapeutic potency of the hydrogen sulfide donors sodium sulfide (Na2S, fast releasing) and sodium thiosulfate (STS, Na2S2O3, slow releasing). The mitoprotection and toxicity of STS and Na2S were investigated on isolated mitochondria and a liver perfusion oxidative stress model by adding text-butyl hydroperoxide and hydrogen sulfide donors. The respiratory capacity of mitochondria, hepatocellular released LDH, glutathione, and lipid-peroxide levels were quantified. In addition, wild-type and cystathionine-γ-lyase knockout mice were subjected to warm selective ischemia-reperfusion injury by clamping the main inflow for 1 h followed by reperfusion of 1 or 24 h. A subset of animals was treated with STS shortly before reperfusion. Glutathione, plasma ALT, and lipid-peroxide levels were investigated alongside mitochondrial changes in structure (electron microscopy) and function (intravital microscopy). Liver tissue necrosis quantified 24 h after reperfusion indicates the net effects of the treatment on the organ. STS refuels and protects the endogenous antioxidant pool during liver ischemia-reperfusion injury. In addition, STS-mediated ROS scavenging significantly reduced lipid peroxidation and mitochondrial damage, resulting in better molecular and histopathological preservation of the liver tissue architecture. STS prevents tissue damage in liver ischemia-reperfusion injury by increasing the liver's antioxidant pool, thereby protecting mitochondrial integrity.

Mechanisms of Ataxia Telangiectasia Mutated (ATM) Control in the DNA Damage Response to Oxidative Stress, Epigenetic Regulation, and Persistent Innate Immune Suppression Following Sepsis.

Cells have evolved extensive signaling mechanisms to maintain redox homeostasis. While basal levels of oxidants are critical for normal signaling, a tipping point is reached when the level of oxidant species exceed cellular antioxidant capabilities. Myriad pathological conditions are characterized by elevated oxidative stress, which can cause alterations in cellular operations and damage to cellular components including nucleic acids. Maintenance of nuclear chromatin are critically important for host survival and eukaryotic organisms possess an elaborately orchestrated response to initiate repair of such DNA damage. Recent evidence indicates links between the cellular antioxidant response, the DNA damage response (DDR), and the epigenetic status of the cell under conditions of elevated oxidative stress. In this emerging model, the cellular response to excessive oxidants may include redox sensors that regulate both the DDR and an orchestrated change to the epigenome in a tightly controlled program that both protects and regulates the nuclear genome. Herein we use sepsis as a model of an inflammatory pathophysiological condition that results in elevated oxidative stress, upregulation of the DDR, and epigenetic reprogramming of hematopoietic stem cells (HSCs) to discuss new evidence for interplay between the antioxidant response, the DNA damage response, and epigenetic status.

Hydrogen sulfide modulates sinusoidal constriction and contributes to hepatic microcirculatory dysfunction during endotoxemia.

Hydrogen sulfide (H2S) affects vascular resistance; however, its effect on the hepatic microcirculation has not been investigated. Hepatic sinusoidal perfusion is dysregulated during sepsis, contributing to liver injury. Therefore, the present study determined the effect of H2S on the hepatic microcirculation and the contribution of endogenous H2S to hepatic microcirculatory dysfunction in an endotoxin model of sepsis. Portal infusion of H2S increased portal pressure in vivo (6.8 ± 0.2 mmHg before H2S vs. 8.6 ± 0.8 mmHg peak during H2S infusion, P < 0.05). Using intravital microscopy, we observed decreased sinusoidal diameter (6.2 ± 0.27 µm before H2S vs. 5.7 ± 0.3 µm after H2S, P < 0.05) and increased sinusoidal heterogeneity during H2S infusion (P < 0.05) and net constriction. Since hepatic H2S levels are elevated during sepsis, we used the cystathionine γ lyase inhibitor DL-propargylglycine (PAG) to determine the contribution of H2S to the hypersensitization of the sinusoid to the vasoconstrictor effect of endothelin-1 (ET-1). PAG treatment significantly attenuated the sinusoidal sensitization to ET-1 in endotoxin-treated animals. ET-1 infusion increased portal pressure to 175% of baseline in endotoxemic animals, which was reduced to 143% following PAG treatment (P < 0.05). PAG abrogated the increase in sinusoidal constriction after ET-1 infusion in LPS-treated rats (30.9% reduction in LPS rats vs. 11.6% in PAG/LPS rats, P < 0.05). Moreover, PAG treatment significantly attenuated the increase in NADH fluorescence following ET-1 exposure during endotoxemia (61 grayscale units LPS vs. 21 units in PAG/LPS, P < 0.05), suggesting an improvement in hepatic oxygen availability. This study is the first to demonstrate a vasoconstrictor action of H2S on the hepatic sinusoid and provides a possible mechanism for the protective effect of PAG treatment during sepsis.

