Elevated ectodomain of type 23 collagen is a novel biomarker of the intestinal epithelium to monitor disease activity in ulcerative colitis and Crohn's disease.

BACKGROUND: Impaired intestinal epithelial barrier is highly affected in inflammatory bowel disease. Transmembrane collagens connecting the epithelial cells to the extracellular matrix have an important role in epithelial cell homeostasis. Thus, we sought to determine whether the transmembrane type 23 collagen could serve as a surrogate marker for disease activity in patients with Crohn's disease and ulcerative colitis. METHODS: We developed an enzyme-linked immunosorbent assay to detect the ectodomain of type 23 collagen (PRO-C23) in serum, followed by evaluation of its levels in both acute and chronic dextran sulphate sodium colitis models in rats and human inflammatory bowel disease cohorts. Serum from 44 Crohn's disease and 29 ulcerative colitis patients with active and inactive disease was included. RESULTS: In the acute and chronic dextran sulphate sodium-induced rat colitis model, the PRO-C23 serum levels were significantly increased after colitis and returned to normal levels after disease remission. Serum levels of PRO-C23 were elevated in Crohn's disease (p < 0.05) and ulcerative colitis (p < 0.001) patients with active disease compared to healthy donors. PRO-C23 differentiated healthy donors from ulcerative colitis (area under the curve [AUC]: 0.81, p = 0.0009) and Crohn's disease (AUC: 0.70, p = 0.0124). PRO-C23 differentiated ulcerative colitis patients with active disease from those in remission (AUC: 0.75, p = 0.0219) and Crohn's disease patients with active disease from those in remission (AUC: 0.68, p = 0.05). CONCLUSION: PRO-C23 was elevated in rats with active colitis, and inflammatory bowel disease patients with active disease. Therefore, PRO-C23 may be used as a surrogate marker for monitoring disease activity in ulcerative colitis and Crohn's disease.

Liver failure complicating segmental hepatic ischaemia induced by a PTFE-coated TIPS stent.

The use of polytetrafluoroethylene (PTFE)-covered prostheses improves trans-jugular intrahepatic porto-systemic shunt (TIPS) patency and decreases the incidence of clinical relapses and re-interventions. Therefore, the improvement provided by covered stents might expand the currently accepted recommendations for TIPS use. Stent-related occlusion of the hepatic vein with consequent ischaemia of the corresponding liver parenchyma emerges as a novel complication reported in at least 5% of patients implanted with coated stents. However, this complication was reported to be mild, without signs or symptoms of liver failure, and self-limiting. We report a case of segmental liver ischaemia following PTFE-covered stent placement resulting in a marked impairment in liver function in a patient with hepatitis C virus cirrhosis implanted because of refractory oesophageal bleeding, thus expanding the severity range of this new procedural complication. Moreover, we discuss the possible involvement of additional pathogenetic mechanisms other than out-flow obstruction in the onset of coated-stent induced congestive liver ischaemia.

Coagulation and fibrosis in chronic liver disease.

In the hepatic tissue repair mechanism, hepatic stellate cells (HSCs) are recruited at the site of injury and their changes reflect paracrine stimulation by all neighbouring cell types, including sinusoidal endothelial cells, Kupffer cells, hepatocytes, platelets and leucocytes. Thrombin converts circulating fibrinogen to fibrin, promotes platelet aggregation, is a potent activator of endothelial cells, acts as a chemoattractant for inflammatory cells and is a mitogen and chemoattractant for fibroblasts and vascular smooth muscle cells. Most of the cellular effects elicited by thrombin are mediated via a family of widely expressed G-protein-coupled receptors termed protease activated receptors (PARs). All known members of the PAR family stimulate cell proliferation/activation in a rat HSC line. Thrombin receptors are constitutively expressed in the liver, and their expression increases in parallel with the severity and/or the duration of liver disease. In human studies, thrombotic risk factors were found to be independently associated with the extent of fibrosis; severity of hepatitis C virus (HCV)-associated liver disease appears to be less in patients with haemophilia when compared with those with HCV alone. Several studies, based mostly on rat models, demonstrate that anticoagulants or antiplatelet agents prevent hepatic necrosis and fibrosis by acting on HSCs. These drugs could be therapeutic agents in patients with chronic liver disease and specific studies should be initiated.

Reliability of transient elastography for the diagnosis of advanced fibrosis in chronic hepatitis C.

