Leptin-mediated reactive oxygen species production does not significantly affect primary mouse hepatocyte functions in vitro.

AIM: Direct and indirect effects of leptin on hepatic stellate cells (HSCs) have been documented in the literature, whereas little is known about leptin's actions on hepatocytes. Leptin mediates its profibrogenic and proinflammatory effects on HSCs in part through the production of intracellular reactive oxygen species (ROS). In this study, we focus our analysis on leptin-induced ROS production in hepatocytes. METHODS: The expression of leptin receptor isoforms on primary mouse liver cells was examined by real-time quantitative-PCR and western blotting. Cultures were exposed to leptin in combination with inhibitors for reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, MAP kinase/ERK kinase 1 (MEK1) or janus kinase 2 (JAK2). ROS levels were quantified by measuring fluorescence. The effects of leptin on hepatocyte functions and programmed cell death were evaluated by fluorescent or luminescent assays. RESULTS: Leptin induced ROS production in primary hepatocytes by 150-450%, compared with a 20-30% increase in HSCs and liver sinusoidal endothelial cells (LSECs). This ROS production could be inhibited by NADPH oxidase, MEK1 and JAK2 inhibitors. Western blotting indicated that mouse HSCs and LSECs mainly express short leptin receptor isoforms, whereas hepatocytes appeared to express both short and long isoform(s). Leptin-induced ROS production in db/db hepatocytes did not differ from wild-type mice. Finally, leptin had no negative influence on primary hepatocyte functions. CONCLUSION: Leptin induced higher ROS levels in primary hepatocytes than in LSECs and HSCs, depending on NADPH oxidase, MEK1 and JAK2 signalling but not on the long leptin receptor isoform. Furthermore, leptin exposure did not influence primary hepatocyte functionality negatively.

Successful isolation of liver progenitor cells by aldehyde dehydrogenase activity in naïve mice.

UNLABELLED: The role of progenitor cells in liver repair and fibrosis has been extensively described, but their purification remains a challenge, hampering their characterization and use in regenerative medicine. To address this issue, we developed an easy and reproducible liver progenitor cell (LPC) isolation strategy based on aldehyde dehydrogenase (ALDH) activity, a common feature shared by many progenitor cells. We demonstrate that a subset of nonparenchymal mouse liver cells displays high levels of ALDH activity, allowing the isolation of these cells by fluorescence-activated cell sorting. Immunocytochemistry and qPCR analyses on freshly isolated ALDH(+) cells reveal an enrichment in cells expressing liver stem cell markers such as EpCAM, CK19, CD133, and Sox9. In culture, the ALDH(+) population can give rise to functional hepatocyte-like cells as illustrated by albumin and urea secretion and cytochrome P450 activity. ALDH1A1 expression can be detected in canals of Hering and bile duct epithelial cells and is increased on liver injury. Finally, we showed that the isolation and differentiation toward hepatocyte-like cells of LPCs with high ALDH activity is also successfully applicable to human liver samples. CONCLUSION: High ALDH activity is a feature of LPCs that can be taken advantage of to isolate these cells from untreated mouse as well as human liver tissues. This novel protocol is practically relevant, because it provides an easy and nontoxic method to isolate liver stem cells from normal tissue for potential therapeutic purposes.

Distinct roles for non-muscle myosin II isoforms in mouse hepatic stellate cells.

BACKGROUND & AIMS: Upon liver injury, hepatic stellate cells (HSCs) undergo dramatic morphological and functional changes including migration and contraction. In the present study, we investigated the role of myosin II isoforms in the development of the contractile phenotype of mouse HSCs, which are considered therapeutic targets to decrease portal hypertension and fibrosis. METHODS: We characterized the expression of myosin IIA and IIB in primary mouse HSCs and addressed their function by gene knock-down using isoform-specific siRNAs. RESULTS: We found that myosin IIA and IIB are differentially expressed and localized and have clearly different functions in HSCs. Myosin IIA is mainly located in the subcortical area of quiescent HSCs and at α-SMA-containing stress fibres after activation, while myosin IIB is located in the cytoplasm and at the edge of protrusions of quiescent HSCs, at stress fibres of activated cells, and at the leading edge of lamellipodia. Knock-down of myosin IIA in HSCs influences cell size and shape, results in the disruption of stress fibres and in a decrease of focal adhesions, and inhibits contractility and intra-cellular Ca(2+) release but increases cell migration. Myosin IIB contributes to the extension of lamellipodia and cell spreading but has no direct role in stress fibres and focal adhesion formation, contraction, or intra-cellular Ca(2+) signalling. CONCLUSIONS: In mouse HSCs, myosin IIA and IIB clearly fulfil distinct roles. Our results provide an insight into the contractile machinery of HSCs, that could be important in the search for new molecules to treat portal hypertension.

