Matrix metalloproteinase-9 inhibition or deletion attenuates portal hypertension in rodents.

Liver cirrhosis and portal hypertension are accompanied by hyperdynamic circulation, angiogenesis and portosystemic collaterals. Matrix metalloproteinases (MMPs) participate in fibrogenesis and angiogenesis, however, whether they can be targeted in cirrhosis treatment is unclear. Therefore, we performed three series of experiments to investigate this issue. Liver cirrhosis was induced by common bile duct ligation (BDL) in Sprague-Dawley rats. Sham-operated rats served as controls. Rats were randomly allocated to receive vehicle, minocycline (a nonselective MMP inhibitor) or SB-3CT (MMP-2 and -9 inhibitor) for 28 days in the first and second series, respectively. MMP-9 knockout mice were used in the third series. The results showed that minocycline ameliorated portal hypertension, hemodynamic abnormalities, reduced collateral shunting, mesenteric vascular density, plasma VEGF level and alleviated liver fibrosis. SB-3CT attenuated portal hypertension, hemodynamic derangements, reduced shunting, mesenteric vascular density, mesenteric VEGF protein expression, and liver fibrosis. Knockout BDL mice had significantly alleviated portal hypertension, liver fibrosis, liver α-SMA and mesenteric eNOS protein expressions compared to wild-type BDL mice. Liver SMAD2 phosphorylation was down-regulated in all series with MMP inhibition or knock-out. In conclusion, MMP-9 inhibition or deletion ameliorated the severity of cirrhosis, portal hypertension, and associated derangements. MMP-9 may be targeted in the treatment of liver cirrhosis.

Folic acid ameliorates homocysteine-induced angiogenesis and portosystemic collaterals in cirrhotic rats.

INTRODUCTION AND OBJECTIVES: Liver cirrhosis is characterized by increased intrahepatic resistance, splanchnic vasodilation/angiogenesis, and formation of portosystemic collateral vessels. Collaterals can cause lethal complications such as gastroesophageal variceal hemorrhage. Homocysteine is linked to vascular dysfunction and angiogenesis and higher levels have been reported in cirrhotic patients. It is also known that folic acid supplementation reverses the effects of homocysteine. However, the treatment effect in cirrhosis has yet to be investigated. MATERIAL AND METHODS: Liver cirrhosis was induced in Sprague-Dawley rats with common bile duct ligation (CBDL). The CBDL rats randomly received (1) vehicle; (2) dl-homocysteine thiolactone (1g/kg/day); (3) dl-homocysteine thiolactone plus folic acid (100mg/kg/day); or (4) folic acid. On the 29th day, hemodynamic parameters, liver and renal biochemistry, protein expressions of proangiogenic factors, mesenteric vascular density and portosystemic shunting were evaluated. RESULTS: In the cirrhotic rats, homocysteine increased mesenteric vascular density and the severity of shunting. It also up-regulated the protein expressions of mesenteric vascular endothelial growth factor (VEGF) and phosphorylated-endothelial nitric oxide synthase (p-eNOS). These effects were reversed by folic acid treatment (P<0.05). CONCLUSION: Folic acid ameliorated the adverse effects of homocysteine in the cirrhotic rats, which may be related to down-regulation of the VEGF-NO signaling pathway.

Glucobrassicin Metabolites Ameliorate the Development of Portal Hypertension and Cirrhosis in Bile Duct-Ligated Rats.

Patients suffering from liver cirrhosis are often complicated with the formation of portosystemic collateral vessels, which is associated with the progression of a splanchnic hyperdynamic circulatory state. Alleviating pathological angiogenesis has thus been proposed to be a feasible treatment strategy. Indole-3-carbinol (C9H9NO, I3C) and 3,3'-diindolymethane (DIM), formed by the breakdown of glucosinolate glucobrassicin, are prevalent in cruciferous vegetables and have anti-angiogenesis properties. We aimed to evaluate their influences on portal hypertension, the severity of mesenteric angiogenesis, and portosystemic collaterals in cirrhosis. Sprague-Dawley rats with common bile duct ligation (CBDL)-induced liver cirrhosis or sham operation (surgical control) were randomly allocated to receive I3C (20 mg/kg/3 day), DIM (5 mg/kg/day) or vehicle for 28 days. The systemic and portal hemodynamics, severity of portosystemic shunting, mesenteric angiogenesis, and mesenteric proangiogenic factors protein expressions were evaluated. Compared to vehicle, both DIM and I3C significantly reduced portal pressure, ameliorated liver fibrosis, and down-regulated mesenteric protein expressions of vascular endothelial growth factor and phosphorylated Akt. DIM significantly down-regulated pErk, and I3C down-regulated NFκB, pIκBα protein expressions, and reduced portosystemic shunting degree. The cruciferous vegetable byproducts I3C and DIM not only exerted a portal hypotensive effect but also ameliorated abnormal angiogenesis and portosystemic collaterals in cirrhotic rats.

