Search

Background and Aims: The chemokines CCL19 and CCL21 bind CCR7, which is involved in the organization of secondary lymphoid tissue and is expressed during chronic tissue inflammation. We investigated the expression of CCL21 and CCR7 in chronic hepatitis C. The effects of CCL21 on hepatic stellate cells (HSCs) were also studied., Methods: Expression of CCL21 was assessed by in situ hybridization and immunohistochemistry. CCR7 on T cells was analyzed by flow cytometry. Cultured human HSCs were studied in their activated phenotype., Results: In patients with chronic hepatitis C, expression of CCL21 and CCR7 was up-regulated. CCL21 was detected in the portal tracts and around inflammatory lymphoid follicles, in proximity to T lymphocytes and dendritic cells, which contributed to expression of this chemokine. Expression of CCR7 was also increased in patients with primary biliary cirrhosis. Intrahepatic CD8(+) T lymphocytes isolated from patients with chronic hepatitis C had a significantly higher percentage of positivity for CCR7 than those from healthy controls, and the expression of CCR7 was associated with that of CXCR3. Cultured HSCs expressed functional CCR7, the activation of which stimulated cell migration and accelerated wound healing in an in vitro model. Exposure of HSCs to CCL21 triggered several signaling pathways, including extracellular signal-regulated kinase, Akt, and nuclear factor kappaB, resulting in induction of proinflammatory genes., Conclusions: Expression of CCL21 during chronic hepatitis C is implicated in the recruitment of T lymphocytes and the organization of inflammatory lymphoid tissue and may promote fibrogenesis in the inflamed areas via activation of CCR7 on HSCs.

Background & Aims: Proliferation and migration of hepatic stellate cells (HSCs) and expression of chemokines are involved in the pathogenesis of liver inflammation and fibrogenesis. Peroxisome proliferator-activated receptor (PPAR)-gamma is a receptor transcription factor that controls growth and differentiation in different tissues. We explored the effects of PPAR-gamma agonists on the biological actions of cultured human HSCs., Methods: HSCs were isolated from normal human liver tissue and used in their myofibroblast-like phenotype or immediately after isolation. Activation of PPAR-gamma was induced with 15-deoxy-Delta(12, 14)-prostaglandin J(2) or with troglitazone., Results: PPAR-gamma agonists dose-dependently inhibited HSC proliferation and chemotaxis induced by platelet-derived growth factor. This effect was independent of changes in postreceptor signaling or expression of c-fos and c-myc and was associated with inhibition of cell cycle progression beyond the G(1) phase. Activation of PPAR-gamma also resulted in a complete inhibition of the expression of monocyte chemotactic protein 1 at the gene and protein levels. Comparison of quiescent and culture-activated HSCs revealed a marked decrease in PPAR-gamma expression in activated cells., Conclusions: Activation of PPAR-gamma modulates profibrogenic and proinflammatory actions in HSCs. Reduced PPAR-gamma expression may contribute to confer an activated phenotype to HSCs.

Background & Aims: Nitrovasodilators have been proposed for the treatment of portal hypertension alone or in combination with beta-blockers. In addition to their vasodilatory properties, nitric oxide (NO) donors may exert direct antifibrogenic properties. We evaluated the effect of nitroglycerin (NTG) and S-nitroso-N-acetyl penicillamine (SNAP) on the mitogenic and chemotactic properties of platelet-derived growth factor (PDGF)-BB and the modulation of the relative intracellular signaling pathways in fully activated human hepatic stellate cells (HSCs), a cell type that plays an active role in liver fibrogenesis and portal hypertension., Methods & Results: Both NTG and SNAP induced a dose-dependent decrease in PDGF-induced DNA synthesis and cell migration, which was associated with a decrease in PDGF-induced intracellular Ca(2+) increase and extracellular signal-regulated kinase (ERK) activity. These effects were not related to activation of the classic soluble guanylate cyclase (sGC)/guanosine 3',5'-cyclic monophosphate pathway; accordingly, Western blot analysis of HSC lysates revealed the absence of the alpha(1)beta(1) ubiquitous subunits of sGC, whereas they were detectable in quiescent HSCs, freshly isolated from normal human liver. Conversely, both NTG and SNAP induced a more than 10-20-fold increase in prostaglandin E(2) in cell supernatants within 1 minute, associated with an increase in intracellular adenosine 3',5'-cyclic monophosphate levels. Accordingly, the inhibitory effects of NO donors on PDGF action and signaling were eliminated after preincubation with ibuprofen., Conclusions: These results suggest that NO donors may exert a direct antifibrogenic action by inhibiting proliferation, motility, and contractility of HSCs in addition to a reduction of fibrillar extracellular matrix accumulation.

