Liver Angiogenesis Research Articles

Liver regeneration and angiogenesis after open and laparoscopic left partial hepatectomy in a porcine experimental model

Liver regeneration and angiogenesis after open and laparoscopic left partial hepatectomy in a porcine experimental model

Sa1005 Antiviral Treatment Eligibility and Treatment Rates in Patients With Chronic Hepatitis B (CHB) At Primary Care, Community and University Referral Clinics: A Comparative Study

Background: Liver cirrhosis is inevitable outcome triggered by consistent chronic inflammation. Recent studies have implied that liver cirrhosis accompanied with angiogenesis. Our previous studies demonstrated that celecoxib could reduce angiogenesis in hepatocellular carcinoma and gastric adenocarcinoma. However, the effect of celecoxib on the antiangiogenesis of cirrhotic liver is still controversial. Objective: To investigate the effect of celecoxib on angiogenesis of cirrhotic liver. Methods: Peritoneal injection of thiacetamide (TAA) was employed to induce liver cirrhosis (200 mg/kg every three days × 16 weeks). 36 male Sprague-Dawley rats were assigned to three groups: group 1 (TAA + celecoxib, n = 12) received TAA plus celecoxib (20 mg/kg/day) by gavage from the initiation of TAA administration on, group 2 (TAA, n = 12) received TAA plus placebo and group 3 (Control, n = 12) received injections of 0.9% saline (1mL i.p., every three days). Serum biochemistry for liver and kidney function parameters and serum prostaglandin E2 (PGE2) were determined. Portal pressure and mean artery pressure were also measured. Histopathological study and vascular casting by scanning electron microscope (SEM) of liver vascular were performed. Additionally, immunohistochemistry (IHC), quantitative real-time PCR (qRT-PCR) and western blot for CD31, vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR2), and Cyclooxygenase-2 (COX-2) were determined. Results: Compared with TAA group, the fibrotic areas of liver tissues in TAA+celecoxib group were significantly decreased by one fold (20.8 ± 1.5% vs. 10.6 ± 0.9%, p , 0.001). Histological sections, vascular casts of hepatic portal vein by SEM, IHC and qRT-PCR for CD31 showed that hepatic fibrosis was accompanied with significant neoangiogenesis in TAA group when compared with Control group (0.1502 ± 0.0143 vs. 0.0325 ± 0.0086 mm2, p , 0.001). Impressively, the increased vascular areas were greatly reduced after celecoxib treatment (0.0485 ± 0.0097 vs. 0.1502 ± 0.0143 mm2, p , 0.001). The up-regulation of VEGF and VEGFR-2, COX-2 and PGE2 induced by TAA administration were significantly inhibited after celecoxib treatment. Compared with TAA group, the portal pressure in TAA+celecoxib group was significantly decreased by 17.8% (14.88 ± 0.84 vs. 12.23 ± 1.09 mmHg, p , 0.001). No significant differences in arterial pressure, heart rate and liver and kidney function parameters were observed among three groups (p . 0.05). Conclusions: Anti-angiogenesis therapy with celecoxib ameliorated hepatic angiogenesis, portal pressure as well as fibrosis. This result suggested that celecoxib would be beneficial for the treatment of liver cirrhosis.

Sa1004 Celecoxib Ameliorated Fibrosis and Portal Pressure Through Antiangiogenesis in Cirrhotic Rats