BACKGROUND: Transient elastography (TE) has received increasing attention as a means to evaluate disease progression in patients with chronic liver disease. AIM: To assess the value of TE for predicting the stage of fibrosis. METHODS: Liver biopsy and TE were performed in 150 consecutive patients with chronic hepatitis C-related hepatitis (92 men and 58 women, age 50.6 (SD 12.5) years on the same day. Necro-inflammatory activity and the degree of steatosis at biopsy were also evaluated. RESULTS: The areas under the curve for the prediction of significant fibrosis (> or = F2), advanced fibrosis (> or = F3) or cirrhosis were 0.91, 0.99 and 0.98, respectively. Calculation of multilevel likelihood ratios showed that values of TE < 6 or > or = 12, < 9 or > or = 12, and < 12 or > or = 18, clearly indicated the absence or presence of significant fibrosis, advanced fibrosis, and cirrhosis, respectively. Intermediate values could not be reliably associated with the absence or presence of the target condition. The presence of inflammation significantly affected TE measurements in patients who did not have cirrhosis (p<0.0001), even after adjusting for the stage of fibrosis. Importantly, TE measurements were not influenced by the degree of steatosis. CONCLUSIONS: TE is more suitable for the identification of patients with advanced fibrosis than of those with cirrhosis or significant fibrosis. In patients in whom likelihood ratios are not optimal and do not provide a reliable indication of the disease stage, liver biopsy should be considered when clinically indicated. Necro-inflammatory activity, but not steatosis, strongly and independently influences TE measurement in patients who do not have cirrhosis.

Non invasive diagnosis of portal hypertension in cirrhotic patients.

The measure of disease progression in chronic liver disease represents a key challenge in any of the different stages of evolution. Indeed, a correct and reliable measure of the stage of the disease has relevant implications for assessing the effectiveness of the current therapeutic regimens and for predicting the occurrence of complication. Accordingly, a current major effort is directed at evaluating methodologies characterized by no or low invasiveness to be employed as clinical discriminators in patients populations potentially requiring invasive assessment. This appears particularly relevant in patients with compensated cirrhosis, where the only reference standard is the measurement of portal pressure by hepatic venous pressure gradient (HVPG). In this particular context, transient elastography (TE) appears to be promising and needs to be further investigated, possibly in combination with other non-invasive methodologies such as serum markers algorithms and/or imaging techniques. On the other hand, the application of non-invasive methods for monitoring the response to vasoactive treatment for the reduction of portal pressure and the prevention of related complications seems at the moment not realistic.

Serological markers of extracellular matrix remodeling predict transplant-free survival in primary sclerosing cholangitis.

BACKGROUND: Primary sclerosing cholangitis is a progressive liver disease with a remarkably variable course. Biomarkers of disease activity or prognostic models predicting outcome at an individual level are currently not established. AIM: To evaluate the prognostic utility of four biomarkers of basement membrane and interstitial extracellular matrix remodeling in patients with primary sclerosing cholangitis. METHODS: Serum samples were available from 138 large-duct primary sclerosing cholangitis patients (of which 102 [74%] with IBD) recruited 2008-2012 and 52 ulcerative colitis patients (controls). The median follow-up time was 2.2 (range 0-4.3) years. Specific biomarkers of type III and V collagen formation (PRO-C3 and PRO-C5, respectively) and type III and IV collagen degradation (C3M and C4M, respectively) were assessed. The Enhanced Liver Fibrosis test, including procollagen type III N-terminal peptide, tissue inhibitor of metalloproteinase-1 and hyaluronic acid was assessed for comparison. RESULTS: All markers were elevated in primary sclerosing cholangitis compared to ulcerative colitis patients (P < 0.001). PRO-C3 showed the largest difference between the two groups with a threefold increase in primary sclerosing cholangitis compared to ulcerative colitis patients. Patients with high baseline serum levels of all markers, except C3M, had shorter survival compared to patients with low baseline serum levels (P < 0.001). Combining PRO-C3 and PRO-C5 the odds ratio for predicting transplant-free survival was 47 compared to the Enhanced Liver Fibrosis test's odds ratio of 11. CONCLUSIONS: Extracellular matrix remodeling is elevated in primary sclerosing cholangitis patients compared to ulcerative colitis patients. Furthermore, the interstitial matrix marker PRO-C3 was identified as a potent prognostic marker and an independent predictor of transplant-free survival in primary sclerosing cholangitis.

