Stellate Cell Contraction Research Articles

Synergic effect of atorvastatin and ambrisentan on sinusoidal and hemodynamic alterations in a rat model of NASH

ABSTRACTIn non-alcoholic steatohepatitis (NASH), decreased nitric oxide and increased endothelin-1 (ET-1, also known as EDN1) released by sinusoidal endothelial cells (LSEC) induce hepatic stellate cell (HSC) contraction and contribute to portal hypertension (PH). Statins improve LSEC function, and ambrisentan is a selective endothelin-receptor-A antagonist. We aimed to analyse the combined effects of atorvastatin and ambrisentan on liver histopathology and hemodynamics, together with assessing the underlying mechanism in a rat NASH model. Diet-induced NASH rats were treated with atorvastatin (10 mg/kg/day), ambrisentan (30 mg/kg/day or 2 mg/kg/day) or a combination of both for 2 weeks. Hemodynamic parameters were registered and liver histology and serum biochemical determinations analysed. Expression of proteins were studied by immunoblotting. Conditioned media experiments were performed with LSEC. HSCs were characterized by RT-PCR, and a collagen lattice contraction assay was performed. Atorvastatin and ambrisentan act synergistically in combination to completely normalize liver hemodynamics and reverse histological NASH by 75%. Atorvastatin reversed the sinusoidal contractile phenotype, thus improving endothelial function, whereas ambrisentan prevented the contractile response in HSCs by blocking ET-1 response. Additionally, ambrisentan also increased eNOS (also known as Nos3) phosphorylation levels in LSEC, via facilitating the stimulation of endothelin-receptor-B in these cells. Furthermore, the serum alanine aminotransferase of the combined treatment group decreased to normal levels, and this group exhibited a restoration of the HSC quiescent phenotype. The combination of atorvastatin and ambrisentan remarkably improves liver histology and PH in a diet-induced NASH model. By recovering LSEC function, together with inhibiting the activation and contraction of HSC, this combined treatment may be an effective treatment for NASH patients.

Regulation of hepatic stellate cell contraction and cirrhotic portal hypertension by Wnt/β-catenin signalling via interaction with Gli1.

Portal hypertension is a lethal complication of cirrhosis. Its mechanism and therapeutic targets remain largely unknown. Hepatic stellate cell (HSC) contraction increases intrahepatic vascular resistance contributing to portal hypertension. We investigated how HSC contraction was regulated by Wnt signalling and the therapeutic implications. Liver tissues from cirrhotic patients were examined. Cirrhotic mice with genetic or pharmacological treatments were used for in vivo assessments, and their primary cells were isolated. Cellular functions and signalling pathways were analysed in human HSC-LX2 cells using real-time PCR, Western blotting, siRNA, luciferase reporter assay, chromatin immunoprecipitation, co-immunoprecipitation and site-directed mutagenesis. Wnt/β-catenin correlated with HSC contraction in human cirrhotic liver. Wnt3a stimulated Smo-independent Gli1 nuclear translocation followed by LARG-mediated RhoA activation leading to HSC contraction. Suppressor of fused (Sufu) negatively mediated Wnt3a-induced Gli1 nuclear translocation. Wnt/β-catenin repressed transcription of Sufu dependent on β-catenin/TCF4 interaction and TCF4 binding to Sufu promoter. Molecular simulation and site-directed mutagenesis identified the β-catenin residues Lys312 and Lys435 critically involved in this interaction. TCF4 binding to the sequence CACACCTTCC at Sufu promoter was required for transrepression of Sufu. In cirrhotic mice, short-term liver-targeting β-catenin deficiency or acute treatment with β-catenin inhibitors reduced portal pressure via restriction of HSC contraction rather than inhibiting HSC activation. Long-term deficiency or treatments also ameliorated liver injury, fibrosis and inflammation. Interaction between Wnt/β-catenin and Smo-independent Gli1 pathways promoted HSC contraction via TCF4-dependent transrepression of Sufu. HSC-specific inhibition of β-catenin may have therapeutic benefits for cirrhotic portal hypertension.

Commentary on "Re-regulation of hepatic stellate cell contraction and cirrhotic portal hypertension by Wnt/β-catenin signaling via interaction with Gli1".

Commentary on "Re-regulation of hepatic stellate cell contraction and cirrhotic portal hypertension by Wnt/β-catenin signaling via interaction with Gli1".