Background: Liver cirrhosis is inevitable outcome triggered by consistent chronic inflammation. Recent studies have implied that liver cirrhosis accompanied with angiogenesis. Our previous studies demonstrated that celecoxib could reduce angiogenesis in hepatocellular carcinoma and gastric adenocarcinoma. However, the effect of celecoxib on the antiangiogenesis of cirrhotic liver is still controversial. Objective: To investigate the effect of celecoxib on angiogenesis of cirrhotic liver. Methods: Peritoneal injection of thiacetamide (TAA) was employed to induce liver cirrhosis (200 mg/kg every three days × 16 weeks). 36 male Sprague-Dawley rats were assigned to three groups: group 1 (TAA + celecoxib, n = 12) received TAA plus celecoxib (20 mg/kg/day) by gavage from the initiation of TAA administration on, group 2 (TAA, n = 12) received TAA plus placebo and group 3 (Control, n = 12) received injections of 0.9% saline (1mL i.p., every three days). Serum biochemistry for liver and kidney function parameters and serum prostaglandin E2 (PGE2) were determined. Portal pressure and mean artery pressure were also measured. Histopathological study and vascular casting by scanning electron microscope (SEM) of liver vascular were performed. Additionally, immunohistochemistry (IHC), quantitative real-time PCR (qRT-PCR) and western blot for CD31, vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR2), and Cyclooxygenase-2 (COX-2) were determined. Results: Compared with TAA group, the fibrotic areas of liver tissues in TAA+celecoxib group were significantly decreased by one fold (20.8 ± 1.5% vs. 10.6 ± 0.9%, p , 0.001). Histological sections, vascular casts of hepatic portal vein by SEM, IHC and qRT-PCR for CD31 showed that hepatic fibrosis was accompanied with significant neoangiogenesis in TAA group when compared with Control group (0.1502 ± 0.0143 vs. 0.0325 ± 0.0086 mm2, p , 0.001). Impressively, the increased vascular areas were greatly reduced after celecoxib treatment (0.0485 ± 0.0097 vs. 0.1502 ± 0.0143 mm2, p , 0.001). The up-regulation of VEGF and VEGFR-2, COX-2 and PGE2 induced by TAA administration were significantly inhibited after celecoxib treatment. Compared with TAA group, the portal pressure in TAA+celecoxib group was significantly decreased by 17.8% (14.88 ± 0.84 vs. 12.23 ± 1.09 mmHg, p , 0.001). No significant differences in arterial pressure, heart rate and liver and kidney function parameters were observed among three groups (p . 0.05). Conclusions: Anti-angiogenesis therapy with celecoxib ameliorated hepatic angiogenesis, portal pressure as well as fibrosis. This result suggested that celecoxib would be beneficial for the treatment of liver cirrhosis.

Sa1006 Effectiveness of Oral Antiviral Therapy for Treatment-Naive Chronic Hepatitis B (CHB) in Routine Clinical Practice

Background: Liver cirrhosis is inevitable outcome triggered by consistent chronic inflammation. Recent studies have implied that liver cirrhosis accompanied with angiogenesis. Our previous studies demonstrated that celecoxib could reduce angiogenesis in hepatocellular carcinoma and gastric adenocarcinoma. However, the effect of celecoxib on the antiangiogenesis of cirrhotic liver is still controversial. Objective: To investigate the effect of celecoxib on angiogenesis of cirrhotic liver. Methods: Peritoneal injection of thiacetamide (TAA) was employed to induce liver cirrhosis (200 mg/kg every three days × 16 weeks). 36 male Sprague-Dawley rats were assigned to three groups: group 1 (TAA + celecoxib, n = 12) received TAA plus celecoxib (20 mg/kg/day) by gavage from the initiation of TAA administration on, group 2 (TAA, n = 12) received TAA plus placebo and group 3 (Control, n = 12) received injections of 0.9% saline (1mL i.p., every three days). Serum biochemistry for liver and kidney function parameters and serum prostaglandin E2 (PGE2) were determined. Portal pressure and mean artery pressure were also measured. Histopathological study and vascular casting by scanning electron microscope (SEM) of liver vascular were performed. Additionally, immunohistochemistry (IHC), quantitative real-time PCR (qRT-PCR) and western blot for CD31, vascular endothelial growth factor (VEGF), VEGF receptor-2 (VEGFR2), and Cyclooxygenase-2 (COX-2) were determined. Results: Compared with TAA group, the fibrotic areas of liver tissues in TAA+celecoxib group were significantly decreased by one fold (20.8 ± 1.5% vs. 10.6 ± 0.9%, p , 0.001). Histological sections, vascular casts of hepatic portal vein by SEM, IHC and qRT-PCR for CD31 showed that hepatic fibrosis was accompanied with significant neoangiogenesis in TAA group when compared with Control group (0.1502 ± 0.0143 vs. 0.0325 ± 0.0086 mm2, p , 0.001). Impressively, the increased vascular areas were greatly reduced after celecoxib treatment (0.0485 ± 0.0097 vs. 0.1502 ± 0.0143 mm2, p , 0.001). The up-regulation of VEGF and VEGFR-2, COX-2 and PGE2 induced by TAA administration were significantly inhibited after celecoxib treatment. Compared with TAA group, the portal pressure in TAA+celecoxib group was significantly decreased by 17.8% (14.88 ± 0.84 vs. 12.23 ± 1.09 mmHg, p , 0.001). No significant differences in arterial pressure, heart rate and liver and kidney function parameters were observed among three groups (p . 0.05). Conclusions: Anti-angiogenesis therapy with celecoxib ameliorated hepatic angiogenesis, portal pressure as well as fibrosis. This result suggested that celecoxib would be beneficial for the treatment of liver cirrhosis.

