The differentiation of prehypertrophic into hypertrophic chondrocytes drives an OA-remodeling program and IL-34 expression.

OBJECTIVES: We hypothesize that chondrocytes from the deepest articular cartilage layer are pivotal in maintaining cartilage integrity and that the modification of their prehypertrophic phenotype to a hypertrophic phenotype will drive cartilage degradation in osteoarthritis. DESIGN: Murine immature articular chondrocytes (iMACs) were successively cultured into three different culture media to induce a progressive hypertrophic differentiation. Chondrocyte were phenotypically characterized by whole-genome microarray analysis. The expression of IL-34 and its receptors PTPRZ1 and CSF1R in chondrocytes and in human osteoarthritis tissues was assessed by RT-qPCR, ELISA and immunohistochemistry. The expression of bone remodeling and angiogenesis factors and the cell response to IL-1ß and IL-34 were investigated by RT-qPCR and ELISA. RESULTS: Whole-genome microarray analysis showed that iMACs, prehypertrophic and hypertrophic chondrocytes each displayed a specific phenotype. IL-1ß induced a stronger catabolic effect in prehypertrophic chondrocytes than in iMACs. Hypertrophic differentiation of prehypertrophic chondrocytes increased Bmp-2 (95%CI [0.78; 1.98]), Bmp-4 (95%CI [0.89; 1.59]), Cxcl12 (95%CI [2.19; 5.41]), CCL2 (95%CI [3.59; 11.86]), Mmp 3 (95%CI [10.29; 32.14]) and Vegf mRNA expression (95%CI [0.20; 1.74]). Microarray analysis identified IL-34, PTPRZ1 and CSFR1 as being strongly overexpressed in hypertrophic chondrocytes. IL-34 was released by human osteoarthritis cartilage; its receptors were expressed in human osteoarthritis tissues. IL-34 stimulated CCL2 and MMP13 in osteoblasts and hypertrophic chondrocytes but not in iMACs or prehypertrophic chondrocytes. CONCLUSION: Our results identify prehypertrophic chondrocytes as being potentially pivotal in the control of cartilage and subchondral bone integrity. Their differentiation into hypertrophic chondrocytes initiates a remodeling program in which IL-34 may be involved.

Nonalcoholic fatty liver disease (NAFLD) in nonobese patients with diabetes: Prevalence and relationships with hemodynamic alterations detected with Doppler sonography().

AIM: To evaluate the prevalence, severity, and hemodynamic features of nonalcoholic fatty liver disease (NAFLD) in nonobese diabetics. METHODS: We studied 100 consecutive nonobese (body mass index [BMI] < 30) patients with type 1 (n = 17) or type 2 (n = 83) diabetes and no known causes of liver disease. Steatosis was diagnosed and graded with ultrasonography. Digital sonographic images of the liver and right kidney were analyzed with dedicated software (HDI-Lab), and the liver/kidney ratio of grey-scale intensity was calculated as an index of the severity of the steatosis. Severity scores ranging from 0 (none) to 5 (severe) were compared with sonographic and Doppler findings (right liver size, portal vein diameter and flow velocity, hepatic and splenic arterial pulsatility indices, hepatic-vein flow profile and A- and S-wave velocities). RESULTS: The prevalence of steatosis was 24% in type I and 80% in type II diabetes (grade 1 in 17%, grade 2 in 34%, grade 3 in 33%, grade 4 in 9%, grade 5 in 7%). In patients with steatosis (especially those with grades 4-5 disease), hepatic volume was increased (p < 0.005). Portal vein diameter was increased in grade 5 steatosis. The hepatic artery pulsatility index was significantly increased, particularly in grades 4 and 5 (p < 0.0001); portal and A-wave velocities were significantly reduced in grades 3-5 (p < 0.001); and the hepatic vein flow profile was altered in 27% (biphasic: 20%, flat: 7%) patients with steatosis, although there was no correlation with severity. CONCLUSIONS: NAFLD is very frequent in nonobese diabetics with type 2 but not type 1 disease, and it is associated with hepatomegaly and liver hemodynamic alterations only when it is severe.

New abdominal collaterals at ultrasound: a clue of progression of portal hypertension.

