Interspecies transcriptomic comparison identifies a potential porto-sinusoidal vascular disorder rat model suitable for in vivo drug testing.

BACKGROUND: Porto-sinusoidal vascular disorder (PSVD) involves a group of rare vascular liver diseases of unknown aetiology that may lead to the development of portal hypertension and its life-threatening complications. Its pathophysiology is not well understood, and animal models described to date do not fully recapitulate human disease. METHODS: We developed three different PSVD rat models by either immunosensitization (repetitive intraportal LPS or intramuscular spleen extract injections) or toxic (Selfox: combination of FOLFOX and a selenium-enriched diet) treatment and characterized them at haemodynamic, histological, biochemical and transcriptional levels. We compared these results to human data. RESULTS: All three models developed significant portal hypertension, while only the LPS and the Selfox models displayed PSVD-specific and nonspecific histological alterations in the absence of cirrhosis. Transcriptional comparison between rat models and human data showed that both LPS and Selfox models recapitulate the main transcriptional alterations observed in humans, especially regarding haemostasis, oxidative phosphorylation and cell cycle regulation. Reproducibility and feasibility was higher for the Selfox model. CONCLUSIONS: The Selfox rat model faithfully reproduces the main alterations described in PSVD. Its use as a preclinical model for drug testing in progressing PSVD can be a significant step forward towards the development of new therapeutic targets for this rare condition.

Spermidine Supplementation Protects the Liver Endothelium from Liver Damage in Mice.

Chronic liver diseases are multifactorial and the need to develop effective therapies is high. Recent studies have shown the potential of ameliorating liver disease progression through protection of the liver endothelium. Polyamine spermidine (SPD) is a caloric restriction mimetic with autophagy-enhancing properties capable of prolonging lifespan and with a proven beneficial effect in cardiovascular disease in mice and humans. We evaluated the use of dietary supplementation with SPD in two models of liver disease (CCl4 and CDAAH diet). We analyzed the effect of SPD on endothelial dysfunction in vitro and in vivo. C57BL/6J mice were supplemented with SPD in the drinking water prior and concomitantly with CCl4 and CDAAH treatments. Endothelial autophagy deficient (Atg7endo) mice were also evaluated. Liver tissue was used to evaluate the impact of SPD prophylaxis on liver damage, endothelial dysfunction, oxidative stress, mitochondrial status, inflammation and liver fibrosis. SPD improved the endothelial response to oxidative injury in vitro and improved the liver endothelial phenotype and protected against liver injury in vivo. SPD reduced the overall liver oxidative stress and improved mitochondrial fitness. The absence of benefits in the Atg7endo mice suggests an autophagy-dependent effect of SPD. This study suggests SPD diet supplementation in early phases of disease protects the liver endothelium from oxidative stress and may be an attractive approach to modify the chronic liver disease course and halt fibrosis progression.

Impact of lifestyle interventions targeting physical exercise and caloric intake on cirrhosis regression in rats.

In patients, advanced cirrhosis only regresses partially once the etiological agent is withdrawn. Animal models for advanced cirrhosis regression are missing. Lifestyle interventions (LIs) have been shown to improve steatosis, inflammation, fibrosis, and portal pressure (PP) in liver disease. We aimed at characterizing cirrhosis regression after etiological agent removal in experimental models of advanced cirrhosis and to study the impact of different LI on it. Advanced cirrhosis was induced in rats either by carbon tetrachloride (CCl4) or by thioacetamide (TAA) administration. Systemic and hepatic hemodynamics, liver fibrosis, hepatic stellate cell (HSC) activation, hepatic macrophage infiltration, and metabolic profile were evaluated after 48 h, 4 wk or 8 wk of etiological agent removal. The impact of LI consisting in caloric restriction (CR) or moderate endurance exercise (MEE) during the 8-wk regression process was analyzed. The effect of MEE was also evaluated in early cirrhotic and in healthy rats. A significant reduction in portal pressure (PP), liver fibrosis, and HSC activation was observed during regression. However, these parameters remained above those in healthy animals. During regression, animals markedly worsened their metabolic profile. CR although preventing those metabolic disturbances did not further reduce PP, hepatic fibrosis, or HSC activation. MEE also prevented metabolic disturbances, without enhancing, but even attenuating the reduction of PP, hepatic fibrosis, and HSC activation achieved by regression. MEE also worsened hepatic fibrosis in early-TAA cirrhosis and in healthy rats.NEW & NOTEWORTHY We have developed two advanced cirrhosis regression experimental models with persistent relevant fibrosis and portal hypertension and an associated deteriorated metabolism that mimic what happens in patients. LI, despite improving metabolism, did not enhance the regression process in our cirrhotic models. CR did not further reduce PP, hepatic fibrosis, or HSC activation. MEE exhibited a profibrogenic effect in the liver blunting cirrhosis regression. One of the potential explanations of this worsening could be ammonia accumulation.

