High glucose increases lysyl oxidase expression and activity in retinal endothelial cells: mechanism for compromised extracellular matrix barrier function.

OBJECTIVE: In diabetes, retinal vascular basement membrane (BM) undergoes significant thickening and compromises vessel function including increased vascular permeability, a prominent lesion of early diabetic retinopathy. In this study we determined whether altered expression and activity of lysyl oxidase (LOX), a cross-linking enzyme, may compromise vascular basement membrane functional integrity under high-glucose (HG) conditions. RESEARCH DESIGN AND METHODS: Rat retinal endothelial cells (RRECs) grown in normal (5 mmol/l) or HG (30 mmol/l glucose) medium for 7 days were assessed for expression of LOX and proLOX by Western blot analysis and LOX enzyme activity. To determine whether HG alters cellular distribution patterns of LOX and proLOX, immunostaining with respective antibodies was performed. Similarly, cells grown in normal or HG medium were subjected to both LOX inhibition with ß-aminopropionitrile (BAPN) and by small interfering RNA knockdown, and respectively examined for cell monolayer permeability. Additionally, retinas of streptozotocin (STZ)-induced diabetic rats were analyzed to determine if diabetes altered LOX expression. RESULTS: Western blot analysis revealed significantly increased LOX and proLOX expression in cells grown in HG medium compared with those grown in normal medium. The increased LOX level was strikingly similar to LOX upegulation in the diabetic retinas. In cells grown in HG medium, LOX activity and cell monolayer permeability was significantly increased, as were LOX and proLOX immunostaining. Small interfering RNA- or BAPN-induced-specific blockage of LOX expression or activity, respectively, reduced cell monolayer permeability. CONCLUSIONS: HG-induced increased LOX expression and activity compromises barrier functional integrity, a prominent lesion of diabetic retinopathy.

Mu (mu) opioid receptor regulation of ethanol-induced dopamine response in the ventral striatum: evidence of genotype specific sexual dimorphic epistasis.

BACKGROUND: Ethanol stimulates the dopaminergic mesoaccumbal pathway, which is thought to play a role in ethanol reinforcement. Mu (mu)-opioid (MOP) receptors modulate accumbal dopamine activity, but it is not clear whether MOP receptors are involved in the mechanism of ethanol-stimulated accumbal dopamine release. METHODS: We investigated the role that MOP receptors play in ethanol (2.0 g/kg)-stimulated accumbal dopamine release by using MOP receptor knockout mice (C57BL/6J-129SvEv and congenic C57BL/6J genotypes) along with blockade of MOP receptors with a mu1 selective antagonist (naloxonazine). RESULTS: Both gene deletion and pharmacological antagonism of the MOP receptor decreased ethanol-stimulated accumbal dopamine release compared with controls with female mice showing a larger effect in the C57BL/6J-129SvEv genotype. However, both male and female mice showed reduced ethanol-stimulated dopamine release in the congenic MOP receptor knockout mice (C57BL/6J). No differences in the time course of dialysate ethanol concentration were found in any of the experiments. CONCLUSIONS: The data demonstrate the existence of a novel interaction between genotype and sex in the regulation of ethanol-stimulated mesolimbic dopamine release by the MOP receptor. This implies that a more complete understanding of the epistatic influences on the MOP receptor and mesolimbic dopamine function may provide more effective pharmacotherapeutic interventions in the treatment of alcoholism.

A randomized phase III study of doxorubicin versus cisplatin/interferon alpha-2b/doxorubicin/fluorouracil (PIAF) combination chemotherapy for unresectable hepatocellular carcinoma.

