慢性肝病不同肝纤维化阶段的肝脏体积及其病理变化特点

目的:测量不同肝纤维化程度慢性肝病患者的肝脏整体与分叶体积,并观察患者肝脏微血管、肝细胞消亡与再生等病理改变,以期了解肝纤维化时肝脏宏观体积变化及其与肝组织微观病理的联系。方法:收集同时进行肝活检组织和腹部磁共振成像检查的慢性乙型肝炎、酒精性脂肪肝、自身免疫性肝病、非酒精性脂肪性肝病、药物性肝病的慢性肝病患者53例。采用Masson染色并根据Ishak肝纤维化分期,将患者分为肝纤维化早期(F1~2)、中期(F3~4)和晚期(F5~6)。应用ITK-SNAP软件测量肝脏和脾脏体积。通过CD31免疫组织化学染色反映肝内血管生成,Ki67与肝细胞核因子4α多重荧光免疫组织化学染色反映肝细胞再生,谷氨酰胺合成酶(GS)染色判断肝实质细胞消亡,原位末端标记法观察肝细胞凋亡。Spearman相关性分析肝体积变化与肝组织病理变化之间的关系。结果:随着肝纤维化进展,总肝体积和右肝叶体积逐渐降低(P<0.05),脾脏体积逐渐升高(P<0.05),CD31、GS的表达逐渐增加(P<0.05),Ki67的表达先升高后下降(P<0.05)。CD31阳性率与右肝叶体积(r=-0.609,P<0.001)和总肝体积(r=﹣0.363,P=0.017)呈负相关。Ki67阳性率与右肝叶体积呈正相关(r=0.423,P=0.018),凋亡细胞的阳性率与总肝体积呈显著负相关(r=-0.860,P<0.001)。GS阳性率与右肝叶体积呈负相关(r=﹣0.440,P=0.002),肝细胞消亡数量与右肝叶体积呈负相关(r=﹣0.476,P=0.013)。CD31阳性染色面积与Ki67阳性染色面积呈负相关(r=﹣0.511,P=0.009)。结论:随肝纤维化进展,患者总肝体积与右肝叶体积缩小,主要与肝组织中肝细胞减少与微血管紊乱有关。

Inducible nitric oxide synthase expression is related to angiogenesis,bcl—2 and cell proliferation in hepatocellular carcinoma

In this study, we examined the expression of inducible nitric oxide s ynthase (iNOS) and vascular endothelial growth factor (VEGF) by immunohistoc hemi cal staining in 76 tissue sections collected from hepatocellular carcinoma (HCC) patients undergoing hepatectomy. Microvascular density (MVD) was determined by counting endothelial cells immunostained using anti-CD34 antibody. We performe d DNA-flow cytometric analyses to elucidate the impact of iNOS and VEGF expressi o n on the cell cycle of HCC. Most of the HCC cells that invaded stroma were mark edly immunostained by iNOS antibody. The iNOS stain intensity of the liver tissu e close to the tumor edge was stronger than that of HCC tissue, and the stronges t was the hepatocytes closer to the tumor tissue. However, iNOS expression in 10 normal hepatic samples was undetectable. VEGF positive expression ratio was 84. 8% in iNOS positive expression cases, and the ratio was 35.3% in negative cases. There was significant correlation (P=0.000) between iNOS and VEGF expressi on. Moreover, iNOS expression was significantly associated with bcl-2 and MVD, but w ithout p53 expression. DNA-flow cytometric analyses showed that combined expres s ion of iNOS and VEGF had significant impact on the cell cycle in HCC. PI (Proli ferating Index) and SPF (S-phase fraction) in the combined positive expression o f iNOS and VEGF group was significantly higher than that in the combined negativ e group. The present findings suggested that iNOS expression was significantly a ssociated with angiogenesis, bcl-2 and cell proliferation of HCC.