Carvedilol Inhibits Angiotensin II-Induced Proliferation and Contraction in Hepatic Stellate Cells through the RhoA/Rho-Kinase Pathway.

Aim Carvedilol is a nonselective beta-blocker used to reduce portal hypertension. This study investigated the effects and potential mechanisms of carvedilol in angiotensin II- (Ang II-) induced hepatic stellate cell (HSC) proliferation and contraction. Methods The effect of carvedilol on HSC proliferation was measured by Cell Counting Kit-8 (CCK-8). Cell cycle progression and apoptosis in HSCs were determined by flow cytometry. A collagen gel assay was used to confirm HSC contraction. The extent of liver fibrosis in mice was evaluated by hematoxylin-eosin (H&E) and Sirius Red staining. Western blot analyses were performed to detect the expression of collagen I, collagen III, α-smooth muscle actin (α-SMA), Ang II type I receptor (AT1R), RhoA, Rho-kinase 2 (ROCK2), and others. Results The results showed that carvedilol inhibited HSC proliferation and arrested the cell cycle at the G0/G1 phase in a dose-dependent manner. Carvedilol also modulated Bcl-2 family proteins and increased apoptosis in Ang II-treated HSCs. Furthermore, carvedilol inhibited HSC contraction induced by Ang II, an effect that was associated with AT1R-mediated RhoA/ROCK2 pathway interference. In addition, carvedilol reduced α-SMA expression and collagen deposition and attenuated liver fibrosis in carbon tetrachloride (CCl4)-treated mice. The in vivo data further confirmed that carvedilol inhibited the expression of angiotensin-converting enzyme (ACE), AT1R, RhoA, and ROCK2. Conclusions The results indicated that carvedilol dose-dependently inhibited Ang II-induced HSC proliferation by impeding cell cycle progression, thus alleviating hepatic fibrosis. Furthermore, carvedilol could inhibit Ang II-induced HSC contraction by interfering with the AT1R-mediated RhoA/ROCK2 pathway.

PORTAL HYPERTENSION TREATMENT WITH CANDESARTAN PLUS PROPRANOLOL FOR NINE MONTHS RESTORES NORMAL PORTAL CIRCULATION HEMODYNAMIC PATTERN

Post HCV liver cirrhosis is one of the most prominent etiologies behind the abnormal portal circulation hemodynamics. It occurs as a result of distorted balance between portal venous flow (PVF) and intrahepatic resistances (IHR). PVF is partially controlled by using both specific and non-specific beta blockers (NSBBs) that have insignificant effects on IHR. Angiotensin recep- tor blockers (ARBs) inhibit the activated hepatic stellate cell (HSC) contraction and thought to reduce the dynamic portion of IHR. The study aimed to slow down the venous blood flow and to reduce the IHR of portal vein vasculature to control sequelae of the enhanced post cirrhosis portal venous turbulence. We evaluated the effects of Candesartan plus propranolol compared to each of them individually in management of portal hypertension (PH). Three groups of 25 patients each, presented with chronic HCV infection and grade II- III esophageal varices (OV), were randomly assigned to one of three treatment regimens: Propranolol or Candesartan or both. Subjects were screened every three month by Doppler Ultrasound for a total of nine months. Damping Index(DI),,pu1se Pulsatiity Index (P), Portal Venous Flow (PVF) Volume, Portal Venous Peak Velocity (PVPV), and Portal Vein Diameter (PVD) were evaluated once every.third month. Our study concluded that combined therapy (Propranolol + Candesartan) induced highly significant improvements that led to restoration of normal values of DI, PI, PVF volume & PVPV overtime compared to monotherapy regimens (P>O.001). Data strongly recommended using Propranolol plus Candesartan' in overtime management of portal hypertension.

Portal Hypertension Treatment with Candesartan plus Propranolol for Nine Months Restores Normal Portal Circulation Hemodynamic Pattern

