Expressions of VEGF,ANG-1,ANG-2 and TSP-1 in cholangiocellular carcinoma and relationship with tumor angiogenesis,differentiation,invasion and metastasis

AIM: To investigate the angiogenesis status,the expression of vascular endothelial growth factor (VEGF),angiopoietin-1 (ANG-1),angiopoietin-2 (ANG-2),thrombospondin-1 (TSP-1) in cholangiocellular carcinoma (CCC) and relationship with tumor angiogenesis,differentiation,invasion and metastasis.METHODS: 33 specimen of surgically resected CCC were investigated.Immunohistochemical staining of CD34,VEGF,ANG-1,ANG-2 and TSP-1 was carried out.RESULTS: The mean MVD was (87.2±52.6)/mm2.VEGF positive expression was found in 75.6% cases;ANG-1 positive expression was observed in 36% cases;ANG-2 positive was detected in 57.6% cases and 45.5% cases exhibited positive TSP-1 expression.VEGF and ANG-2 expressions were found to be associated with significant higher level of MVD (P<0.01 and P<0.05,respectively).TSP-1 expression was found to be associated with significant low level of MVD (P<0.01).Positive TSP-1 expression was also found to be associated with higher level of intrahepatic metastasis (46.7% vs 5.6%,P<0.05).CONCLUSION: Considerable angiogenesis compared to other solid tumors can be observed in CCC.VEGF and ANG-2 might play a proangiogenic role and TSP-1 may play an inhibitory role.Although TSP-1 may increase the intrahepatic metastasis of CCC,neither MVD levels nor the expression of VEGF,ANG-1,or ANG-2 is associated with tumor differentiation,invasion and metastasis.     

Significance of expression of thrombospondin-1 and receptor-CD36 in hepatocellular carcinoma

AIM: To study the expression of thrombospondin-1 (TSP-1) and receptor-CD36, and investigate the relationship between tumor invasive capability and microvessel density and thrombospondin-1. METHODS: 43 hepatocellular carcinoma (HCC) cases were under investigation. Tissues from tumor, corresponding adjacent non-HCC tissue were stained with CD34 to show the MVD. TSP-1 and CD36 were examined by immunohistochemistry (SP) and RT-PCR. Relationship between clinical pathological features and above parameters was analyed. RESULTS: The staining of TSP-1 in HCC tissue is significantly lower than that in corresponding adjacent non-HCC tissue. Expression of TSP-1 was correlated to tumor thrombi, capsule, tumor invasive capability and CD36. CD36 was also correlated to tumor thrombi and tumor invasive capability. MVD was significantly higher in TSP-1, CD36 positive group than that in negative group. CONCLUSION: TSP-1 inhibits the growth, invasion and angiogenesis in HCC. TSP-1 may take effect through CD36.     

Canstatin: a novel inhibitor of angiogenesis

As a human basement membrane-derived inhibitor of angiogenesis and tumor growth, canstatin has been paid great attention since it was isolated and identified in 2000. Canstatin significantly inhibited human endothelial cell migration and proliferation and induced apoptosis, suggesting that it might be a powerful and potential therapeutic molecule for atherosclerosis, unstable angina and tumor.     

Establishing a three-dimensional angiogenesis model in vitro and secretion of MCP-1 and VEGF by tumor cells