Homocysteine deteriorates intrahepatic derangement and portal-systemic collaterals in cirrhotic rats.

In liver cirrhosis, the altered levels of vasoactive substances, especially endothelin-1 (ET-1) and nitric oxide (NO) lead to elevated intrahepatic resistance, increased portal-systemic collaterals and abnormal intra- and extra-hepatic vascular responsiveness. These derangements aggravate portal hypertension-related complications such as gastro-oesophageal variceal bleeding. Homocysteine, a substance implicated in cardiovascular diseases, has been found with influences on vasoresponsiveness and angiogenesis. However, their relevant effects in liver cirrhosis have not been investigated. In the present study, liver cirrhosis was induced by common bile duct ligation (BDL) in Sprague-Dawley rats. In acute study, the results showed that homocysteine enhanced hepatic vasoconstriction to ET-1 but decreased portal-systemic collateral vasocontractility to arginine vasopressin (AVP). Homocysteine down-regulated hepatic phosphorylated endothelial NO synthase (p-eNOS) and p-Akt protein expressions. Inducible NOS (iNOS) and cyclooxygenase (COX)-2 expressions were up-regulated by homocysteine in splenorenal shunt (SRS), the most prominent intra-abdominal collateral vessel. In chronic study, BDL or thioacetamide (TAA) rats received homocysteine or vehicle for 14 days. The results revealed that homocysteine increased hepatic collagen fibre deposition and fibrotic factors expressions in both BDL- and TAA-induced liver fibrotic rats. Portal-systemic shunting and expressions of mesenteric angiogenetic factors [vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), PDGF receptor ß (PDGFRß) and p-eNOS] were also increased in BDL rats. In conclusion, homocysteine is harmful to vascular derangements and liver fibrosis in cirrhosis.

Caffeine ameliorates hemodynamic derangements and portosystemic collaterals in cirrhotic rats.

UNLABELLED: Portal hypertension (PH), a pathophysiological derangement of liver cirrhosis, is characterized by hyperdynamic circulation, angiogenesis, and portosystemic collaterals. These may lead to lethal complications, such as variceal bleeding. Caffeine has been noted for its effects on liver inflammation, fibrogenesis, and vasoreactiveness. However, the relevant influences of caffeine in cirrhosis and PH have not been addressed. Spraque-Dawley rats with common bile duct ligation-induced cirrhosis or sham operation received prophylactic or therapeutic caffeine treatment (50 mg/kg/day, the first or 15th day since operation, respectively) for 28 days. Compared to vehicle (distilled water), caffeine decreased cardiac index, increased systemic vascular resistance, reduced portal pressure (PP), superior mesenteric artery flow, mesenteric vascular density, portosystemic shunting (PSS), intrahepatic angiogenesis, and fibrosis without affecting liver and renal biochemistry. The beneficial effects were reversed by selective adenosine A1 agonist N6-cyclopentyladenosine (CPA) or A2A agonist GCS21680. Both prophylactic and therapeutic caffeine treatment decreased portal resistance and PP in thioacetamide (200mg/kg, thrice-weekly for 8 weeks)-induced cirrhotic rats. Caffeine down-regulated endothelial nitric oxide synthase, vascular endothelial growth factor (VEGF), phospho-VEGFR2, and phospho-Akt mesenteric protein expression. Caffeine adversely affected viability of hepatic stellate and sinusoidal endothelial cells, which was reversed by CPA and GCS21680. On the other hand, caffeine did not modify vascular response to vasoconstrictors in splanchnic, hepatic, and collateral vascular beds. CONCLUSIONS: Caffeine decreased PP, ameliorated hyperdynamic circulation, PSS, mesenteric angiogenesis, hepatic angiogenesis, and fibrosis in cirrhotic rats. Caffeine may be a feasible candidate to ameliorate PH-related complications in cirrhosis.