BACKGROUND: Abdominal ultrasound can detect non-invasively the presence of abdominal portal-systemic collaterals in patients with liver cirrhosis. Abdominal portal-systemic collaterals may be protective from the formation and growth of oesophageal varices, but available data are inconclusive. AIM: We aimed at investigating the relationship between abdominal portal-systemic collaterals and variceal formation and growth. METHODS: We studied 126 cirrhotic patients without (n=43) or with small (n=83) oesophageal varices who entered a protocol of serial ultrasonographic and endoscopic examinations for a median of 55 months. Presence and kind of abdominal portal-systemic collaterals was recorded on first ultrasonography and on each control thereafter. RESULTS: At inclusion, abdominal portal-systemic collaterals were found in 19/43 patients without varices and in 23/83 patients with small varices (NS). There was no difference in variceal formation and growth between patients with and without abdominal portal-systemic collaterals at inclusion. However, patients developing new abdominal portal-systemic collaterals during follow-up had a significantly higher rate of variceal formation (56.2% vs. 22.2%; p=0.024) and growth (52.9% vs. 30.6%; p=0.041) compared with patients with unchanged ultrasonography. CONCLUSIONS: Abdominal collaterals are not protective from the formation or growth of oesophageal varices. Conversely, new abdominal portal-systemic collaterals emergence is a non-invasive clue of formation and progression of varices. Therefore, endoscopy is probably indicated whenever new abdominal portal-systemic collaterals are detected in cirrhotic patients.

Thrombosis of the portal venous system.

Portal vein thrombosis (PVT) is a rare cause of portal hypertension. Its diagnosis has been facilitated by improvements in imaging techniques, in particular Doppler sonography. The prevalence is about 1% in the general population, but much higher rates are observed in patients with hepatic cirrhosis (7%, range 0.6-17%), particularly those who also have hepatocellular carcinoma (HCC) (35%). The most common causes of PVT are myeloproliferative disorders, deficiencies of anticoagulant proteins, prothrombotic gene mutations, cirrhosis with portal hypertension, and HCC. Its development often requires the presence of two or more risk factors (local and/or systemic), e.g., a genetically determined thrombophilic state plus an infectious episode or abdominal surgery. It is clinically useful to distinguish between cirrhotic and noncirrhotic forms. Portal vein thrombosis is also traditionally classified as acute or chronic, but this distinction is often difficult. Color Doppler ultrasound is the first-line imaging study for diagnosis of PVT; magnetic resonance angiography and CT angiography are valid alternatives. The main complications are ischemic intestinal necrosis (in acute PVT) and esophageal varices (in chronic cases); the natural history of the latter differs depending on whether or not the thrombosis is associated with cirrhosis. The treatment of choice for PVT has never been adequately investigated. It is currently based on the use of anticoagulants associated, in some cases, with thrombolytics, but experience with the latter agents is too limited to draw any definite conclusions. In chronic thrombosis (even forms associated with cirrhosis), anticoagulant therapy is recommended and possibly, beta-blockers as well. Naturally, treatment of the underlying pathology is essential.

Portal vein arterialization: a new surgical option against acute liver failure?

Survival rates of patients with acute liver failure (ALF) without transplantation are poor. Supporting these patients until an organ becomes available or until their own liver is able to regenerate itself, avoiding transplantation, is a major goal in the treatment of ALF. We report our clinical experience of portal vein arterialization in one case of massive liver necrosis after liver transplantation and in two patients with ALF caused by idiosyncratic drug reaction and mushroom intoxication. Portal vein arterialization, at least in two cases, was a turning point in the course of the disease since a close temporal association between surgery and clinical improvement was clearly evident. We believe that this novel approach, which should promote liver regeneration by providing an additional oxygen supply to the liver, may disclose a new possibility in the treatment of ALF and prompt new clinical and experimental research.

Different haemodynamic effects of a single dose of long-acting isosorbide-5-mononitrate in healthy subjects and patients with cirrhotic portal hypertension.

BACKGROUND: The action pathways of nitrates are hypothesised to be deranged in cirrhosis. AIM: In order to confirm it, the acute haemodynamic effects of isosorbide-5-mononitrate in cirrhotic patients and controls was investigated. PATIENT: Nine cirrhotics and nine healthy controls. METHODS: Evaluation in the fasting state, 90 min after isosorbide-5-mononitrate or placebo (double-blind on two different days) and then 30 and 120 min after eating a standard meal. Various systemic and splanchnic haemodynamic parameters, including arterial impedance, assessed as Doppler pulsatility index, were measured. RESULTS: isosorbide-5-mononitrate reduced arterial pressure and increased heart rate and mesenteric pulsatility index both in controls and in cirrhotics, whereas the following parameters behaved differently in the two groups (P < 0.05): hepatic pulsatility index decreased (-9%) and the portal velocity increased (+13%) in controls, whereas hepatic pulsatility increased (+18%) and portal velocity decreased (-18%) in cirrhotics. The two groups presented a similar pattern of changes in most variables under placebo after a meal. In controls, the administration of isosorbide-5-mononitrate blunted the postprandial mesenteric vasodilation and related changes in splanchnic and systemic circulation, expected at 30 min, in comparison to those observed under placebo. In cirrhotics, instead, the postprandial pattern was similar under placebo and isosorbide-5-mononitrate. CONCLUSIONS: The acute administration of isosorbide-5-mononitrate produces different haemodynamic effects in healthy and diseased livers, both in the fasting state and after a meal, consistent with the hypothesis of a deranged response of the intrahepatic microcirculation to nitrates in cirrhosis.

