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Background & Aims: Ductular reaction expansion is associated with poor prognosis in patients with advanced liver disease. However, the mechanisms promoting biliary cell proliferation are largely unknown. Here, we identify neutrophils as drivers of biliary cell proliferation and the defective wound-healing response., Methods: The intrahepatic localization of neutrophils was evaluated in patients with chronic liver disease. Neutrophil dynamics were analyzed by intravital microscopy and neutrophil-labeling assays in DDC-treated mice. Neutrophil depletion or inhibition of recruitment was achieved using a Ly6g antibody or a CXCR1/2 inhibitor, respectively. Mice deficient in PAD4 (peptidyl arginine deiminase 4) and ELANE/NE (neutrophil elastase) were used to investigate the mechanisms underlying ductular reaction expansion., Results: In this study we describe a population of ductular reaction-associated neutrophils (DRANs), which are in direct contact with biliary epithelial cells in chronic liver diseases and whose numbers increased in parallel with disease progression. We show that DRANs are immobilized at the site of ductular reaction for a prolonged period of time. In addition, liver neutrophils display a unique phenotypic and transcriptomic profile, showing a decreased phagocytic capacity and increased oxidative burst. Depletion of neutrophils or inhibition of their recruitment reduces DRANs and the expansion of ductular reaction, while mitigating liver fibrosis and angiogenesis. Mechanistically, neutrophils deficient in PAD4 and ELANE abrogate neutrophil-induced biliary cell proliferation, thus indicating the role of neutrophil extracellular traps and elastase release in ductular reaction expansion., Conclusions: Overall, our study reveals the accumulation of DRANs as a hallmark of advanced liver disease and a potential therapeutic target to mitigate ductular reaction and the maladaptive wound-healing response., Impact and Implications: Our results indicate that neutrophils are highly plastic and can have an extended lifespan. Moreover, we identify a new role of neutrophils as triggers of expansion of the biliary epithelium. Overall, the results of this study indicate that ductular reaction-associated neutrophils (or DRANs) are new players in the maladaptive tissue-healing response in chronic liver injury and may be a potential target for therapeutic interventions to reduce ductular reaction expansion and promote tissue repair in advanced liver disease., (Copyright © 2023 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Proteases are the most abundant enzyme gene family in vertebrates and they execute essential functions in all living organisms. Their main role is to hydrolase the peptide bond within proteins, a process also called proteolysis. Contrary to the conventional paradigm, proteases are not only random catalytic devices, but can perform highly selective and targeted cleavage of specific substrates, finely modulating multiple essential cellular processes. Lysosomal protease cathepsins comprise 3 families of proteases that preferentially act within acidic cellular compartments, but they can also be found in other cellular locations. They can operate alone or as part of signalling cascades and regulatory circuits, playing important roles in apoptosis, extracellular matrix remodelling, hepatic stellate cell activation, autophagy and metastasis, contributing to the initiation, development and progression of liver disease. In this review, we comprehensively summarise current knowledge on the role of lysosomal cathepsins in liver disease, with a particular emphasis on liver fibrosis, non-alcoholic fatty liver disease and hepatocellular carcinoma., Competing Interests: Conflict of interest The authors have no conflict of interest to disclose. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Portal hypertension is the main cause of complications in patients with chronic liver disease. Over the past 25 years, progress in the understanding of the pathophysiology of portal hypertension was followed by the introduction of an effective pharmacological therapy, consisting mainly of treatments aimed at correcting the increased splanchnic blood flow. It is only recently that this paradigm has been changed. Progress in our knowledge of the mechanisms of increased resistance to portal blood flow, of the formation of portal-systemic collaterals, and of mechanisms other than vasodilatation maintaining the increased splanchnic blood flow have opened entirely new perspectives for developing more effective treatment strategies. This is the aim of the current review, which focuses on: (a) the modulation of hepatic vascular resistance by correcting the increased hepatic vascular tone due to hepatic endothelial dysfunction, and (b) correcting the abnormal angiogenesis associated with portal hypertension, which contributes to liver inflammation and fibrogenesis, to the hyperkinetic splanchnic circulation, and to the formation of portal-systemic collaterals and varices., (Copyright 2010 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Angiogenesis and disruption of liver vascular architecture have been linked to progression to cirrhosis and liver cancer (HCC) in chronic liver diseases, which contributes both to increased hepatic vascular resistance and portal hypertension and to decreased hepatocyte perfusion. On the other hand, recent evidence shows that angiogenesis modulates the formation of portal-systemic collaterals and the increased splanchnic blood flow which are involved in the life threatening complications of cirrhosis. Finally, angiogenesis plays a key role in the growth of tumours, suggesting that interference with angiogenesis may prevent or delay the development of HCC. This review summarizes current knowledge on the molecular mechanisms of liver angiogenesis and on the consequences of angiogenesis in chronic liver disease. On the other hand, it presents the different strategies that have been used in experimental models to counteract excessive angiogenesis and its potential role in preventing transition to cirrhosis, development of portal hypertension and its consequences, and its application in the treatment of hepatocellular carcinoma.