Interleukin-27 expression following infection with the murine gammaherpesvirus 68.

IL-27 is a heterodimeric cytokine composed of p28 and Epstein Barr virus induced gene 3 (Ebi3) protein subunits. In the present study, we questioned whether murine gammaherpesvirus 68 (HV-68) could induce expression of Ebi3, p28, and IL-27 in this mouse model of an EBV-like infection. Cultured macrophages and dendritic cells exposed to HV-68 upregulated p28 mRNA expression and increased secretion of the p28 and IL-27 (p28+Ebi3) proteins. B220(+) and CD11b(+) cells also upregulated p28 mRNA expression following in vivo infection with this virus. Surprisingly, no significant increases in p28 or IL-27 protein production were observed in vivo during the acute or mononucleosis phases of the disease. The possibility that HV-68-induced upregulation of p28 mRNA expression primed cells for IL-27 secretion was suggested by the ability of a TLR4 agonist to augment cytokine production. When cultured macrophages and dendritic cells were exposed to virus plus a suboptimal dose of LPS, increased levels of p28 protein expression were observed. More importantly, when latently infected mice were challenged with a sublethal dose of LPS, augmented p28 and IL-27 protein production occurred. Using a model of sepsis, mice latently infected with HV-68 had exaggerated p28 protein production when compared to mice that were singularly infected or subjected to cecal ligation and puncture. Taken together, these studies define expression of HV-68 induced IL-27, and suggest that mice latently infected with this gammaherpesvirus will have exaggerated responses when confronted with other stimuli capable of inducing this member of the IL-12 family of cytokines.

Role of Kupffer cells in vascular stress genes during trauma and sepsis.

BACKGROUND: Sepsis remains a serious complication after trauma. Although hepatic microvascular dysfunction occurs during trauma and sepsis, the mechanism responsible is unclear. Since Kupffer cells can provide the signals that regulate the hepatic response in inflammation and contribute to multiple organ failure, this study investigated the role of Kupffer cells in the imbalance of the expression of vasoactive and inflammatory mediators during trauma and sepsis. MATERIALS AND METHODS: The Kupffer cells were inactivated by gadolinium chloride (GdCl(3), 7.5 mg/kg body weight, i.v.) 1 and 2 d before surgery. The animals then underwent femur fracture (FFx) followed 48 h later by cecal ligation and puncture (CLP). After 24 h, blood was obtained to determine the alanine aminotransferase (ALT) activity. Liver samples were also taken for RT-PCR analysis of the mRNA for genes of interest. RESULTS: Serum ALT levels increased in FFx and CLP. This increase was potentiated by FFx + CLP and was attenuated by GdCl(3). The mRNA expression levels in the FFx showed no change in the ET(A), ET(B), iNOS, and HO-1, and showed a slight increase of 2.6-fold, 2.2-fold, and 2.8-fold for ET-1, eNOS, and TNF-alpha, respectively. The ET-1 mRNA expression level increased after CLP and FFx + CLP. The ET(A) mRNA level showed no change, whereas the ET(B) transcripts increased after CLP. This increase was potentiated after FFx + CLP, and was attenuated by GdCl(3). After CLP alone, iNOS, eNOS, and TNF-alpha mRNA expression levels were increased 20.3-fold, 5.8-fold, and 11.9-fold, respectively. This increase was potentiated after FFx + CLP, and was attenuated by GdCl(3). HO-1 mRNA expression significantly increased after FFx + CLP, and this increase was attenuated by GdCl(3). CONCLUSIONS: Mild trauma alone causes little change in the expression of vasoactive mediators while sequential injury potentiates the imbalanced gene expression induced by sepsis. Kupffer cells might be essential for hepatic microvascular dysfunction after sequential stress.