Significance and relationship between infiltrating inflammatory cell and tumor angiogenesis in hepatocellular carcinoma tissues

AIM: To investigate the relationship between infiltrating inflammatory cell and tumor angiogenesis in hepatocellular carcinoma (HCC) tissues and their clinicopathological features.METHODS: The paraffin-embedded specimens from 70 cases with HCC were stained using EliVision immunohistochemistry with mAbs against CD68, tryptase,and CD34. The counts of tumor-associated macrophage (TAM), mast cell (MC) and tumor microvessel (MV) were performed in the tissue sections.RESULTS: The mean counts of TAM, MC, and MV in HCC tissues were significantly higher than those in pericarcinomatous liver tissues (TAM: 69.31± 11.58 vs 40.23±10.36; MC: 16.74±5.67 vs 7.59±4.18; MV:70.11±12.45 vs 38.52± 11.16, P＜0.01). The MV count in the patients with metastasis was markedly higher than that with non-metastasis (P＜0.01). In addition, the MC count in the patients with poorly differentiated HCC was obviously higher than that with well differentiated HCC (P＜ 0.01). The correlation analysis showed that the TAM count was significantly correlated with the count of MV(r=0.712, P＜0.01), and the MC count was obviously correlated with the MV count (r= 0.336, P＜ 0.05).CONCLUSION: TAM and MC might be closely related to the enhancement of tumor angiogenesis. The MV count might be associated with tumor invasion and metastasis.Moreover, the MC count might be associated with tumor differentiation and prognosis of HCC.

Fumagillin treatment of hepatocellular carcinoma in rats: An in vivo study of antiangiogenesis

AIM: To investigate the effect and possible mechanisms of antiangiogenesis therapy for HCC in rats.METHODS: Adult male LEW/SsN rats were divided into 3groups, 25 animals each. Group A was the control group.Groups B and C were given diethylnitrosamine, 5 mg/kg/d.In addition, group C rats received an intraperitoneal injection of fumagillin, 30 mg/(kg.d). Five animals in each group were killed at 6th, 12th, 18th, 20th and 24th wk to evaluate the development of HCC and metastasis. Weight of the rats, liver tumors, and number of organs involved by HCC were measured at each stage. We compared methionine aminopeptidase-2 (MetAP-2) mRNA, Bcl-2mRNA, telomerase mRNA, and telomerase activity at 24th wk in the liver tissue of group A rats and tumor tissue of HCC from group B and C rats.RESULTS: No HCC developed in group A, but tumors were present in group B and C rats by the 18th wk. At wk 20 and 24, the median liver weight in group B was 0.64 g (range:0.58-0.70 g) and 0.79 g (range: 0.70-0.90 g) (P = 0.04),and that in group C was 0.37 g (range: 0.35-0.42 g) and 0.39 g (range: 0.35-0.47 g) (P = 0.67). The liver weight in group C rats was significantly lower than that in group B rats (P = 0.009). At the same time, the median metastasis score (number of organ systems involved) was 3 (range2-3)in group B, and 1 (range 1-2) in group C, a significant difference between the groups (P = 0.007, 0.004). The levels of MetAP-2 mRNA were significantly higher in groups B and C than in group A (P = 0.025), and significantly higher in group C than in group B (P = 0.047). The level of Bcl-2 mRNA was significantly higher in group B than in group A (P = 0.024), but lower in group C than in group B, although not significantly (P = 0.072). Telomerase mRNA was significantly higher in group B than in group A (P = 0.025), but significantly lower in group C than in group B (P = 0.016). The same inter-group relationship was also true for telomerase activity (P = 0.025 and 0.046).CONCLUSION: Fumagillin effectively inhibits both liver tumor growth and metastasis in rats in vivo. A possible mechanism is fumagillin-induced inhibition of MetAP-2,which plays an essential role in endothelial cell proliferation.Inhibition of MetAP-2 also results in inhibition of Bcl-2and telomerase activity.

Genistein inhibits invasive potential of human hepatocellular carcinoma by altering cell cycle, apoptosis, and angiogenesis