Background/aims: The transforming growth factor-beta (TGF-beta) induces apoptosis in hepatocytes through an oxidative stress process. Here, we have analyzed the role of different NADPH oxidase isoforms in the intracellular signalling induced by TGF-beta in hepatocytes, to later explore whether this mechanism is altered in liver tumor cells., Methods: Primary cultures of rat and human hepatocytes, HepG2 and Hep3B cells were used in in vitro studies to analyze the TGF-beta response., Results: TGF-beta-induced apoptosis in rat hepatocytes does not require Rac-dependent NADPH oxidases. TGF-beta upregulates the Rac-independent Nox4, which correlates with its pro-apoptotic activity. Regulation of Nox4 occurs at the transcriptional level and is counteracted by intracellular survival signals. siRNA targeted knock-down of Nox4 attenuates NADPH oxidase activity, caspase activation and cell death in rat hepatocytes. NOX4 upregulation by TGF-beta is also observed in human hepatocytes, coincident with apoptosis. In human hepatocellular carcinoma (HCC) cell lines, NOX4 upregulation by TGF-beta is only observed in cells that are sensitive to its cytotoxic effect, such as Hep3B cells. siRNA targeted knock-down of NOX4 in these cells impairs TGF-beta-induced apoptosis., Conclusions: Upregulation of NOX4 by TGF-beta is required for its pro-apoptotic activity in hepatocytes. Impairment of this TGF-beta-induced response might confer apoptosis resistance in HCC cells.

Background/aims: Tetrahydrobiopterin is an essential cofactor for NOS enzymes to synthesize NO. It has been suggested that reduced intrahepatic tetrahydrobiopterin decreases intrahepatic NO and contributes to increase hepatic vascular resistance and portal pressure in cirrhosis. The main aim of the study was to evaluate the effect of tetrahydrobiopterin supplementation in portal pressure in CCl4 cirrhotic rats., Methods: Cirrhotic rats received vehicle or tetrahydrobiopterin (10mg/kg/day i.p.) for 3 days. Hepatic and systemic hemodynamics and hepatic tetrahydrobiopterin, NOS activity and cGMP levels were measured. In addition, hepatic and systemic hemodynamics were evaluated in normal rats in which tetrahydrobiopterin deficiency was induced by administrating 2,4-diamino-6-hydroxy-pyrimidine (DAHP) for 8h., Results: In cirrhotic rats, tetrahydrobiopterin administration increased liver NOS activity and cGMP levels and markedly and significantly reduced portal pressure. Amelioration of portal hypertension was associated with a normalization of arterial pressure. In normal rats DAHP decreased hepatic tetrahydrobiopterin and NOS activity and increased hepatic vascular tone. These effects of DAHP administration were corrected by tetrahydrobiopterin supplementation., Conclusions: The present study shows that tetrahydrobiopterin markedly reduces portal hypertension and improves systemic hemodynamics in cirrhotic rats. These data support the concept that tetrahydrobiopterin supplementation may represent a new therapeutic strategy for portal hypertension.

Background/aims: This study aimed to investigate: (i) whether the hyperkinetic circulation that develops after portacaval shunt is associated with decreased vascular sensitivity to vasoconstrictors and (ii) the role of nitric oxide on its pathogenesis., Methods: Portacaval-shunted and sham-operated rats received long-term treatment with the nitric oxide inhibitor L-NAME (osmotic minipump) or its inactive enantiomer D-NAME. Measurements of arterial pressure, cardiac output and superior mesenteric artery blood flow (transit-time flow probe) were done 4 days later in baseline conditions and after increasing doses of methoxamine. Peripheral and superior mesenteric vascular resistance were calculated., Results: Portacaval shunted rats showed a significantly lower peripheral and superior mesenteric vascular resistance and a significant reduction in their response to incremental doses of methoxamine than sham-operated controls. Chronic nitric oxide inhibition attenuated the systemic but not the splanchnic vasodilatation and totally corrected the hyposensitivity to methoxamine of portacaval-shunted rats. However, they still had a significantly lower peripheral and superior mesenteric vascular resistance than sham-operated rats., Conclusions: This study shows that the splanchnic and systemic hyporesponsiveness to methoxamine observed in portacaval-shunted rats could be explained by an excess of nitric oxide. However, other factors may be involved in maintaining splanchnic and systemic vasodilatation despite NO-inhibition.

