Prevention of fibrosis progression in CCl4-treated rats: role of the hepatic endocannabinoid and apelin systems.

Endocannabinoids behave as antifibrogenic agents by interacting with cannabinoid CB2 receptors, whereas the apelin (AP) system acts as a proangiogenic and profibrogenic mediator in the liver. This study assessed the effect of long-term stimulation of CB2 receptors or AP receptor (APJ) blockade on fibrosis progression in rats under a non-discontinued fibrosis induction program. The study was performed in control and CCl(4)-treated rats for 13 weeks. Fibrosis-induced rats received a CB2 receptor agonist (R,S)-3-(2-iodo-5-nitrobenzoyl)-1-(1-methyl-2-piperidinylmethyl)-1H-indole (AM1241) (1 mg/kg b.wt.), an APJ antagonist [Ala(13)]-apelin-13 sequence: Gln-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Ala (F13A) (75 µg/kg b.wt.), or vehicle daily during the last 5 weeks of the CCl(4) inhalation program. Mean arterial pressure (MAP), portal pressure (PP), hepatic collagen content, angiogenesis, cell infiltrate, and mRNA expression of a panel of fibrosis-related genes were measured in all animals. Fibrosis-induced rats showed increased hepatic collagen content, reduced MAP, portal hypertension, and increased expression of the assessed messengers in comparison with control rats. However, fibrotic rats treated with either AM1241 or F13A had reduced hepatic collagen content, improved MAP and PP, ameliorated cell viability, and reduced angiogenesis and cell infiltrate compared with untreated fibrotic rats. These results were associated with attenuated induction of platelet-derived growth factor receptor ß, α-smooth muscle actin, matrix metalloproteinases, and tissue inhibitors of matrix metalloproteinase. CB2 receptor stimulation or APJ blockade prevents fibrosis progression in CCl(4)-treated rats. The mechanisms underlying these phenomena are coincident despite the marked dissimilarities between the CB2 and APJ signaling pathways, thus opening new avenues for preventing fibrosis progression in liver diseases.

Hypoxia and proinflammatory factors upregulate apelin receptor expression in human stellate cells and hepatocytes.

BACKGROUND: The activation of the apelin receptor (APJ) plays a major role in both angiogenic and fibrogenic response to chronic liver injury. However, the mechanisms that govern the induction of APJ expression have not been clarified so far. METHODS: The regulation and the role of APJ in cultured human liver cells were investigated. Tissular expression of APJ and α-smooth muscle actin was analysed by immunocolocalisation in human cirrhotic liver and in control samples. mRNA and protein expression of APJ were analysed in two cell lines, LX-2 (as hepatic stellate cells, HSCs) and HepG2 (as hepatocytes), under hypoxic conditions or after exposure to proinflammatory or profibrogenic factors. Additionally, both hepatic cell lines were stimulated with apelin to assess cell survival and the expression of angiogenic factors. RESULTS: The APJ-positive signal was negligible in control livers. In contrast, APJ was highly expressed in HSCs and slightly expressed in hepatocytes of human cirrhotic liver. Sustained hypoxia and lipopolysaccharide stimulated the expression of APJ in LX-2 cells. Moreover, hypoxia, tumour necrosis factor α and angiotensin II induced the expression of APJ in HepG2 cells. Activation of APJ stimulated angiopoietin-1 expression and cell survival in LX-2 cells and, in turn, triggered the synthesis of vascular endothelial growth factor type A and platelet-derived growth factor-BB in HepG2 cells. CONCLUSIONS: These results suggest that hypoxia and inflammatory factors could play a major role in the activation of the hepatic apelin system leading to angiogenic and fibroproliferative response occurring in chronic liver disease.

Apelin mediates the induction of profibrogenic genes in human hepatic stellate cells.