Advances in therapeutic options for portal hypertension.

Portal hypertension represents one of the major clinical consequences of chronic liver disease, having a deep impact on patients' prognosis and survival. Its pathophysiology defines a pathological increase in the intrahepatic vascular resistance as the primary factor in its development, being subsequently aggravated by a paradoxical increase in portal blood inflow. Although extensive preclinical and clinical research in the field has been developed in recent decades, no effective treatment targeting its primary mechanism has been defined. The present review critically summarizes the current knowledge in portal hypertension therapeutics, focusing on those strategies driven by the disease pathophysiology and underlying cellular mechanisms.

Simvastatin Prevents Progression of Acute on Chronic Liver Failure in Rats With Cirrhosis and Portal Hypertension.

BACKGROUND & AIMS: Cirrhosis and its clinical consequences can be aggravated by bacterial infections, ultimately leading to the development of acute on chronic liver failure (ACLF), characterized by acute decompensation, organ failure, and high mortality within 28 days. Little is known about cellular and molecular mechanisms of ACLF in patients with cirrhosis, so no therapeutic options are available. We developed a sepsis-associated preclinical model of ACLF to facilitate studies of pathogenesis and evaluate the protective effects of simvastatin. METHODS: Male Wistar rats inhaled CCl4 until they developed cirrhosis (at 10 weeks) or cirrhosis with ascites (at 15-16 weeks). Male Sprague-Dawley rats received bile-duct ligation for 28 days or intraperitoneal thioacetamide for 10 weeks to induce cirrhosis. After induction of cirrhosis, some rats received a single injection of lipopolysaccharide (LPS) to induce ACLF; some were given simvastatin or vehicle (control) 4 hours or 24 hours before induction of ACLF. We collected data on changes in hepatic and systemic hemodynamics, hepatic microvascular phenotype and function, and survival times. Liver tissues and plasma were collected and analyzed by immunoblots, quantitative polymerase chain reaction, immuno(fluoro)histochemistry and immunoassays. RESULTS: Administration of LPS aggravated portal hypertension in rats with cirrhosis by increasing the severity of intrahepatic microvascular dysfunction, exacerbating hepatic inflammation, increasing oxidative stress, and recruiting hepatic stellate cells and neutrophils. Rats with cirrhosis given LPS had significantly shorter survival times than rats with cirrhosis given the control. Simvastatin prevented most of ACLF-derived complications and increased survival times. Simvastatin appeared to increase hepatic sinusoidal function and reduce portal hypertension and markers of inflammation and oxidation. The drug significantly reduced levels of transaminases, total bilirubin, and ammonia, as well as LPS-mediated activation of hepatic stellate cells in liver tissues of rats with cirrhosis. CONCLUSIONS: In studies of rats with cirrhosis, we found administration of LPS to promote development of ACLF, aggravating the complications of chronic liver disease and decreasing survival times. Simvastatin reduced LPS-induced inflammation and liver damage in rats with ACLF, supporting its use in treatment of patients with advanced chronic liver disease.