Post HCV liver cirrhosis is one of the most prominent etiologies behind the abnormal portal circulation hemodynamics. It occurs as a result of distorted balance between portal venous flow (PVF) and intrahepatic resistances (IHR). PVF is partially controlled by using both specific and non-specific beta blockers (NSBBs) that have insignificant effects on IHR. Angiotensin recep- tor blockers (ARBs) inhibit the activated hepatic stellate cell (HSC) contraction and thought to reduce the dynamic portion of IHR. The study aimed to slow down the venous blood flow and to reduce the IHR of portal vein vasculature to control sequelae of the enhanced post cirrhosis portal venous turbulence. We evaluated the effects of Candesartan plus propranolol compared to each of them individually in management of portal hypertension (PH). Three groups of 25 patients each, presented with chronic HCV infection and grade II- III esophageal varices (OV), were randomly assigned to one of three treatment regimens: Propranolol or Candesartan or both. Subjects were screened every three month by Doppler Ultrasound for a total of nine months. Damping Index(DI),,pu1se Pulsatiity Index (P), Portal Venous Flow (PVF) Volume, Portal Venous Peak Velocity (PVPV), and Portal Vein Diameter (PVD) were evaluated once every.third month. Our study concluded that combined therapy (Propranolol + Candesartan) induced highly significant improvements that led to restoration of normal values of DI, PI, PVF volume & PVPV overtime compared to monotherapy regimens (P>O.001). Data strongly recommended using Propranolol plus Candesartan' in overtime management of portal hypertension.

Dihydroartemisinin counteracts fibrotic portal hypertension via farnesoid X receptor-dependent inhibition of hepatic stellate cell contraction.

Portal hypertension is a frequent pathological symptom occurring especially in hepatic fibrosis and cirrhosis. Current paradigms indicate that inhibition of hepatic stellate cell (HSC) activation and contraction is anticipated to be an attractive therapeutic strategy, because activated HSC dominantly facilitates an increase in intrahepatic vein pressure through secreting extracellular matrix and contracting. Our previous in vitro study indicated that dihydroartemisinin (DHA) inhibited contractility of cultured HSC by activating intracellular farnesoid X receptor (FXR). However, the effect of DHA on fibrosis-related portal hypertension still requires clarification. In this study, gain- and loss-of-function models of FXR in HSC were established to investigate the mechanisms underlying DHA protection against chronic CCl4 -caused hepatic fibrosis and portal hypertension. Immunofluorescence staining visually showed a decrease in FXR expression in CCl4 -administrated rat HSC but an increase in that in DHA-treated rat HSC. Serum diagnostics and morphological analyses consistently indicated that DHA exhibited hepatoprotective effects on CCl4 -induced liver injury. DHA also reduced CCl4 -caused inflammatory mediator expression and inflammatory cell infiltration. These improvements were further enhanced by INT-747 but weakened by Z-guggulsterone. Noteworthily, DHA, analogous to INT-747, significantly lowered portal vein pressure and suppressed fibrogenesis. Experiments on mice using FXR shRNA lentivirus consolidated the results above. Mechanistically, inhibition of HSC activation and contraction was found as a cellular basis for DHA to relieve portal hypertension. These findings demonstrated that DHA attenuated portal hypertension in fibrotic rodents possibly by targeting HSC contraction via a FXR activation-dependent mechanism. FXR could be a target molecule for reducing portal hypertension during hepatic fibrosis.

Murine junctional adhesion molecules JAM-B and JAM-C mediate endothelial and stellate cell interactions during hepatic fibrosis

ABSTRACTClassical junctional adhesion molecules JAM-A, JAM-B and JAM-C influence vascular permeability, cell polarity as well as leukocyte recruitment and immigration into inflamed tissue. As the vasculature becomes remodelled in chronically injured, fibrotic livers we aimed to determine distribution and role of junctional adhesion molecules during this pathological process. Therefore, livers of naïve or carbon tetrachloride-treated mice were analyzed by immunohistochemistry to localize all 3 classical junctional adhesion molecules. Hepatic stellate cells and endothelial cells were isolated and subjected to immunocytochemistry and flow cytometry to determine localization and functionality of JAM-B and JAM-C. Cells were further used to perform contractility and migration assays and to study endothelial tubulogenesis and pericytic coverage by hepatic stellate cells. We found that in healthy tissue, JAM-A was ubiquitously expressed whereas JAM-B and JAM-C were restricted to the vasculature. During fibrosis, JAM-B and JAM-C levels increased in endothelial cells and JAM-C was de novo generated in myofibroblastic hepatic stellate cells. Soluble JAM-C blocked contractility but increased motility in hepatic stellate cells. Furthermore, soluble JAM-C reduced endothelial tubulogenesis and endothelial cell/stellate cell interaction. Thus, during liver fibrogenesis, JAM-B and JAM-C expression increase on the vascular endothelium. More importantly, JAM-C appears on myofibroblastic hepatic stellate cells linking them as pericytes to JAM-B positive endothelial cells. This JAM-B/JAM-C mediated interaction between endothelial cells and stellate cells stabilizes vessel walls and may control the sinusoidal diameter. Increased hepatic stellate cell contraction mediated by JAM-C/JAM-C interaction may cause intrahepatic vasoconstriction, which is a major complication in liver cirrhosis.