UNLABELLED: Apelin is a peptide with relevant functions in angiogenesis and inflammation. Recent studies have demonstrated that apelin is overexpressed in hepatic stellate cells (HSCs) of cirrhotic rats. Moreover, patients with cirrhosis show high circulating levels of this peptide. We evaluated the role of endogenous apelin system in fibrogenesis-related gene induction in human HSCs. Messenger expression and immunolocalization of apelin were analyzed in human cirrhotic liver and in control samples. Apelin expression was analyzed in a human HSC line (LX-2) under hypoxic conditions or in the presence of proinflammatory or profibrogenic stimuli. LX-2 cells were stimulated with apelin, and a selected profile of fibrogenesis-related genes was quantified. In vivo inactivation of apelin was analyzed in the liver of fibrotic rats after administrating specific blockers of the molecules triggering apelin induction. Apelin was overexpressed in HSCs from human cirrhotic liver. Neither hypoxia nor proinflammatory substances induced the expression of apelin in LX-2. By contrast, both profibrogenic molecules angiotensin II (AII) and endothelin-1 (ET-1) enhanced apelin expression in these cells. Apelin increased the synthesis of collagen-I and platelet-derived growth factor receptor ß (PDGFRß) in LX-2. AII and ET-1 stimulated collagen-I and PDGFRß expression, and this induction was drastically reduced when apelin receptor was blocked in these cells. In accordance, AII or ET-1 receptor antagonists reduced the hepatic synthesis of apelin, collagen-I, and PDGFRß in fibrotic rats. CONCLUSIONS: apelin mediates some of the fibrogenic effects triggered by AII and ET-1, thus suggesting that apelin could be an important mediator of fibrogenesis in human liver disease.

Cannabinoides endógenos en la enfermedad hepática: muchos dardos para una sola diana / Endogenous cannabinoids in liver disease: Many darts for a single target

Los cannabinoides (CB) endógenos son moléculas lipídicas capaces de mimetizar los efectos producidos por el Δ9-tetrahidrocannabinol, el compuesto causante de los efectos psicológicos de la marihuana. Los endocannabinoides son derivados del ácido araquidónico y participan en numerosos efectos fisiológicos. Esta familia de sustancias está formada por la anandamida (araquidoniletanolamida), el 2-araquidonilglicerol, el éter de noladina y la virodamina. La interacción de estas sustancias con los receptores de CB1 y de CB2 da lugar a la mayoría de sus efectos biológicos. El sistema endocannabinoide está implicado en la patogénesis de la disfunción vascular que se produce en la enfermedad hepática avanzada y desempeña un papel importante en la patogénesis de la hipertensión portal y de la fibrosis hepática. Además, este sistema también se encuentra alterado en otros procesos asociados a la disfunción hepática, como la encefalopatía, la obesidad y la esteatosis. Este sistema podría representar una nueva diana terapéutica para la fibrosis y la hipertensión portal (AU)

Endogenous cannabinoids are ubiquitous lipid-signaling molecules able to partially mimic the actions produced by Δ9-tetrahydrocannabinol, the compound responsible for most of the psychological effects of marijuana. Endocannabinoids are derived from arachidonic acid and are involved in many physiological effects. This family of substances includes anandamide (arachidonylethanolamide), 2-arachydonylglycerol, noladin ether and virodhamine. The interaction of these substances with CB1 and CB2 receptors results in most of their biological effects. The endocannabinoid system is involved in the pathogenesis of the cardiovascular dysfunction occurring in advanced liver disease and also plays a role in the pathogenesis of portal hypertension and liver fibrosis. Moreover, this system is also altered in other processes associated with hepatic dysfunction, including encephalopathy, obesity and steatosis. These findings indicate that the endocannabinoid system may open new avenues for the therapeutic regulation of fibrosis and portal hypertension in advanced liver disease (AU)

[Endogenous cannabinoids in liver disease: Many darts for a single target]. / Cannabinoides endógenos en la enfermedad hepática: muchos dardos para una sola diana.

Endogenous cannabinoids are ubiquitous lipid-signaling molecules able to partially mimic the actions produced by Delta(9)-tetrahydrocannabinol, the compound responsible for most of the psychological effects of marijuana. Endocannabinoids are derived from arachidonic acid and are involved in many physiological effects. This family of substances includes anandamide (arachidonylethanolamide), 2-arachydonylglycerol, noladin ether and virodhamine. The interaction of these substances with CB1 and CB2 receptors results in most of their biological effects. The endocannabinoid system is involved in the pathogenesis of the cardiovascular dysfunction occurring in advanced liver disease and also plays a role in the pathogenesis of portal hypertension and liver fibrosis. Moreover, this system is also altered in other processes associated with hepatic dysfunction, including encephalopathy, obesity and steatosis. These findings indicate that the endocannabinoid system may open new avenues for the therapeutic regulation of fibrosis and portal hypertension in advanced liver disease.

Hypoxia induces B-type natriuretic peptide release in cell lines derived from human cardiomyocytes.