Review article: percutaneous treatment of hepatocellular carcinoma.

In early stage hepatocellular carcinoma (HCC), liver transplantation, surgical resection and percutaneous techniques are classified as radical treatments, and may be offered to about 25% of all patients with HCC evaluated in referral centres. The restricted inclusion criteria for surgical resection and the shortage of liver donors for transplantation have stimulated an increasing demand for minimally invasive treatments able to achieve effective and reproducible percutaneous tumour ablation, with less associated morbidity and lower cost than other interventions. Among percutaneous techniques, ethanol injection has proven to be highly effective in single HCC up to 3 cm, with a rate of complete response of 80%, being well tolerated and with a limited risk of minor complication. In larger and/or multinodular HCC the efficacy is reduced to 50% of complete response in nodules between 3 and 5 cm, and to lower rate in larger tumours. Alternative options to ethanol injection have been recently proposed, including radiofrequency, microwave and laser thermal ablation, aimed to extend the necrotic area thus improving the rate of complete response. To date, radiofrequency is the most used technique, with a reported rate of complete response of 90-98% in nodules smaller than 3 cm, and with the advantage of fewer sessions, otherwise counteracted by a higher rate of side-effects. Microwave and laser are promising technologies, but only few clinical data are available. Randomized controlled trials are needed in order to assess treatment response, long-term survival, rate of complication and cost-efficacy of newer technologies in comparison to ethanol injection.

Influence of the spleen on portal haemodynamics: a non-invasive study with Doppler ultrasound in chronic liver disease and haematological disorders.

BACKGROUND: Splanchnic haemodynamic parameters for the differential diagnosis of splenomegalies of different origins are still suboptimal and the role of spleen enlargement in cirrhosis remains controversial. In an attempt to elucidate these questions, we assessed splanchnic haemodynamics in chronic liver diseases and various other disorders with splenomegaly. METHODS: Study groups comprised: (i) patients with chronic liver disease (89 with cirrhosis, 35 with chronic hepatitis), (ii) patients with splenomegaly without relevant portal hypertension (14 with haematological splenomegaly and 25 liver transplant recipients without complications), (iii) 15 patients with arterial hypertension, (iv) 22 healthy controls. In all subjects, spleen size, portal flow parameters and splenic artery resistance index were measured using duplex-Doppler ultrasound. RESULTS: Splenic artery resistance index was significantly and selectively increased in patients with cirrhosis (0.63, whereas all other group means ranged between 0.53 and 0.56; P < 0.01). Portal flow velocity was significantly decreased in cirrhosis (P < 0.01). The combination of these two parameters provided an accuracy of 87.5% in distinguishing portal hypertensive from haematological splenomegaly. In patients with cirrhosis, the degree of spleen enlargement was positively correlated with increasing portal flow volume, portal vein diameter and variceal size, whereas splenic resistance index and portal velocity did not differ in connection with spleen size. CONCLUSIONS: Splenoportal Doppler sonography provides specific findings in cirrhosis and may therefore be a useful tool in differentiating between splenomegaly of portal hypertensive or haematological origin. In patients with cirrhosis, the presence of splenomegaly is associated with the presence of larger oesophageal varices.

Abnormal brain energy metabolism shown by in vivo phosphorus magnetic resonance spectroscopy in patients with chronic liver disease.

We used phosphorus magnetic resonance spectroscopy (31P-MRS) to assess in vivo the brain bioenergetics of 28 patients with liver cirrhosis. Seven had clinical hepatic encephalopathy (HE), nine hepatocellular carcinoma. 31P-MRS was performed by the DRESS localisation technique on occipital lobes. Brain phosphocreatine was significantly reduced in patients with or without overt HE, and inorganic phosphate was increased in both groups of patients. The cytosolic phosphorylation potential (PP), the relative rate of oxidative metabolism and the regulatory [ADP] were all abnormal. Brain PP was inversely correlated with serum ammonia concentration only in patients without liver cancer. The degree of bioenergetic failure was significantly higher in the presence of overt encephalopathy. We conclude that patients with liver cirrhosis had a derangement of brain energy metabolism, and that 31P-MRS offers a non-invasive method for investigating the underlying mechanisms of HE, with relevant implications in the identification and management of this condition.