Improved preservation of warm ischemic livers by hypothermic machine perfusion with supplemented University of Wisconsin solution.

Hypothermic machine perfusion (HMP) has the potential to improve recovery and preservation of Donation after Cardiac Death (DCD) livers, including uncontrolled DCD livers. However, current perfusion solutions lack the needed substrates to improve energy recovery and minimize hepatic injury, if warm ischemic time (WIT) is extended. This proof-of-concept study tested the hypothesis that the University of Wisconsin (UW) solution supplemented with anaplerotic substrates, calcium chloride, thromboxane A2 inhibitor, and antioxidants could improve HMP preservation and minimize reperfusion injury of warm ischemic livers. Preflushed rat livers subjected to 60 min WIT were preserved for 5 h with standard UW or supplemented UW (SUW) solution. Post preservation hepatic functions and viability were assessed during isolated perfusion with Krebs-Henseleit solution. Livers preserved with SUW showed significantly (p < .001) improved recovery of tissue ATP levels (micromol/g liver), 2.06 +/- 0.10 (mean +/- SE), as compared to the UW group, 0.70 +/- 0.10, and the level was 80% of that of fresh control livers (2.60 +/- 0.13). At the end of 1 h of rewarming, lactate dehydrogenase (U/L) in the perfusate was significantly (p < .05) lower in the SUW group (429 +/- 58) as compared to ischemia-reperfusion (IR) (781 +/- 12) and the UW group (1151 +/- 83). Bile production (microg/min/g liver) was significantly (p < .05) higher in the SUW group (280 +/- 13) as compared to the IR (224 +/- 24) and the UW group (114 +/- 14). The tissue edema formation assessed by tissue wet-dry ratio was significantly (p < .05) higher in UW group. Histology showed well-preserved hepatic structure in the SUW group. In conclusion, this study suggests that HMP with SUW solution has the potential to restore and preserve livers with extended WIT.

S-adenosyl-L-methionine attenuates oxidative stress and hepatic stellate cell activation in an ethanol-LPS-induced fibrotic rat model.

Previous studies report S-adenosyl-L-methionine (SAMe) can exert hepatoprotective effects. At present, the role of SAMe in affecting the activation and/or proliferation of hepatic stellate cells (HSCs) during alcohol-induced fibrotic disease progression is poorly understood. In the human disease state, chronic ethanol intake increases hepatic exposure to LPS and magnifies the hepatic insult leading to fibrosis and cirrhosis. In this study, we developed a "2-hit" ethanol-LPS fibrotic liver rat model with which to investigate the effects of SAMe as a hepatic antifibrotic treatment. Male rats were maintained on liquid diets containing either ethanol or isocalorically matched controls for 8 weeks. Animals received ethanol alone (E), ethanol concomitant with twice weekly LPS injections (EL), or ethanol, LPS, and daily SAMe injections. When using this model, SAMe-treated animals demonstrated significantly decreased fibrosis, oxidative stress, steatosis, and improved liver function versus the EL group. In addition, the EL group showed increased HSC activation, an effect that was abrogated by the addition of SAMe. Analysis of the transforming growth factor-beta (TGF-beta) signaling pathways demonstrated increased hepatic TGF-beta and Smad3 messenger RNA expression in the E and EL groups, which was inhibited in the presence of SAMe. Conversely, SAMe led to increased Smad7 (an inhibitor of TGF-beta signaling) messenger RNA expression. These data demonstrate chronic ethanol feeding combined with LPS induces liver fibrosis, and the addition of SAMe significantly reduces hepatic injury and fibrosis through inhibition of oxidative stress and HSC activation.