Cultured human liver fat-storing cells produce monocyte chemotactic protein-1. Regulation by proinflammatory cytokines.

Monocytes infiltrate the portal space during chronic liver inflammation. Monocyte chemotactic protein-1 (MCP-1) is a cytokine that induces monocyte chemotaxis and activation. We investigated if human liver fat-storing cells (FSC) secrete MCP-1, and the mechanisms that regulate MCP-1 production. Unstimulated FSC secrete MCP-1 as measured by radioimmunoassay as well as a chemotactic assay and express mRNA that encodes for this cytokine. A two- to threefold increase in MCP-1 secretion was observed when FSC were treated with either interleukin-1 alpha (IL-1 alpha) or interferon-gamma (IFN-gamma). Tumor necrosis factor-alpha (TNF alpha) also increased MCP-1 secretion, although to a lesser extent (1.6-fold). Northern blot analysis showed that IL-1 alpha and IFN-gamma strongly increase the levels of mRNA that encodes for MCP-1, whereas TNF alpha appears to be a weaker stimulus. Analysis of FSC-conditioned medium by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting revealed three bands of MCP-1 that most likely represent isoforms of different apparent molecular weights. Pretreatment of FSC with H-7, a protein kinase C inhibitor, blocked cytokine-induced increase in both MCP-1 gene expression and secretion. To determine the potential role of MCP-1 in vivo, we also analyzed normal and pathologic human liver tissue. Northern blot analysis showed that MCP-1 mRNA expression is more abundant in liver tissue obtained from patients with chronic active hepatitis compared with normal liver tissue. These studies indicate that MCP-1 secreted by FSC is stimulated by proinflammatory cytokines and that MCP-1 gene expression is upregulated in chronic inflammatory liver disease. MCP-1 released by FSC may participate in the recruitment and activation of monocytes at sites of liver injury.

Effects of platelet-derived growth factor and other polypeptide mitogens on DNA synthesis and growth of cultured rat liver fat-storing cells.

In vitro and in vivo studies suggest that liver fat-storing cells (FSC) may play an important role in the development of liver fibrosis. We explored the effects of platelet-derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor (TGF)-alpha and TGF-beta, and basic fibroblast growth factor (bFGF) on DNA synthesis and growth of rat liver FSC. PDGF, EGF, TGF-alpha, and bFGF induced a dose-dependent increase in DNA synthesis with a peak effect at 24 h. PDGF produced the most striking effect with a maximum 18-fold increase over control. EGF, TGF-alpha, and bFGF elicited a maximum three- to fourfold increase in DNA synthesis. Analysis of growth curves revealed a similar pattern of potency of the growth factors. TGF-beta did not affect DNA synthesis of FSC; however, TGF-beta markedly potentiated the stimulatory effects of both EGF and PDGF. FSC showed high specific binding of 125I-PDGF and Scatchard analysis revealed high affinity receptors with an apparent Kd of 2.3 x 10(-10) M. Our data suggest that PDGF is a key mitogen for FSC and that the coordinate release of other growth factors together with PDGF by inflammatory cells represents a potent potential stimulus for FSC proliferation in conditions of chronic self-perpetuating liver inflammation.

Expression of platelet-derived growth factor receptors in normal and diseased human kidney. An immunohistochemistry and in situ hybridization study.

We studied the expression of PDGF-alpha and -beta receptors in 10 normal and 40 pathologic human kidneys (five minimal change disease, five membranous nephropathy, 25 IgA nephropathy, five lupus nephritis), by both immunohistochemistry and in situ hybridization techniques. In normal-appearing kidneys, both PDGF-alpha and -beta receptors were expressed at the glomerular and interstitial level, the latter receptor more intensely than the former. The distribution and degree of expression of both receptors in nonproliferative glomerulonephritides were comparable with those found in normal-appearing kidneys. PDGF-beta receptor gene and protein expression were upregulated in proliferative nephritides both at the glomerular and the interstitial level and strictly correlated with the grade of histologic lesions. Finally, PDGF beta receptor expression was observed at a low level in normal-appearing renal vessels, and strikingly increased in injured arteries. Diseased kidneys displayed only a slight increase of PDGF-alpha receptor expression, chiefly at the interstitial level. Noteworthy, a few cases of lupus nephritis showed a moderate increase of PDGF-alpha receptor also at the glomerular level. These data establish PDGF-beta receptor activation as a candidate for driving glomerular and interstitial proliferation and, probably, expansion of extracellular matrix in proliferative glomerulonephritis, while the role of PDGF-alpha receptor activation at the renal level remains to be elucidated.