Value of splanchnic Doppler ultrasound in the diagnosis of portal hypertension.

The accuracy of various Doppler parameters of portal circulation in the diagnosis of relevant portal hypertension (presence of gastroesophageal varices) was prospectively validated. The following parameters were compared in 51 patients with chronic liver disease (40 with cirrhosis and 11 with chronic hepatitis): portal vein flow velocity and congestion index, hepatic and splenic arteries resistance indexes (RI), modified liver vascular index (portal flow velocity/hepatic artery RI) and portal hypertension index, a new index calculated as: [(hepatic artery RI x 0.69) x (splenic artery RI x 0.87)]/portal vein flow velocity. Highest accuracy was achieved by the splenic artery RI and the portal hypertension index (both around 75%) at cut-offs, respectively, of 0.60 and 12 cm/s(-1), which appeared to be, therefore, the most favorable parameters for the clinical practice. Their use may limit the need for endoscopy to search for varices.

Influence of liver fibrosis on hepatic artery Doppler resistance index in chronic hepatitis of viral origin.

BACKGROUND: Hepatic arterial Doppler sonography is increasingly being used in liver diagnostics. The determinants of the elevation of hepatic artery impedance indexes in chronic liver disease, however, have still not been fully clarified. The aim of the present study was to investigate the relationship between histological alterations and liver circulation in chronic hepatitis. METHODS: Hepatic artery resistance index and portal flow velocity were measured using Doppler sonography in 47 patients with chronic hepatitis of viral origin diagnosed at histopathology. The patients were divided into two groups, those with mild and those with severe alterations, in accordance with the various histological parameters of the Knodell scoring system. RESULTS: Hepatic artery resistance index and age were higher in patients with more severe liver fibrosis (respectively 0.638 +/- 0.084 and 39.0 +/- 10.9 (years) in mild fibrosis versus 0.687 +/- 0.060 and 49.4 +/- 14.4 (years) in severe fibrosis; P < 0.05 for both), whereas no difference between the two groups was found for the other histological features (degeneration, inflammation and necrosis), nor for portal flow velocity. CONCLUSIONS: The increase in hepatic artery resistance index appears to be influenced by the extent of fibrous tissue deposition in the liver, determined by chronic inflammation and repair and, secondly, by ageing.

Influence of esophageal varices and spontaneous portal-systemic shunts on postprandial splanchnic hemodynamics.

OBJECTIVE: The aim of the study was to assess postprandial splanchnic hemodynamic changes in cirrhosis in relation to variceal status. METHODS: In 9 healthy controls and 56 patients with liver cirrhosis, stratified according to variceal status and presence of spontaneous portal-systemic shunts, the portal vein diameter and flow velocity, the congestion index of the portal vein, and the resistive index of the superior mesenteric artery (SMA-RI) were studied by Doppler ultrasound before and 30, 60, and 120 min after the intake of a standard meal. Comparison of postprandial parameters with basal ones was done within each group by paired t test and among groups by ANOVA and Duncan test. RESULTS: Healthy controls and cirrhotic patients without varices showed similar significant splanchnic hemodynamic changes, namely a reduction of SMA-RI (-13% at 30 min) and a consequent increase in portal vein diameter (respectively, +32% and +17% in the two groups) and velocity (+66% and +51%). A significant reduction of SMA-RI was also found in patients with varices, irrespective of the variceal size (range, -7 to -11%), but the expected portal vein dilation and velocity increase were progressively blunted with the increase of variceal size (range, 0-5% for diameter and 5-19% for velocity). Patients with spontaneous portal-systemic shunts showed a response similar to that of patients with large varices. Significant modification of the congestion index of the portal vein did not occur in any group. CONCLUSIONS: Our results show that the hemodynamic response to meal in patients with liver cirrhosis is influenced by the presence and size of esophageal varices and the presence of spontaneous portal-systemic shunts.

Surveillance programme of cirrhotic patients for early diagnosis and treatment of hepatocellular carcinoma: a cost effectiveness analysis.