Background & Aims: Hepatocellular carcinoma is a common complication in liver cirrhosis. The integrin alpha6 beta1, a receptor for the laminin family of extracellular matrix proteins, has been found to be overexpressed in hepatocarcinoma. In an effort to further characterize the involvement of alpha6 beta1-integrin in hepatocarcinoma progression and to study alpha6 beta1-mediated functions, a human hepatocarcinoma cell line, HepG2, that express high surface levels of alpha6 beta1 and uses only this integrin to mediate adhesion on laminin was identified., Methods: To assess the role of alpha6 beta1 in these cells, a cytoplasmic domain deletion mutant of the beta4-integrin subunit by complementary DNA transfection was expressed. The expression of the mutant beta4 subunit in association with endogenous alpha6 showed a dominant-negative effect on alpha6 beta1 expression., Results: Stable transfectants of HepG2 that expressed the mutant beta4 subunit showed a reduced ability to adhere and migrate on laminin matrices and to invade Matrigel. Furthermore, transfected cells showed significantly lower growth rates and reduced anchorage-independent growth compared with mock-transfected cells., Conclusions: These findings on the expression and function of alpha6 beta1 in hepatocarcinoma cells emphasize the potential contribution of this laminin receptor in the neoplastic transformation of hepatocytes.

Background/aims: Platelet-activating factor (PAF) is an important mediator of proinflammatory cell-to-cell interactions with powerful vasoactive properties. We evaluated the biosynthesis of PAF by cultured human fat-storing cells (FSC), liver-specific pericytes involved in the inflammatory and fibrogenic process of liver tissue., Methods: PAF synthesis was evaluated by measuring [3H]acetate incorporation under basal conditions and upon stimulation with A23187, thrombin, and lipopolysaccharide. Further analysis of PAF species synthesized by FSC was performed using gas chromatography/mass spectrometry., Results: All stimuli induced a significant increase of basal PAF synthesis by FSC. Further analysis showed that > 50% of the newly synthesized PAF species was secreted whereas the remaining fraction was cell-associated. PAF species produced by FSC were able to induce aggregation of rabbit washed platelets with an effectiveness correspondent to 10(-9) mol/L authentic PAF. Gas chromatography/mass spectrometry analysis revealed that a large percentage (74%) of PAF-like lipids synthesized by FSC consisted of 1O-acyl PAF. Finally, stimulation of FSC with PAF caused an increase in cytosolic free calcium, thus suggesting a possible involvement of this pericyte in the well-known effects of PAF on portal pressure., Conclusions: These results expand the available knowledge concerning the role of PAF in conditions characterized by extensive activation and damage of the liver sinusoidal endothelium and decreased hepatic scavenger activity.

Background: Patients with advanced cirrhosis show defective platelet aggregation, which is dependent, at least in part, on intrinsic platelet abnormalities. The aim of this study was to evaluate the activating and inhibitory pathways of platelet signal transduction in cirrhotic patients., Methods: Twelve cirrhotic patients and 12 control subjects participated in this study. Measurements were performed on washed platelets., Results: Thrombin-stimulated inositol 1,4,5-trisphosphate production was reduced fivefold, and the increase in cytosolic calcium concentration was significantly lower in platelets from cirrhotic patients following stimulation with thrombin, platelet activating factor, or U-46619. In addition, the activity of the platelet Na+/H+ antiporter, evaluated after an acid load, was significantly lower in platelets from cirrhotic patients (0.90 +/- 0.19 vs. 1.37 +/- 0.16 delta pHi/min, P = 0.07). Cirrhotic patients also showed a significantly increased basal intraplatelet content of both 5'-cyclic adenosine monophosphate (cAMP) (2724 +/- 330 vs. 1561 +/- 258 fmol/10(8) platelets, P < 0.05) and 5'-cyclic guanosine monophosphate (cGMP) (217 +/- 18 vs. 159 +/- 29 fmol/10(8) platelets, P < 0.05)., Conclusions: Our results indicate that in platelets from cirrhotic patients, defective early signal transduction is associated with an increase in platelet cAMP and cGMP, thus revealing new mechanisms contributing to the defective platelet function in this disease.