2'-Hydroxyflavanone ameliorates mesenteric angiogenesis and portal-systemic collaterals in rats with liver fibrosis.

BACKGROUND AND AIM: Portal-systemic collaterals lead to dreadful consequences in patients with cirrhosis. Angiogenesis participates in the development of liver fibrosis, hyperdynamic circulation, and portal-systemic collaterals. 2'-Hydroxyflavanone (2'-HF), one of the citrus fruits flavonoids, is known to have antiangiogenesis effect without adverse response. However, the relevant effects in liver fibrosis have not been surveyed. METHODS: Male Wistar rats received thioacetamide (TAA, 100 mg/kg tiw, i.p.) for 6 weeks to induce liver fibrosis. On the 29th to 42nd day, rats randomly received 2'-HF (100 mg/kg, qod, i.p.) or vehicle (corn oil). On the 43rd day, after hemodynamic measurements, the followings were surveyed: (i) severity of collaterals; (ii) mesenteric angiogenesis; (iii) mesenteric proangiogenic factors protein expressions; (iv) Mesenteric vascular endothelial cells apoptosis; and (v) Mesenteric expressions of proteins regulating apoptosis. RESULTS: Compared with the vehicle group, 2'-HF did not significantly change body weight, mean arterial pressure, heart rate, and portal pressure in TAA rats. 2'-HF significantly alleviated the severity of collaterals, but the mesenteric phospho-ERK, ERK, phospho-Akt, Akt, COX1, COX2, VEGF, and VEGFR-2 protein expressions were not altered. The apoptotic index of 2'-HF group was significantly higher and the mesenteric protein expressions of pro-apoptotic factors, NFkB 50, NFkB 65, Bax, phospho-p53, 17 kD cleaved caspase 3, and 17 kD casepase 3 were up-regulated. CONCLUSIONS: 2'-HF does not influence the hemodynamics but alleviated the severity of collaterals in rats with liver fibrosis and early portal hypertension. This is, at least partly, attributed to enhanced apoptosis of mesenteric vascular endothelial cells.

The Impact of Spironolactone on the Severity of Portal-Systemic Collaterals and Hepatic Encephalopathy in Cirrhotic Rats.

Liver cirrhosis and portal hypertension are accompanied by portal-systemic collaterals formation and lethal complications. Angiogenesis participates in the development of collaterals. Spironolactone is an aldosterone receptor antagonist used to control fluid overload in cirrhotic patients although recent studies suggest that it also inhibits angiogenesis. This study investigated the effect of spironolactone on abnormal angiogenesis and portal-systemic collaterals in cirrhosis. Liver cirrhosis was induced in Sprague-Dawley rats by common bile duct ligation (BDL), and sham-operated rats were the controls. The BDL and sham rats received spironolactone (20 mg/kg/d, oral gavage) or vehicle from day 15 to 28 after the operations. Spironolactone did not influence the portal and systemic hemodynamic, and the renal and hepatic biochemistry data, but it significantly ameliorated hepatic fibrosis, portal-systemic shunting, and mesenteric angiogenesis. Plasma vascular endothelial growth factor (VEGF) levels and the mesenteric protein expression of VEGF and phosphor-vascular endothelial growth factor receptor 2 (VEGFR-2) decreased in the spironolactone group. Spironolactone did not affect motor activity or plasma ammonia levels. The down-regulation of VEGF pathway participates, albeit partly, in the antiangiogenic effect of spironolactone. Thus, spironolactone treatment in patients with liver cirrhosis may provide additional benefits aside from ascites control.

The ability of 17 ß-estradiol to attenuate intrahepatic vasoconstriction to endothelin-1 in female rats is lost in cirrhosis.