Fat-storing cells as liver-specific pericytes. Spatial dynamics of agonist-stimulated intracellular calcium transients.

Liver perisinusoidal fat-storing cells (FSC) show morphological and ultrastructural characteristics similar to pericytes regulating local blood flow in other organs. In the present study we have analyzed whether FSC respond to local vasoconstrictors such as thrombin, angiotensin-II, and endothelin-1 with an increase in intracellular free calcium concentration ([Ca2+]i) coupled with effective cell contraction. All agonists tested induced a rapid and dose-dependent increase in [Ca2+]i followed by a sustained phase lasting several minutes in confluent monolayers of Fura-2-loaded human FSC. Pharmacological studies performed using different Ca2+ channel blockers indicated that, at least for thrombin and angiotensin-II, the sustained phase is due to the opening of voltage-sensitive membrane Ca2+ channels. To analyze the temporal and spatial dynamics of Ca2+ release in response to these agonists, we performed experiments on individual Fura-2-loaded human FSC using a dual wavelength, radiometric video imaging system. The rise in [Ca2+]i was exclusively localized to the cytoplasm, particularly in the branching processes. Increases in [Ca2+]i more than four-fold were associated with a simultaneous and transient reduction of cell area indicating reversible cell contraction. Our results indicate that the Ca(2+)-dependent contraction of human FSC in vitro may reflect a potential role in regulating sinusoidal blood flow in vivo.

HNE interacts directly with JNK isoforms in human hepatic stellate cells.

4-Hydroxy-2,3-nonenal (HNE) is an aldehydic end product of lipid peroxidation which has been detected in vivo in clinical and experimental conditions of chronic liver damage. HNE has been shown to stimulate procollagen type I gene expression and synthesis in human hepatic stellate cells (hHSC) which are known to play a key role in liver fibrosis. In this study we investigated the molecular mechanisms underlying HNE actions in cultured hHSC. HNE, at doses compatible with those detected in vivo, lead to an early generation of nuclear HNE-protein adducts of 46, 54, and 66 kD, respectively, as revealed by using a monoclonal antibody specific for HNE-histidine adducts. This observation is related to the lack of crucial HNE-metabolizing enzymatic activities in hHSC. Kinetics of appearance of these nuclear adducts suggested translocation of cytosolic proteins. The p46 and p54 isoforms of c-Jun amino-terminal kinase (JNKs) were identified as HNE targets and were activated by this aldehyde. A biphasic increase in AP-1 DNA binding activity, associated with increased mRNA levels of c-jun, was also observed in response to HNE. HNE did not affect the Ras/ERK pathway, c-fos expression, DNA synthesis, or NF-kappaB binding. This study identifies a novel mechanism linking oxidative stress to nuclear signaling in hHSC. This mechanism is not based on redox sensors and is stimulated by concentrations of HNE compatible with those detected in vivo, and thus may be relevant during chronic liver diseases.

Novel insights into the biology and physiology of the Ito cell.

Ito cells, perisinusoidal mesenchymal elements with possible pericytic functions within the liver, recently have been shown to play multiple physiological and pathophysiological roles. In particular, several in vivo and in vitro studies have clearly indicated that Ito cells play a relevant role in the progression of liver fibrogenesis. More recently, attention has been focussed on the mechanisms leading to Ito cell activation, proliferation and synthesis of extracellular matrix components. Among other soluble factors potentially involved in these processes, transforming growth factor-beta 1 and platelet-derived growth factor have been shown to act in a paracrine, and possibly autocrine, fashion on Ito cells, thus perpetuating their activated state. Finally, other studies have shown that Ito cells could play an active role in chronic liver tissue inflammation by promoting chemotaxis of infiltrating inflammatory cells.

Secretion of insulin-like growth factor-I and binding proteins by rat liver fat-storing cells: regulatory role of platelet-derived growth factor.