Obeticholic acid, a farnesoid X receptor agonist, improves portal hypertension by two distinct pathways in cirrhotic rats

The farnesoid X receptor (FXR) is a nuclear bile acid receptor involved in bile acid homeostasis, hepatic and intestinal inflammation, liver fibrosis, and cardiovascular disease. We studied the effect of short-term treatment with obeticholic acid (INT-747), a potent selective FXR agonist, on intrahepatic hemodynamic dysfunction and signaling pathways in different rat models of cirrhotic portal hypertension (PHT). For this, thioacetamide (TAA)-intoxicated and bile-duct-ligated (BDL) rats were used as models. After gavage of two doses of 30 mg/kg of INT-747 or vehicle within 24 hours, in vivo hemodynamics were assessed. Additionally, we evaluated the direct effect of INT-747 on total intrahepatic vascular resistance (IHVR) and intrahepatic vascular tone (endothelial dysfunction and hyperresponsiveness to methoxamine) by means of an in situ liver perfusion system and on hepatic stellate cell contraction in vitro. FXR expression and involved intrahepatic vasoactive pathways (e.g., endothelial nitric oxide synthase [eNOS], Rho-kinase, and dimethylarginine dimethylaminohydrolase [DDAH]) were analyzed by immunohistochemistry, reverse-transcriptase polymerase chain reaction, or western blotting. In both cirrhotic models, FXR expression was decreased. Treatment with INT-747 in TAA and BDL reactivated the FXR downstream signaling pathway and decreased portal pressure by lowering total IHVR without deleterious systemic hypotension. In the perfused TAA and BDL cirrhotic liver, INT-747 improved endothelial vasorelaxation capacity, but not hyperresponsiveness. In both groups, this was associated with an increased eNOS activity, which, in TAA, related to down-regulation of Rho-kinase and in BDL to up-regulation of DDAH-2. FXR agonist INT-747 improves PHT in two different rat models of cirrhosis by decreasing IHVR. This hemodynamic effect relates to increased intrahepatic eNOS activity by pathways that differ depending on the etiology of cirrhosis.

Effects of candesartan and propranolol combination therapy versus propranolol monotherapy in reducing portal hypertension.

Background/AimsAngiotensin receptor blockers (ARBs) inhibit activated hepatic stellate cell contraction and are thought to reduce the dynamic portion of intrahepatic resistance. This study compared the effects of combined treatment using the ARB candesartan and propranolol versus propranolol monotherapy on portal pressure in patients with cirrhosis in a prospective, randomized controlled trial.MethodsBetween January 2008 and July 2009, 53 cirrhotic patients with clinically significant portal hypertension were randomized to receive either candesartan and propranolol combination therapy (26 patients) or propranolol monotherapy (27 patients). Before and 3 months after the administration of the planned medication, the hepatic venous pressure gradient (HVPG) was assessed in both groups. The dose of propranolol was subsequently increased from 20 mg bid until the target heart rate was reached, and the candesartan dose was fixed at 8 mg qd. The primary endpoint was the HVPG response rate; patients with an HVPG reduction of >20% of the baseline value or to <12 mmHg were defined as responders.ResultsThe mean portal pressure declined significantly in both groups, from 16 mmHg (range, 12-28 mmHg) to 13.5 mmHg (range, 6-20 mmHg) in the combination group (P<0.05), and from 17 mmHg (range, 12-27 mmHg) to 14 mmHg (range, 7-25 mmHg) in the propranolol monotherapy group (P<0.05). However, the medication-induced pressure reduction did not differ significantly between the two groups [3.5 mmHg (range, -3-11 mmHg) vs. 3 mmHg (range, -8-10 mmHg), P=0.674]. The response rate (55.6% vs. 61.5%, P=0.435) and the reductions in mean blood pressure or heart rate also did not differ significantly between the combination and monotherapy groups.ConclusionsThe addition of candesartan (an ARB) to propranolol confers no benefit relative to classical propranolol monotherapy for the treatment of portal hypertension, and is thus not recommended.