Peroxisomal and microsomal lipid pathways associated with resistance to hepatic steatosis and reduced pro-inflammatory state.

Accumulation of fat in the liver increases the risk to develop fibrosis and cirrhosis and is associated with development of the metabolic syndrome. Here, to identify genes or gene pathways that may underlie the genetic susceptibility to fat accumulation in liver, we studied A/J and C57Bl/6 mice that are resistant and sensitive to diet-induced hepatosteatosis and obesity, respectively. We performed comparative transcriptomic and lipidomic analysis of the livers of both strains of mice fed a high fat diet for 2, 10, and 30 days. We found that resistance to steatosis in A/J mice was associated with the following: (i) a coordinated up-regulation of 10 genes controlling peroxisome biogenesis and ß-oxidation; (ii) an increased expression of the elongase Elovl5 and desaturases Fads1 and Fads2. In agreement with these observations, peroxisomal ß-oxidation was increased in livers of A/J mice, and lipidomic analysis showed increased concentrations of long chain fatty acid-containing triglycerides, arachidonic acid-containing lysophosphatidylcholine, and 2-arachidonylglycerol, a cannabinoid receptor agonist. We found that the anti-inflammatory CB2 receptor was the main hepatic cannabinoid receptor, which was highly expressed in Kupffer cells. We further found that A/J mice had a lower pro-inflammatory state as determined by lower plasma levels and IL-1ß and granulocyte-CSF and reduced hepatic expression of their mRNAs, which were found only in Kupffer cells. This suggests that increased 2-arachidonylglycerol production may limit Kupffer cell activity. Collectively, our data suggest that genetic variations in the expression of peroxisomal ß-oxidation genes and of genes controlling the production of an anti-inflammatory lipid may underlie the differential susceptibility to diet-induced hepatic steatosis and pro-inflammatory state.

Advanced glycation end products induce production of reactive oxygen species via the activation of NADPH oxidase in murine hepatic stellate cells.

BACKGROUND & AIMS: Advanced glycation end products are known to play an important role in the metabolic syndrome and were recently suggested to contribute to liver fibrosis development. However, little is known about the effect of advanced glycation end products on hepatic stellate cells, the major contributors to liver fibrosis development. We therefore studied the effect of advanced glycation end products on reactive oxygen species generation, a main feature for the activation hepatic stellate cells. METHODS: Three different types of advanced glycation end products were generated by BSA incubation with different substrates. The presence of advanced glycation end product receptors was examined by RTq-PCR, immunofluorescence and western blotting. Reactive oxygen species production was measured using DCFH-DA. RESULTS: Hepatic stellate cells express five advanced glycation end product receptors: Galectin-3, CD36, SR-AI, SR-BI and RAGE. All receptors, except SR-BI, showed up-regulation during HSC activation. All three advanced glycation end product types induced reactive oxygen species generation. DPI and NSC, a NADPH oxidase and a Rac1 inhibitor respectively, inhibited reactive oxygen species production. Rottlerin, a molecule often used as a PKCdelta inhibitor, also abrogated reactive oxygen species production. SiRNA mediated knockdown of p47(phox), Rac1 and PKCdelta decreased reactive oxygen species production induced by advanced glycation end products, establishing a role for these proteins in reactive oxygen species induction. CONCLUSIONS: The demonstration of advanced glycation end product-induced reactive oxygen species generation in hepatic stellate cells unveils a potential new route through which advanced glycation end products induce liver fibrosis in the metabolic syndrome.

Blebbistatin inhibits contraction and accelerates migration in mouse hepatic stellate cells.

