Transcriptomic study in explanted liver from a patient with acute intermittent porphyria.

Acute intermittent porphyria (AIP) is a rare disease caused by a deficiency of hydroxymethylbilane synthase (HMBS), the third enzyme of the heme-synthesis pathway. Decreased enzymatic activity in the liver induces an overproduction of heme-precursors and acute neurological attacks. We report a 36-years-old female with AIP with a long-term history of severe, disabling, recurrent attacks, who underwent curative liver transplantation. Tissue samples from the explant were obtained for transcriptome analysis. Whole RNA was extracted and 16 gene-transcripts were selected and investigated by quantitative polymerase chain reaction. These included nine genes encoding enzymes that consecutively catalyze heme-synthesis and catabolism in the liver (ALAS1; ALAD; HMBS; UROS; UROD; CPOX; PPOX; FECH; HMOX1). Additionally, we studied genes related to inflammation (IL6; TNF) insulin signaling (PGC-1α; IGF-1; FOXO-1) and tryptophan metabolism (TDO2; IDO). Transcripts of eight house-keeping genes were co-measured for normalization. All transcripts were also measured in five control samples from healthy living liver donors. The transcriptome of the controls showed important differences between the various genes, with the first two genes of the heme-synthesis pathway, ALAS1 and ALAD showing strikingly high mRNA levels compared to the consecutive HMBS gene. Transcripts of several genes significantly differed in the AIP liver compared to controls. Transcripts of HMOX1 and UROS were increased in the AIP liver whereas transcripts of UROD; CPOX, PPOX, and TDO2 were decreased. ALAS1 expression was not increased, possibly due to hemin administered to the patient before transplantation. These results highlight several transcriptomic changes related to heme homeostasis in AIP.

Regression of fibrosis after chronic stimulation of cannabinoid CB2 receptor in cirrhotic rats.

Two cannabinoid (CB) receptor subtypes, CB1 and CB2, have been cloned and characterized. Among other activities, receptor activation by cannabinoid ligands may result in pro- or antifibrogenic effects depending on their interaction with CB1 or CB2, respectively. In the current study, we investigated whether selective activation of hepatic CB2 modifies collagen abundance in cirrhotic rats with ascites. mRNA and protein expression of CB receptors in the liver of control and cirrhotic rats was assessed by reverse transcription-polymerase chain reaction, Western blot, and immunohistochemistry. The effect of chronically activating the CB2 receptor was investigated in cirrhotic rats with ascites treated daily (9 days) with the CB2 receptor-selective agonist 3-(1,1-dimethylbutyl)-1-deoxy-Delta(8)-tetrahydrocannabinol (JWH-133). At the end of treatment, mean arterial pressure and portal pressure were measured, and liver samples were obtained to evaluate infiltrate of mononuclear cells, hepatic apoptosis, alpha-smooth muscle actin (SMA) expression, collagen content, and matrix metalloproteinase (MMP)-2 abundance in all animals. JWH-133 improved arterial pressure, decreased the inflammatory infiltrate, reduced the number of activated stellate cells, increased apoptosis in nonparenchymal cells located in the margin of the septa, and decreased fibrosis compared with cirrhotic rats treated with vehicle. This was associated with decreased alpha-SMA and collagen I and increased MMP-2 in the hepatic tissue of cirrhotic rats treated with the CB2 agonist compared with untreated cirrhotic animals. Therefore, selective activation of hepatic CB2 receptors significantly reduces hepatic collagen content in rats with pre-existing cirrhosis, thus raising the possibility of using selective CB2 agonists for the treatment of hepatic fibrosis in human cirrhosis.

Antiangiogenic treatment with sunitinib ameliorates inflammatory infiltrate, fibrosis, and portal pressure in cirrhotic rats.

UNLABELLED: Liver cirrhosis is a very complex disease in which several pathological processes such as inflammation, fibrosis, and pathological angiogenesis are closely integrated. We hypothesized that treatment with pharmacological agents with multiple mechanisms of action will produce superior results to those achieved by only targeting individual mechanisms. This study thus evaluates the therapeutic use of the multitargeted receptor tyrosine kinase inhibitor Sunitinib (SU11248). The in vitro effects of SU11248 were evaluated in the human hepatic stellate cell line LX-2 by measuring cell viability. The in vivo effects of SU11248 treatment were monitored in the livers of cirrhotic rats by measuring angiogenesis, inflammatory infiltrate, fibrosis, alpha-smooth muscle actin (alpha-SMA) accumulation, differential gene expression by microarrays, and portal pressure. Cirrhosis progression was associated with a significant enhancement of vascular density and expression of vascular endothelial growth factor-A, angiopoietin-1, angiopoietin-2, and placental growth factor in cirrhotic livers. The newly formed hepatic vasculature expressed vascular cellular adhesion molecule 1 and intercellular adhesion molecule 1. Interestingly, the expression of these adhesion molecules was adjacent to areas of local inflammatory infiltration. SU11248 treatment resulted in a significant decrease in hepatic vascular density, inflammatory infiltrate, alpha-SMA abundance, LX-2 viability, collagen expression, and portal pressure. CONCLUSION: These results suggest that multitargeted therapies against angiogenesis, inflammation, and fibrosis merit consideration in the treatment of cirrhosis.