AIM： To study the in vitro and in vivo inhibitory effects of genistein on invasive potential of Bel 7402 hepatocellular carcinoma （HCC） cells and to explore the underlying mechanism. METHODS： Bel 7402 HCC cells were exposed to genistein. The invasive activity of tumor cells was assayed in transwell cell culture chamber, p125^FAK expression and cell cycle were evaluated by a functional assay. Cell apoptosis analysis was performed with TUNEL method. In addition, bilateral subrenal capsule xenograft transplantation of HCC was performed in 10 nude mice. Genistein was injected and the invasion of HCC into the renal parenchyma was observed. Nicrovessels with immunohistochemical staining were detected. RESULTS： Genistein significantly inhibited the growth of Bel 7402 cells, the inhibitory rate of tumor cells was 26 -42%. The invasive potential of Bel 7402 cells in vitro was significantly inhibited, the inhibitory rate was 11- 28%. Genistein caused G2/M cell cycle arrest, S phase decreased significantly. The occurrence of apoptosis in genistein group increased significantly. The expression of p125^FAK in 5 μg/mL genistein group （15.26±0.16%） and 10 μg/mL genistein group （12.89±0.36%） was significantly lower than that in the control group （19.75± 1.12%, P〈0.05）. Tumor growth in genistein-treated nude mice was significantly retarded in comparison to control mice, the inhibitory rate of tumor growth was about 20%. Genistein also significantly inhibited the invasion of Bel 7402 cells into the renal parenchyma of nude mice with xenograft transplant. The positive unit value of microvessels in genistein-treated group （10.422 ±0.807） was significantly lower than that in control group （22.330 ± 5.696, P〈 0.01）. CONCLUSION： Genistein can effectively inhibit the invasive potential of Bel 7402 HCC cells by altering cell cycle, apoptosis and angiogenesis, inhibition of focal adhesion kinase may play a significant role in this process.

Anti-cancer and anti-angiogenic effects of curcumin and tetrahydrocurcumin on implanted hepatocellular carcinoma in nude mice

AIM: To determine the effect of tetrahydrocurcumin (THC) on tumor angiogenesis compared with curcumin (CUR) by using both in vitro and in vivo models of human hepatocellular carcinoma cell line (HepG2). METHODS: The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) assay was used for testing the anti-proliferating activities of CUR and THC. In male BALB/c nude mice, 2 × 106 human HepG2 cells were inoculated onto a dorsal skin-fold chamber. One day after HepG2 inoculation, the experimental groups were fed oral daily with CUR or THC (300 mg/kg or 3000 mg/kg). On d 7, 14 and 21, the tumor microvasculature was observed using fluorescence videomicroscopy and capillary vascularity (CV) was measured. RESULTS: Pathological angiogenic features including microvascular dilatation, tortuosity, and hyper-permeability were observed. CUR and THC could attenuate these pathologic features. In HepG2-groups, the CV were significantly increased on d 7 (52.43%), 14 (69.17%), and 21 (74.08%), as compared to controls (33.04%,P < 0.001). Treatment with CUR and THC resulted in significant decrease in the CV (P < 0.005 and P < 0.001, respectively). In particular, the anti-angiogenic effects of CUR and THC were dose-dependent manner. However, the beneficial effect of THC treatment than CUR was observed, in particular, from the 21 d CV (44.96% and 52.86%, P < 0.05). CONCLUSION: THC expressed its anti-angiogenesis without any cytotoxic activities to HepG2 cells even at the highest doses. It is suggested that anti-angiogenic properties of CUR and THC represent a common potential mechanism for their anti-cancer actions.

