Intrahepatic angiogenesis increases portal hypertension in hepatitis B patients with cirrhosis

Aim

It remains unclear whether intrahepatic angiogenesis increases portal hypertension (PH) in hepatitis B with cirrhosis. We aim to investigate the relationship between intrahepatic angiogenesis and PH in hepatitis B patients with cirrhosis.

Methods

Sixty hepatitis B patients with cirrhosis and 40 healthy subjects were included in this study. Angiogenesis markers vascular endothelial growth factor receptor‐2 (VEGFR2), von Willebrand factor (vWF), and fibrosis marker α‐smooth muscle actin (α‐SMA) were observed by immunohistochemistry. Sirius Red staining was also used to determine liver fibrosis. Correlations between levels of intrahepatic angiogenesis and Child–Pugh classes, liver fibrosis degree, and portal vein pressure were examined. We also analyzed the relationship between levels of intrahepatic angiogenesis and complications of PH, including esophageal varices (EV), ascites, and hypersplenism.

Results

Correlation was observed between the levels of VEGFR2 (r = 0.590, P < 0.01), vWF (r = 0.524, P < 0.01) in tissue, and Child–Pugh classes. Significant correlations were observed between levels of VEGFR2 and α‐SMA (r = 0.710, P < 0.01), VEGFR2 and Sirius Red (r = 0.841, P < 0.01), vWF and α‐SMA (r = 0.768, P < 0.01), and vWF and Sirius Red (r = 0.825, P < 0.01). Patients with hepatic venous pressure gradient (HVPG) ≥12 mmHg showed higher levels of VEGFR2 and vWF expression compared to those with (HVPG) <12 mmHg (2.60 ± 1.28% vs. 1.09 ± 0.73%; 5.85 ± 2.45% vs. 2.31 ± 1.34%, P < 0.01), respectively. Moreover, complications of PH, including size of esophageal varices (P < 0.01), presence of ascites (P < 0.01), and spleen volume (P < 0.01) were significantly affected by the levels of intrahepatic angiogenesis.

Conclusion

Intrahepatic angiogenesis increases PH in hepatitis B patients with cirrhosis. The study provides the potential ways to intervene in the progresses for therapeutic benefits in cirrhosis and PH.

     

Simvastatin effects on portal‐systemic collaterals of portal hypertensive rats

Background and Aim: Portal‐systemic collateral vascular resistance and vasoconstrictor responsiveness are crucial in portal hypertension and variceal bleeding control. Statins enhance vasodilators production, but their influence on collaterals is unknown. This study aimed to survey the effect of simvastatin on collaterals. Methods: Partially portal vein‐ligated rats received oral simvastatin (20 mg/kg/day) or distilled water from ?2 to +7 day of ligation. After hemodynamic measurements on the eighth postoperative day, baseline perfusion pressure (i.e. an index of collateral vascular resistance) and arginine vasopressin (AVP, 0.1 nM–0.1 µM) responsiveness were evaluated with an in situ perfusion model for collateral vascular beds. RT‐PCR of endothelial NO synthase (eNOS), inducible NOS (iNOS), cyclooxygenase‐1 (COX‐1), COX‐2, thromboxane A₂ synthase (TXA₂‐S) and prostacyclin synthase genes was performed in parallel groups for splenorenal shunt (SRS), the most prominent intra‐abdominal collateral vessel. To determine the acute effects of simvastatin, collateral AVP response was assessed with vehicle or simvastatin. SRS RT‐PCR of eNOS, iNOS, COX‐1, COX‐2 and TXA₂‐S, and measurements of perfusate nitrite/nitrate, 6‐keto‐PGF1_α and TXB₂ levels were performed in parallel groups without AVP. Results: Acute simvastatin administration enhanced SRS eNOS expression and elevated perfusate nitrite/nitrate and 6‐keto‐PGF1_α concentrations. Chronic simvastatin treatment reduced baseline collateral vascular resistance and portal pressure and enhanced SRS eNOS, COX‐2 and TXA₂‐S mRNA expression. Neither acute nor chronic simvastatin administration influenced collateral AVP responsiveness. Conclusion: Simvastatin reduces portal‐systemic collateral vascular resistance and portal pressure in portal hypertensive rats. This may be related to the enhanced portal‐systemic collateral vascular NO and prostacyclin activities.     

Increased angiogenesis and permeability in the mesenteric microvasculature of rats with cirrhosis and portal hypertension: an in vivo study.