Terutroban, a TP-receptor antagonist, reduces portal pressure in cirrhotic rats

Increased production of vasoconstrictive prostanoids, such as thromboxane A2 (TXA2 ), contributes to endothelial dysfunction and increased hepatic vascular tone in cirrhosis. TXA2 induces vasoconstriction by way of activation of the thromboxane-A2 /prostaglandin-endoperoxide (TP) receptor. This study investigated whether terutroban, a specific TP receptor blocker, decreases hepatic vascular tone and portal pressure in rats with cirrhosis due to carbon tetrachloride (CCl4 ) or bile duct ligation (BDL). Hepatic and systemic hemodynamics, endothelial dysfunction, liver fibrosis, hepatic Rho-kinase activity (a marker of hepatic stellate cell contraction), and the endothelial nitric oxide synthase (eNOS) signaling pathway were measured in CCl4 and BDL cirrhotic rats treated with terutroban (30 mg/kg/day) or its vehicle for 2 weeks. Terutroban reduced portal pressure in both models without producing significant changes in portal blood flow, suggesting a reduction in hepatic vascular resistance. Terutroban did not significantly change arterial pressure in CCl4 -cirrhotic rats but decreased it significantly in BDL-cirrhotic rats. In livers from CCl4 and BDL-cirrhotic terutroban-treated rats, endothelial dysfunction was improved and Rho-kinase activity was significantly reduced. In CCl4 -cirrhotic rats, terutroban reduced liver fibrosis and decreased alpha smooth muscle actin (α-SMA), collagen-I, and transforming growth factor beta messenger RNA (mRNA) expression without significant changes in the eNOS pathway. In contrast, no change in liver fibrosis was observed in BDL-cirrhotic rats but an increase in the eNOS pathway. Our data indicate that TP-receptor blockade with terutroban decreases portal pressure in cirrhosis. This effect is due to decreased hepatic resistance, which in CCl4 -cirrhotic rats was linked to decreased hepatic fibrosis, but not in BDL rats, in which the main mediator appeared to be an enhanced eNOS-dependent vasodilatation, which was not liver-selective, as it was associated with decreased arterial pressure. The potential use of terutroban for portal hypertension requires further investigation.

CXCL12 induces hepatic stellate cell contraction through a calcium-independent pathway

Liver fibrosis, with subsequent development of cirrhosis and ultimately portal hypertension, results in the death of patients with end-stage liver disease if liver transplantation is not performed. Hepatic stellate cells (HSCs), central mediators of liver fibrosis, resemble tissue pericytes and regulate intrahepatic blood flow by modulating pericapillary resistance. Therefore, HSCs can contribute to portal hypertension in patients with chronic liver disease (CLD). We have previously demonstrated that activated HSCs express functional chemokine receptor, CXCR4, and that receptor engagement by its ligand, CXCL12, which is increased in patients with CLD, leads to further stellate cell activation in a CXCR4-specific manner. We therefore hypothesized that CXCL12 promotes HSC contraction in a CXCR4-dependent manner. Stimulation of HSCs on collagen gel lattices with CXCL12 led to gel contraction and myosin light chain (MLC) phosphorylation, which was blocked by addition of AMD3100, a CXCR4 small molecule inhibitor. These effects were further mediated by the Rho kinase pathway since both Rho kinase knockdown or Y-27632, a Rho kinase inhibitor, blocked CXCL12 induced phosphorylation of MLC and gel contraction. BAPTA-AM, a calcium chelator, had no effect, indicating that this pathway is calcium sensitive but not calcium dependent. In conclusion, CXCL12 promotes stellate cell contractility in a predominantly calcium-independent fashion. Our data demonstrates a novel role of CXCL12 in stellate cell contraction and the availability of small molecule inhibitors of the CXCL12/CXCR4 axis justifies further investigation into its potential as therapeutic target for portal hypertension.