Serum level of soluble vascular cell adhesion molecule in patients with hepatocellular carcinoma and its association with severity of liver disease

Background. VCAM-1 (soluble vascular cell adhesion molecule-1) plays a role in liver angiogenesis. Hepatocellular carcinoma (HCC) has important angiogenic activity, so expression of VCAM-1 may be pathogenic. Aim. To assess the association between serum VCAM-1 (sVCAM-1) levels and features of tumour and liver disease in patients with and without HCC, and to study the influence of HCC treatment on sVCAM-1 levels.Material and methods. Concentrations in peripheral (sVCAM-1-P) and hepatic (sVCAM-1-H) veins were analysed using ELISA in 134 consecutive patients with chronic liver disease between May 2004 and February 2006, who underwent a splanchnic haemodynamic study. Of these patients, 58 had HCC.Results. sVCAM-1-P and sVCAM-1-H were well correlated in both groups. No association was found between sVCAM-1-H and tumour features. No differences were observed in sVCAM-1-H between HCC and non-HCC cirrhotic patients. There was a significant linear association between Child-Pugh stage and sVCAM-1-H in HCC-patients (Child-Pugh A [2,485 ± 1,294 ng/mL] vs. Child-Pugh B [3,408 ± 1,338 ng/mL] vs. Child-Pugh C [4,096 ± 862 ng/ mL]; p = 0.007). Seven non-cirrhotic HCC patients had a significantly lower sVCAM-1-H than cirrhotic HCC patients. Treatment of HCC leads to an increase in sVCAM-1-H levels although this was not associated with the necrosis response to treatment.Conclusions. sVCAM-1 levels are more closely associated with the severity of underlying liver disease than with the presence of HCC. sVCAM-1 levels are not associated with tumour features or invasiveness; therefore, sVCAM-1 does not seem to play an important role in the angiogenic processes of HCC.

Abnormal liver differentiation and excessive angiogenesis in mice lacking Runx3

Runt-related transcription factor 3 (Runx3) is essential for normal mouse development, and Runx3 knock-out (KO) mice (FVB strain), which die within 24 h after birth, show various organ defects, such as lung hyperplasia. For proper early liver development, angiogenesis and liver cell differentiation mechanisms are necessary in mammals. Previous studies have reported that various signaling molecules, such as vascular endothelial growth factor (VEGF), von Willebrand factor (vWF) and cluster of differentiation 31 (CD31), are closely related to angiogenesis in the developing liver. Proper expression levels of molecules that induce liver cell differentiation, such as phosphorylated Smad2 (pSmad2), nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB), Wilms tumor-1 (WT-1) and CD90 (Thy-1), are necessary for fetal liver development. To confirm the pathogenesis of liver defects caused by the loss of function of Runx3, the localization of proliferating cells was examined in wild-type and Runx3 KO mouse livers at postnatal day 1 (PN1). Specimens were also stained for various liver differentiation markers to confirm the function of Runx3. Moreover, gene expression level was examined by real-time quantitative polymerase chain reaction (RT-qPCR). Our results indicate that VEGF, vWF, CD31, pSmad2, NF-kB, WT-1 and Thy-1 were markedly up-regulated by the loss of Runx3. Therefore, our results indicate that liver development is controlled by Runx3. Clarifying the mechanisms of angiogenesis and liver differentiation might aid in the design of efficient and safe antiangiogenic therapy and gene therapy for liver disorders.