Background: Several alterations of polymorphonuclear leukocyte (PMN) function were found in alcoholic cirrhotics that may contribute to augmented susceptibility to infections. We evaluated function and synthesis of lipid mediators in PMN obtained from nonalcoholic cirrhotics., Methods: We evaluated the phagocytic and chemotactic response together with superoxide anion (O2-), leukotriene B4, (LTB4) and platelet-activating factor (PAF) production in response to different stimuli in PMN from nonalcoholic cirrhotics as compared with controls., Results: PMN from cirrhotics showed, after stimulation with opsonized zymosan (STZ) and phorbol-12-myristate-13-acetate, a reduced capacity to produce O2- when compared with controls. [3H]acetate incorporation into PAF was significantly higher in PMN obtained from controls in respect to cirrhotics. Gas chromatography/mass spectrometry analysis confirmed a reduced PAF synthesis by PMN obtained from cirrhotics. LTB4 production from PMN, after stimulation with calcium ionophore (A23187) and STZ, was significantly reduced in cirrhotics. [3H]arachidonic acid release from prelabeled PMN, measured upon stimulation with A23187 and STZ, was higher in controls than in cirrhotics., Conclusions: An altered synthesis of LTB4 and PAF is associated with an impaired O2- production by PMN in nonalcoholic cirrhosis. Reduced synthesis of lipid mediators may be related to an altered phospholipase A, activity.

This study was undertaken to investigate the role of increased renal thromboxane (TX) A2 production in modulating renal hemodynamics and sodium and water retention in cirrhotic patients with ascites. In a randomized, double-blind, placebo-controlled, crossover trial, 15 nonazotemic cirrhotic patients with ascites and elevated urinary TXB2 excretion received the thromboxane-receptor antagonist ONO-3708 (3 micrograms.kg-1.min-1) in a 4-hour continuous infusion. Administration of ONO-3708 significantly blocked TXA2 receptors; bleeding time showed a twofold increase (432 +/- 65 vs. 131 +/- 17 seconds; P less than 0.005), and platelet aggregation to U-46619 (an agonist of TXA2 receptors) was abolished in all patients studied. The drug induced a significant increase in free water clearance (3.06 +/- 0.70 vs. 1.72 +/- 0.57 mL/min; P less than 0.001) and diuresis (4.74 +/- 0.79 vs. 3.94 +/- 0.66 mL/min; P less than 0.05) compared with placebo, as well as a significant (14%) increase in renal plasma flow. The increases in both free water clearance and diuresis induced by ONO-3708 were directly related to basal urinary TXB2 excretion. These results suggest a role for renal TXA2 as a modulator of water handling in cirrhotic patients with ascites.

The mechanisms underlying the defective platelet function in cirrhotic patients were investigated. Eleven cirrhotic patients with mild disease (group 1), 20 patients with severe cirrhosis (group 2), and 31 controls were studied. Platelet aggregation was significantly reduced in cirrhotics compared with controls. Compared with controls, cirrhotic patients in group 2 showed a significant reduction in the total content of adenosine triphosphate (57.8 +/- 7.8 vs. 26.1 +/- 6.3 mumol/10(11) platelets; P less than 0.05), 5-hydroxytryptamine (285 +/- 26 vs. 104 +/- 38 nmol/10(11) platelets; P less than 0.05), beta-thromboglobulin (2129 +/- 120 vs. 1223 +/- 161 ng/10(8) platelets; P less than 0.01), and platelet factor 4 (1389 +/- 108 vs. 805 +/- 176 ng/10(8) platelets; P less than 0.05). In patients with severe disease, an increase in plasma beta-thromboglobulin-platelet factor 4 ratio, an index of in vivo platelet activation, was observed (controls, 3.50 +/- 0.50; group 1, 4.02 +/- 0.80; and group 2, 6.59 +/- 1.15). Our data indicate the existence of a platelet storage pool defect, which may favor the bleeding tendency of cirrhotic patients.