Cicletanine stimulates eNOS phosphorylation and NO production via Akt and MAP kinase/Erk signaling in sinusoidal endothelial cells

The function of the endothelial isoform of nitric oxide synthase (eNOS) and production of nitric oxide (NO) is altered in a number of disease states. Pharmacological approaches to enhancing NO synthesis and thus perhaps endothelial function could have substantial benefits in patients. We analyzed the effect of cicletanine, a synthetic pyridine with potent vasodilatory characteristics, on eNOS function and NO production in normal (liver) and injured rat sinusoidal endothelial cells, and we studied the effect of cicletanine-induced NO on stellate cell contraction and portal pressure in an in vivo model of liver injury. Sinusoidal endothelial cells were isolated from normal and injured rat livers. After exposure to cicletanine, eNOS phosphorylation, NO synthesis, and the signaling pathway regulating eNOS activation were measured. Cicletanine led to an increase in eNOS (Ser¹¹⁷⁷) phosphorylation, cytochrome c reductase activity, L-arginine conversion to L-citrulline, as well as NO production. The mechanism of the effect of cicletanine appeared to be via the protein kinase B (Akt) and MAP kinase/Erk signaling pathways. Additionally, cicletanine improved NO synthesis in injured sinusoidal endothelial cells. NO production induced by cicletanine in sinusoidal endothelial cells increased protein kinase G (PKG) activity as well as relaxation of stellate cells. Finally, administration of cicletanine to mice with portal hypertension induced by bile duct ligation led to reduction of portal pressure. The data indicate that cicletanine might improve eNOS activity in injured sinusoidal endothelial cells and likely activates hepatic stellate cell NO/PKG signaling. It raises the possibility that cicletanine could improve intrahepatic vascular function in portal hypertensive patients.

Aldosterone induction of hepatic stellate cell contraction through activation of RhoA/ROCK-2 signaling pathway

The RhoA/ROCK-2 signaling pathway is necessary for activated hepatic stellate cell (HSC) contraction. HSC contraction plays an important role in the pathogenesis of cirrhosis and portal hypertension. This study investigated whether aldosterone contributes to HSC contraction by activation of the RhoA/ROCK-2 signaling pathway. Primary HSCs were isolated from Sprague-Dawley rats via in situ pronase/collagenase perfusion. We found that aldosterone enhanced the contraction of a collagen lattice seeded with HSCs. This induced contraction was suppressed by the mineralcorticoid receptor (MR) inhibitor spironolactone, the ROCK-2 inhibitor Y27632, and the angiotensin II type 1 receptor (AT1R) inhibitor irbesartan. Moreover, actin fiber staining showed that aldosterone significantly increased actin fiber formation in HSCs. Pre-incubating with spironolactone, Y27632, or irbesartan inhibited the aldosterone-induced actin fiber reorganization. Molecularly, the effect of aldosterone on activation of HSC contraction was mediated by phosphorylated myosin light chain (P-MLC) through the RhoA/ROCK-2 signaling pathway. All these inhibitors had the ability to block aldosterone-induced protein expressions in the RhoA/ROCK-2/P-MLC cascade in HSCs. Taken together, our current study suggests that aldosterone induces contraction of activated HSCs through the activation of the RhoA/ROCK-2 signaling pathway. This finding may provide a potential therapeutic target for control of cirrhosis and portal hypertension.

Tumor necrosis factor α stimulates endothelin-1 synthesis in rat hepatic stellate cells in hepatic wound healing through a novel IKK/JNK pathway

Endothelin-1 (ET-1), a potent vasoconstrictor peptide up-regulated during wound healing and fibrosis, induces myofibroblasts to contract tissue. Here we have used a liver injury model to test the hypothesis that TNFα may be an important stimulator of ET-1 production in hepatic wound healing. We examined primary rat hepatic stellate cells, isolated from either normal or injured livers and used standard methodology to measure preproET-1 mRNA and mature ET-1 peptide, specific kinases, and preproET-1 promoter activity. Chromatin immunoprecipitation analysis was used to determine basal binding of transcription factors to the preproET-1 promoter. TNFα induced preproET-1 expression in activated hepatic stellate cells in a c-Jun N-terminal kinase (JNK)/AP-1-dependent fashion. TNFα activated JNK through an IκB kinase (IKK) pathway, which activated the transcriptional factor, c-Jun, leading to preproET-1 promoter mediated ET-1 transcription. The TNFα mediated induction of ET-1 synthesis also had functional effects, specifically mediating autocrine induced stellate cell contraction. TNFα stimulated activated stellate cells to produce ET-1 via a novel IKK–JNK-dependent signaling pathway. The resulting autocrine functional effects of ET-1 are likely to be important in the wound-healing process.