Background/aims: It has been suggested that mechanical pumping of portal blood to the liver may correct portal hypertension while increasing portal flow to the liver, which may enhance liver function in cirrhosis. However, the hemodynamic effects of this procedure are unknown. The present study investigated these issues in rats with portal hypertension due to portal vein stenosis., Methods: Mechanical pumping of portal blood to the liver was established by an extracorporeal shunt bypassing the portal vein stenosis, connected to a continuous withdrawal/infusion pump. Portal pressure, portal-systemic shunting (mesenteric injection of Cr-51 microspheres, n = 10), mesenteric artery blood flow (perivascular Transonic flowmeter, n = 7) and systemic hemodynamics and regional blood flows (left ventricle injection of Ce-141 microspheres, n = 15), were measured at pumping rates of 0, 3 and 6 ml.min-1., Results: Mechanical pumping of portal blood to the liver caused a marked decrease in portal pressure (from 17 +/- 1 to 12.6 +/- 1 and 9.4 +/- .9 mmHg at pumping rates of 0, 3 and 6 ml.min-1) and portal-systemic shunting (from 97 +/- 4 to 70 +/- 4 and 51 +/- 6% respectively) (p < 0.001). However, there were no significant changes in mesenteric artery flow (5.5 +/- 3 vs 5.6 +/- 3 ml.min-1.100 g-1), suggesting that all blood pumped to the liver was withdrawn from that circulating through the collaterals. Moreover, there were no changes in mean arterial pressure, cardiac index, peripheral resistance and splanchnic arteriolar resistance., Conclusions: The short-term mechanical pumping of portal blood to the liver effectively decreases portal pressure and portal-systemic shunting and has no significant effects on systemic and splanchnic hemodynamics in portal hypertensive rats.

The present study investigated the hemodynamic effects of nicardipine, a new calcium channel blocker, and placebo in 14 patients with cirrhosis. Sixty minutes after nicardipine administration (20 mg orally; n = 8), there was a significant increase in hepatic blood flow (25 +/- 21%; p < 0.05) and azygos blood flow (33 +/- 40%; p < 0.05) but no significant change in the hepatic venous pressure gradient. As a result of the increase in hepatic blood flow and the lack of change in the hepatic venous pressure gradient, nicardipine significantly reduced hepatic sinusoidal resistance (-14 +/- 15%; p < 0.05). Enhanced liver perfusion was associated with a significant increase in the hepatic clearance of indocyanine green (from 241 +/- 81 to 265 +/- 92 ml/min, p < 0.05). A mild, well-tolerated decrease in mean arterial pressure (-10 +/- 6%, p < 0.05), without significant changes in cardiac output, systemic vascular resistance and heart rate, was also observed. Placebo administration (n = 6) did not cause significant changes in systemic or hepatic hemodynamics. The results of the present study show that nicardipine, unlike other calcium channel blockers, effectively increases hepatic blood flow and the hepatic clearance of indocyanine green in patients with cirrhosis. The acute beneficial effects of nicardipine should be confirmed in chronic studies.

To alleviate the risk of variceal bleeding, the portal pressure gradient--usually evaluated as the hepatic venous pressure gradient (HVPG)--must be reduced to < or = 12 mmHg. Although beta-blocking agents are accepted therapy for preventing first or subsequent bleeding episodes, propranolol therapy decreases final HVPG to < or = 12 mmHg in only 12% of patients, while only 24% of patients have a > or = 20% reduction in HVPG and nearly 40% show no reduction in HVPG. This has stimulated research on alternative or additional treatments. Nitrates such as isosorbide dinitrate reduce portal pressure by decreasing resistance to portal and collateral blood flow and by promoting reflex splanchnic vasoconstriction. However, while nitrates are effective in the acute situation, tolerance leading to refractoriness develops over the long term unless they are combined with diuretics or other agents in the treatment of portal hypertension. Propranolol and isosorbide-5-mononitrate combined cause a substantially greater reduction in HVPG than monotherapy with either drug in both acute and long-term use. Presumably concomitant isosorbide-5-mononitrate administration opposes the increase in portal resistance induced by propranolol. Spironolactone, which has been shown to lower HVPG in patients with cirrhosis, produces a reduction in plasma volume that attenuates the increased cardiac output associated with cirrhosis and triggers vasoactive mechanisms that decrease splanchnic blood flow. Potentially, spironolactone may maintain and enhance the decrease in portal pressure achieved by nitrates or propranolol. Triple therapy with a beta-blocker, a nitrate and spironolactone may be feasible.(ABSTRACT TRUNCATED AT 250 WORDS)