Nonsteroidal antiinflammatory drugs impair renal function in susceptible patients with cirrhosis and ascites. A new antiinflammatory drug, sulindac, is reported not to affect renal function. To evaluate its renal-sparing mechanism, sulindac was administered for 5 days and ibuprofen for 1 day to 10 patients and paraaminohippurate and inulin clearances, serum and urine eicosanoids, and serum and urine sulindac metabolites were monitored. Ibuprofen reduced renal clearances in the 5 subjects with greatest sodium retention, whereas sulindac had no effect. Plasma concentration of the active sulfide metabolite was markedly increased in liver patients, and this concentration correlated with the inhibition of serum thromboxane (r = 0.75, p = 0.01). The percent inhibition of serum thromboxane with sulindac administration correlated with the inhibition of urinary eicosanoids (r = 0.68-0.81, all p less than 0.02). Ibuprofen was generally a more potent inhibitor of serum and urine eicosanoids. Thus, a major factor in the renal-sparing effect of sulindac appears to be its less potent inhibition of renal and extrarenal cyclooxygenase systems.

Furosemide occasionally causes azotemia in patients with ascites, independently of induced volume depletion. To define this effect, we measured renal clearances in patients with chronic liver disease and ascites and in nonascitic controls. Furosemide (80 mg i.v.) transiently increased p-aminohippurate clearance in controls (from 693 +/- 67 to 928 +/- 93 ml/min) and in 11 patients with ascites (from 418 +/- 81 to 526 +/- 80 ml/min). In contrast, in 13 patients with ascites, p-aminohippurate clearance fell by 34% (from 545 +/- 51 to 360 +/- 24 ml/min) within 20 min and by 41% within 60 min, and inulin clearance fell by 19% at 20 min and by 30% at 60 min. The renal effects lasted approximately 4 h. The renal response could not be predicted by renin activity, urinary prostaglandin excretion, urinary sodium, or clinical characteristics. In all 14 patients who received oral furosemide, p-aminohippurate clearance fell within 90 min (by 24%) and remained suppressed for at least 4 h. These immediate effects of furosemide on renal perfusion may contribute to azotemia in some patients with ascites.

We investigated the effect of a continuous infusion (50 micrograms as an initial bolus followed by a maintenance infusion at a rate of 0.1 micrograms/min.kg body wt for 45 min) of synthetic human atrial natriuretic factor (hANF) on renal hemodynamics and the renin-angiotensin-aldosterone system in 15 cirrhotic patients with ascites. Basal hANF levels were higher in cirrhotic patients when compared with normal values. Human atrial natriuretic factor infusion induced a significant decrease in mean blood pressure (from 77.8 +/- 1.1 to 68.6 +/- 1.5 mmHg, p less than 0.001) and a significant increase in heart rate (from 76.4 +/- 2.7 to 89.8 +/- 2.4 beats/min, p less than 0.001) in the patients studied. A remarkable increase in natriuresis (i.e., greater than or equal to 200 muEq/min) was observed in 5 patients (responders), whereas the infusion did not modify sodium excretion (i.e., less than or equal to 20 muEq/min) in 6 patients (nonresponders) and induced an intermediate response in 4 patients. Human atrial natriuretic factor-induced natriuresis was related to changes in renal hemodynamics that occurred during hANF infusion. In responders, the extent of the natriuretic response paralleled the increase of effective renal plasma flow and glomerular filtration rate; in non-responders the absent natriuretic response was associated with an evident reduction of these parameters. The reduction of blood pressure was similar in responders and nonresponders, but in the latter group it was followed by a marked increase of plasma renin activity and heart rate. It is likely that in nonresponders the natriuretic effect of hANF was blunted by the hemodynamic and hormonal changes triggered by the concomitant hANF-induced hypotension. This probably occurs in the presence of a greater reduction of effective arterial blood volume, as suggested by the higher baseline levels of plasma renin activity and the inability to increase free water excretion after a water load observed in nonresponders.

To assess the effects of intrarenal thromboxane A2 generation on furosemide-induced sodium and water excretion we administered furosemide (40 mg i.v.) to 8 nonazotemic cirrhotic patients with ascites and 8 healthy subjects before and after the administration of OKY 046 (200 mg twice orally), a powerful thromboxane-synthase inhibitor. Selective thromboxane-synthase inhibition significantly reduced basal and postfurosemide (1 h) urinary thromboxane B2 excretion in healthy subjects (65% before and 62% after furosemide) as well as in cirrhotic patients (52% before and 67% after furosemide) without affecting urinary prostaglandin E2 and 6-keto prostaglandin F1 alpha excretion. During the first hour after furosemide administration, OKY 046 administration significantly enhanced postfurosemide water excretion (milliliters per minute) in both healthy subjects (from 8.5 +/- 2.0 to 11.6 +/- 2.1, p less than 0.001) and cirrhotic patients (from 1.1 +/- 0.8 to 4.2 +/- 0.5, p less than 0.005), whereas furesemide-induced natriuresis (microequivalents per minute) was significantly increased only in the latter group (from 973 +/- 125 to 1405 +/- 121, p less than 0.05). Our data indicate that intrarenal thromboxane A2 generation, elicited by furosemide administration, may reduce the effects of the drug on water and sodium diuresis. Such a reduction seems to be more marked in the presence of an activated intrarenal prostaglandin system, suggesting that renal thromboxane A2 may represent an additional factor in conditioning the impaired responsiveness to furosemide, which is frequently observed in cirrhotic patients with ascites.