BACKGROUND: Hepatocellular carcinoma (HCC) is a major cause of death in cirrhotic patients. This neoplasm is associated with liver cirrhosis (LC) in more than 90% of cases. Early diagnosis and treatment of HCC are expected to improve survival of patients. AIMS: To assess the cost effectiveness of a surveillance programme of patients with LC for the early diagnosis and treatment of HCC. PATIENTS: A cohort of 313 Italian patients with LC were enrolled in the surveillance programme between March 1989 and November 1991. In the same period, 104 consecutive patients with incidentally detected HCC were referred to our centre and served as a control group. METHODS: Surveillance was based on ultrasonography (US) and alpha fetoprotein (AFP) determinations repeated at six month intervals. Risk factors for HCC were assessed by multivariate analysis (Cox model). Outcome measures analysed were: (1) number and size of tumours; (2) eligibility for treatment; and (3) survival of patients. Economic issues were: (1) overall cost of surveillance programme; (2) cost per treatable HCC; and (3) cost per year of life saved (if any). Costs were assessed according to charges for procedures at our university hospital. RESULTS: Surveillance lasted a mean of 56 (31) months (range 6-100). During the follow up, 61 patients (19.5%) developed HCC (unifocal at US in 49 cases), with an incidence of 4.1% per year of follow up. AFP, Child-Pugh classes B and C, and male sex were detected as independent risk factors for developing HCC. Only 42 (68.9%) of 61 liver tumours were treated by surgical resection, orthotopic liver transplantation, or local therapy. The cumulative survival rate of the 61 patients with liver tumours detected in the surveillance programme was significantly longer than that of controls (p=0.02) and multivariate analysis showed an association between surveillance and survival. The overall cost of the surveillance programme was US$753 226, the cost per treatable HCC was US$17 934, and the cost for year of life saved was US$112 993. CONCLUSION: Our surveillance policy of patients with LC requires a large number of resources and offers little benefit in terms of patient survival. The decision whether to adopt a surveillance policy towards HCC should rely on the prevalence of the disease in the population and on the resources of a particular country.

Left ventricular volumes in liver cirrhosis.

BACKGROUND: Patients with alcoholic cirrhosis have left ventricular dimensions similar to controls. Few data have been reported in patients with cirrhosis of viral origin. AIM: To assess left ventricular dimensions in patients with pure viral cirrhosis. PATIENTS AND METHODS: Thirty patients with virus-related cirrhosis, 23 patients with alcoholic cirrhosis and 12 healthy controls were submitted to measurement of left ventricular volumes, cardiac output, mean arterial pressure and total peripheral resistance. RESULTS: Patients with cirrhosis showed a similar increase in cardiac index and heart rate and reduction of mean arterial pressure and peripheral vascular resistance in comparison to controls, irrespective of the aetiology. Left ventricular end systolic volume index was lower (p<0.01) and ejection fraction higher (p<0.01) in virus-related cirrhotic patients [mean +/- SD, respectively 12.4+/-4.1 ml/sqm and 77.9%) in comparison both to controls (21.5+/-6.3 ml/sqm and 66.8%) and alcoholics (20.6+/-7.0 ml/sqm and 68.8%). End diastolic volume index was not significantly different between the three groups. CONCLUSIONS: Our findings indicate smaller left ventricular volumes and higher ejection fraction in pure virus-related cirrhosis than in alcoholic cirrhosis and controls. Since peripheral haemodynamics proved similar in virus- and alcohol-related cirrhosis, a subclinical alcohol cardiomyopathy may be hypothesised to account for the absence of such left ventricular pattern in alcoholic patients.

Measurement of oxidative stress in human liver by EPR spin-probe technique.

A method for the measurement of reactive oxygen species (ROS) in human hepatic tissue has been developed. The method is based on the EPR detection of the nitroxide radical produced by reaction of the hydroxylamine spin-probe bis(1-hydroxy-2,2,6,6-tetramethyl-4-piperidinyl)decandioate with ROS generated under pseudo-physiologic conditions in fine needle biopsies of healthy (10 controls) and diseased (22 patients) human liver. Measures of malonaldehyde in 9 liver biopsies (3 controls and 6 patients) have also been obtained by high pressure liquid chromatography and values parallel those obtained by the spin-probe technique. The amount of ROS found in healthy human liver (median = 1.8 x 10(-11) mol/mg) was significantly lower than values found in liver affected by hepatitis B (median=5.8 x 10(-10) mol/mg; p < 0.02) or by hepatitis C (median = 2.7 x 10(-9) mol/mg; p < 0.003) as well as compared to some other non-viral liver diseases (NVLD): autoimmune hepatitis, primary biliary cirrhosis, primary schlerosing cholangitis (median = 9.8 x 10(-9) mol/mg; p < 0.005). NVLD also showed significantly higher ROS levels compared to hepatitis B (p < 0.04) and hepatitis C (p < 0.04). The mechanism, potentiality and limitations of our method are discussed.