Portal hypertensive response to kinin

Portal hypertension is the most common complication of chronic liver diseases, such as cirrhosis. The increased intrahepatic vascular resistance seen in hepatic disease is due to changes in cellular architecture and active contraction of stellate cells. In this article, we review the historical aspects of the kallikrein-kinin system, the role of bradykinin in the development of disease, and our main findings regarding the role of this nonapeptide in normal and experimental models of hepatic injury using the isolated rat liver perfusion model (mono and bivascular) and isolated liver cells. We demonstrated that: 1) the increase in intrahepatic vascular resistance induced by bradykinin is mediated by B2 receptors, involving sinusoidal endothelial and stellate cells, and is preserved in the presence of inflammation, fibrosis, and cirrhosis; 2) the hepatic arterial hypertensive response to bradykinin is calcium-independent and mediated by eicosanoids; 3) bradykinin does not have vasodilating effect on the pre-constricted perfused rat liver; and, 4) after exertion of its hypertensive effect, bradykinin is degraded by angiotensin converting enzyme. In conclusion, the hypertensive response to BK is mediated by the B2 receptor in normal and pathological situations. The B1 receptor is expressed more strongly in regenerating and cirrhotic livers, and its role is currently under investigation.

Fibronectin Stimulates Endothelin-1 Synthesis in Rat Hepatic Myofibroblasts via a Src/ERK-Regulated Signaling Pathway

Liver injury serves as an excellent model of wound healing, characterized by increased synthesis of various cytokines and peptides, including the vasoactive peptide endothelin-1. In the liver, wound healing is mediated by effector cells such as hepatic stellate cells, which cause tissue contraction. Endothelin-1 has autocrine effects on stellate cells and induces their contractile activities. We explored the role of various extracellular matrix components, particularly of fibronectin, in regulating endothelin-1 production during liver injury. Hepatic stellate cells were isolated from normal and injured rats. Real-time polymerase chain reaction immunoblot and enzyme-linked immunosorbent assay analyses were used to measure specific variables, including endothelin-1 production. Preproendothelin-1 promoter activity was determined by a luciferase assay. Stellate cell contraction was measured by a gel contraction assay. Fibronectin stimulated transcription of preproendothelin-1 messenger RNA and expression of endothelin through an integrin-dependent pathway in activated hepatic stellate cells. In these cells, fibronectin induced phosphorylation/activation of extracellular signal-regulated kinase (ERK) through a Shc- and Src-dependent mechanism; ERK activation was required for fibronectin-induced endothelin-1 expression. Fibronectin stimulation by stellate cells induced expression of smooth muscle alpha-actin and endothelin-1-mediated autocrine stellate cell contraction. Stellate cells isolated from injured livers of rats exhibited increased basal phosphorylation levels of Src, Shc, and ERK, as well as increased endothelin-1 synthesis. Fibronectin stimulates activated stellate cells to produce endothelin-1 and contract, via an ERK-dependent signaling pathway. The resulting autocrine functional effects of endothelin-1 are likely to be important in the wound-healing process in injured liver.

Role of Rho-Rock pathways induced by angiotensin II in hepatic stellate cell contraction