Inhibitory effects of salvianolic acid B on CCl4-induced hepatic fibrosis through regulating NF-κB/IκBα signaling

Ethnopharmacological relevanceHepatic fibrosis, a precursor of liver cirrhosis, is a consequence of severe liver damage that occurs in many patients with chronic liver diseases. Salvianolic acid B (SA-B) is one of water soluble compounds derived from Salvia miltiorrhiza Bunge (Danshen in Chinese) widely used for chronic liver diseases. In this study we investigated the protective effects of SA-B on CCl4-induced hepatic fibrosis. Materials and methodsHepatic fibrosis in rats was induced by carbon tetrachloride (CCl4). Rats were divided into four groups, including normal controls (N group), model (M group), low SA-B of 10mg/kg body weight (L group), or high SA-B of 20mg/kg body weight (H group). After 6 weeks, macroscopic features of the liver and weight ratio of liver to body were measured. Liver fibrosis of the rats was evaluated by HE and Massion staining. Activities of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and total bilirubin (TBIL) were checked with automated biochemistry analyzer. Serum levels of hyaluronic acid (HA), type IV collagen (IV-C), Laminin (LN) and procollagen III peptide (PIIIP) were detected by radioimmunoassay (RIA). The expression of NF-κB and IκBα was detected by western blotting. ResultsSA-B was shown to reduce CCl4-induced hepatic fibrosis in rats. The serum levels of ALT, AST, and TBIL were significantly lower in the SA-B treatment groups than in the M group. Compared the M group, the serum levels of HA, LN, IV-C and PIIIP were decreased markedly after treatment with SA-B, especially in the H group. Treatment with SA-B at 10–20mg/kg (L and N groups, respectively) dose-dependently decreased the expression of NF-κB in the nucleolus and increased the expression levels of NF-κB and IκBα protein in the cytoplasm compared to that of the M group. ConclusionsThis study reveals that SA-B could prevent the progression of liver angiogenesis and alleviate liver fibrosis possibly by regulating the expression of NF-κB and IκBα.

Angiogenesis: a phenomenon which aggravates chronic liver disease progression.

It has become increasingly clear that angiogenesis occurring during chronic wound healing and fibrogenesis provides a key contribution to disease progression and complications. The association of fibrogenesis and angiogenesis should be regarded as crucial in the modern evaluation of liver disease progression and in the search for therapeutic targets. Physiological hepatic angiogenesis occurs during liver regeneration, contributing to the formation of new functional sinusoids. Pathological angiogenesis in liver is characterized by intrahepatic vascular remodeling with capillarization of the sinusoids and development of intrahepatic shunts, which lead to increased hepatic resistance and decreased effective hepatocyte perfusion. The problem of angiogenesis in chronic hepatitis C and nonalcoholic fatty liver disease has not been fully resolved. This manuscript briefly describes pathogenesis of new blood vessel formation in chronic hepatitis and potential role of angiogenesis in disease progression.

Increased number and function of endothelial progenitor cells stimulate angiogenesis by resident liver sinusoidal endothelial cells (SECs) in cirrhosis through paracrine factors

Recent studies have shown a pathological role of angiogenesis in the progression of chronic liver diseases (CLDs). The present study focused on numbers and angiogenic functions of circulating endothelial progenitor cells (EPCs) in patients with cirrhosis. Circulating EPCs were counted by flow-cytometry, and correlated with different parameters of liver disease. They were cultured in patients and controls to compare colony-formation, proliferation and tube formation. Interactions of EPCs with hepatic stellate cells (HSCs) and sinusoidal endothelial cells (SECs) were examined by indirect and direct co-cultures in presence of EPCs and EPC-conditioned medium, respectively. ELISA and inhibition assays were performed to assess the role of EPC-derived angiogenic factors. The number of circulating EPCs was substantially higher in cirrhotic patients compared to controls (p<0.05), and showed good correlation with hepatic disease severity. Functional assays revealed that colonies and proliferation of EPCs were significantly increased in patients compared to controls (p<0.05). Direct and indirect co-cultures of patients' EPCs showed an increase in tube formation by SECs as compared to that observed with control EPCs (p<0.05). There was, however, no tube formation in HSC-EPC co-cultures. Levels of PDGF-BB and VEGF were substantially increased in patients' EPC media and inhibition of these factors by neutralizing antibodies led to a significant reduction in SECs proliferation. Mobilization and proliferation of EPCs are significantly enhanced in cirrhotic patients in comparison to controls. EPCs may play an important paracrine role in liver angiogenesis by stimulating resident SECs in cirrhosis.