Endothelial precursor cells promote angiogenesis in hepatocellular carcinoma

AIM:To investigate the role of bone marrow-derived endothelial progenitor cells(EPCs) in the angiogenesis of hepatocellular carcinoma(HCC).METHODS:The bone marrow of HCC mice was reconstructed by transplanting green fluorescent protein(GFP) + bone marrow cells.The concentration of circulating EPCs was determined by colony-forming assays and fluorescence-activated cell sorting.Serum and tissue levels of vascular endothelial growth factor(VEGF) and colony-stimulating factor(CSF) were quantified by enzyme-linked immunosorbent assay.The distribution of EPCs in tumor and tumor-free tissues was detected by immunohistochemistry and real-time polymerase chain reaction.The incorporation of EPCs into hepatic vessels was examined by immunofluorescence and immunohistochemistry.The proportion of EPCs in vessels was then calculated.RESULTS:The HCC model was successful established.The flow cytometry analysis showed the mean percentage of CD133CD34 and CD133VEGFR2 double positive cells in HCC mice was 0.45% ± 0.16% and 0.20% ± 0.09% respectively.These values are much higher than in the sham-operation group(0.11% ± 0.13%,0.05% ± 0.11%,n = 9) at 14 d after modeling.At 21 d,the mean percentage of circulating CD133CD34 and CD133VEGFR2 cells is 0.23% ± 0.19%,0.25% ± 0.15% in HCC model vs 0.05% ± 0.04%,0.12% ± 0.11% in control.Compared to the transient increase observed in controls,the higher level of circulating EPCs were induced by HCC.In addition,the level of serum VEGF and CSF increased gradually in HCC,reaching its peak 14 d after modeling,then slowly decreased.Consecutive sections stained for the CD133 and CD34 antigens showed that the CD133+ and CD34+ VEGFR2 cells were mostly recruited to HCC tissue and concentrated in tumor microvessels.Under fluorescence microscopy,the bone-marrow(BM)-derived cells labeled with GFP were concentrated in the same area.The relative levels of CD133 and CD34 gene expression were elevated in tumors,around 5.0 and 3.8 times that of the tumor free area.In frozen liver sections from HCC mice,cells co-expressing CD133 and VEGFR2 were identified by immunohistochemical staining using anti-CD133 and VEGFR2 antibodies.In tumor tissue,the double-positive cells were incorporated into vessel walls.In immunofluorescent staining.These CD31 and GFP double positive cells are direct evidence that tumor vascular endothelial cells(VECs) come partly from BM-derived EPCs.The proportion of GFP CD31 double positive VECs(out of all VECs) on day 21 was around 35.3% ± 21.2%.This is much higher than the value recorded on day 7 group(17.1% ± 8.9%).The expression of intercellular adhesion molecule 1,vascular adhesion molecule 1,and VEGF was higher in tumor areas than in tumor-free tissues.CONCLUSION:Mobilized EPCs were found to participate in tumor vasculogenesis of HCC.Inhibiting EPC mobilization or recruitment to tumor tissue may be an efficient strategy for treating HCC.

Effect of grape proanthocyanidins on tumor growth and angiogenesis in H22 liver cancer xenograft model

Objective: The aim of this study was to investigate the effect of grape proanthocyanidins(GPC) on the growth and angiogenesis of hepatocellular carcinoma H22 cells xenograft in mice. Methods: The xenograft model was established using injected subcutaneously H22 cells into the right axilla of the mice. Each group was treated with different doses of GPC and Endostar. All these treatments were maintained for 10 days, and mice were sacrificed. The xenograft tumors in mice were measured. The proliferation activity level of H22 cells was determined by MTT assay, and the levels of vascular endothelial growth factor(VEGF) protein were examined by immunohistochemistry. Results: When treated with 50, 100 and 200 mg/kg of GPC and Endostar, the tumor inhibition rates were 13.17%, 23.37%, 36.15% and 14.71%, respectively. The tumor weight of xenograft was significantly lighter in high GPC group than the control group(P < 0.05). The ODs in GPC groups were 0.835, 0.666 and 0.519, respectively. The absorbances in middle and high GPC groups were statistically significant, compared with control group(P < 0.01). Immunohistochemical technique showed the expression of VEGF of the GPC groups was downregulated significantly compared with the control group(P < 0.01). Conclusion: GPC can inhibit the growth of hepatocellular carcinoma H22 cell xenograft in mice. The inhibition of angiogenesis by the down-regulation of VEGF expression may play a key role in the anti-neoplastic effect of GPC.

PSG9 promotes angiogenesis by stimulating VEGFA production and is associated with poor prognosis in hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is a common malignant solid tumor characterized by rich vascularization. Pregnancy-specific glycoprotein 9(PSG9) is a member of the carcinoembryonic antigen(CEA)/PSG family and is produced at high levels during pregnancy. We previously identified PSG9 as an HCC-related protein. However, the expression of PSG9 and its regulation during HCC carcinogenesis remain poorly explored. In the present study, we first found that the levels of PSG9 protein were significantly increased in the plasma of HCC patients. PSG9 overexpression also increased the proliferation ability of an HCC cell line. High expression of PSG9 was associated with angiogenesis by accelerating VEGFA expression. In addition, Cox's proportional hazards model analysis revealed that the plasma level of PSG9 was an independent prognostic factor for overall survival. We propose that PSG9 is a novel indicator of prognosis in patients with HCC and could serve as a novel therapeutic target for HCC. Furthermore,our results indicate that PSG9 protein may facilitate the development of HCC by fostering angiogenesis via promoting VEGFA production in cancer cells.