To investigate the mechanism of Ca(2+)-independent pathways mediated by Rho-kinase in contraction of hepatic stellate cells (HSCs) induced by angiotonin II (Ang II). Human HSCs of the line HSC-T6 were cultured and randomly divided into 6 groups: negative control group, Ang II group treated by Ang II10 micromol/L for 15 min, Ang II + irbesartan (Ang II receptor inhibitor) group, exposed to irbesartan for 60 min prior to Ang II treatment, Ang II + Y27632 (Rho kinase specific inhibitor) exposed to Y27632 for 60 min prior to Ang II treatment, Ang II + ML-7 (myosin light chain kinase specific inhibitor) + saturo (protein kinase C specific inhibitor) group exposed to stauro for 60 min prior to Ang II treatment, and Ang II + Y27632 + ML-7 + stauro group, exposed to Y27632 and stauro for 60 min prior to Ang II treatment. The cell contraction was detected by silicone-rubber-membrane cultivation directly. The protein levels of MLC and phosphorylated MLC were detected by Western blotting 5, 15, 30, 60, and 120 min after Ang II treatment. RT-PCR was used to detect the expression of Rock2, RhoAGTP, and RhoGEF in Ca(2+)-independent pathways mediated by Rho-kinase. The silicone-rubber-membrane covered by Ang II treated HSCs showed obvious wrinkles indicating the contraction of HSCs. The ratios of phosphorylated MLC protein at the time pints 5, 15, 30, 60, and 120 min of the Ang II group to the control group (0 min) were 11.7 +/- 0.1, 26.9 +/- 0.1, 11.2 +/- 0.1, 4.1 +/- 0.1, and 1.0 +/- 0.1, showing that Ang II increased the phosphorylated MLC protein level time-dependently with the peak level at the time point of 15 minutes. The levels of phosphorylated MLC protein of the Ang II + irbesartan and Ang II + Y27632 groups were (1.12 +/- 0.09)and (1.22 +/- 0.10) respectively, both significantly lower than that of the Ang II group (1.33 +/- 0.06, both P < 0.01). The level of phosphorylated MLC protein of the Ang I + ML-7 + stauro group was (1.43 +/- 0.09), significantly higher than that of the Ang II + Y27632 group (0.64 +/- 0.04, P < 0. 01). The level of phosphorylated MLC protein of the Ang II + Y27632 + ML-7 + stauro group was (0.64 +/- 0.04), significantly lower than that of the Ang II group (P = 0. 003). The mRNA expression levels of Rock2, RhoAGTP, and RhoGEF of the Ang II group were (0.36 +/- 0.01), (0.80 +/- 0.01), and(0.65 +/- 0.11)respectively, all significantly higher than those of the control group [(0.12 +/- 0.01), (0.40 +/- 0.02), and (0.33 +/- 0.09) respectively, all P = 0. 000], and the mRNA expression levels of Rock2, RhoAGTP, and RhoGEF of the Ang II + irbesartan + group were (0.21 +/- 0.02), (0.62 +/- 0.02), and (0.41 +/- 0.10) respectively, all significantly lower than those of the control group. The mRNA expression levels of Rock2 (0.15 +/- 0.01) and RhoGEF (0.28 +/- 0.08) were lower, but The mRNA expression level of RhoAGTP (1.14 +/- 0.02) was higher in the Ang II + Y27632 group. The mRNA expression levels of Rock2, RhoAGTP, and RhoGEF of the Ang II + ML-7 + stauro group were (0.22 +/- 0.01), (0.55 +/- 0.03), and (0.44 +/- 0.10) respectively, all significantly higher than those of the control group. The mRNA expression levels of Rock2, RhoAGTP, and RhoGEF of the Ang II + Y27632 + ML-7 + stauro group were (0.23 +/- 0.01), (0.83 +/- 0.02), and (0.69 +/- 0.08) respectively, all significantly higher than those of the Ang II + ML-7 + stauro group. Ang II induces HSCs contraction in Ca(2+)-independent pathways mediated by Rho-kinase.

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.

The methionine connection: Homocysteine and hydrogen sulfide exert opposite effects on hepatic microcirculation in rats

Increased intrahepatic resistance in cirrhotic livers is caused by endothelial dysfunction and impaired formation of two gaseous vasodilators, nitric oxide (NO) and hydrogen sulfide (H(2)S). Homocysteine, a sulfur-containing amino acid and H(2)S precursor, is formed from hepatic methionine metabolism. In the systemic circulation, hyperhomocystenemia impairs vasodilation and NO production from endothelial cells. Increased blood levels of homocysteine are common in patients with liver cirrhosis. In this study, we demonstrate that acute liver perfusion with homocysteine impairs NO formation and intrahepatic vascular relaxation induced by acetylcholine in methoxamine-precontracted normal livers (7.3% +/- 3.0% versus 26% +/- 2.7%; P < 0.0001). In rats with mild, diet-induced hyperhomocystenemia, the vasodilating activity of acetylcholine was markedly attenuated, and incremental increases in flow induced a greater percentage of increases in perfusion pressure than in control livers. Compared with normal rats, animals rendered cirrhotic by 12 weeks' administration of carbon tetrachloride exhibited a greater percentage of increments in perfusion pressure in response to shear stress (P < 0.05), and intrahepatic resistance to incremental increases in flow was further enhanced by homocysteine (P < 0.05). In normal hyperhomocysteinemic and cirrhotic rat livers, endothelial dysfunction caused by homocysteine was reversed by perfusion of the livers with sodium sulfide. Homocysteine reduced NO release from sinusoidal endothelial cells and also caused hepatic stellate cell contraction; this suggests a dual mechanism of action, with the latter effect being counteracted by H(2)S. Impaired vasodilation and hepatic stellate cell contraction caused by homocysteine contribute to the dynamic component of portal hypertension.