B-type natriuretic peptide (BNP) is a peptide hormone of myocardial origin with significant cardioprotective properties. Patients with myocardial ischemia present with high levels of BNP in plasma and elevated expression in the myocardium. However, the molecular mechanisms of BNP induction in the ischemic myocardium are not well understood. The aim of the investigation was to assess whether myocardial hypoxia induces the production of BNP in human ventricular myocytes. To test the hypothesis that reduced oxygen tension can directly stimulate BNP gene expression and release in the absence of hemodynamic or neurohormonal stimuli, we used an in vitro model system of cultured human ventricular myocytes (AC16 cells). Cells were cultured under normoxic (21% O(2)) or hypoxic (5% O(2)) conditions for up to 48 h. The accumulation of BNP, atrial natriuretic peptide (ANP), and vascular endothelial growth factor (VEGF) was then measured. Hypoxia stimulated the protein release of BNP and VEGF but not ANP. In concordance, the increased mRNA levels of BNP and VEGF but not ANP were found on culturing AC16 cells under hypoxic conditions. The analysis of the transcriptional activity of the hypoxia-inducible factor 1 (HIF-1) in nuclear extracts showed that HIF-1 activity was induced under hypoxic conditions. Finally, the treatment of AC16 cells with the HIF-1 inhibitor rotenone in hypoxia inhibited BNP and VEGF release. In conclusion, these data indicate that hypoxia induces the synthesis and secretion of BNP in human ventricular myocytes, likely through HIF-1-enhanced transcriptional activity.

The hepatic apelin system: a new therapeutic target for liver disease.

UNLABELLED: Apelin is a peptide that plays an important role in heart physiology and pathophysiology, inflammation, and angiogenesis. We evaluated whether the endogenous apelin system is involved in the pathogenesis of the hepatic remodeling and cardiovascular and renal complications occurring in advanced liver disease. The circulating levels of apelin, the messenger RNA (mRNA) and protein expression of apelin and apelin receptor, the immunohistological detection of apelin and apelin receptor, and the effects induced by the chronic administration of an apelin receptor antagonist on fibrosis and vessel density were evaluated in rats with cirrhosis and ascites and in control rats. The serum levels of apelin in patients with cirrhosis were also measured. Apelin levels were higher in rats with cirrhosis than in controls. Apelin mRNA showed a four-fold rise only in hepatic tissue, but not in the lung, heart, or kidney of rats with cirrhosis. These animals also showed hepatic apelin receptor mRNA levels 300 times higher than controls. Apelin was highly expressed by stellate cells, whereas apelin receptor was overexpressed in the hepatic parenchyma of animals with cirrhosis. Rats with cirrhosis treated with the apelin receptor antagonist showed diminished hepatic fibrosis and vessel density, improved cardiovascular performance, and renal function and lost ascites. Human patients also showed a marked increase in apelin levels. CONCLUSION: The selective hepatic activation of the apelin system, together with the drop in fibrosis and neoangiogenesis and the improvement in cardiovascular and excretory function resulting from apelin receptor blockade, points to the hepatic apelin system as a novel therapeutic target in liver disease.

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.

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.

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.

Ascites from cirrhotic patients induces angiogenesis through the phosphoinositide 3-kinase/Akt signaling pathway.

BACKGROUND/AIMS: Ascites in patients with cirrhosis is associated with worsening of systemic hemodynamics. Thus, the aim of this study was to investigate the biological activity of ascites on endothelial cells. METHODS: Human umbilical vein endothelial cells (HUVECs) were used to investigate the angiogenic activity of ascites obtained from cirrhotic patients. RESULTS: Ascites-induced Akt activation, cell migration and tube formation in HUVECs. The pretreatment of HUVECs with the phosphatidylinositide 3-kinase (PI3-kinase) inhibitor LY294002, resulted in a decrease in chemotaxis and cell tube formation induced by ascites. Moreover, the inhibition of Akt activity in HUVECs by transduction of an inactive phosphorylation Akt mutant (AA-Akt), blocked tube formation. These angiogenic effects of ascites were also operative in vivo showing a PI3-kinase activation dependence in the angiogenesis induced by ascites. In addition, the preincubation of ascites with anti-fibronectin antibody led to a significant decrease in HUVECs migration, cell tube formation and in vivo angiogenesis. CONCLUSIONS: These results confirm the novel concept that ascites is a bioactive fluid which can modify vascular properties through the activation of the PI3-kinase/Akt pathway in endothelial cells. Furthermore, our results demonstrated that this ascites-induced mechanism is mediated, at least in part, by fibronectin.

A novel sodium overload test predicting ascites decompensation in rats with CCl4-induced cirrhosis.