BACKGROUND: Single-agent doxorubicin has been widely used to treat unresectable hepatocellular carcinoma (HCC), but the response rate is low (< 20%) and there is no convincing evidence for improved survival. Cisplatin, interferon, doxorubicin, and fluorouracil (PIAF) used in combination, by contrast, has shown promise in a phase II study. We compared doxorubicin to PIAF in patients with unresectable HCC in a phase III trial. METHODS: Patients with histologically confirmed unresectable HCC were randomly assigned to receive either doxorubicin or PIAF every 3 weeks, for up to six cycles. The primary endpoint was overall survival, and secondary endpoints were response rate and toxicity. Survival differences were calculated using the Kaplan-Meier method. Treatment groups were compared for differences in the incidence of adverse events using chi-square tests. All statistical tests were two-sided. RESULTS: The median survival of the doxorubicin and PIAF groups was 6.83 months (95% confidence [CI] = 4.80 to 9.56) and 8.67 months (95% CI = 6.36 to 12.00), respectively (P = 0.83). The hazard ratio for death from any cause in the PIAF compared with the doxorubicin groups was 0.97 (95% CI = 0.71 to 1.32). Eighty-six of the 94 patients receiving doxorubicin and 91 of the 94 receiving PIAF were assessable for response. The overall response rates in the doxorubicin and PIAF groups were 10.5% (95% CI = 3.9% to 16.9%) and 20.9% (95% CI = 12.5% to 29.2%), respectively. Neutropenia, thrombocytopenia, and hypokalemia were statistically significantly more common in patients treated with PIAF than in patients treated with doxorubicin. CONCLUSION: Although patients on PIAF had a higher overall response rate and better survival than patients on doxorubicin, the differences were not statistically significant. PIAF was also associated with increased treatment-related toxicity. The prognosis of patients with unresectable HCC remains poor.

Ethanol increases extracellular dopamine concentration in the ventral striatum in C57BL/6 mice.

BACKGROUND: Mesolimbic dopamine is thought to play a role in the reinforcing properties of ethanol, but ethanol-induced changes in extracellular dopamine in the ventral striatum have not been well characterized in mouse models. METHODS: Two experiments were used to characterize the pharmacodynamic response of ethanol in the ventral striatum in C57BL/6 mice. The first experiment determined the effect of ethanol on ventral striatal dopamine in male and female mice after intraperitoneal injection of either 2.0 g/kg ethanol or saline. The second experiment was a replication in males, except that the mice were habituated to intraperitoneal injections before the dialysis experiment. RESULTS: Distinct patterns of dopamine activity in response to ethanol were demonstrated in male and female C57BL/6 mice. A significant increase in dialysate dopamine relative to saline injection was observed in females but not in males. With habituation to intraperitoneal injection before the dialysis experiment, ethanol administration caused a significant dopamine response in males as well. A linear decline was observed in dialysate ethanol concentrations after the peak concentration was reached. Concurrent analysis of the time course of dopamine and ethanol content showed that the dopamine response declined significantly faster than the ethanol concentrations. CONCLUSIONS: The C57BL/6 mouse strain is a justifiable model system for studying the mechanisms involved in ethanol regulation of mesolimbic dopamine activity. Habituation to intraperitoneal injection should be used in male C57BL/6 mice for experiments in which the dopamine response is measured after intraperitoneal injection of a drug. The dissociation between dopamine and ethanol may indicate an acute neural adaptation to ethanol-induced dopamine response in the ventral striatum after a single ethanol injection.

Characterization of probe and tissue factors that influence interpretation of quantitative microdialysis experiments for dopamine.

Two quantitative methods, the Lönnroth (no-net-flux) and variation of perfusion flow rate methods, were used to investigate the influence of the probe and tissue on dopamine microdialysis measurements. In vivo measurements were made in the nucleus accumbens of awake, freely moving rats on two consecutive days of dialysis. The results of the no-net-flux study showed that there was no statistically significant difference in extraction fraction at a perfusion flow rate of 2.0 microl/min between in vitro in a well-stirred solution and in vivo measured during 2 days of continuous dialysis. Also, varying the perfusate flow rate over the range 0.25-2.0 microl/min produced a variation in the extraction fraction that was the same in vitro and in vivo. These results indicate that the extraction fraction for dopamine over the 2 days was dominated by the properties of the probe. The negligible influence of the tissue on dopamine extraction fraction was probably due to the high basal activity of the dopamine transporter in vivo. Therefore, the extraction fraction is unlikely to be sensitive to increases in dopamine uptake in the vicinity of the probe. The apparent extracellular dopamine concentration increased by 37% on the second day of dialysis while the calcium-dependence of basal dialysate dopamine levels declined by 20%. These findings are consistent with a decrease in physiological viability of the dopamine nerve terminals surrounding the probe during a long-term experiment.