Sustained liver regeneration after portal vein embolization – A human molecular pilot study

BackgroundPortal vein embolization is a treatment option to achieve a sufficient future remnant liver volume for patients with central liver tumours requiring an extended resection with an extensive parenchymal loss. However, molecular mechanisms of this intervention are up to now poorly understood. The objective of this prospective pilot study was the characterization of molecular events leading to late hypertrophy of the non-embolized liver tissue in the human liver. MethodsLiver tissue of ten patients was collected before and intraoperatively more than one month after embolization. Investigation of molecular features was performed by pangenomic chips, polymerase chain reaction, immunostaining of proliferation marker Ki-67 and immunofluorescence measurements. ResultsSignificantly elevated genes hint towards angiogenesis and signalling by insulin-like growth factor and associated binding proteins. Increased transcript levels of activator protein 1 complex members like c-jun were reflecting potential molecular events of liver growth after embolization. Immunofluorescence data confirmed a predominant upregulation of β-catenin and c-jun (p<0.1) supported by Ki-67 (p<0.05) in the non-embolized liver. In silico analysis of transcriptomic dysplasia and hepatocellular carcinoma data showed divergent signatures compared to embolization. ConclusionsOur findings indicate a sustained regeneration after portal vein embolization reflected in hyperplasia and angiogenesis in the human liver and provide novel molecular mechanisms of interlobe crosstalk.

Tumor and Non-Tumor Liver Angiogenesis Is Traced and Evaluated by Hepatic Arterial Ultrasound in Murine Models

We studied the relationships between hepatic and mesenteric mean blood-flow velocities (mBFVs) measured by ultrasound imaging and (1) downstream tumor angiogenesis during liver metastasis induced by spleen injection of LS174 human colon cells overexpressing the antiangiogenic Netrin4 (LS174-NT4) or not (LS174-WT) and (2) downstream normal angiogenesis during hepatic regeneration after 50% hepatectomy. Liver volume and mBFVs were measured before and after surgery, at day 30 in the first model and at days 2, 7 and 16 in the second model. LS174-NT-4 vs. LS174-WT mice presented fewer metastases (25% vs. 90%, p < 0.001) and decreased hepatic mBFVs (16.5 ± 0.8 vs. 21.8 ± 1.4 cm s−1, p < 0.01), without difference in mesenteric mBFVs. After partial hepatectomy, hepatic and mesenteric mBFVs increased at day 7, from 12.4 ± 1.7 and 11.8 ± 2.6 to 19.1 ± 1.8 and 17.5 ± 2.4 cm s−1, respectively, (p < 0.01) then returned to baseline as liver volume. Duplex Doppler ultrasonography reliably assesses normal or tumor angiogenesis and may provide follow-up functional evaluation.

Chemokine Cxcl9 attenuates liver fibrosis-associated angiogenesis in mice

Recent data suggest that the chemokine receptor CXCR3 is functionally involved in fibroproliferative disorders, including liver fibrosis. Neoangiogenesis is an important pathophysiological feature of liver scarring, but a functional role of angiostatic CXCR3 chemokines in this process is unclear. We therefore investigated neoangiogenesis in carbon tetrachloride (CCl(4))-induced liver fibrosis in Cxcr3(-/-) and wildtype mice by histological, molecular, and functional imaging methods. Furthermore, we assessed the direct role of vascular endothelial growth factor (VEGF) overexpression on liver angiogenesis and the fibroproliferative response using a Tet-inducible bitransgenic mouse model. The feasibility of attenuation of angiogenesis and associated liver fibrosis by therapeutic treatment with the angiostatic chemokine Cxcl9 was systematically analyzed in vitro and in vivo. The results demonstrate that fibrosis progression in Cxcr3(-/-) mice was strongly linked to enhanced neoangiogenesis and VEGF/VEGFR2 expression compared with wildtype littermates. Systemic VEGF overexpression led to a fibrogenic response within the liver and was associated with a significantly increased Cxcl9 expression. In vitro, Cxcl9 displayed strong antiproliferative and antimigratory effects on VEGF-stimulated endothelial cells and stellate cells by way of reduced VEGFR2 (KDR), phospholipase Cγ (PLCγ), and extracellular signal-regulated kinase (ERK) phosphorylation, identifying this chemokine as a direct counter-regulatory molecule of VEGF signaling within the liver. Accordingly, systemic administration of Cxcl9 led to a strong attenuation of neoangiogenesis and experimental liver fibrosis in vivo. The results identify direct angiostatic and antifibrotic effects of the Cxcr3 ligand Cxcl9 in a model of experimental liver fibrosis. The amelioration of liver damage by systemic application of Cxcl9 might offer a novel therapeutic approach for chronic liver diseases associated with increased neoangiogenesis.