BACKGROUND: In vivo evidence for angiogenesis in the splanchnic vasodilation in portal hypertension (PHT) and cirrhosis is lacking. Vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) are mediators of angiogenesis. The present study visualises in vivo structural changes (angiogenesis and vascular hyperpermeability) and examines the presence of VEGF and eNOS in the mesenteric microvasculature of animal models of PHT with and without cirrhosis. METHODS: Portal hypertension was induced by partial portal vein ligation (PPVL) and cirrhosis was induced by common bile duct ligation (CBDL) in rats. The mesenteric microcirculation was examined by intravital microscopy. Expression of VEGF, eNOS and CD31 in mesenteric tissue were studied by immunohistochemistry. RESULTS: An increased mesenteric angiogenesis was observed in PPVL and CBDL rats compared with Sham-operated and control rats, as shown by intravital microscopy and CD 31 staining. VEGF and eNOS expression was higher in CBDL and PPVL rats compared with control groups and correlated positively with vascular density. Macromolecular leakage was increased in cirrhotic rats compared with control and PPVL rats. CONCLUSION: Our study provides in vivo evidence of an increased angiogenesis in the mesenteric microvasculature of animal models of PHT and cirrhosis. Increased VEGF and eNOS expression in the mesentery of PPVL and CBDL rats may suggest their contribution. Microvascular permeability in the mesenteric vessels was only increased in cirrhotic rats.     

Update on implications and mechanisms of angiogenesis in liver fibrosis

Liver fibrosis occurs as a compensatory response to the process of tissue repair in a wide range of chronic liver injures. It is characterized by excessive deposition of extracellular matrix in liver tissues. As the pathogenesis progresses without effective management, it will lead to formation of liver fiber nodules and disruption of normal liver structure and function, finally culminating in cirrhosis and hepatocellular carcinoma. A new discovery shows that liver angiogenesis is strictly associated with, and may even favor fibrogenic progression of chronic liver diseases. Recent basic and clinical investigations also demonstrate that liver fibrogenesis is accompanied by pathological angiogenesis and sinusoidal remodeling, which critically determine the pathogenesis and prognosis of liver fibrosis. Inhibition of pathological angiogenesis is considered to be a new strategy for the treatment of liver fibrosis. This review summarizes current knowledge on the process of angiogenesis, the relationships between angiogenesis and liver fibrosis, and on the molecular mechanisms of liver angiogenesis. On the other hand, it also presents the different strategies that have been used in experimental models to counteract excessive angiogenesis and the role of angiogenesis in the prevention and treatment of liver fibrosis.     

Overexpression of NK2 promotes liver fibrosis in carbon tetrachloride‐induced chronic liver injury

Background/Aims: Hepatocyte growth factor (HGF) inhibits liver fibrosis induced by carbon tetrachloride (CCl₄) in animal models. NK2 is a natural splice variant of HGF, but its in vivo function remains to be elucidated. We investigated the in vivo effects of NK2 on CCl₄‐induced liver fibrosis. Methods: NK2 transgenic mice and wild‐type (WT) mice were injected intraperitoneally with CCl₄ twice a week. The extent of hepatic fibrosis was evaluated by Azan–Mallory staining. Expression levels of mRNAs of transforming growth factor‐β1 (TGF‐β1) and matrix metalloproteinase‐13 (MMP‐13) were examined by real‐time polymerase chain reaction. The protein levels of α‐smooth muscle actin (α‐SMA), c‐Met and its phosphorylation were determined by Western blot analysis. Results: Liver fibrosis was significantly more severe in NK2 transgenic mice than in WT mice. CCl₄ administration increased the expression levels of TGF‐β1 mRNA and α‐SMA protein, and decreased the expression of MMP‐13 mRNA in livers of NK2 transgenic mice compared with those of WT mice. c‐Met protein expression in the liver was compatible with the degree of fibrosis. As for c‐Met activation, no difference was found between NK2 and WT livers. Conclusion: Overexpression of NK2 acts as an antagonist of HGF and promotes liver fibrosis in CCl₄‐induced chronic liver injury.     

Hepatocyte growth factor gene transfer with naked plasmid DNA ameliorates dimethylnitrosamine-induced liver fibrosis in rats

Aim: Hepatocyte growth factor (HGF) has various biological properties, including antifibrogenic activity. In the present study, we tested the efficacy of HGF gene therapy using naked plasmid DNA in dimethylnitrosamine (DMN)-induced liver fibrosis in a rat model. Methods: Naked plasmid DNA encoding human HGF was injected once, together with a hypertonic solution, into the hepatic artery after DMN treatment on three consecutive days per week for 3 weeks. Naked plasmid DNA encoding beta-galactosidase was injected similarly in the DMN-treated control rats. DMN treatment was continued once weekly after gene transfer for additional 3 weeks. Results: The human HGF protein expression was detected in livers transfected with human HGF naked plasmid DNA, gradually decreasing by day 21. The expression of the endogenous rat HGF protein was also upregulated after human HGF gene transfer. Phosphorylation of c-Met, a HGF receptor, was detected only in livers transfected with human HGF plasmid DNA. Fibrosis was attenuated significantly in livers transfected with the human HGF plasmid. Attenuation wasaccompanied by decreased expression of alpha-smooth muscle actin. Increased portal vein pressure after treatment with DMN was suppressed significantly by HGF gene transfer. The upregulated hepatic protein expression of transforming growth factor-beta (TGF-beta) in response to DMN was markedly attenuated by HGF gene transfer accompanied by the increased protein expression for matrix metalloproteinases (MMP)-3 and -13. Conclusion: The hepatic arterial injection of human naked plasmid HGF DNA was effective in suppressing liver fibrosis induced in rats by DMN. The mechanisms by which HGF expression attenuated liver fibrosis may include the suppression of hepatic TGF-beta expression and the induction of MMP expression.     