Insulin-like growth factor (IGF-I) is synthesized in multiple organs, including the liver, and may play a role in tissue growth and repair. We report that liver fat-storing cells (FSC) secrete IGF-I immunoreactivity in the culture medium. Secretion of IGF-I immunoreactivity was blocked in the presence of cycloheximide, suggesting de novo synthesis. Culture medium conditioned by FSC was concentrated and applied to a Sephadex G100 column equilibrated in a denaturing buffer. Two major species with apparent mol wts of 7.5 and greater than 25 k were identified by IGF-I RIA. Reverse phase HPLC of the 7.5 kilodalton species (the size of IGF-I) showed that it eluted in a single peak. To determine whether the higher mol wt species possessed IGF-I binding activity, appropriate fractions were desalted, incubated with [125I]IGF-I for 2 h at 30 C and applied to a Sephadex G100 column equilibrated in a nondissociating buffer. The major peak of radioactivity was confined to a high mol wt region. Western blot ligand analysis revealed the presence of two insulin-like growth factor binding proteins of approximately 28 and 31 kilodaltons. Platelet-derived growth factor, a potent mitogen for FSC, resulted in a 230% increase in release of IGF-I immunoreactivity that could be accounted for by an increase in IGF-I binding activity. In addition IGF-I increased DNA synthesis in FSC and this effect was additive to that of platelet-derived growth factor. IGF-I treatment also resulted in an increase in cell number. IGF-I and insulin-like growth factor binding proteins secreted by FSC may play a role in the hepatic tissue response to injury via autocrine and/or paracrine mechanisms.

Modulation of hepatic fibrogenesis by antioxidants.

The oxidative damage of liver tissue appears to favour a fibrogenic process, through the stimulation of TGF beta 1 and procollagen gene expression. The latter effect has been also showed by a well defined end-product of lipid peroxidation, 4-hydroxy-2,3-nonenal in human Ito cell culture. "In vivo" and "in vitro" data give evidence of a strong down modulation of TGF beta 1 and collagen expression afforded by supplementation with different antioxidant molecules.

Involvement of phosphatidylinositol 3-kinase in the activation of extracellular signal-regulated kinase by PDGF in hepatic stellate cells.

Phosphatidylinositol 3-kinase (PI 3-K) is a lipid and protein kinase which associates with the activated platelet-derived growth factor (PDGF) receptor and other tyrosine kinases. We studied the effects of wortmannin, a selective inhibitor of PI 3-K, on the activation of extracellular-signal regulated kinase (ERK) by PDGF in cultured hepatic stellate cells, mesenchymal cells responsible for extracellular matrix synthesis within the liver. Incubation with 100 nM wortmannin, a dose which almost completely blocks PI 3-K, resulted in 50% reduction of ERK activity. Direct inhibition of ERK by wortmannin could not be considered responsible for this effect, since wortmannin did not inhibit ERK activity in vitro. Rather, inhibition of PI 3-K acts on the kinase cascade that leads to ERK activation, since PDGF-dependent phosphorylation of ERK was found to be reduced after incubation with wortmannin. Wortmannin also inhibited the increase in c-fos mRNA induced by PDGF, which is dependent on ERK activation. The results of this study show that in hepatic stellate cells PI 3-K is involved in ERK activation, although it is not necessary. These data indicate cross-talk between PI 3-K and the Ras/ERK pathway in PDGF-stimulated cells.

Integrin-mediated stimulation of monocyte chemotactic protein-1 expression.

We investigated whether activation of integrin receptors could modulate the expression of monocyte chemotactic protein-1 (MCP-1) in human hepatic stellate cells (HSC), mesenchymal cells responsible for extracellular matrix synthesis within the liver. When compared to non-adherent cells, HSC plated on collagen types I or IV, or fibronectin, showed increased MCP-1 gene expression and protein secretion in the conditioned medium. Increased MCP-1 secretion was also observed when cells were plated on dishes coated with a monoclonal antibody directed against the beta1-integrin subunit, demonstrating that ligation of beta1-integrins is sufficient to stimulate MCP-1 expression. Conversely, integrin-independent cell adhesion on poly-L-lysine did not modify MCP-1 secretion. Disruption of the actin cytoskeleton by cytochalasin D blocked the collagen-dependent increase in MCP-1 secretion. Chemotactic assay of HSC-conditioned medium showed that HSC plated on collagen secrete higher amounts of chemotactic factors for lymphomonocytes, and that MCP-1 accounts for the great majority of this effect. These findings indicate a novel mechanism of MCP-1 regulation possibly relevant in those conditions where HSC interact with an altered extracellular matrix.