BACKGROUND AND PURPOSE: Blebbistatin, an inhibitor of myosin-II-specific ATPase, has been used to inhibit contraction of invertebrate and mammalian muscle preparations containing non-muscle myosin. Activated hepatic stellate cells have contractile properties and play an important role in the pathophysiology of liver fibrosis and portal hypertension. Therefore, hepatic stellate cells are considered as therapeutic target cells. In the present study, we studied the effect of blebbistatin during the transition of mouse hepatic stellate cells into contractile myofibroblasts. EXPERIMENTAL APPROACH: Effects of blebbistatin on cell morphology were evaluated by phase contrast microscopy. Cell stress fibres and focal adhesions were investigated by dual immunofluorescence staining and visualized using fluorescence microscopy. Contractile force generation was examined by silicone wrinkle formation assays and collagen gel contraction assays. Intracellular Ca(2+) release in response to endothelin-1 was measured by using Fluo-4. Cell migration was measured by wound healing experiments. KEY RESULTS: In culture-activated hepatic stellate cells, blebbistatin was found to change both cell morphology and function. In the presence of blebbistatin, stellate cells became smaller, acquired a dendritic morphology and had less myosin IIA-containing stress fibres and vinculin-containing focal adhesions. Moreover, blebbistatin impaired silicone wrinkle formation, reduced collagen gel contraction and blocked endothelin-1-induced intracellular Ca(2+) release. Finally, it promoted wound-induced cell migration. CONCLUSIONS AND IMPLICATIONS: By inhibiting myosin II ATPase, blebbistatin has profound effects on the morphology and function of activated hepatic stellate cells. Our data suggest that myosin II could be a therapeutic target in the treatment of liver fibrosis and portal hypertension.

The quest for liver progenitor cells: a practical point of view.

Many chronic liver diseases can lead to hepatic dysfunction with organ failure. At present, orthotopic liver transplantation represents the benchmark therapy of terminal liver disease. However this practice is limited by shortage of donor grafts, the need for lifelong immunosuppression and very demanding state-of-the-art surgery. For this reason, new therapies have been developed to restore liver function, primarily in the form of hepatocyte transplantation and artificial liver support devices. While already offered in very specialized centers, both of these modalities still remain experimental. Recently, liver progenitor cells have shown great promise for cell therapy, and consequently they have attracted a lot of attention as an alternative or supportive tool for liver transplantation. These liver progenitor cells are quiescent in the healthy liver and become activated in certain liver diseases in which the regenerative capacity of mature hepatocytes and/or cholangiocytes is impaired. Although reports describing liver progenitor cells are numerous, they have not led to a consensus on the identity of the liver progenitor cell. In this review, we will discuss some of the characteristics of these cells and the different ways that have been used to obtain these from rodents. We will also highlight the challenges that researchers are facing in their quest to identify and use liver progenitor cells.

Chronic administration of valproic acid inhibits activation of mouse hepatic stellate cells in vitro and in vivo.

UNLABELLED: Hepatic stellate cell (HSC) activation is a pivotal step in the pathogenesis of liver fibrosis. The clarification of this transdifferentiation process is therefore important for the development of effective therapies for fibrosis. We analyzed the effect of a histone deacetylase inhibitor, valproic acid (VPA), on mouse HSC transdifferentiation in vitro and in vivo. The exposure of freshly isolated mouse HSCs to 2.5 mM VPA led to increased histone H4 acetylation and inhibited cell proliferation. Expression of stellate cell activation markers analyzed by quantitative polymerase chain reaction and western blotting revealed that treatment with VPA inhibited the induction of activation markers such as Acta2, Lox, Spp1, and Myh11. Treatment of mice with VPA decreased collagen deposition and in vivo activation of stellate cells in the livers of CCl(4)-treated mice. Class I histone deacetylase silencing through RNA interference in mouse HSCs only partially mimicked treatment with VPA. CONCLUSION: Chronic administration of VPA results in a marked decrease in stellate cell activation both in vitro and in vivo. We hypothesize that the VPA effect results partially from class I histone deacetylase inhibition, but that also non-histone deacetylase class I VPA targets are involved in the stellate cell activation process.

Role of epigenetics in liver-specific gene transcription, hepatocyte differentiation and stem cell reprogrammation.

Controlling both growth and differentiation of stem cells and their differentiated somatic progeny is a challenge in numerous fields, from preclinical drug development to clinical therapy. Recently, new insights into the underlying molecular mechanisms have unveiled key regulatory roles of epigenetic marks driving cellular pluripotency, differentiation and self-renewal/proliferation. Indeed, the transcription of genes, governing cell-fate decisions during development and maintenance of a cell's differentiated status in adult life, critically depends on the chromatin accessibility of transcription factors to genomic regulatory and coding regions. In this review, we discuss the epigenetic control of (liver-specific) gene-transcription and the intricate interplay between chromatin modulation, including histone (de)acetylation and DNA (de)methylation, and liver-enriched transcription factors. Special attention is paid to their role in directing hepatic differentiation of primary hepatocytes and stem cells in vitro.