Hepatic arterial flow becomes the primary supply of sinusoids following partial portal vein ligation in rats.

BACKGROUND AND AIM: Partial portal vein ligation (PPVL) is a commonly used procedure to induce prehepatic portal hypertension in animal models. The aim of this study was to test the hypothesis that the hepatic arterial flow becomes the primary source feeding the sinusoids in the liver after PPVL. METHODS: Sprague-Dawley rats underwent either sham operation or partial portal vein ligation (PPVL). The number of vessels in the liver at 2 weeks postoperatively was determined by factor VIII immunolocalization and the gene expression of angiogenic factors was assessed by RT-PCR. The total hepatic arterial supply to the liver was measured using the fluorescent microsphere injection technique. To further test the hypothesis, two additional groups of rats underwent hepatic artery ligation (HAL) or PPVL plus HAL (PPHAL). The integrity of hepatic microcirculation was then evaluated in all four groups by intravital microscopy. RESULTS: At 2 weeks after operation, the number of vessels detected by factor VIII staining was significantly higher in PPVL compared to sham. Densitometric analysis of RT-PCR bands revealed a significant increase of vascular endothelial growth factor gene expression in PPVL compared to sham. Arterial flow to the liver measured by fluorescent microspheres was increased by 190% in PPVL compared to sham. When all four groups were compared, no prominent histological abnormality was observed in sham, HAL, and PPVL groups; however, PPHAL livers showed focal necrosis and inflammatory cell infiltration around the portal triads. Additionally, only the PPHAL livers showed a decreased sinusoidal diameter and significantly lower perfusion index (PPHAL 42.9+/-6.1; sham 85.7+/-7.0, PPVL 80.2+/-6.5, HAL 70.9+/-4.5). CONCLUSIONS: These results suggest that the hepatic artery flow becomes the primary source for the blood supply of sinusoids and the compensatory change in the hepatic arterial system plays a critical role in maintaining microcirculatory perfusion following the restriction of the portal vein flow by PPVL.

Bosentan inhibits tumor vascularization and bone metastasis in an immunocompetent skin-fold chamber model of breast carcinoma cell metastasis.

Angiogenic factors including endothelin-1 (ET-1) play a key role in the progression of breast metastases to bone. We investigated the impact of ET-1 on the development of bone metastases in an immunocompetent murine skin-fold chamber model. Murine mammary carcinoma 4T1 was injected in a skin-fold chamber implanted on CB6 mice along with bone explants. Furthermore, mice were treated with or without a dual selective antagonist of both ET-1 receptors. The progression of the vascularization within the chamber was monitored over time by intravital microscopy (IVM). The tumor growth and the development of bone metastases were assessed by cytokeratin-19 gene expression and histological studies. Results indicate that this new model associated with IVM allows for the continuous monitoring of the change in vascularization associated with the development of bone metastases. Additionally, treatment with an antagonist of both ET-1 receptors was associated with the presence of significantly less vessels near the tumor mass compared to control mice. These changes were correlated with smaller tumor masses and reduced bone invasion (P < 0.05). Thus, in an immunocompetent murine model of breast carcinoma metastases to bone, our data support the hypothesis that vascularization plays a role in tumor development and progression and that ET-1 specifically modulates the angiogenesis associated with breast metastases to the bone.

Remote trauma sensitizes hepatic microcirculation to endothelin via caveolin inhibition of eNOS activity.