AIM: To establish a three-dimensional angiogenesis model in vitro for observing the influence of tumor cells on angiogenesis, and to explore its possible molecular mechanism. METHODS: Human umbilical vein endothelial cells (HUVECs) and mouse endothelial cells SVEC4-10EE2 were coated on the surface of beads, and then mixed with fibrinogen and seeded in cell culture plates containing thrombin. The cultured endothelial cells coated on the beads grew into a vessel-like structure in a three-dimensional space containing fibrin condensate to establish an in vitro three-dimensional angiogenesis model. Tumor cells MDA-MB-231 and E0771 were co-cultured in the model to observe the effect of tumor cells on angiogenesis in vitro,respectively. The concentrations of monocyte chemoattractant protein-1 (MCP-1) and vascular endothelial growth factor (VEGF) in the tumor cells culture supernatant were measured by ELISA. An antagonist of CCR2 (receptor of MCP-1), along with SU5416 (an inhibitor of VEGF receptor), were added to the tumor cell co-culture system. The supernatant of the tumor cells was collected as a conditioned medium, which was then added to the angiogenesis system of HUVECs or SVEC4-10EE2 cultured individually. The effect of conditioned medium on angiogenesis was observed under the conditions of with or without SU5416, as well as with or without CCR2 antagonist. RESULTS: Under normal condition, HUVECs and SVEC4-10EE2 formed a multi-cellular vascular structure the three-dimensional angiogenesis model in vitro. The co-culture of MDA-MB-231 (E0771) cells significantly promoted in the formation of membrane-like structures. The ELISA results showed that the levels of MCP-1 and VEGF in the supernatant of tumor cells were significantly elevated (P<0.05). MCP-1 promoted the formation of membrane tube in three-dimensional culture system in vitro. After adding the antagonists of MCP-1 receptor CCR2 and VEGF (SU5416), the angiogenesis was significantly inhibited (P<0.05). At the same time, conditioned medium promoted the formation of extracorporeal membranes in the endothelial cells. The promoting effect was blocked by CCR2 antagonists and SU5416 (P<0.05). CONCLUSION: The in vitro three-dimensional angiogenesis model is successfully established. Tumor cells significantly promote the formation of membrane tubes. The effect of tumor cells might be related to MCP-1 and VEGF secretion.     

MCP-1/CCR2 signaling pathway mediates alcohol-induced angiogenesis in breast cancer

AIM To investigate the role of monocyte chemoattractant protein-1 (MCP-1) and its receptor CC chemokine receptor 2 (CCR2) in ethanol-promoted breast cancer angiogenesis and the underlying mechanism. METH?ODS: A mouse model of transplanted breast tumor with moderate alcohol consumption was established. The correlations between the expression of MCP-1/CCR2 and the expression of angiogenesis markers [platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial growth factor (VEGF)] in tumor tissues were examined by immunohistochemistry. In vitro, a 3D tumor-endothelial co-culture system was established to observe tumor angiogenesis and the role of MCP-1/CCR2 signaling pathway in alcohol-mediated angiogenesis. The cell migration ability was detected to clarify whether MCP-1/CCR2 enhanced cell mobility to form new vessels. RESULTS MCP-1 and CCR2 were both highly expressed in the breast tumor tissues of tumor-bearing mice consuming alcohol, and their expression levels were consistent with the angiogenic markers PECAM-1 and VEGF (P<0.05). The interaction between mouse breast cancer E0771 cells and endothelial cells was observed to promote angiogenesis in the 3D tumor-endothelial co-culture system with or without alcohol stimulation. MCP-1 promoted this kind of tumor angiogenesis, while CCR2 antagonist effectively inhibited the tumor angiogenesis and especially blocked alcohol-induced angiogenesis. Activation of MCP-1/CCR2 signaling pathway enhanced the migration ability of endothelial cells. CONCLUSION The MCP-1/CCR2 signaling pathway plays an important role in promoting the angiogenesis of breast cancer stimulated by alcohol. The mechanism might be that MCP-1 improves the migration of endothelial cells and then promotes angiogenesis.     

Expression and significance of thrombospondin-1 in myocardium of type 2 diabetic cardiomyopathy rats