Factors predicting response and survival in 149 patients with unresectable hepatocellular carcinoma treated by combination cisplatin, interferon-alpha, doxorubicin and 5-fluorouracil chemotherapy.

BACKGROUND: The objective of the current study was to identify patient and disease related factors that influence response and survival for patients with unresectable hepatocellular carcinoma (HCC) who received a systemic combination chemotherapy consisting of cisplatin, alpha-interferon, doxorubicin, and 5-fluorouracil (PIAF). METHODS: From July 1996 to February 1999, 149 patients with unresectable HCC were treated with PIAF: cisplatin (20mg/m2 intravenously, Days 1-4), doxorubicin (40mg/m2 intravenously, Day 1), 5-fluorouracil (400mg/m2 intravenously, Days 1-4), and alpha-interferon (5MU/m2 subcutaneously, Days 1-4), once every 3 weeks up to a maximum of six cycles. Univariate and multivariate analyses of patient and disease characteristics were used to identify factors predicting response and survival. RESULTS: The objective response rate according to conventional criteria was 16.8% (complete response in 3 out of 149 patients, or 2%, 95% confidence interval [CI] 0-4.3%; partial response in 22 out of 149 patients, or 14.8%, 95% CI 9-20%). The median survival time was 30.9 weeks (95% CI 22.1 to 40). Significant independent predictors of an objective response were: absence of cirrhosis (P = 0.006), low bilirubin level (P = 0.006), and positive hepatitis C serology (P = 0.025). The following factors were related to a shorter survival time: high Okuda stage (P = 0.001), vascular involvement (P = 0.018), and cirrhosis (P = 0.008). Good risk patients (absence of cirrhosis and total bilirubin < or = 0.6mg/dL) had an objective response rate of 50%. CONCLUSIONS. Patients with unresectable HCC who also have normal total bilirubin and non-cirrhotic livers have a better chance of response and prolonged survival after treatment with systemic PIAF.

Construction of the Chinese University Prognostic Index for hepatocellular carcinoma and comparison with the TNM staging system, the Okuda staging system, and the Cancer of the Liver Italian Program staging system: a study based on 926 patients.

BACKGROUND: The current TNM staging system for patients with hepatocellular carcinoma (HCC) does not include liver function parameters and does not provide a precise prognosis for patients in different risk groups. The objectives of this study were to construct a new prognostic index for patients with hepatocellular carcinoma, the Chinese University Prognostic Index (CUPI), and to compare it with existing staging systems in terms of their ability to classify patients into different risk group. METHODS: From 1996 to 1998, 926 ethnic Chinese patients who were diagnosed with HCC (mainly hepatitis B-associated) at a single institution were recruited prospectively into this study. A multivariate analysis on 19 patient characteristics was performed using a Cox regression model to identify independent prognostic factors. Weights were derived from the regression coefficients of various factors to construct the CUPI. Patients were classified according to different staging systems. Survival curves were plotted with the Kaplan-Meier method and were compared by using a log-rank test. RESULTS: Both the TNM staging system and the Okuda staging system had prognostic significance, but the significance was lower for the Cancer of the Liver Italian Program (CLIP) prognostic score among the patients in the study population. The CUPI was constructed by adding the following factors into the TNM staging system: total bilirubin, ascites, alkaline phosphatase, alpha fetoprotein, and asymptomatic disease on presentation. The new CUPI characterized three risk groups with highly significant differences in survival during the whole period of follow-up (P < 0.00001) and was more discriminant than the other systems. CONCLUSIONS: In the study population of patients with mainly hepatitis B-associated HCC, the CUPI was more discriminant than the TNM staging system, the Okuda staging systems, or the CLIP prognostic score in classifying patients into different risk groups and was better at predicting survival. The CUPI needs to be validated by different cohorts of patients before it can be recommended for general use.