Inhibition by pentoxifylline of extracellular signal-regulated kinase activation by platelet-derived growth factor in hepatic stellate cells.

1. It has been proposed that pentoxifylline (PTF) acts an antifibrogenic agent by reducing the synthesis of extracellular matrix components, and this possibility has been confirmed in animal models of hepatic fibrosis. In this study the effects of PTF on the proliferation of extracellular matrix producing cells induced by platelet-derived growth factor (PDGF) were evaluated. The study was performed on hepatic stellate cells, currently indicated as the major source of extracellular matrix in fibrotic liver. 2. PTF caused a dose-dependent reduction of PDGF-induced mitogenesis with an IC50 of 170 microM, identical to the EC50 for the increase in intracellular cyclic AMP levels. Preincubation with PTF did not affect either PDGF-receptor autophosphorylation or phosphotidylinositol 3-kinase activity, whereas it markedly reduced PDGF-stimulated extracellular signal-regulated kinase (ERK) activity and ERK isoform phosphorylation. PTF also reduced PDGF-induced c-fos mRNA expression, which is dependent on activation of the RAS/ERK pathway. In addition, the PDGF-induced increase in cytsolic-free calcium was almost completely prevented by pretreating the cells with PTF. 3. The results of the present study indicate that PTF, in addition to its effect on collagen deposition and degradation, may exert an antifibrogenic effect by reducing the PDGF-induced proliferation of extracellular matrix producing cells. This effect appears to be mediated by a reduction of PDGF-stimulated ERK activity as well as of other intracellular signalling pathways such as the PDGF-induced elevation of cytosolic-free calcium.

Effect of pentoxifylline on the degradation of procollagen type I produced by human hepatic stellate cells in response to transforming growth factor-beta 1.

1. Pentoxifylline (PTF) may act as a potential antifibrogenic agent by inhibiting cell proliferation and/or collagen deposition in cell type(s) responsible for the accumulation of extracellular matrix. The aim of the present study was to investigate at which level PTF may affect synthesis and degradation of type I collagen in human hepatic stellate cells (HSCs), a key source of connective tissue in fibrotic liver. 2. Procollagen type I synthesis and release were evaluated in cells maintained in serum free/insulin free medium for 48 h and then stimulated with transforming growth factor-beta 1 (TGF-beta 1) for different time periods in the presence or absence of PTF. TGF-beta 1 caused an upregulation of procollagen I mRNA levels with a peak increase after 3-6 h of stimulation. This effect was followed by an increase in both the cell associated and the extracellular levels of the corresponding protein, with a peak effect at 9-12 h after the addition of TGF-beta 1. Co-incubation with PTF slightly but consistently reduced basal as well as stimulated procollagen I mRNA levels, with negligible effects on the cell-associated expression of the corresponding protein. Conversely, PTF dose-dependently reduced procollagen type I levels detected in supernatants from unstimulated and stimulated cells. 3. Pulse-chase experiments employing L-[3H]-proline revealed that PTF was able to induce significantly the degradation of procollagen, mainly in the extracellular compartment. We next analysed the effect of PTF on the major pathway involved in type I collagen degradation. PTF did not affect the expression of metalloproteinase 1 (MMP-1) mRNA both in basal and stimulated conditions, whereas it markedly reduced the expression of tissue inhibitor of metalloproteinase 1 (TIMP-1) mRNA. Accordingly incubation with PTF increased the levels of 'activated MMP-1' in cell supernatants in both basal and stimulated conditions. 4. These results suggest that the antifibrogenic action of PTF on human HSCs is mainly mediated by extracellular collagen degradation rather than by a reduction of collagen synthesis.

Arachidonic acid derivatives and renal function in liver cirrhosis.