AIM: To investigate the expression and significance of thrombospondin-1 (TSP-1) in left ventricular myocardium of type 2 diabetic cardiomyopathy (DCM).METHODS: The rat model of DCM was established by eating a high-fat diet together with injection of low dose streptozocin (30 mg/kg) intrapertoneally.After 12 weeks,the content of collagen was quantified by Masson staining.The mRNA level of TSP-1 was determined by quantification real-time RT-PCR,while the protein level of TSP-1 was analyzed by Western blotting and immunohistochemistry.RESULTS: Compared with the control group,the content of collagen in the DCM group was increased greatly (11.01±3.05 vs 16.92±3.18,P<0.01).The mRNA and protein expressions of TSP-1 were significantly higher than those in control group (0.0089±0.0034 vs 0.0141±0.0037,P<0.05；96.38±16.80 vs 129.98±16.96,P<0.05).In DCM group,the mRNA and protein expressions of TSP-1 showed significantly positive correlations with the levels of fasting blood glucose and collagen (r=0.762,P<0.01; r=0.717,P<0.05; r=0.735,P<0.01; r=0.750,P<0.01).There was a significantly positive correlation of TSP-1 mRNA level with LVEDP (r=0.658,P<0.05).In contrast,there was a significantly negative correlation of TSP-1 protein with LVSP and -dp/dtmax (r=-0.605,P<0.05; r=-0.694,P<0.05).There was a significantly positive correlation of TSP-1 protein with LVEDP (r=0.716,P<0.05).There was a significantly negative correlation of TSP-1 protein with LVSP and -dp/dtmax (r=-0.633,P<0.05; r=-0.669,P<0.05).CONCLUSION: The increased expression of TSP-1 may play an important role in the development of myocardial interstitial fibrosis in DCM.     

Recent advances in angiogenesis and tumor angiogenesis inhibitor in prostate cancer

Angiogenesis plays a key role in progression of prostate cancer. Antigiogenesis becomes a new treament target for prastate cancer. In this review, we focus on the current knowledge of angiogenesis and tumor angiogenesis inhibitor in prastate cancer.     

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AIM:To investigate the effects of plasminogen kringle 5(K5) deletion mutantⅠ(K5 mut1) on the neovascularization and growth of hepatocarcinoma in vivo. METHODS:K5 mut1 was expressed in E. coli BL-21(DE3) induced by IPTG and purified by Ni2+-His Bind Resin affinity chromatography. The purity and identity of K5 mut1 was examined by SDS-PAGE and Western blotting analysis. K5 mut1 activity was evaluated in vivo, HepA-grafed hepatocarcinoma mouse model was established by subcutaneously injection of mouse hepatoma cells(1×106) into the oxter of mice, and the mice were then divided into different groups and treated with PBS and K5 mut1 at different doses, respectively. The tumor suppressing rate and micro vascular density (MVD) in hepatoma tissues were assayed. RESULTS:The purity of recombinant K5 mut1 was over 90% according to the analysis of SDS-PAGE and Western blotting analysis. K5 mut1 significantly inhibited the growth of tumor in a HepA-grafed hepatocarcinoma mouse model and decreased microvessel density (MVD) in hepatoma tissues in a dose-dependent manner, compared with PBS control group. CONCLUSION:K5 mut1 exhibits anti-tumor effect in a HepA-grafted hepatocarcinoma mice model by the anti-angiogenic activity. These results support the conclusion that K5 mut1 may be a promising angiogenesis inhibitor and tumor suppressor with considerable therapeutic potential in liver cancer.     

Effects of thrombospondin 1 on rat cardiac fibroblasts induced by transforming growth factor β1

AIM:To investigate the effects of thrombospondin 1 on transforming growth factor β₁ induced rat cardiac fibroblasts (CFs). METHODS: CFs of neonatal Sprague -Dawley (SD) rats were isolated with the method of digestion and differential anchoring velocity. The proliferation and collagen synthesis of rat CFs were observed with MTT and hydroxyproline. The expression of TSP-1mRNA was analyzed by RT-PCR.RESULTS: The dose and time-dependent effects of TGF-β₁ were observed. Expression of TSP-1 was increased significantly (P<0.01). Stimulation of CFs with TGF-β₁ (20 μg/L, 24 h) significantly increased CFs proliferation and collagen synthesis (P<0.01). TSP-1 antisense oligonucleotide effectively inhibited TGF-β₁ induced CFs proliferation and collagen synthesis (P<0.01).CONCLUSION:The proliferation and collagen synthesis of CFs induced by TGF-β₁ are inhibited by TSP-1 antisense oligonucleotide, which may exert helpful effect on anti-fibrosis.     