Mitochondria-targeted antioxidant mitoquinone deactivates human and rat hepatic stellate cells and reduces portal hypertension in cirrhotic rats.

BACKGROUND & AIMS: In cirrhosis, activated hepatic stellate cells (HSC) play a major role in increasing intrahepatic vascular resistance and developing portal hypertension. We have shown that cirrhotic livers have increased reactive oxygen species (ROS), and that antioxidant therapy decreases portal pressure. Considering that mitochondria produce many of these ROS, our aim was to assess the effects of the oral mitochondria-targeted antioxidant mitoquinone on hepatic oxidative stress, HSC phenotype, liver fibrosis and portal hypertension. METHODS: Ex vivo: Hepatic stellate cells phenotype was analysed in human precision-cut liver slices in response to mitoquinone or vehicle. In vitro: Mitochondrial oxidative stress was analysed in different cell type of livers from control and cirrhotic rats. HSC phenotype, proliferation and viability were assessed in LX2, and in primary human and rat HSC treated with mitoquinone or vehicle. In vivo: CCl4 - and thioacetamide-cirrhotic rats were treated with mitoquinone (5 mg/kg/day) or the vehicle compound, DecylTPP, for 2 weeks, followed by measurement of oxidative stress, systemic and hepatic haemodynamic, liver fibrosis, HSC phenotype and liver inflammation. RESULTS: Mitoquinone deactivated human and rat HSC, decreased their proliferation but with no effects on viability. In CCl4 -cirrhotic rats, mitoquinone decreased hepatic oxidative stress, improved HSC phenotype, reduced intrahepatic vascular resistance and diminished liver fibrosis. These effects were associated with a significant reduction in portal pressure without changes in arterial pressure. These results were further confirmed in the thioacetamide-cirrhotic model. CONCLUSION: We propose mitochondria-targeted antioxidants as a novel treatment approach against portal hypertension and cirrhosis.

The anticoagulant rivaroxaban lowers portal hypertension in cirrhotic rats mainly by deactivating hepatic stellate cells.

In cirrhosis, increased intrahepatic vascular resistance (IHVR) is the primary factor for portal hypertension (PH) development. Hepatic stellate cells (HSCs) play a major role increasing IHVR because, when activated, they are contractile and promote fibrogenesis. Protease-activated receptors (PARs) can activate HSCs through thrombin and factor Xa, which are known PAR agonists, and cause microthrombosis in liver microcirculation. This study investigates the effects of the oral anticoagulant, rivaroxaban (RVXB), a direct antifactor Xa, on HSC phenotype, liver fibrosis (LF), liver microthrombosis, and PH in cirrhotic rats. Hepatic and systemic hemodynamic, nitric oxide (NO) bioavailability, LF, HSC activation, and microthrombosis were evaluated in CCl4 and thioacetamide-cirrhotic rats treated with RVXB (20 mg/kg/day) or its vehicle for 2 weeks. RVXB significantly decreased portal pressure (PP) in both models of cirrhosis without changes in portal blood flow, suggesting a reduction in IHVR. RVXB reduced oxidative stress, improved NO bioavailability, and ameliorated endothelial dysfunction. Rivaroxaban deactivated HSC, with decreased alpha-smooth muscle actin and mRNA expression of other HSC activation markers. Despite this marked improvement in HSC phenotype, no significant changes in LF were identified. RVXB markedly reduced fibrin deposition, suggesting reduced intrahepatic microthrombosis. CONCLUSION: RVXB decreases PP in two rat models of cirrhosis. This effect is mostly associated with decreased IHVR, enhanced NO bioavailability, HSC deactivation, and reduced intrahepatic microthrombosis. Our findings suggest that RVXB deserves further evaluation as a potential treatment for cirrhotic PH. (Hepatology 2017;65:2031-2044).

Local indicators of climate change: The potential contribution of local knowledge to climate research.

