Josefa Ros

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

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, α‐smooth muscle actin (α‐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, α‐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. (HEPATOLOGY 2007.)

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-Δ8-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, α-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 α-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.

Increased anandamide induced relaxation in mesenteric arteries of cirrhotic rats: role of cannabinoid and vanilloid receptors

Background and aims: Anandamide is an endocannabinoid that evokes hypotension by interaction with peripheral cannabinoid CB1 receptors and with the perivascular transient receptor potential vanilloid type 1 protein (TRPV1). As anandamide has been implicated in the vasodilated state in advanced cirrhosis, the study investigated whether the mesenteric bed from cirrhotic rats has an altered and selective vasodilator response to anandamide. Methods: We assessed vascular sensitivity to anandamide, mRNA and protein expression of cannabinoid CB1 receptor and TRPV1 receptor, and the topographical distribution of cannabinoid CB1 receptors in resistance mesenteric arteries of cirrhotic and control rats. Results: Mesenteric vessels of cirrhotic animals displayed greater sensitivity to anandamide than control vessels. This vasodilator response was reverted by CB1 or TRPV1 receptor blockade, but not after endothelium denudation or nitric oxide inhibition. Anandamide had no effect on distal femoral arteries. CB1 and TRPV1 receptor protein was higher in cirrhotic than in control vessels. Neither CB1 mRNA nor protein was detected in femoral arteries. Immunochemistry showed that CB1 receptors were mainly in the adventitia and in the endothelial monolayer, with higher expression observed in vessels of cirrhotic rats than in controls. Conclusions: These results indicate that anandamide is a selective splanchnic vasodilator in cirrhosis which predominantly acts via interaction with two different types of receptors, CB1 and TRPV1 receptors, which are mainly located in perivascular sensory nerve terminals of the mesenteric resistance arteries of these animals.

The hepatic apelin system: A new therapeutic target for liver disease

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. (HEPATOLOGY 2008.)

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.

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.

Altered biosynthesis of leukotrienes and lipoxins and host defense disorders in patients with cirrhosis and ascites

BACKGROUND & AIMS Advanced cirrhosis is associated with impaired leukocyte function, but the mechanism underlying this host defense alteration is unknown. The aim of this study was to investigate the lipoxygenase pathway of arachidonic acid metabolism and its influence in leukocyte trafficking in patients with cirrhosis and ascites. METHODS Neutrophils (polymorphonuclear leukocytes [PMN]) were isolated from patients with cirrhosis and ascites and healthy subjects, and 5-lipoxygenase (5-LO) messenger RNA levels and 5-LO-derived products were measured. The effect of leukotrienes (LT) and lipoxins (LX) on PMN adhesion and migration was also assessed. RESULTS PMN from patients with cirrhosis showed increased 5-LO messenger RNA expression. However, in vitro generation of LTB4, cysteinyl-containing LT and LX was significantly decreased in cirrhotic patients. Interestingly, a close relationship between the activity of the renin-angiotensin system and LXA4 biosynthesis was observed both in vitro and in vivo. PMN isolated from cirrhotic patients with ascites showed significantly decreased adhesion and migration in response to LTB4. LXA4 did not provoke PMN adhesion and migration, but rather abrogated the differences between control and cirrhotic PMN. Cirrhotic monocytes showed marked impairment in adherence to laminin when stimulated with either LTB4 or LXA4. CONCLUSIONS These results show the existence of altered biosynthesis of LT and LX and defective response to these lipoxygenase products in leukocytes from patients with cirrhosis and ascites. This abnormality may be relevant to the pathogenesis of host defense disorders in chronic liver disease.

Effect of bacterial lipopolysaccharide on endothelin-1 production in human vascular endothelial cells

BACKGROUND/AIMS The plasma levels of endothelin (ET) are 2-5 fold higher in patients with cirrhosis than in healthy subjects. It has been proposed that endotoxemia could be a mechanism responsible for this phenomenon. However, investigations in rats with cirrhosis indicate that a differential regulation for prepro ET-1 mRNA expression occurs in the liver tissue of these animals but not in the aorta or other organs. The aim of the study was to investigate the effect of bacterial lipopolysaccharide (LPS) on endothelin-1 synthesis and release in cultured human vascular endothelial cells (HUVEC). METHODS Confluent HUVEC at passage levels 3 and 4 were exposed to increasing doses of LPS (1-1000 ng/ml) for 4 h at 37 degrees C and prepro ET-1 mRNA accumulation and big ET-1 and ET-1 concentrations in the conditioned medium were measured. RESULTS Endotoxin had a dual effect on HUVEC. LPS at doses ranging between 250 and 1000 ng/ml induced a progressive diminution in ET-1 concentration in the culture medium. However, lower LPS concentrations dose-dependently increased big ET-1 and ET-1 release by HUVEC without altering prepro ET-1 mRNA expression. CONCLUSIONS These results suggest that low LPS concentrations promote ET-1 release in HUVEC by a post-transcriptional mechanism located upstream of big ET-1 in the biosynthetic pathway of ET-1. These findings could explain the existence of high circulating levels of ET-1 in cirrhosis in spite of transcriptional activation of prepro ET-1 mRNA only occurring in the liver.

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.

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.