To assess the possible role of increased renal thromboxane A2 (TXA2) synthesis in nonazotemic patients with cirrhosis and ascites and to establish the potential beneficial effect of inhibitors of renal TXA2 production in this clinical setting, we administered OKY 046, a selective TXA2 synthase inhibitor, 200 mg t.i.d. for 5 days, to 9 nonazotemic cirrhotic patients with ascites and avid sodium retention. OKY 046 inhibited platelet TXA2 production, as expressed by serum thromboxane B2 (TXB2) concentration, by approximately 85% (p less than 0.001 vs. baseline values) and reduced urinary TXB2 excretion by 72% (p less than 0.01). A significant increase of approximately 19% in inulin clearance was observed during the treatment (from 61.0 +/- 8.42 to 72.7 +/- 7.45 ml/min, p less than 0.05), whereas renal blood flow was unchanged (from 408.50 +/- 19.97 to 424.50 +/- 30.84 ml/min). Drug administration did not affect positive sodium balance [sodium excretion was 4.67 +/- 1.22 mEq/day before drug administration and 6.26 +/- 1.05 mEq/day during drug administration (on day 7)], plasma renin activity, plasma aldosterone concentration, or the urinary excretion of prostaglandin E2, 6-keto prostaglandin F1 alpha, or prostaglandin F2 alpha. These results suggest that renal TXA2 synthesis contributes to the regulation of renal hemodynamics in nonazotemic cirrhotic patients with ascites and avid sodium retention, but it does not seem to affect sodium balance.

Some patients with chronic alcoholic liver disease and ascites have an impaired natriuretic response to furosemide. To elucidate the mechanism of this diuretic resistance, we measured para-aminohippurate and inulin clearances and urinary excretion of electrolytes, prostaglandin E2, and furosemide after intravenous administration of 80 mg of furosemide in 26 patients. The natriuretic response was variable (3.3-172 mEq/h) and was unrelated to basal sodium excretion, renal clearances, or urinary prostaglandin E2. Natriuresis correlated negatively with plasma aldosterone (r = -0.54, p less than 0.01), and strongly with urinary furosemide (range 5.5-76 mg/h, r = 0.71, p less than 0.001). As urinary furosemide excretion reflects the amount of furosemide reaching the active site on the luminal side of the tubule, the data demonstrate markedly reduced amounts of furosemide at its primary site of action in patients with diuretic resistance. Plasma furosemide was higher in patients with reduced furosemide excretion and impaired natriuresis, suggesting that the defect was an impairment of furosemide transport into the tubule. Thus, a major factor in diuretic resistance is altered furosemide pharmacokinetics.

Urinary excretion rates of prostaglandin (PG) E2, PGF2 alpha, 6-keto-PGF1 alpha, and thromboxane (TX) B2 were evaluated in three groups of cirrhotic patients [without ascites (group 1, 13 cases), with ascites and normal renal function (group 2, 15 cases), and with ascites and renal failure (group 3, 5 cases)] and in 14 healthy controls. All urinary arachidonate metabolites were significantly increased in group 2 patients. Patients with renal failure showed lower PGE2, PGF2 alpha, and TXB2 values than those from group 2; PGF2 alpha values were also lower than controls. Platelet TXA2 production during whole blood clotting was significantly reduced in all groups of patients. Administration of low-dose aspirin and sulindac, two cyclooxygenase inhibitors selectively sparing renal cyclooxygenase activity, effectively inhibited platelet TXA2 production without affecting urinary TXB2 excretion, thus ruling out platelets as a possible source of urinary TXB2. We conclude that patients with ascites and normal renal function show an overall activation of the renal PG system. Renal production of vasodilating PGE2 and PGI2 may be involved in supporting renal function in these patients. A reduced platelet synthesis of proaggregatory TXA2 also occurs in cirrhotic patients. This may play a role in the bleeding tendency of cirrhosis.