PURPOSE: Angiogenesis is a critical determinant of tumor growth and the development of metastasis. Angiostatin and endostatin have been used in a variety of in vitro and in vivo animal models as effective inhibitors of angiogenesis. However, human angiostatin and endostatin have not been tested against an intact human tissue target in vitro to determine its ability to achieve an antiangiogenic response. We performed our study to determine if human angiostatin and endostatin would inhibit the development of an angiogenic response (initiation) and to determine the subsequent growth (angiogenic index) of human vessels in a dose-dependent manner with a human placental vein angiogenesis model (HPVAM). METHODS: We used full thickness human placental vein discs cultured in three-dimensional fibrin-thrombin clots with an overlay of liquid media. Human angiostatin and endostatin were evaluated in concentrations ranging from 10-9 M to 10-4 M. A positive control containing 20% fetal bovine serum and a negative control using heparin and hydrocortisone 21-phosphate were also tested. RESULTS: Human angiostatin did not inhibit the initiation of an angiogenic response and the subsequent development of the angiogenic response (angiogenic index) at any concentration. Human endostatin significantly inhibited the initiation rate of an angiogenic response at a concentration of 10-4 M (p<0.001) and the subsequent development of an angiogenic response (angiogenic index) from a concentrations of 10-5 M to 10-4 M (p<0.001, p<0.001, respectively). CONCLUSION: We conclude that a very high concentration of human endostatin can inhibit the angiogenic response in human vascular tissue and that human angiostatin will not inhibit angiogenesis of normal human blood vessels in vitroThese results suggest that human endostatin has a more powerful antiangiogenic effect than human angiostatin, but we need further investigations of human angiostatin against an intact human tissue target.
Angiostatins* ; Blood Vessels ; Endostatins* ; Heparin ; Humans* ; Hydrocortisone ; Models, Animal ; Neoplasm Metastasis ; Veins

Angiostatin is derived from enzymatic degradation of plasminogen and it has endogenous anti-angiogenic properties. Although tumor cells, macrophages, platelets, and neutrophils generate high amount of angiostatin, its expression is increased in inflammatory conditions. Moreover, angiostatin binds to integrin alpha(v)beta(3), ATP synthase, and angiomotin, which expressed on neutrophils. Activated neutrophils are essential to innate immune response, but also cause tissue damage through production of reactive oxygen species (ROS) and increase lifespan. In this article, it suggests several mechanism of angiostatin as immune regulator for neutrophils in inflammatory conditions; complex with integrin alpha(v)beta(3) and F(1)F(0) ATP synthase on lipid raft, attenuate polarization, and ROS production. These data provide possible exploit of double-edged role of neutrophils in acute inflammatory pathologies to preserve beneficial effect and minimize tissue damage.
Adenosine Triphosphate ; Angiostatins* ; Apoptosis ; Immunity, Innate ; Integrin alphaVbeta3 ; Macrophages ; Neutrophil Activation* ; Neutrophils* ; Pathology ; Plasminogen ; Reactive Oxygen Species

A recombinant strain of Pichia pastoris with a phenotype of Muts was used to produce angiostatin in a 5-L fermentor. The methanol utilization ability of the present strain was weak, which resulted in extremely low growth rate and angiostatin productivity during the expression phase with methanol as the sole carbon source. To enhance the cell density and angiostatin expression level, mixed-carbon-source of glycerol-methanol was used in the expression phase. The methanol concentration was well controlled at 5 g/L by a methanol sensor and control system, and glycerol was continuously fed into the fermentor to achieve a higher cell density. 120 g/L of cells and 39 mg/L of angiostatin were reached at the end of fermentation which lasted 110 h. The mean specific cell growth rate in the expression phase was 0.01 h(-1), and the mean specific angiostatin productivity was 0.006 mg/(g x h). According to the data obtained in several runs of fermentation in which glycerol was fed at different rates, a higher mean specific angiostatin productivity was reached at the mean specific cell growth rate of 0.012 h(-1). To avoid the repression of angiostatin expression caused by residual glycerol and ethanol accumulation due to overfeeding of glycerol, glycerol addition was controlled to produce continuous oscillations in dissolved oxygen, because the change of dissolved oxygen concentration could deliver the information of available carbon source in the fermentation broth. Controlled glycerol feeding also avoided the problem of oxygen limitation brought by high cell density, and thus decreased the cooling requirement of the fermentor. Cell density reached 150 g/L at the end of fermentation, and angiostatin level reached 108 mg/L after an expression period of 96 h when the mean specific growth rate was maintained at 0.012 h(-1) by using the glycerol feeding strategy to result in the oscillations in dissolved oxygen. The mean specific angiostatin productivity was improved to 0.02 mg/(g x h). The apparent cell yield on glycerol and methanol were respectively 0.69 g/g and 0.93 g/g, higher than those in the fermentation without using the feeding strategy with dissolved oxygen as the indicator of metabolism.
Angiostatins ; genetics ; metabolism ; physiology ; Biotechnology ; methods ; Carbon ; metabolism ; Fermentation ; physiology ; Glycerol ; metabolism ; Methanol ; metabolism ; Oxygen ; metabolism ; Pichia ; genetics ; metabolism