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

Introduction and ObjectivesLiver 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 methodsLiver cirrhosis was induced in Sprague-Dawley rats with common bile duct ligation (CBDL). The CBDL rats randomly received (1) vehicle; (2) dl-homocysteine thiolactone (1 g/kg/day); (3) dl-homocysteine thiolactone plus folic acid (100 mg/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.ResultsIn 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).ConclusionFolic acid ameliorated the adverse effects of homocysteine in the cirrhotic rats, which may be related to down-regulation of the VEGF-NO signaling pathway.     

Cannabinoid receptor 2 agonist ameliorates mesenteric angiogenesis and portosystemic collaterals in cirrhotic rats

Angiogenesis in liver cirrhosis leads to splanchnic hyperemia, increased portal inflow, and portosystemic collaterals formation, which may induce lethal complications, such as gastroesophageal variceal hemorrhage and hepatic encephalopathy. Cannabinoids (CBs) inhibit angiogenesis, but the relevant influences in cirrhosis are unknown. In this study, Spraque-Dawley rats received common bile duct ligation (BDL) to induce cirrhosis. BDL rats received vehicle, arachidonyl-2-chloroethylamide (cannabinoid receptor type 1 [CB(1) ] agonist), JWH-015 (cannabinoid receptor type 2 [CB(2) ] agonist), and AM630 (CB(2) antagonist) from days 35 to 42 days after BDL. On the 43rd day, hemodynamics, presence of CB receptors, severity of portosystemic shunting, mesenteric vascular density, vascular endothelial growth factor (VEGF), VEGFR-1, VEGFR-2, phospho-VEGFR-2, cyclooxygenase (COX)-1, COX-2, and endothelial nitric oxide synthase (eNOS) expressions as well as plasma VEGF levels were evaluated. Results showed that CB(1) and CB(2) receptors were present in left adrenal veins of sham rats, splenorenal shunts (the most prominent intra-abdominal shunts) of BDL rats, and mesentery of sham and BDL rats. CB(2) receptor was up-regulated in splenorenal shunts of BDL rats. Both acute and chronic JWH-015 treatment reduced portal pressure and superior mesenteric arterial blood flow. Compared with vehicle, JWH-015 significantly alleviated portosystemic shunting and mesenteric vascular density in BDL rats, but not in sham rats. The concomitant use of JWH-015 and AM630 abolished JWH-015 effects. JWH-133, another CB(2) agonist, mimicked the JWH-015 effects. JWH-015 decreased mesenteric COX-1, COX-2 messenger RNA expressions, and COX-1, COX-2, eNOS protein expressions. Furthermore, JWH-015 decreased intrahepatic angiogenesis and fibrosis. CONCLUSIONS: CB(2) agonist alleviates portal hypertension (PH), severity of portosystemic collaterals and mesenteric angiogenesis, intrahepatic angiogenesis, and fibrosis in cirrhotic rats. The mechanism is, at least partly, through COX and NOS down-regulation. CBs may be targeted in the control of PH and portosystemic collaterals.     

All‐trans‐retinoic acid ameliorates carbon tetrachloride‐induced liver fibrosis in mice through modulating cytokine production

Background/Aims: Liver fibrosis with any aetiology, induced by the transdifferentiation and proliferation of hepatic stellate cells (HSCs) to produce collagen, is characterized by progressive worsening in liver function, leading to a high incidence of death. We have recently reported that all‐trans‐retinoic acid (ATRA) suppresses the transdifferentiation and proliferation of lung fibroblasts and prevents radiation‐ or bleomycin‐induced lung fibrosis. Methods: We examined the impact of ATRA on carbon tetrachloride (CCl₄)‐induced liver fibrosis. We performed histological examinations and quantitative measurements of transforming growth factor (TGF)‐β1 and interleukin (IL)‐6 in CCl₄‐treated mouse liver tissues with or without the administration of ATRA, and investigated the effect of ATRA on the production of the cytokines in quiescent and activated HSCs. Results: CCl₄‐induced liver fibrosis was attenuated in histology by intraperitoneal administration of ATRA, and the overall survival rate at 12 weeks was 26.5% without ATRA (n=25), whereas it was 75.0% (n=24) in the treatment group (P=0.0187). In vitro studies disclosed that the administration of ATRA reduced (i) the production of TGF‐β1, IL‐6 and collagen from HSCs, (ii) TGF‐β‐dependent transdifferentiation of the cells and IL‐6‐dependent cell proliferation and (iii) the activities of nuclear factor‐κB p65 and p38mitogen‐activated protein kinase, which stimulate the production of TGF‐β1 and IL‐6, which could be the mechanism underlying the preventive effect of ATRA on liver fibrosis. Conclusions: Our findings indicate that ATRA ameliorates liver fibrosis. As the oral administration of the drug results in good compliance, ATRA could be a novel approach in the treatment of liver fibrosis.     