BACKGROUND/AIMS: We aimed to develop a non-invasive test to identify the initial alterations of sodium homeostasis and prospectively predict decompensation in preascitic cirrhotic rats. METHODS: The sodium overload test (SOT) was performed in control (CT) and CCl4-induced cirrhotic rats (CH) by calculating the percentage of sodium excreted in the urine after NaCl oral administration (0.5 g/kg). Liver fibrosis was quantified by image cytometry. RESULTS: From the 8th week of CCl4 intoxication, while the daily sodium balance did not change in CH and CT, SOT became significantly lower in the former (62.1+/-13.2 vs 78.8+/-13.2%; P=0.035). At sacrifice, ascites was only present in one animal. The degree of liver fibrosis correlated with SOT. In subsequent experiments, 17 cirrhotic rats developed ascites between the 9th and 14th weeks. SOT remained stable up to 3 weeks before ascites appearance, while it fell significantly to 35+/-19 and 26+/-21% at 2 and 1 week before ascites diagnosis, respectively. Nearly all the rats (95%) with a SOT<60% developed ascites within 3 weeks. CONCLUSIONS: In preascitic cirrhotic rats, SOT unveils sodium metabolism abnormalities earlier than the daily sodium balance and prospectively predicts ascites appearance, identifying rats in a homogeneous stage of cirrhosis, which is essential in pathophysiological studies on sodium retention.

Sodium retention in cirrhotic rats is associated with increased renal abundance of sodium transporter proteins.

BACKGROUND: Liver cirrhosis with ascites is associated with a decrease in renal sodium excretion and therefore sodium retention. METHODS: In this paper, we utilize transporter-specific antibodies to address the hypothesis that dysregulation of one or more sodium transporters or channels is associated with sodium chloride (NaCl) retention in a rat model of cirrhosis induced by repeated exposure to carbon tetrachloride. Age-matched controls and cirrhotic rats were pair fed to ensure identical NaCl and water intake for 4 days prior to euthanasia for quantitative immunoblotting studies. RESULTS AND CONCLUSION: The rats manifested marked extracellular fluid volume expansion with massive ascites. Plasma aldosterone levels were markedly elevated. Analysis of immunoblots revealed marked increases in the abundances of both of the major aldosterone-sensitive apical transport proteins of the renal tubule, namely the thiazide-sensitive NaCl cotransporter NCC and the epithelial sodium channel alpha subunit (alpha-ENaC). These results are consistent with an important role for hyperaldosteronism in the pathogenesis of sodium retention and ascites formation in cirrhosis. In addition, we observed a large decrease in cortical NHE3 abundance (proximal tubule) and a large increase in NKCC2 abundance (thick ascending limb), potentially shifting premacula densa sodium absorption from proximal tubule to loop of Henle (which powers urinary concentration and dilution).

Adrenomedullin and vascular endothelial growth factor production by follicular fluid macrophages and granulosa cells.

BACKGROUND: Human follicular fluid contains several substances, such as cytokines and growth factors, which may affect follicular growth and maturation. The present study examines the relative contribution of macrophages and granulosa cells in the production of vascular endothelial growth factor (VEGF) and adrenomedullin in the human ovulatory follicle. METHODS: Both follicular fluid samples and blood samples were obtained at the time of oocyte retrieval following ovarian stimulation from 20 women undergoing IVF treatment because of male infertility. Human follicular fluid macrophages and luteinized granulosa cells were obtained from pooled follicular fluid of individual patients. Accumulation of VEGF and adrenomedullin in the culture medium of the isolated macrophages and human granulosa cells was determined at variable time intervals ranging from 0 to 48 h. Plasma and follicular fluid concentrations of VEGF and adrenomedullin were also measured. RESULTS: The follicular fluid concentrations of VEGF and adrenomedullin were significantly higher than those found in plasma. After 48 h, accumulation of VEGF in the culture medium of follicular fluid macrophages was significantly higher than that released in the culture medium of luteinized granulosa cells. In contrast, the production rate of adrenomedullin by follicular fluid macrophages was similar to that found in granulosa cells. VEGF secreted by follicular fluid macrophages increased progressively within 48 h of cell culture. A similar response pattern was observed with the culture medium of luteinized granulosa cells, but with lower production rates. CONCLUSIONS: This study suggests for the first time that both luteinized granulosa cells and macrophages actively secrete VEGF and adrenomedullin into follicular fluid in the human ovary.

Transduction of the liver with activated Akt normalizes portal pressure in cirrhotic rats.