Systemic antigen cross-presented by liver sinusoidal endothelial cells induces liver-specific CD8 T-cell retention and tolerization.

UNLABELLED: Peripheral CD8 T-cell tolerance can be generated outside lymphatic tissue in the liver, but the course of events leading to tolerogenic interaction of hepatic cell populations with circulating T-cells remain largely undefined. Here we demonstrate that preferential uptake of systemically circulating antigen by murine liver sinusoidal endothelial cells (LSECs), and not by other antigen-presenting cells in the liver or spleen, leads to cross-presentation on major histocompatibility complex (MHC) I molecules, which causes rapid antigen-specific naïve CD8 T-cell retention in the liver but not in other organs. Using bone-marrow chimeras and a novel transgenic mouse model (Tie2-H-2K(b) mice) with endothelial cell-specific MHC I expression, we provide evidence that cross-presentation by organ-resident and radiation-resistant LSECs in vivo was both essential and sufficient to cause antigen-specific retention of naïve CD8 T-cells under noninflammatory conditions. This was followed by sustained CD8 T-cell proliferation and expansion in vivo, but ultimately led to the development of T-cell tolerance. CONCLUSION: Our results show that cross-presentation of circulating antigens by LSECs caused antigen-specific retention of naïve CD8 T-cells and identify antigen-specific T-cell adhesion as the first step in the induction of T-cell tolerance.

The interleukin-17 pathway is involved in human alcoholic liver disease.

UNLABELLED: Immune dysregulations in alcoholic liver diseases are still unclear, especially regarding alcoholic hepatitis inflammatory burst. Interleukin-17 (IL-17) is known to enhance neutrophil recruitment. We studied the IL-17 pathway in alcoholic cirrhosis and alcoholic hepatitis. Patients with alcoholic liver disease were compared with patients with chronic hepatitis C virus (HCV) infection or autoimmune liver disease and with healthy controls. IL-17 plasma levels and peripheral blood mononuclear cell secretion were assessed by enzyme-linked immunosorbent assay (ELISA) and T cell phenotype by flow cytometry. IL-17 staining and co-staining with CD3 and myeloperoxidase were performed on liver biopsy specimens. IL-17 receptor expression was studied on liver biopsies and in human hepatic stellate cells as well as their response to recombinant IL-17 by chemotaxis assays. IL-17 plasma levels were dramatically increased in alcoholic liver disease patients. Peripheral blood mononuclear cells of patients with alcoholic liver disease produced higher amounts of IL-17, and their CD4(+) T lymphocytes disclosed an IL-17-secreting phenotype. In the liver, IL-17-secreting cells contributed to inflammatory infiltrates in alcoholic cirrhosis, and alcoholic hepatitis foci disclosed many IL-17(+) cells, including T lymphocytes and neutrophils. In alcoholic liver disease, liver IL-17(+) cells infiltrates correlated to model for end-stage liver disease score, and in alcoholic hepatitis to modified discriminant function. IL-17 receptor was expressed in alcoholic liver disease by hepatic stellate cells, and these cells recruited neutrophils after IL-17 stimulation in a dose-dependent manner through IL-8 and growth related oncogen alpha (GRO-alpha) secretion in vitro. CONCLUSION: Human alcoholic liver disease is characterized by the activation of the IL-17 pathway. In alcoholic hepatitis, liver infiltration with IL-17-secreting cell infiltrates is a key feature that might contribute to liver neutrophil recruitment. (Clinical trials number NCT00610597).

Vinculin and cellular retinol-binding protein-1 are markers for quiescent and activated hepatic stellate cells in formalin-fixed paraffin embedded human liver.

Hepatic stellate cells (HSCs) have important roles in the pathogenesis of liver fibrosis and cirrhosis. As response to chronic injury HSCs are activated and change from quiescent into myofibroblast-like cells. Several HSC-specific markers have been described in rat or mouse models. The aim of our work was to identify the best marker(s) for human HSCs. To this end we used the automated high throughput NexES IHC staining device (Ventana Medical Systems) to incubate sections under standardized conditions. Formalin fixed paraffin embedded (FFPE) normal and diseased human livers were studied. With immunohistochemistry we examined the expression of synemin, desmin, vimentin, vinculin, neurotrophin-3 (NT-3), alpha-smooth muscle actin (alpha-SMA), cellular retinol-binding protein-1 (CRBP-1), glial fibrillary acidic protein (GFAP), cysteine- and glycine-rich protein 2 (CRP2), and cytoglobin/stellate cell activation-associated protein (cygb/STAP). This is the first study in which a series of HSC markers is compared on serial FFPE human tissues. CRBP-1 clearly stains lobular HSCs without reacting with smooth muscle cells (SMCs) and shows variable cholangiocyte positivity. Vinculin has a similar staining pattern as CRBP-1 but additionally stains SMCs, and (myo)fibroblasts. In conclusion, we therefore propose to use CRBP-1 and/or vinculin to stain HSCs in human liver tissues.