Advances in endostatin research

Endostatin, a 20 kD (184 aa) C-teminal fragment of collagenⅩⅧ, is the most potent inhibitor of tumor angiogenesis described so for. Endostatin was initially isolated from a murine hemangioendothelioma cell line (EOMA). Purified recombinant murine endostatin generated in E. coli bacteria injected as unfolded suspension, inhibited the growth of a varity of metastatic and primary tumors in mice. However, its widespread application has been hampered by difficulties in the large-scale production of the antiangiogenic proteins. The limitation may be resolved by in vivo delivery and expression of the antitangiogenic gene. This review summarized the advances in endostain research in recent years including structure, the mechanism of generating, function and therapy.     

Inhibition of 17-DMAG on VEGF expression in transplanted human gastric cancer in nude mice

AIM: To observe the anti-tumor effects of heat shock protein 90(HSP90) inhibitor 17-dimethylaminoethylamino-17 demethoxygeldanamycin (17-DMAG) on the tumor growth and angiogenesis in implanted gastric cancer nude mouse model. METHODS: Human gastric cancer cell HGC-27 was subcutaneous inoculation into the nude mice to develop a tumor model. Ten days later, 24 mice with implanted tumor were randomly divided into 3 groups: 17-DMAG group (receiving 17-DMAG at dose of 25 mg/kg), control group (treated with NS at dose of 10 mL/kg) and 5-fluorouracil(5-FU) group (treated with 5-FU at dose of 20 mg/kg). Four weeks after treatment, the tumor volume and weight, and the inhibitory rates of tumor growth were evaluated. In the meantime, the expression of CD31 was detected by immunohistochemical staining. The expression of vascular endothelial growth factor (VEGF) was determined by Western blotting. RESULTS: The size of xenografts in 17-DMAG treatment group was (288.10±23.32)mm³, and that in 5-FU treatment group was (366.37±26.42)mm³, both were significantly smaller than that in control group (957.66±117.51)mm³. The tumor weight in 17-DMAG treatment group was (0.41±0.02)g, significantly less than that in control group (1.12±0.08)g. The inhibitory rate of 17-DMAG was 63%. A significant decease of MVD in 17-DMAG group (21.72±1.24) was observed as compared to 5-FU group (36.70±1.51) and control group (37.78±1.68). The expression of VEGF in 17-DMAG group (15.39±4.37) was significantly lower than that in 5-FU group (26.11±6.26) and control group (36.45±7.45). CONCLUSION: HSP90 inhibitor 17-DMAG suppresses the expression of VEGF and the angiogenesis of the gastric cancer to inhibit the tumor growth.     

Relationship between peritubular capillary injury and renal interstitial fibrosis in mice with unilateral ureteral obstruction

AIM: To observe the injury of peritubular capillary (PTC), hypoxia and interstitial fibrosis after unilateral ureteral obstruction (UUO), and to explore the effects of PTC injury and hypoxia on interstitial fibrosis in mouse model of UUO. METHODS: Forty-eight male KM mice were randomly divided into control group and UUO group. On the 1st, 3rd, 7th and 14th days, 6 mice in each group were sacrificed. The changes of pathomorphism in the kidney were observed by HE and Masson staining. The expression levels of thrombospondin-1 (TSP-1), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α), and PTC density were detected by immunohistochemistry. The protein expression of VEGF was also determined by Western blotting. RESULTS: No histological abnormalities of the kidneys were observed in sham-operated mice. The expression of TSP-1 was increased 1 day after UUO, and significantly increased on the 3rd, 7th and 14th days(P<0.05). The expression of VEGF was obviously decreased(P<0.05). PTC density was gradually decreased. The expression of HIF-1α was gradually increased, and renal interstitial area was gradually expanded. PTC density was negatively correlated with the expression of TSP-1 and HIF-1α (r=-0.874 and r=-0.930, respectively). VEGF expression was positively correlated with PTC density (r=0.745). PTC density was negatively correlated with the area of renal fibrosis (r=-0.787). HIF-1α expression was positively correlated with the area of renal fibrosis (r=0.835, P<0.05). CONCLUSION: In mouse UUO model, the expression of TSP-1 is increased. The expression of VEGF is reduced. The peritubular capillary injury and tissue hypoxia are aggravated, and renal interstitial fibrosis area is expanded. Ischemia and hypoxia may play an important role in the progression of UUO.     