Liver angiogenesis: tumor host interaction in non-metastatic colorectal cancer

Objective Angiogenesis is a crucial step for tumor growth and progression. Changes of liver angiogenesis (without metastatic invasion) in response to primary tumors are not known. The aim of the study was to investigate the liver angiogenesis in non-metastatic colorectal cancer (CRC).

Chinese herbal medicine Xiayuxue Decoction inhibits liver angiogenesis in rats with carbon tetrachloride-induced liver fibrosis

To evaluate the effects of Xiayuxue Decoction, a compound traditional Chinese medicine, on liver angiogenesis in rats with carbon tetrachloride (CCl(4))-induced liver fibrosis. Liver cirrhosis was induced by intraperitoneal injection of 50% CCl(4)-olive oil solution at the dose of 1 mL/kg body weight, twice per week for 9 consecutive weeks. After 3- and 6-week injection, 6 rats in the normal group and 6 rats in the model group were randomly sacrificed for dynamic observation. The survival rats of model group were randomly divided into model group (n=15) and Xiayuxue Decoction group (n=11). Six normal rats were used as a normal control. Xiayuxue Decoction was administered orally starting from the 7th week for 3 weeks. At the end of the ninth week, animals were sacrificed and liver tissues were harvested to measure histological changes, activities of matrix metalloproteinase-2 (MMP-2) and MMP-9 and protein expressions of platelet endothelial cell adhesion molecule-1 (CD31), von Willebrand factor (vWF), vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-2 (VEGFR2), complement decay-accelerating factor (DAF) and α-smooth muscle actin (α-SMA) in the liver tissues. Compared with the normal group, liver injury, fatty degeneration and collagen deposition were evidently observed in the model group and protein expressions of CD31, vWF, VEGF, VEGFR2, DAF and α-SMA were gradually increased. In addition, the activities of MMP-2 and MMP-9 in liver tissues were enhanced in the model group (P<0.01). Compared with 9-week model group, liver injury, fatty degeneration and collagen deposition were markedly inhibited by Xiayuxue Decoction; protein expressions of CD31, vWF, VEGF, VEGFR2,α-SMA and DAF and activities of MMP-2 and MMP-9 in the liver tissues were decreased in the Xiayuxue Decoction group (P<0.01). The angiogenesis is evident and aggravating gradually during the progression of liver cirrhosis induced by CCl(4). Xiayuxue Decoction inhibits the angiogenesis by decreasing the activities of MMP-2 and MMP-9, inhibiting the activation of hepatic stellate cells, and damaging the new vessel integrality.

Expression of genes involved in rat liver angiogenesis after ischaemia and reperfusion: effects of ischaemic pre- and post-conditioning

BackgroundDuring surgery, ischaemic pre- (IPC) and post-conditioning (IPO) protects the liver against ischaemia/reperfusion injuries (I/R-injuries). The impact of ischaemic conditioning on liver regeneration has been less well studied. Angiogenesis is an important part of liver regeneration after hepatectomy. The aim of the present study was to investigate the effect of ischaemia/reperfusion and ischaemic conditioning on the expression of genes with angiogenic potential in a model of rat liver ischaemia. MethodsA model of total liver ischaemia (30min) and reperfusion (30min) was employed using Wistar rats. Rats were randomized into five groups: (C) control (IRI) ischaemic, IPC, IPO and IPC + IPO. Liver enzymes were sampled at the end of reperfusion. Liver biopsies were analysed using cDNA microarrays. ResultsAlanine aminotransferase (ALT) increased significantly in all the ischaemic groups compared with controls (P= 0.000). Searching databases 99 genes involved in rat liver angiogenesis were identified. Compared with group (C) the number of genes significantly up-regulated was as follows: IRI (n= 5), IPC (n= 24), IPO (n= 33) and IPC + IPO (n= 18). No genes were down-regulated in the four groups compared with controls. ConclusionIschaemic conditioning, as demonstrated in the present study, seems to be potent activators of angiogenic genes. This might be favourable to the regenerating liver.