Local knowledge has been proposed as a place-based tool to ground-truth climate models and to narrow their geographic sensitivity. To assess the potential role of local knowledge in our quest to understand better climate change and its impacts, we first need to critically review the strengths and weaknesses of local knowledge of climate change and the potential complementarity with scientific knowledge. With this aim, we conducted a systematic, quantitative meta-analysis of published peer-reviewed documents reporting local indicators of climate change (including both local observations of climate change and observed impacts on the biophysical and the social systems). Overall, primary data on the topic are not abundant, the methodological development is incipient, and the geographical extent is unbalanced. On the 98 case studies documented, we recorded the mention of 746 local indicators of climate change, mostly corresponding to local observations of climate change (40%), but also to observed impacts on the physical (23%), the biological (19%), and the socioeconomic (18%) systems. Our results suggest that, even if local observations of climate change are the most frequently reported type of change, the rich and fine-grained knowledge in relation to impacts on biophysical systems could provide more original contributions to our understanding of climate change at local scale.

ß-Cell dedifferentiation, reduced duct cell plasticity, and impaired ß-cell mass regeneration in middle-aged rats.

Limitations in ß-cell regeneration potential in middle-aged animals could contribute to the increased risk to develop diabetes associated with aging. We investigated ß-cell regeneration of middle-aged Wistar rats in response to two different regenerative stimuli: partial pancreatectomy (Px + V) and gastrin administration (Px + G). Pancreatic remnants were analyzed 3 and 14 days after surgery. ß-Cell mass increased in young animals after Px and was further increased after gastrin treatment. In contrast, ß-cell mass did not change after Px or after gastrin treatment in middle-aged rats. ß-Cell replication and individual ß-cell size were similarly increased after Px in young and middle-aged animals, and ß-cell apoptosis was not modified. Nuclear immunolocalization of neurog3 or nkx6.1 in regenerative duct cells, markers of duct cell plasticity, was increased in young but not in middle-aged Px rats. The pancreatic progenitor-associated transcription factors neurog3 and sox9 were upregulated in islet ß-cells of middle-aged rats and further increased after Px. The percentage of chromogranin A+/hormone islet cells was significantly increased in the pancreases of middle-aged Px rats. In summary, the potential for compensatory ß-cell hyperplasia and hypertrophy was retained in middle-aged rats, but ß-cell dedifferentiation and impaired duct cell plasticity limited ß-cell regeneration.

Enoxaparin reduces hepatic vascular resistance and portal pressure in cirrhotic rats.

BACKGROUND & AIMS: Increased hepatic vascular resistance due to fibrosis and elevated hepatic vascular tone is the primary factor in the development of portal hypertension. Heparin may decrease fibrosis by inhibiting intrahepatic microthrombosis and thrombin-mediated hepatic stellate cell activation. In addition, heparin enhances eNOS activity, which may reduce hepatic vascular tone. Our study aimed at evaluating the effects of acute, short-, long-term and preventive enoxaparin administration on hepatic and systemic hemodynamics, liver fibrosis and nitric oxide availability in cirrhotic rats. METHODS: Enoxaparin (1.8 mg/kg subcutaneously), or its vehicle, was administered to CCl4-cirrhotic rats 24h and 1h before the study (acute), daily for 1 week (short-term) or daily for 3 weeks (long-term) and to thioacetamide-cirrhotic rats daily for 3 weeks with/without thioacetamide (preventive/long-term, respectively). Mean arterial pressure, portal pressure, portal blood flow, hepatic vascular resistance and molecular/cellular mechanisms were evaluated. RESULTS: No significant changes in hemodynamic parameters were observed in acute administration. However, one-week, three-week and preventive treatments significantly decreased portal pressure mainly due to a decrease in hepatic vascular resistance without significant changes in mean arterial pressure. These findings were associated with significant reductions in liver fibrosis, hepatic stellate cell activation, and desmin expression. Moreover, a reduction in fibrin deposition was observed in enoxaparin-treated rats, suggesting reduced intrahepatic microthrombosis. CONCLUSION: Enoxaparin reduces portal pressure in cirrhotic rats by improving the structural component of increased liver resistance. These findings describe the potentially beneficial effects of enoxaparin beyond the treatment/prevention of portal vein thrombosis in cirrhosis, which deserve further investigation.