Screening of amide analogues of Trichostatin A in cultures of primary rat hepatocytes: search for potent and safe HDAC inhibitors.

The vast majority of preclinical studies of HDAC inhibitors (HDAC-I) focus on the drug-target (cancer) cell interaction, whereas little attention is paid to the effects on non-target healthy cells, which could provide decisive information to eliminate potential cytotoxic compounds at a very early stage during drug development. In the current study we used cultures of primary rat hepatocytes as a read out system to select for the most potent HDAC-I in the group of structural analogues of an archetypal HDAC-I, namely Trichostatin A. This kind of approach allowed selecting compounds with high biological activity and with no apparent toxicity towards cultured hepatocytes.

Multidrug resistance-associated proteins are crucial for the viability of activated rat hepatic stellate cells.

UNLABELLED: Hepatic stellate cells (HSCs) survive and proliferate in the chronically injured liver. ATP-binding cassette (ABC) transporters play a crucial role in cell viability by transporting toxic metabolites or xenobiotics out of the cell. ABC transporter expression in HSCs and its relevance to cell viability and/or activation have not been reported so far. The aim of this study was to investigate the expression, regulation, and function of multidrug resistance-associated protein (Mrp)-type and multidrug resistance protein (Mdr)-type ABC transporters in activated rat HSCs. Rat HSCs were exposed to cytokines or oxidative stress. ABC transporter expression was determined by quantitative polymerase chain reaction and immunohistochemistry. HSCs were exposed to the Mdr inhibitors verapamil and PSC-833 and the Mrp inhibitor MK571. Mdr and Mrp transporter function was evaluated with flow cytometry. Apoptosis was determined by activated caspase-3 and acridine orange staining, and necrosis was determined by Sytox green nuclear staining. An in vivo model of carbon tetrachloride (CCl(4))-induced liver fibrosis was used. With respect to hepatocytes, activated HSCs expressed high levels of Mrp1 and comparable levels of Mrp3, Mrp4, Mdr1a, and Mdr1b but not the hepatocyte-specific transporters bile salt export pump, Mrp2, and Mrp6. Mrp1 protein staining correlated with desmin staining in livers from CCl(4)-treated rats. Mrp1 expression increased upon activation of HSCs. Cytokines induced Mdr1b expression only. Oxidative stress was not a major regulator of Mdr and Mrp transporter expression. Activated HSCs became necrotic when exposed to the Mrp inhibitors. CONCLUSION: Activated HSCs contain relatively high levels of Mrp1. Mrp-type transporters are required for the viability of activated HSCs. Mrp-dependent export of endogenous metabolites is important for the survival of activated HSCs in chronic liver diseases.

Regulation of sinusoidal perfusion in portal hypertension.

Portal hypertension, a major complication of cirrhosis, is caused by both increased portal blood flow and augmented intrahepatic vascular resistance. Even though the latter is primarily caused by anatomical changes, it has become clear that dynamic factors contribute to the increased hepatic vascular resistance. The hepatic sinusoid is the narrowest vascular structure within the liver and is the principal site of blood flow regulation. The anatomical location of hepatic stellate cells, which embrace the sinusoids, provides a favorable arrangement for sinusoidal constriction, and for control of sinusoidal vascular tone and blood flow. Hepatic stellate cells possess the essential contractile apparatus for cell contraction and relaxation. Moreover, the mechanisms of stellate cell contraction are better understood, and many substances which influence contractility have been identified, providing a rationale and opportunity for targeting these cells in the treatment of portal hypertension in cirrhosis.

CRBP-I in the renal tubulointerstitial compartment of healthy rats and rats with renal fibrosis.