Sustained aquaretic effect of the V2-AVP receptor antagonist, RWJ-351647, in cirrhotic rats with ascites and water retention.

1 A disturbance in body water homeostasis is a common feature in advanced cirrhosis. This disturbance is always associated with the existence of ascites and is characterized by an inability to adjust the amount of water excreted in the urine to the amount of water ingested. Vasopressin (AVP) is of major importance in the pathogenesis of water retention and hyponatremia in cirrhosis. 2 The current study assessed the renal, hormonal and hemodynamic effects induced by 10-day chronic oral administration of RWJ-351647 (0.5 mg kg(-1) daily), a new nonpeptide V(2)-AVP antagonist, in rats with CCl(4)-induced cirrhosis, ascites and severe water retention. Urine volume (UV), urine osmolality and sodium and potassium excretion were measured daily. At the end of the study, systemic hemodynamic parameters were also assessed. 3 Long-term administration of RWJ-351647 has an aquaretic effect in rats with cirrhosis, ascites, water retention and hypo-osmolality. It increases UV (ANOVA: F=7.32, P<0.0001) and reduces urine osmolality (ANOVA: F=12.69, P<0.0001) throughout the entire period of treatment, thereby leading to a greater renal ability to excrete a water load at the end of the 10-day treatment period (the percentage of water load excreted improved from 30+/-8 to 92+/-21%, P<0.025). 4 The nonpeptide AVP V(2)-receptor antagonist RWJ-351647 also increased sodium excretion without affecting creatinine clearance and blood pressure. 5 These data suggest that RWJ-351647 could be therapeutically useful in the treatment of water retention in human cirrhosis.

Impaired extracellular matrix degradation in aortic vessels of cirrhotic rats.

BACKGROUND/AIMS: Thinning of the vascular wall occurs in conductance vessels of cirrhotic rats. Increased nitric oxide synthase (NOS) activity has been involved in the pathogenesis of this phenomenon. Therefore, we assessed the NO-regulated cell signaling pathways participating in vascular remodeling in cirrhosis. METHODS: Aortas were obtained from 15 control and 15 cirrhotic rats. Phosphorylated p38 MAPK and ERK1/2 were used to evaluate the activation of cell MAPK signaling pathways. Extracellular matrix (ECM) turnover was estimated by measuring matrix metalloproteinases (MMPs) activity and protein expression of collagen IV, MMP-2, MMP-9 and tissue inhibitor of MMPs (TIMP)-2. Thereafter, 12 control and 12 cirrhotic rats received Nomega-nitro-L-arginine-methyl-ester or vehicle daily for 11 weeks. RESULTS: Cirrhotic vessels showed a reduction in ERK1/2 phosphorylation, lower MMP activity, decreased MMPs expression and higher collagen IV and TIMP-2 abundance, compared to control rats. Chronic NOS inhibition normalized ERK1/2 phosphorylation and MMPs activity, increased MMPs abundance and decreased TIMP-2 expression in cirrhotic rats. CONCLUSIONS: Vascular remodeling in cirrhotic rats is mediated by down-regulation of cell growth and impaired ERK1/2 activation and subsequent imbalance of ECM turnover. These results further stress the importance of vascular NO overactivity in the reduction of vascular wall thickness in cirrhosis.

Microarray analysis of endothelial differentially expressed genes in liver of cirrhotic rats.

BACKGROUND & AIMS: There is a long-standing interest in the identification of endothelial-specific pathways for therapeutic targeting in cirrhosis. Therefore, the aim of this study was to evaluate differences in gene expression patterns between liver endothelial cells (LECs) from control and cirrhotic rats by using microarrays. METHODS: LECs were obtained by isopycnic centrifugation. LECs gene expression was then analyzed on high-density oligonucleotide microarrays. RESULTS: Analysis of gene expression revealed that most of the differentially expressed mRNA in cirrhosis are associated with extracellular matrix remodeling, inflammation, antioxidant/stress response, and cell signaling. CONCLUSIONS: The collective expression changes observed within some functional groups of genes indicate that LECs in cirrhotic livers may contribute to lymphangiogenesis, enhancement of fibrogenesis and inflammatory processes, changes in cell-cell interaction with up-regulation of adherens junction proteins, and alterations in the intrahepatic vascular tone because of the down-regulation of genes involved in vasodilatation.