BACKGROUND & AIMS: Portal hypertension in cirrhosis is secondary to an increase in hepatic resistance that occurs mainly through collagen deposition. However, recent evidence points to a major contribution by other factors, such as an intrahepatic reduction in nitric oxide production. Akt is a major activator of the endothelial nitric oxide synthase (eNOS) enzyme, but its potential role in intrahepatic resistance in cirrhosis is unknown. For this reason the aims of the present study were to determine whether there is an impaired Akt activation in cirrhotic livers and how this phenomenon relates to the decrease in NO production associated with portal hypertension. METHODS: Cirrhosis was induced in rats by carbon tetrachloride inhalation. Protein abundance and phosphorylation status of Akt and eNOS were examined by Western blotting. The role of Akt in the liver of cirrhotic rats was investigated through infection with adenoviruses encoding either beta-galactosidase (beta-gal) or constitutively active Akt (myr-Akt). RESULTS: The liver of cirrhotic animals showed a significant reduction in Akt and eNOS phosphorylation. Adenoviral delivery of myr-Akt restored eNOS phosphorylation and increased the intrahepatic concentration of guanosine 3',5'-cyclic monophosphate. These events were associated with normalization in portal pressure and a significant increase in mean arterial pressure after 3 days of adenoviral infection. In contrast, transduction of livers with beta-gal did not produce any change in these hemodynamic parameters. CONCLUSIONS: myr-Akt gene therapy restored Akt activation and NO production in the cirrhotic liver, suggesting that this therapy may be useful for the treatment of portal hypertension.

Effect of the V1a/V2-AVP receptor antagonist, Conivaptan, on renal water metabolism and systemic hemodynamics in rats with cirrhosis and ascites.

BACKGROUND: Selective V(2)-AVP receptor antagonists are effective in inducing aquaresis in humans and rats with cirrhosis, hyponatremia and water retention. However, it is unknown whether dual V(1a)/V(2)-AVP antagonists are also efficacious as aquaretic agents under these conditions. This is important, particularly considering that blockade of V(1a)-AVP receptors could aggravate cardiocirculatory function in decompensated cirrhosis. AIMS: To evaluate the renal, hormonal and hemodynamic effects induced by the chronic oral administration of the V(1a)/V(2)-AVP antagonist, Conivaptan, in rats with CCl(4)-induced cirrhosis, ascites and severe water retention. METHODS: We assessed the aquaretic efficacy of 10-day chronic oral administration of Conivaptan (0.5mg/kg body weight (bw)) in cirrhotic rats with hyponatremia and water retention. Urine volume (UV), osmolality (UOsm), and sodium excretion (U(Na)V) were measured daily. At the end of the study arterial pressure was also measured. RESULTS: Conivaptan produced an acute increase in UV, a reduction in UOsm and, at the end of the investigation, cirrhotic rats receiving the V(1a)/V(2)-AVP receptor antagonist did not show hyponatremia or hypoosmolality. Conivaptan also normalized U(Na)V without affecting creatinine clearance and arterial pressure. CONCLUSIONS: Dual V(1a)/V(2)-receptor antagonists may be therapeutically useful for the treatment of water retention and dilutional hyponatremia in human cirrhosis.

Nitric oxide synthase 3-dependent vascular remodeling and circulatory dysfunction in cirrhosis.

Vascular remodeling is an active process that consists in important modifications in the vessel wall. Endothelium-derived nitric oxide (NO) plays a major role in this phenomenon. We assessed wall thickness (WT), total wall area (TWA), lumen diameter, and total nuclei number/cross-section (TN) in cirrhotic rats with ascites and in control rats. A second group of cirrhotic rats received the NO synthesis inhibitor, L-NAME, or vehicle daily for 11 weeks and systemic hemodynamics, arterial compliance, aortic NO synthase 3 (NOS3) protein expression, and vascular morphology were analyzed. Cirrhotic vessels showed a significant reduction in WT, TWA, and TN as compared to control vessels. Long-term inhibition of NOS activity in cirrhotic rats resulted in a significant increase in WT, TWA, and TN as compared to cirrhotic rats receiving vehicle. NOS3 protein abundance was higher in aortic vessels of nontreated cirrhotic animals than in controls. This difference was abolished by chronic treatment with L-NAME. NOS inhibition in cirrhotic rats resulted in higher arterial pressure and peripheral resistance and lower arterial compliance than cirrhotic rats receiving vehicle. Therefore, vascular remodeling in cirrhosis with ascites is a generalized process with significant functional consequences that can be negatively modulated by long-term inhibition of NOS activity.