VEGF表达与鼻咽癌血管生成、瘤细胞增殖及预后的关系

目的探讨血管内皮生长因子（VEGF）表达与鼻咽癌（NPC）血管生成、肿瘤细胞增殖及预后的关系。方法选取有明确病理诊断、临床资料完整并随访5a以上的86例NPC患者活检标本,采用免疫组织化学技术检测活检标本中VEGF、微血管密度（iMVD）和增殖细胞核抗原（PCNA）的表达,并分析其与临床病理及预后的关系。结果全组VEGF阳性表达率69．9％（60／86）,与iMVD、PCNA呈显著正相关,与组织学分级、原发灶范围、淋巴结转移、临床分期及生存期亦具相关性。结论NPC组织中VEGF过表达可能参与NPC发生发展,可作为判断NPC恶性程度、侵袭能力和预后的指标。     

硝酸异山梨酯对大鼠缺血心肌血管新生的影响

目的观察硝酸异山梨酯对急性心肌梗死(AMI)大鼠缺血心肌组织形态、梗死面积及毛细血管新生的影响。方法Wistar大鼠90只建立AMI模型后,随机分为5组,分别为正常组,假手术组,模型组,硝酸异山梨酯低剂量组(IDL组)和硝酸异山梨酯高剂量组(IDH组),每组18只。大鼠饲养7、14天后各随机处死9只,截取心肌组织,进行光镜、电镜观察,利用NBT染色法测定心肌梗死面积,应用免疫组织化学法测定Ⅷ因子,进而计算缺血心肌微血管密度(MVD),通过RT-PCR技术检测血管内皮生长因子(VEGF)及碱性成纤维细胞生长因子(bFGF)基因表达情况。结果光镜下观察,缺血区可见炎细胞浸润、心肌肿胀、梗死区纤维化。与模型组比较,IDL组和IDH组7、14天心肌梗死面积明显缩小,MVD明显增加(P<0.05);正常组、假手术组VEGF mRNA、bFGF mRNA表达有所减少,而IDH组则有所增加。结论硝酸异山梨酯可明显缩小AMI大鼠梗死面积,促进缺血心肌的毛细血管新生,对心肌缺血损伤具有保护作用。     

Pravastatin administration does not induce detrimental effects on hemodynamics and collaterals of portal hypertensive rats

Background and Aims: The 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase inhibitor can enhance endothelial nitric oxide synthase expression and induce vasodilatation. The vasodilatory effect may be detrimental to portal‐systemic collaterals due to aggravating the shunting degrees. The present study investigated the effects of pravastatin, a HMG‐CoA reductase inhibitor, on the collateral vascular responsiveness to endothelin‐1 (ET‐1) and portal‐systemic shunting in portal hypertensive rats. Methods: The partial portal vein‐ligated (PVL) rats received either pravastatin (25 mg/kg per day) or distilled water since 2 days prior to until 7 days after ligation. On the 8^th day following hemodynamic measurements, the collateral vascular responsiveness to ET‐1 was evaluated by an in situ collateral perfusion model. The shunting degrees of collaterals were evaluated by constructing vascular flow‐pressure curves and color microsphere study, respectively. PVL rats underwent pre‐incubation with: (i) Krebs solution (control); or Krebs solution plus (ii) 2 × 10^?5 M pravastatin; (iii) pravastatin + N^ω‐nitro‐L‐arginine (10^?4 M); and (iv) pravastatin + indomethacin (10^?5 M), followed by ET‐1 (10^?10–10^?7 M) administration to evaluate the collateral vascular responsiveness. Results: In chronic study, pravastatin did not modify systemic and portal hemodynamics and collateral vascular responsiveness to ET‐1. The resistances of flow‐pressure curves and the microsphere study demonstrated similar shunting degrees between both groups. Furthermore, pravastatin pre‐incubation didn't reduce collateral perfusion pressure to ET‐1. Conclusion: Chronic pravastatin administration does not induce detrimental effects on hemodynamics and collaterals in PVL rats, nor does it influence the shunting degree. In addition, it does not modify the vasoconstrictive effect of ET‐1 on the collaterals of PVL rats.