Resveratrol improves intrahepatic endothelial dysfunction and reduces hepatic fibrosis and portal pressure in cirrhotic rats.

BACKGROUND & AIMS: Resveratrol, a polyphenol found in a variety of fruits, exerts a wide range of beneficial effects on the endothelium, regulates multiple vasoactive substances and decreases oxidative stress, factors involved in the pathophysiology of portal hypertension. Our study aimed at evaluating the effects of resveratrol on hepatic and systemic hemodynamics, hepatic endothelial dysfunction, and hepatic fibrosis in CCl4 cirrhotic rats. METHODS: Resveratrol (10 and 20 mg/kg/day) or its vehicle was administered to cirrhotic rats for two weeks and hepatic and systemic hemodynamics were measured. Moreover, we evaluated endothelial function by dose-relaxation curves to acetylcholine, hepatic NO bioavailability and TXA2 production. We also evaluated liver fibrosis by Sirius Red staining of liver sections, collagen-1, NFκB, TGFß mRNA expression, and desmin and α-smooth muscle actin (α-SMA) protein expression, as a surrogate of hepatic stellate cell activation. RESULTS: Resveratrol administration significantly decreased portal pressure compared to vehicle (12.1 ± 0.9 vs. 14.3 ± 2.2 mmHg; p <0.05) without significant changes in systemic hemodynamics. Reduction in portal pressure was associated with an improved vasodilatory response to acetylcholine, with decreased TXA2 production, increased endothelial NO, and with a significant reduction in liver fibrosis. The decrease in hepatic fibrosis was associated with a reduced collagen-1, TGFß, NFκB mRNA expression and desmin and α-SMA protein expression. CONCLUSIONS: Resveratrol administration reduces portal pressure, hepatic stellate cell activation and liver fibrosis, and improves hepatic endothelial dysfunction in cirrhotic rats, suggesting it may be a useful dietary supplement in the treatment of portal hypertension in patients with cirrhosis.

Gastrin treatment stimulates ß-cell regeneration and improves glucose tolerance in 95% pancreatectomized rats.

ß-Cell mass reduction is a central aspect in the development of type 1 and type 2 diabetes, and substitution or regeneration of the lost ß-cells is a potentially curative treatment of diabetes. To study the effects of gastrin on ß-cell mass in rats with 95% pancreatectomy (95%-Px), a model of pancreatic regeneration, rats underwent 95% Px or sham Px and were treated with [15 leu] gastrin-17 (Px+G and S+G) or vehicle (Px+V and S+V) for 15 d. In 95% Px rats, gastrin treatment reduced hyperglycemia (280 ± 52 mg vs. 436 ± 51 mg/dl, P < 0.05), and increased ß-cell mass (1.15 ± 0.15 mg)) compared with vehicle-treated rats (0.67 ± 0.15 mg, P < 0.05). Gastrin treatment induced ß-cell regeneration by enhancing ß-cell neogenesis (increased number of extraislet ß-cells in Px+G: 0.42 ± 0.05 cells/mm(2) vs. Px+V: 0.27 ± 0.07 cells/mm(2), P < 0.05, and pancreatic and duodenal homeobox 1 expression in ductal cells of Px+G: 1.21 ± 0.38% vs. Px+V: 0.23 ± 0.10%, P < 0.05) and replication (Px+G: 1.65 ± 0.26% vs. S+V: 0.64 ± 0.14%; P < 0.05). In addition, reduced ß-cell apoptosis contributed to the increased ß-cell mass in gastrin-treated rats (Px+G: 0.07 ± 0.02%, Px+V: 0.23 ± 0.05%; P < 0.05). Gastrin action on ß-cell regeneration and survival increased ß-cell mass and improved glucose tolerance in 95% Px rats, supporting a potential role of gastrin in the treatment of diabetes.