Effect of microRNA-155 on regulation of angiogenesis in diabetic rats with cerebral ischemic injury

AIM: To evaluate the effect of microRNA-155(miRNA-155) on the regulation of angiogenesis in diabetic rats with cerebral ischemic injury. METHODS: Adult male Sprague-Dawley rats were randomly divided into 5 groups:sham group, cerebral ischemia group, diabetic cerebral ischemia group, diabetic cerebral ischemia+miRNA-155 inhibitors group and diabetic cerebral ischemia+scramble group. Diabetes model was made by injection of streptozocin and permanent cerebral ischemic model was developed by suture-occluded method. The scores of neurological deficit and infarct volume were estimated at 24 h after cerebral ischemia. miRNA-155 level was detected by real-time polymerase chain reaction. The expression of platelet endothelial cell adhesion molecule-1(PECAM-1/CD31) and vascular endothelial growth factor(VEGF) was detected by Western blotting. RESULTS: miRNA-155 inhibitor significantly reduced miRNA-155 levels in the ischemic cortex(P<0.05), improved the scores of neurological deficit, reduced infarction size and upregulated the levels of CD31 and VEGF(P<0.05). CONCLUSION: miRNA-155 has a critical role in the regulation of angiogenesis in diabetic rats with cerebral ischemia. Down-regulation of miRNA-155 using miRNA-155 inhibitor attenuates brain infarct injury in diabetic rats.     

Effect of miR-18a on angiogenesis of human aortic endothelial cells

AIM: To study the effect of miR-18a on angiogenesis of human aortic artery endothelial cells. METHODS: After 10 nmol/L miR-18a mimics or 20 nmol/L inhibitor was transfected into human aortic endothelial cells, the expression level of miR-18a was determined by qRT-PCR, and the capacities of endothelial cell angiogenesis, such as migration, adhesion and tube formation, were observed. RESULTS: Forty-eight hours after transfection with miR-18a mimics, the expression of miR-18a was as 608 folds as the control (P<0.05), but decreased to 31% of the control level in miR-18a inhibitor transfection group (P<0.05). Compared with control group, the numbers of endothelial cells, which migrated to the lower Transwell chamber and formed capillary-like tubes, declined by 60% and 52%, respectively, in miR-18a mimics group (P<0.01), and they increased by 100% and 84%, respectively, in miR-18a inhibitor transfection group (P<0.05, P<0.01). In addition, the inhibitor treatment group displayed more potent adhesion capacity, 43% higher than that in control group (P<0.05). CONCLUSION: miR-18a is involved in angiogenesis of human arterial endothelial cells, and might be a potential molecular therapeutic target of vascular diseases.     

Effect of Yiqi Huoxue Jiedu Fang on the growth,metastasis and angiogenesis of Lewis lung carcinomas

AIM: To study the effect of Yiqi Huoxue Jiedu Fang (YHJ), composed of ginsenoside, penex notogingseng and berberin, on tumor growth and metastasis and to explore its mechanism.METHODS: Murine Lewis lung carcinoma transplant model was established and mice were treated with YHJ by intraperitoneal injection. After 10 days, the inhibitory rate of tumor, pathology of tumor and PCNA of tumor cells were detected. After 20 days, numbers of metastatic foci on lung surface and microvessel density (MVD) were determined. Expression of VEGF in tumor and serum were also analyzed by immunohistochemical test and ELISA, respectively. RESULTS: YHJ reduced the weight of tumor and the amount of metastatic foci. The inhibitory rates of tumor at high and low dose of YHJ (24 mg·kg_^-1·d_^-1, 12 mg·kg_^-1·d_^-1) were 48.29% and 37.26%, and the number of metastatic foci was 1.67 and 3.50, while control was 6.44. Furthermore, PCNA of tumor cells, MVD of tumor and VEGF expression in serum and tumor were decreased in YHJ treatment goup as compared with control. CONCLUSION: YHJ remarkably inhibits Lewis lung carcinoma growth and metastasis in mice. Its mechanism may be related to inhibition of angiogenesis.     