Prostaglandins (PGs) are arachidonic acid (AA) derivatives via the PG endoperoxyde H synthase (PGHS) complex. Two PGHS isoforms have been recognized, constitutive (PGHS-1) and inducible (PGHS-2), respectively. Within the kidney, vascular endothelium mainly produces PGI2; the whole glomerulus synthesizes several prostanoids, the predominant AA metabolite in humans being PGI2; tubules and medullary interstitial cells produce mainly PGE2. Renal PGs modulates the action of other hormones and autacoids involved in the regulation of renal hemodynamics, glomerular filtration and the renal handling of sodium and water. Renal PGs are, at least in part, excreted into urine. Measurement of urinary PGs or their metabolites has been found to provide a reliable estimation of basal as well as stimulated PG synthesis. Patients with cirrhosis of the liver show an increased renal synthesis of vasodilating PGs, as indicated by the high urinary excretion of PGs and/or their metabolites. Administration of nonsteroidal anti-inflammatory drugs (NSAIDs) to these patients causes a profound reduction in renal blood flow and glomerular filtration rate, a reduction in sodium excretion, and an impairment of free water clearance. These data clearly indicate that the increased renal synthesis of vasodilating PGs has a relevant role in maintaining renal hemodynamics, sodium and water excretion in a clinical setting characterized by a reduction of effective plasma volume and a striking activation of the major vasoconstricting systems, namely the renin-angiotensin-aldosterone, the sympathetic nervous system, and vasopressin. Patients with hepato-renal syndrome have a reduced renal synthesis of vasodilating PGE2 in the setting of a striking activation of endogenous vasoconstrictors and a maintained or increased renal production of thromboxane A2. Therefore, an imbalance between vasoconstricting systems and the renal vasodilator PGE2 was proposed to explain the renal failure observed in this condition. The urinary excretion of 2-3-dinor 6-keto-PGF1 alpha, an index of systemic PGI2 synthesis, is increased in patients with cirrhosis and hyperdynamic circulation, thus raising the possibility that systemic synthesis of PGI2 may contribute to the arterial vasodilatation of these patients. Finally, administration of exogenous prostanoids to patients with cirrhosis is not effective either in ameliorating renal function or in preventing the deleterious effect of NSAIDs.

Liver fibrosis: from the bench to clinical targets.

Progressive liver fibrosis is the main cause of organ failure in chronic liver diseases of any aetiology. Fibrosis develops with different spatial patterns and is a consequence of different prevalent mechanisms according to the diverse causes of parenchymal damage. Indeed, fibrosis, observed as a consequence of chronic viral infection is initially concentrated within and around the portal tract, while fibrosis secondary to toxic/metabolic damage is located mainly in the centrolobular areas. In addition, it is increasingly evident that different cell types are involved in the deposition of fibrillar extracellular matrix during active hepatic fibrogenesis: hepatic stellate cells are mainly involved when hepatocellular damage is limited or concentrated within the liver lobule, whereas portal myofibroblasts and fibroblasts provide a predominant contribution when the damage is located in the proximity of the portal tracts. In the later stages of evolution (septal fibrosis) it is likely that all extracellular matrix-producing cells contribute to fibrogenesis. Recruitment and activation of extracellular matrix-producing cells to the site of tissue damage can be due to different major mechanisms: (1) Chronic activation of the tissue repair process. In this case, as a consequence of the reiterated damage, accumulation of fibrillar extracellular matrix reflects the impossibility of an effective remodelling and regeneration. (2) Effect of oxidative stress products, including reactive oxygen intermediates and reactive aldehydes. These products, whose concentration become critical in toxic/metabolic liver injury, are able to induce the synthesis of fibrillar extracellular matrix even in the absence of significant hepatocyte damage and inflammation. (3) Derangement of normal the epithelial/mesenchymal interaction. This typically occurs in all conditions characterised by cholangiocyte damage/proliferation, where a consensual proliferation of extracellular matrix-producing cells and progressive fibrogenesis is commonly observed. A major advancement towards the understanding of the molecular mechanisms of fibrogenesis is derived from a consistent number of in vitro studies investigating the biological role of growth factors/cytokines and other soluble factors and their intracellular signalling pathways. The relevance of these factors has been confirmed by studies performed on animal models and by studies performed on pathological human liver. Along these lines, the elucidation of a consistent number of cellular and molecular mechanisms responsible for the progression of liver fibrosis has provided sound basis for the development of pharmacological strategies able to modulate this important pathophysiological process. Finally, there are several clinically relevant issues that need re-evaluation and/or further investigation, and in particular: (1) the need of an accurate and effective monitoring of the fibrotic progression of chronic liver diseases and of the effectiveness of the currently proposed treatments; (2) the identification of general or individual factors potentially relevant for a faster progression of the disease.