Abstract 2469: Combination of branched-amino acids and ACE inhibitor attenuates the insulin resistance-based hepatocarcinogenesis via angiogenesis suppression

Abstract A strong correlation between insulin resistance (IR) and hepatocellular carcinoma (HCC) has recently been identified, although little is known about the possible mechanisms involved. IR is a consistent finding in patients with type 2 diabetes mellitus (DM), and a close interaction between DM and HCC has been documented. Since neovascularization plays an important role in HCC including hepatocarcinogenesis, an angiostatic therapy would be a promising approach for chemoprevention against HCC. The aims of this study were to elucidate the role of angiogenesis in IR-based hepatocarcinogenesis, and to elucidate the therapeutic effect of combination of clinically used branched-chain amino acids (BCAA) and angiotensin-converting enzyme inhibitor (ACE-I), in conjunction with neovascularization. The combined effects on the development of liver enzyme-altered pre-neoplastic lesions, angiogenesis, and several indices were elucidated in obese diabetic rats. We also performed several sets of in-vitro experiments to examine the mechanisms involved. The IR status directly accelerated hepatocarcinogenesis, and treatment with both BCAA and ACE-I markedly inhibited the development of pre-neoplastic lesions under the condition of IR along with suppression of angiogenesis and VEGF expression in the liver. The combination treatment with BCAA and ACE-I exerted more potent inhibitory effects than single-agent treatments. Our in-vitro study demonstrated that BCAA and ACE-I inhibited the endothelial tubular formation almost in parallel with suppression of development of pre-neoplastic lesions. This combination regimen with BCAA and ACE-I showed a marked chemopreventive effect against hepatocarcinogenesis along with suppression of neovascularization and VEGF expression in obese diabetic rat as compared with either single-agent treatment. These inhibitory effects were achieved at clinically comparable low doses. Since both BCAA and ACE-I are widely used in the clinical practice, this combination therapy may represent a potential new strategy for chemoprevention against IR-based HCC in the future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2469.

Development of Arterial Blood Supply in Experimental Liver Metastases

In this study, we present a mechanism for the development of arterial blood supply in experimental liver metastases. To analyze the arterialization process of experimental liver metastases, we elucidated a few key questions regarding the blood supply of hepatic lobules in mice. The microvasculature of the mouse liver is characterized by numerous arterioportal anastomoses and arterial terminations at the base of the lobules. These terminations supply one hepatic microcirculatory subunit per lobule, which we call an arterial hepatic microcirculatory subunit (aHMS). The process of arterialization can be divided into the following steps: 1) distortion of the aHMS by metastasis; 2) initial fusion of the sinusoids of the aHMS at the tumor parenchyma interface; 3) fusion of the sinusoids located at the base of the aHMSs, which leads to the disruption of the vascular sphincter (burst pipe); 4) incorporation of the dilated artery and the fused sinusoids into the tumor; and 5) further development of the tumor vasculature (arterial tree) by proliferation, remodeling, and continuous incorporation of fused sinusoids at the tumor-parenchyma interface. This process leads to the inevitable arterialization of liver metastases above the 2000- to 2500-mum size, regardless of the origin and growth pattern of the tumor.

Pharmacological inhibition of integrin αvβ3 aggravates experimental liver fibrosis and suppresses hepatic angiogenesis #