巴西菇麦角甾醇抗肿瘤活性及作用机理初探

采用小鼠S180移植瘤实验研究了巴西菇子实体中麦角甾醇的体内抗肿瘤活性,并通过巨噬细胞吞噬功能测定(中性红法)、脾淋巴细胞增殖功能检测(MTT法)、体外抑杀肿瘤细胞(MTT法)以及鸡胚绒毛尿囊膜(CAM)等试验初步探讨了麦角甾醇抗肿瘤活性的作用机理.结果表明,巴西菇子实体中提取的麦角甾醇在剂量10 mg·kg-1·d-...     

Angiogenesis and its regulation mechanism in S180 transplanted tumor of mice

AIM: To investigate the angiogenesis in the process of sarcoma 180 (S180) tumor transplantation and changes of regulator factors, and explore the possible mechanism. METHODS: The S180 transplanted tumor in the Km mouse was used to detect the tumor angiogenesis by immunohistochemical examination of FⅧ. The levels of VEGF (V) and endostatin (E) in serum and the homogenate of tumor tissue were measured by ELISA and EIA, and the correlation between tumor weight and microvessel count (MVC) and morphology in tumor was also analyzed by multiple ANNOVA method. RESULTS: MVC, the relative count of total vessels and relative total vessel area increased with the development of transplanted S180. VEGF level in tumor tissue were higher at the 10th and 15th day than the 5th day after tumor transplantation. Endostatin in the tumor tissue and serum both reached the highest level at the 15th day, V/E ratio did not changed in this process. Furthermore, MVC, average vessel area and relative total area had a significant correlation with tumor weight. CONCLUSION: MVC increases in the development of S180 transplantation tumor and is related with the tumor weight; the positive regulator of angiogenesis in the tumor tissue is up-regulated during tumor growth, and the regulators in the tumor tissue maintains a relative balance.     

Angiogenesis: molecular mechanism and related diseases

Angiogenesis is the process of capillary formation from the existing blood vessels, which is regulated by many cytokines. Balance of these cytokines plays an important role in angiogenesis. Unbalance of these cytokines, leading to excessive or insufficient blood vessel, relates to a variety of diseases, such as tumor, ophthalmic diseases and wound healing. Recently, it has been observed that angiogenesis is also involved in Parkinson's disease and Alzheimer's disease. This review mainly discusses the molecular mechanism of angiogenesis and related diseases, and emphasizes the value of targeting angiogenesis as a strategy to develop drugs for those diseases.     

Inhibitory effect of Shenci capsule in combination with DDP on angiogenesis in mice with Lewis lung cancer

AIM: To investigate the inhibitory effect of Shenci capsule, a Chinese medicine, in combination with cisplatin (DDP) on the tumor growth and angiogenesis in mouse Lewis lung cancer.METHODS: Forty mice with Lewis lung cancer were randomly divided into 4 groups: Shenci capsule group, Shenci capsule in combination with DDP group, DDP group and control group. Shenci capsule, Shenci capsule in combination with DDP,DDP and sodium chloride wene given, respectively. After 21 days, the weight of subcutaneous tumors was measured, and the tumor inhibition rate was calculated. The microvessel density (MVD) and the protein level of vascular endothelial growth factor (VEGF) were detected by immunohistochemical(IHC) staining. The mRNA expression levels of kinase insert domain-containing receptor (KDR) and the receptor of VEGF were detected by FQ-PCR. RESULTS: The tumor growth inhibitory rate in Shenci capsule in combination with DDP group (63.99%) was higher than that in Shenci capsule group, DDP group and control group (P<0.01). The tumor MVD, VEGF protein and KDR mRNA expression level in Shenci capsule in combination with DDP group were lower than those in the above three groups (P<0.01). CONCLUSION: Shenci capsule in combination with DDP have addition or synergistic effects to inhibit the tumor growth and angiogenesis in mouse Lewis lung cancer. Down-regulation of VEGF and its receptor KDR may be one of the mechanisms that combined use of these two drugs for inhibiting angiogenesis.