BACKGROUND AND RATIONALE: The control of Endothelin-1 (ET-1)-mediated intrahepatic vasoconstriction in cirrhosis is beneficial for the alleviation of relevant complications. Cirrhosis is accompanied by hypogonadism and altered sex hormone status. Besides, sex hormones have vasoactive effects, but it is unknown if they influence vascular function in cirrhosis. This study aimed to investigate the roles of sex hormones in hepatic vascular reactions to ET-1 in cirrhosis. Liver cirrhosis was induced in Spraque-Dawley male and female rats with common bile duct ligation (BDL). Sham-operated (Sham) rats were controls. On the 43rd day after operations, intrahepatic vascular concentration-response curves to ET-1 were obtained with the following preincubatioins: 1) vehicle; 2) 17ß-estradiol; 3) progesterone; 4) testosterone. Livers from sham and BDL rats were dissected for real-time polymerase chain reaction analysis of estrogen, progesterone and testosterone receptors. RESULTS: Compared with sham males perfused with vehicle, sham females presented higher perfusion pressure changes to ET-1 which was reversed only by 17 ß-estradiol. In cirrhosis, compared with males, 17 ß-estradiol no longer attenuated vascular responsiveness to ET-1 in females. In females, BDL rats had lower hepatic estrogen receptor α(ERßα) mRNA expression than that in sham rats. CONCLUSIONS: The sham females showed a stronger intrahepatic vascular constrictive effect to ET-1 than sham males, which could be reversed by 17ß-estradiol. However, the influence of 17 ß-estradiol was lost in cirrhotic females, which may be attributed, at least partly, to intrahepatic ER α down-regulation in females with cirrhosis.

The effects of proton pump inhibitor on hepatic vascular responsiveness and hemodynamics in cirrhotic rats.

BACKGROUND: Liver cirrhosis is associated with increased intrahepatic resistance due to hepatic fibrosis and exaggerated vasoconstriction. Recent studies have indicated that proton pump inhibitors (PPIs), in addition to acid suppression, modulate vasoactive substances and vasoresponsiveness. PPIs are frequently prescribed in patients with cirrhosis due to a higher prevalence of peptic ulcers, however other impacts are unknown. METHODS: Liver cirrhosis was induced in Sprague-Dawley rats with common bile duct ligation (BDL). On the 29th day after BDL and after hemodynamic measurements, the intrahepatic vascular responsiveness to high concentrations of endothelin-1 (ET-1) was evaluated after preincubation with (1) Krebs solution (vehicle), (2) esomeprazole (30 µM), or (3) esomeprazole plus Nω-nitro l-arginine (NNA, a non-selective NO synthase (NOS) inhibitor, 10-4 M). After perfusion, the hepatic protein expressions of endothelial NOS (eNOS), inducible NOS (iNOS), cyclooxygenase (COX)-1, COX-2, endothelin-1, DDAH-1 (dimethylarginine dimethylaminohydrolase-1, ADMA inhibitor), DDAH-2, ADMA (asymmetrical dimethyl arginine, NOS inhibitor) were evaluated. In the chronic model, the BDL rats received (1) vehicle; or (2) esomeprazole (3.6 mg/kg/day, oral gavage) from the 1st to 28th day after BDL. On the 29th day and after hemodynamic measurements, plasma liver biochemistry and liver fibrosis were evaluated. RESULTS: Esomeprazole did not affect hepatic ET-1 vasoresponsiveness. The hepatic protein expressions of the aforementioned factors were not significantly different among the groups. There were no significant differences in hemodynamics, liver biochemistry and hepatic fibrosis after chronic esomeprazole administration. CONCLUSION: PPIs did not affect hepatic vasoresponsiveness or the release of vasoactive substances. Furthermore, they did not influence hemodynamics, liver biochemistry or severity of hepatic fibrosis in the cirrhotic rats.

Ascorbate lacks significant influence in rats with bile duct ligation-induced liver injury.