This study addresses the microvascular mechanisms by which a remote, mild stress such as blunt trauma sensitizes the liver to injury. Rats received closed femur fracture (FFx), and 24 h later livers were isolated and perfused at a similar starting flow rate for assessment of vascular response to endothelin-1 (ET-1). Sinusoidal volumetric flow (QS), red blood cell velocity (VRBC), and sinusoidal diameter (Ds) were determined by intravital microscopy. Baseline portal resistance in livers from FFx rats was not changed. The FFx group showed a lower baseline VRBC (322.9 +/- 26.4 and 207.3 +/- 17.2 microm/s in sham and FFx,) and QS (28.4 +/- 4.2 and 17.6 +/- 2.1 pL/s in sham and FFx, P < 0.05). ET-1 caused a decrease in the VRBC in sham but no change after FFx. In contrast, Ds was unchanged by ET-1 in sham but decreased in FFx (10.3 +/- 0.4 to 10.7 +/- 0.5 vs. 10.6 +/- 0.4 to 9.0 +/- 0.4 microm at 10 min in sham and FFx groups, P < 0.05). The overall result of these changes was a greater decrease in sinusoidal flow in FFx compared with sham. There was no significant change in mRNA for ET-1, endothelin A (ETA) receptor, or iNOS (inducible nitric oxide synthase) in FFx compared with sham. However, endothelin B (ETB) receptor mRNA and eNOS (endothelial nitric oxide synthase) mRNA were increased in the FFx group (ETB, 54.81 +/- 8.08 in sham vs. 83.28 +/- 8.19 in FFx; eNOS, 56.11 +/- 2.53 in sham vs. 83.31 +/- 5.51 in FFx; P < 0.05) while the levels of these proteins remained unchanged. Caveolin-1 (cav-1) protein levels were elevated in FFx, and coimmunoprecipitation with both ETB and eNOS showed increased associations with these proteins, suggesting a possible inactivation of eNOS. The eNOS activity was also blunted in FFx animals in the presence of increased cav-1 expression. Taken together, these results demonstrate that remote trauma sensitizes the liver to the sinusoidal constrictor effect of ET-1. We propose that this hyperresponsiveness occurs as a result of uncoupling of the ETB receptor from eNOS activity mediated by interaction of eNOS and possibly the ETB receptor with increased caveolin-1. This vascular sensitization that occurs after FFx may contribute to the exacerbation of injury during subsequent stresses.

Pronlonged hypothermic machine perfusion preserves hepatocellular function but potentiates endothelial cell dysfunction in rat livers.

BACKGROUND: Although hypothermic machine perfusion (HMP) preservation has been shown to improve organ function and to expand the organ donor pool, problems still exist with the current HMP technology for liver preservation. The present study was conducted to investigate endothelial and hepatocellular functions following extended HMP (> r =24 hr) in rat liver model. METHODS: Following 24-hour hypothermic HMP with University of Wisconsin (UW) solution or 24-hour simple cold storage (SCS), livers were reperfused with Krebs-Henseleit buffer solution at 37 degree C for 30 minutes. Hepatocyte damage and function were assessed by measuring lactate dehydrogenase (LDH) activity, bile production, and indocyanine green (ICG) extraction. Sinusoidal endothelial cell (SEC) function and permeability were determined by hyaluronic acid (HA) uptake and multiple indicator dilution (MID) method, respectively. RESULTS: After 24-hour hypothermic preservation, HMP livers showed lower released LDH levels, higher bile flow rate, and greater hepatic ICG uptake compared with SCS livers. However, LDH levels became significantly higher in HMP than in SCS after 30 minutes of warm perfusion. The increased enzyme levels were accompanied by a significant increase in endothelial permeability to albumin and a decrease in hyaluronic acid uptake in HMP compared to SCS. Liver wet/dry weight ratio confirmed a greater edema in HMP livers than SCS livers. CONCLUSION: These results suggest that 24-hour hypothermic HMP may help preservation of hepatocyte function, but endothelial cell dysfunction during the cold preservation may play a key role in hepatocyte dysfunction and parenchymal cell death upon reperfusion.

Glucagon increases gap junctional intercellular communication via cAMP in the isolated perfused rat liver.