The vitronectin receptor integrin alphavbeta3 promotes angiogenesis by mediating migration and proliferation of endothelial cells, but also drives fibrogenic activation of hepatic stellate cells (HSCs) in vitro. Expecting antifibrotic synergism, we studied the effect of alphavbeta3 inhibition in two in vivo models of liver fibrogenesis. Liver fibrosis was induced in rats by way of bile duct ligation (BDL) for 6 weeks or thioacetamide (TAA) injections for 12 weeks. A specific alphavbeta3 (alphavbeta5) inhibitor (Cilengitide) was given intraperitoneally twice daily at 15 mg/kg during BDL or after TAA administration. Liver collagen was determined as hydroxyproline, and gene expression was quantified by way of quantitative polymerase chain reaction. Liver angiogenesis, macrophage infiltration, and hypoxia were assessed by way of CD31, CD68 and hypoxia-inducible factor-1alpha immunostaining. Cilengitide decreased overall vessel formation. This was significant in portal areas of BDL and septal areas of TAA fibrotic rats and was associated with a significant increase of liver collagen by 31% (BDL) and 27% (TAA), and up-regulation of profibrogenic genes and matrix metalloproteinase-13. Treatment increased gamma glutamyl transpeptidase in both models, while other serum markers remained unchanged. alphavbeta3 inhibition resulted in mild liver hypoxia, as evidenced by up-regulation of hypoxia-inducible genes. Liver infiltration by macrophages/Kupffer cells was not affected, although increases in tumor necrosis factor alpha, interleukin-18, and cyclooxygenase-2 messenger RNA indicated modest macrophage activation. Specific inhibition of integrin alphavbeta3 (alphavbeta5) in vivo decreased angiogenesis but worsened biliary (BDL) and septal (TAA) fibrosis, despite its antifibrogenic effect on HSCs in vitro. Angiogenesis inhibitors should be used with caution in patients with hepatic fibrosis.

Protein profile in hepatitis B virus replicating rat primary hepatocytes and HepG2 cells by iTRAQ‐coupled 2‐D LC‐MS/MS analysis: Insights on liver angiogenesis

Hepatitis B virus (HBV) infection and in particular Hepatitis B Virus X Protein have been shown to modulate angiogenesis. However, a comprehensive and coordinated mechanism in the HBV-induced angiogenesis remains to be established. In this study, transient transfection of replicative HBV genome was carried out in rat primary hepatocytes (RPHs) as well as HepG2 cells. Angiogenesis was assessed by tube formation assay. 2-D LC-MS/MS analysis was used to detect differentially expressed proteins in cells, supporting HBV replication compared with those transfected with the empty vector. A cell-based HBV replication was established in both RPHs and HepG2 cells. HBV replication-induced angiogenesis was indicated by tube formation of endothelial cells cultured in condition medium from RPHs or HepG2 cells supporting HBV replication. Enzymes associated with angiogenesis, namely fumarate hydratase and tryptophanyl-tRNA synthetase, were identified by 2-D LC-MS/MS analysis in HBV replicating RPHs and HepG2 cells. Our results indicated that the application of quantitative proteomics based on iTRAQ can be an effective approach to evaluate the effects of HBV replication on liver angiogenesis. The angiogenesis-associated proteins identified in our study may eventually lead to novel anti-angiogenic hepatocellular carcinoma cancer therapy based on tumor vascular targeting or be the markers for hepatocellular carcinoma diagnosis.

Hyperbaric oxygen preconditioning promotes angiogenesis in rat liver after partial hepatectomy

Hyperbaric oxygen preconditioning (HBO-PC) increases the level of HIF-1α (hypoxia inducible factor-1α) and its target gene VEGF (vascular endothelial growth factor) which is involved in angiogenesis. Liver regeneration is an angiogenesis-dependent process. We hypothesized that HIF-1α and VEGF mediated the angiogenesis effect of HBO-PC on regenerating rat liver. Male Sprague Dawley rats received HBO-PC followed by 70% partial hepatectomy. Proliferation of hepatocytes and endothelial cells was evaluated by BrdU (bromodeoxyuridine) staining. Microvascular density was assessed by immunohistochemistry. mRNA expression of HIF-1α was assessed by quantitative RT-PCR and protein levels of HIF-1α and VEGF were assessed by western blot. HIF-1α DNA-binding activity was determined with an ELISA-based kit. HBO-PC increased the proliferation index of endothelial cells and microvascular density at 48 h after partial hepatectomy. The protein level and DNA-binding activity of HIF-1α and the protein level of VEGF were increased by HBO-PC before and after partial hepatectomy. Partial hepatectomy alone also increased proliferation index and the expressions of HIF-1α and VEGF. Our results indicated that the angiogenesis effect of HBO-PC on liver after partial hepatectomy could be achieved by increased HIF-1α activity and VEGF expression. However, the angiogenic effect of HBO-PC is moderate and HBO-PC failed to produce additional effect on the enhancement of HIF-1α and VEGF induced by partial hepatectomy alone.