BACKGROUND: Liver inflammation may induce fibrogenesis, cirrhosis and portal hypertension. Liver cirrhosis is characterized by increased intrahepatic resistance and enhanced vasoconstrictive response. The splanchnic vasodilatation, angiogenesis and portosystemic collaterals formation further bring about lethal complications. Ascorbate is a potent antioxidant with anti-inflammation, anti-fibrosis, and anti-angiogenesis effects. However, the relevant influences in chronic liver injury have not been sufficiently explored. METHODS: Chronic liver injury was induced in Spraque-Dawley rats with common bile duct ligation (BDL). Ascorbate (250 mg/kg/day, oral gavage) or vehicle was administered starting on the 1st day after operation. On the 8th (hepatitis) and 29th (cirrhosis) day, serum biochemistry parameters, hepatic concentrations of lipid peroxidation-related substances, protein expressions of α-SMA, TGF-ß, iNOS, eNOS, p-eNOS-Ser1177, p-eNOS-Thr496, VEGF, VEGFR2, p-VEGFR2, and liver histology were evaluated. In three series of paralleled groups, rats treated with 28-day ascorbate or vehicle received hemodynamic measurements, hepatic and collateral vasoresponsiveness perfusion experiments, mesenteric CD31 immunofluorescence staining, and Western blot analyses of mesenteric VEGF, VEGFR2, pVEGFR2, PDGF, PDGFß, COX1, COX2, eNOS, p-eNOS-Thr495, p-eNOS-Ser1177 protein expressions. In another series, the severity of portosystemic shunting was evaluated. RESULTS: Ascorbate did not influence hepatitis, oxidative stress, fibrosis, and hemodynamic parameters in BDL rats. The intrahepatic and collateral vasoresponsiveness were not affected, either from direct incubation or acute treatment with ascorbate. Furthermore, the mesenteric angiogenesis and severity of shunting were not influenced. CONCLUSION: The oxidative stress, fibrosis, hemodynamic derangements, angiogenesis and vascular functional changes in BDL-induced chronic liver injury may be too overwhelming to be modulated by ascorbate.

Role of cyclooxygenase isoforms in encephalopathy of cirrhotic rats.

BACKGROUND: Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome secondary to acute or chronic liver failure. However, its pathophysiology remains obscure. Recently, we found that the inhibition of cyclooxygenase by indomethacin aggravated HE in rats with thioacetamide-induced acute hepatic failure, suggesting a pivotal role of cyclooxygenase in HE. This study was aimed at surveying the roles of cyclooxygenase isoforms responsible for prostaglandins synthesis, cyclooxygenase-1 (COX1) and COX2, in cirrhotic rats with HE. METHODS: Liver cirrhosis was induced (using formalin) in male Sprague-Dawley rats with bile duct ligation (FBDL). Sham-operated rats served as the surgical controls. The severity of HE was assessed by motor activity counts. Plasma 6-keto-prostaglandin-F1α [6-keto-PGF1α; a relatively stable metabolite of prostacyclin (PGI2)], alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALK-P), and total bilirubin were measured. Hepatic mRNA expressions of COX1 and COX2 were tested. RESULTS: The FBDL group showed lower motor activity counts than the sham group in total (1472 ± 156 vs. 2174 ± 262 counts/30 min, p = 0.034), ambulatory (824 ± 99 vs. 1443 ± 206 counts/30 min, p = 0.014), and vertical movement (431 ± 69 vs. 849 ± 145 counts/30 min, p = 0.018). The mRNA expression of hepatic COX2 was significantly higher in the FBDL group. Plasma ALK-P and bilirubin levels were negatively correlated with total movements, respectively (both p < 0.05). In addition, hepatic COX2 mRNA expression was positively correlated with AST, ALK-P, total bilirubin, and 6-keto-PGF1α (all p < 0.05), but not correlated with total movements. CONCLUSION: Hepatic COX2 expression and PGI2 production are enhanced in cirrhotic rats, but the correlation with HE is not remarkable. Cyclooxygenase and PGI2 may not play important roles in HE in the setting of chronic liver failure.

Molecular mechanisms of circulatory dysfunction in cirrhotic portal hypertension.

Acute or chronic insults to the liver are usually followed by a tissue repairing process. Unfortunately, this action, in most cases, is not effective enough to restore the normal hepatic structure and function. Instead, fibrogenesis and regenerative nodules formation ensue, which are relatively nonfunctioning. The common final stage of the process is liver cirrhosis with increased intrahepatic resistance to portal venous blood flow. Throughout the entire course, the extrahepatic circulatory dysfunction, including increased splanchnic blood flow, elevated portal venous blood flow and pressure, decreased splanchnic and peripheral vascular resistance, tachycardia, and increased cardiac output, are noted and denoted as portal hypertension with hyperdynamic circulatory dysfunction. When such a condition is established, patients may suffer from fatal complications such as gastroesophageal variceal hemorrhage, hepatic encephalopathy, or hepatorenal syndrome. The cause of such a circulatory dysfunction is not fully elucidated. Nevertheless, clarification of the pathophysiology definitely contributes to the control of portal hypertension-related complications. Herein, the molecular mechanism of this intriguing disaster is reviewed and discussed.