BACKGROUND: Cellular retinol-binding protein I (CRBP-I), a member of the intracellular lipid-binding protein (iLBP) superfamily, is a specific marker of quiescent stellate cells in the healthy human liver. In the diseased fibrotic/cirrhotic liver, portal and septal myofibroblasts acquire CRBP-I expression, while activated hepatic stellate cells maintain their CRBP-I expression. Here, we investigate the distribution of CRBP-I in the renal cortex of healthy rats and rats with renal fibrosis. METHODS: Kidneys of healthy and adriamycin-treated rats were studied by immunohistochemistry, using antibodies against CRBP-I, desmin, vimentin and alpha-smooth muscle actin (alpha-SMA). Double stainings were done with immunofluorescence. Western blotting was performed to semi-quantify the expression levels of vimentin, desmin, alpha-SMA and CRBP-I. RESULTS: In the normal rat kidney, the convoluted proximal tubular epithelial cells express CRBP-I; no expression is found in the interstitium, nor in the glomeruli. In the adriamycin-induced fibrotic rat kidney, CRBP-I expression diminishes in the convoluted proximal tubular epithelial cells, whereas peritubular myofibroblasts in the interstitium acquire CRBP-I expression. CONCLUSIONS: In the tubulointerstitial compartment of the adriamycin-induced fibrotic rat kidney, CRBP-I is expressed in a different pattern than in the healthy rat kidney. As the convoluted proximal tubular epithelial cells dedifferentiate during fibrosis, CRBP-I expression decreases. Furthermore, de novo expression of CRBP-I is found in activated myofibroblast-like cells in the interstitium of adriamycin-treated rats. CRBP-I is therefore a useful marker to identify a subpopulation of activated/ myodifferentiated fibroblasts in the rat kidney.

Influence of somatostatin and octreotide on liver microcirculation in an experimental mouse model of cirrhosis studied by intravital fluorescence microscopy.

BACKGROUND AND AIMS: Chronic liver damage causes hepatic stellate cell (HSC) activation and contraction, leading to intrahepatic microvascular and structural changes. In vitro endothelin-1 (ET-1)-induced contraction of HSCs can be reduced by somatostatin (SST); however, intrahepatic in vivo effects have never been studied. METHODS: Sinusoidal diameter was measured by intravital fluorescence microscopy in carbon tetrachloride (CCl(4)) and control mice before and after an intravenous (IV) bolus and after 0, 5, 10 and 15 min of an IV infusion of saline, 8 microg/kg/h SST or 8 microg/kg/h octreotide. RESULTS: The baseline sinusoidal diameter in CCl(4) mice (3.01+/-0.05 microm) was significantly smaller than that in controls (4.37+/-0.06 microm). The sinusoidal diameter increased significantly in both groups after a bolus (27, 16% respectively) and following 5 min of SST IV infusion (28, 14% respectively). The percentage increase was significantly higher in CCl(4) mice as compared with controls. This dilatory effect continued for at least 15 min. SST did not influence the mean arterial blood pressure (MAP) and portal venous inflow. In none of the groups did octreotide or saline have any influence on sinusoidal diameters, MAP and portal venous inflow. CONCLUSIONS: Sinusoidal diameter in cirrhotic mice is significantly smaller than that in controls. SST causes significant sinusoidal dilation following a bolus and for at least 15 min of IV infusion. Octreotide does not have any influence on liver sinusoids. These results demonstrate for the first time the in vivo dilatory effect of SST on liver sinusoids.

Expression of somatostatin receptors in splanchnic blood vessels of normal and cirrhotic rats.

BACKGROUND/AIMS: Somatostatin has been used for over two decades to treat acute variceal bleeding. Although it is assumed that somatostatin lowers portal pressure by constriction of the splanchnic arteries, little is known about the expression of somatostatin receptors (SSTR) in splanchnic blood vessels. In this study we investigated SSTR expression in splanchnic blood vessels from normal and cirrhotic rats. METHODS/RESULTS: Cirrhosis was induced by intraperitoneal injection of 50 mg thioacetamide twice a week for 14 weeks. In portal vein, mesenteric artery and aorta of normal and cirrhotic rats, mRNA for the five known SSTR was measured by quantitative reverse transcriptase-polymerase chain reaction. SSTR subtypes 1, 2, 3 and 4 were expressed, but subtype 5 was undetectable. In the portal vein of cirrhotic animals, SSTR1 was significantly down-regulated as compared with controls. Otherwise, no major differences in receptor expression between normal and cirrhotic animals were observed. Using immunohistochemistry, we identified all five receptors, although the staining of receptor 5 was very weak. CONCLUSION: All five SSTR are expressed in splanchnic blood vessels. Our results suggest that cirrhosis reduces expression of SSTR1 in portal vein. In other vessels, no major differences between the normal and cirrhotic state were noted.