The effects of glucagon on the subacinar distribution of hepatic transmembrane potentials were studied in the perfused fasted rat liver. The livers were perfused with a Krebs-Henseleit buffer, and membrane potentials of matched periportal and pericentral hepatocytes were determined using glass microelectrodes. Lactate- and pyruvate-induced glucose production and O2 uptake were potentiated by 10(-8) M glucagon. Twenty-five micromoles 8-bromoadenosine 3',5'cyclic monophosphate (8-BrcAMP) exhibited stimulatory effects similar, in terms of glucose production and O2 uptake to those of glucagon. Octanol (0.1 and 0.5 mM) had no effect on glucose production but reversibly increased O2 uptake by 16% to 30% over all experiments. Under basal conditions (no exogenous substrate) hepatocyte membrane potentials averaged approximately -27 mV, and no gradients were found between periportal and pericentral hepatocytes. Addition of lactate and pyruvate produced hyperpolarization in all hepatocytes. However, there was a small but statistically significant gradient produced across the hepatic acinus in membrane potential, i.e., the hyperpolarization was higher in the periportal region compared with the pericentral region. Glucagon and 8-BrcAMP induced marked hyperpolarization in periportal and pericentral hepatocytes with no gradients across the acinus. Although no changes were found under basal and lactate plus pyruvate, 0.5 mM octanol induced heterogeneity of membrane potential during glucagon and 8-BrcAMP stimulation. Our findings suggest that glucagon-induced homogeneity of membrane potential may be mediated by increased gap junctional coupling. In addition, cAMP may be responsible for the increase in the intercellular communication during glucagon stimulation.

Ex-vivo study of flow dynamics and endothelial cell structure during extended hypothermic machine perfusion preservation of livers.

Liver transplantation is often the only effective treatment for end stage liver diseases resulting from cirrhosis, hepatitis, progressive jaundice, and biliary atresia. Hypothermic machine perfusion (HMP) preservation may enhance donor pool by extending preservation time and reclaiming marginal donor livers including those from non-heart beating donors (NHBD), as demonstrated in the kidney. However, current HMP protocols have not been successful in improving extended preservation of livers and the major cause of preservation injury remains unknown. An intravital microscopy study was conducted to understand the flow dynamics of sinusoidal perfusion during 24h HMP with cold modified University of Wisconsin (UW) solution. Fluorescein isothiocynate (FITC) labeled albumin was utilized to visualize microvascular space and FITC labeled red blood cells (RBCs) were used to visualize flow dynamics during HMP. A heterogeneous flow pattern with regions of red cell stasis was observed after 24-h HMP. To examine the cause of red cell stasis, intravital and confocal microscopy studies of endothelial cells (ECs) structure labeled with DiI acetylated low-density lipoprotein (DiI acLDL) were conducted. These studies suggest that morphological changes in EC structures occurred during 24h HMP, which may cause obstruction to the sinusoidal flow. Histological findings confirm these results. As a result, heterogeneous flow pattern, red cell stasis, and edema occur, which may lead to the failure of these tissues following extended HMP.

Hepatic vascular response to elevated intraperitoneal pressure in the rat.

The rat is increasingly being used to study the physiological response to elevated intra-abdominal pressure (IAP) during laparoscopic surgery. Although decreased portal venous flow associated with the elevated IAP has been reported in large animals, little information is available in rats. Furthermore, the relative blood flow changes in the hepatic artery and portal vein have not been reported. Therefore, this study was performed to elucidate the change in systemic and splanchnic circulation, including hepatic arterial and portal venular flow, during pneumoperitoneum in rats. Sprague-Dawley rats were assigned into either a ventilated or nonventilated group and then subjected to various levels of IAP (0, 5, 10, and 20 mm Hg) using carbon dioxide gas. At each pressure, both cardiac output and splanchnic organ flow were determined using fluorescent microspheres. There was no obvious hemodynamic difference between the ventilated and nonventilated groups. Mean arterial pressure and cardiac index were significantly lower with 20 mm Hg of IAP compared to 0 mm Hg in both groups. Flow to the spleen, stomach, duodenum, total intestine, and portal vein was all decreased by increasing IAP (P < 0.05 at 20 mm Hg compared to 0 mm Hg) and was significantly correlated to the decrease in cardiac index. However, the hepatic arterial flow was relatively preserved throughout all levels of IAP, suggesting activation of the hepatic arterial buffer response. We conclude that the decreased splanchnic flow during pneumoperitoneum largely depends on the decreased cardiac index. Hepatic artery flow, however, is selectively preserved and may provide protection for liver function during sustained elevations in IAP.