Angiogenesis is recognized as a critical factor in the growth of tumor cells and plays a key role in the tumor metastasis. Recent studies for antiangiogenic substances are getting popular. The angiostatin, one of the antiangiogenic substances, leads to the increased apoptosis of the tumor cells by inhibiting the neovascularization of the tumor. The angiostatin was identified as the internal fragments of the plasminogen which has no antiangiogenic activity. By hydrolysis of the plasminogen, the angiostatin can be produced. In this study, we constructed the SFV-derived DNA vector by employing the cytomegalovirus immediate early enhancer/ promoter (CMV). This vector makes it possible to transfect the cells with DNA without the in vitro transcription process. The C-myc epitope and polyhistidine residue sequences were placed in downstream of the angiostatin gene to make it eligible to detect the expressed protein. The murine Ig kappa-chain V-J2-C signal sequence was placed in upstream to secrete the expressed protein from the cells. We confirmed the expression of angiostatin in the BHK-21 cells using DNA-based SFV replicon.
Angiostatins/analysis/*genetics ; Animals ; Base Sequence ; Cell Line ; Cricetinae ; DNA Primers ; Gene Expression Regulation, Viral ; Immunohistochemistry ; Kidney ; Plasmids ; Replicon/*genetics ; Semliki forest virus/*genetics ; Transfection

OBJECTIVECastrated rats exhibit significant shrinkage of the ventral prostate and apoptosis of prostatic cells, which can be attributed to the reduced blood supply to the prostate. But what causes the blood decrease in the prostate remains unknown. This study aims to explore the molecular mechanism of the changes in the microcirculation of the ventral prostate of rats following castration.

METHODSWe randomized 24 male adult rats into 6 groups of equal number, and collected their ventral prostates at 0, 1/2, 1, 2, 3 and 7 d, respectively, after castration. Then we observed the changes of the microvessels under the transmission electron microscope, detected the apoptosis of endothelial cells by TUNEL, and determined the expressions of VEGF, endostatin, angiostatin and angiopoietin-2 by Western blot.

RESULTSThe castrated rats showed dramatic changes in the microvessels of the ventral prostate, obvious apoptosis of the endothelial cells, down-regulated expression of VEGF, and up-regulated expressions of endostatin and angiostatin, while angiopoietin-2 remained unchanged.

CONCLUSIONThe decreased level of VEGF and increased levels of endostatin and angiostatin might underlie the mechanism of the changes in the microcirculation of the ventral prostate of rats following castration.

Angiopoietin-2 ; metabolism ; Angiostatins ; metabolism ; Animals ; Endostatins ; metabolism ; In Situ Nick-End Labeling ; Male ; Microcirculation ; Orchiectomy ; Prostate ; blood supply ; Rats ; Rats, Sprague-Dawley ; Vascular Endothelial Growth Factor A ; metabolism

OBJECTIVETo investigate the relationship between TSP-1, Angiostatin and Endostatin serum concentrations and progression of pancreatic adenocarcinoma.

METHODSFifty-six patients with suspected pancreatic cancer were enrolled in the study and divided into resectable group (n = 32) and unresectable group (n = 24) according to evaluation and staging with dual phase helical CT. Histopathologic examinations included postoperative final pathology and preoperative fine needle biopsies. Peripheral blood concentrations of antiangiogenic factors Angiostatin, Endostatin and TSP-1 were detected by using ELISA methods, selecting samples of health people as a control.

RESULTSSerum concentrations of antiangiogenic factors in pancreatic cancer group were significantly higher than those in health group (P < 0.01). Serum concentrations of Endostatin, Angiostatin and TSP-1 were significantly increased in unresectable group, and highly expressed in patients whom tumor sizes were greater than 2 cm and tumor invaded peripancreatic great vessels (P < 0.05). After operation, serum concentrations of Endostatin, Angiostatin and TSP-1 significantly decreased (P < 0.05). There were no significant difference between I, II stage group and III, IV group.

CONCLUSIONSDetection of serum concentrations of antiangiogenic factors may be used to evaluate the resectability of pancreatic cancer and may play important roles in growth, invasion and metastasis of pancreatic cancer.

Adenocarcinoma ; blood ; pathology ; surgery ; Adult ; Aged ; Angiostatins ; blood ; Disease Progression ; Endostatins ; blood ; Female ; Humans ; Male ; Middle Aged ; Pancreatic Neoplasms ; blood ; pathology ; surgery ; Thrombospondin 1 ; blood ; Treatment Outcome

In order to explore the new methods of biological treatment of human gliomas, this project is to study the biological properties of gliomas from four different aspects, the results show that there is a IL-6 autocrine loop in human gliomas and the growth of gliomas will be inhibited when the autocrine loop is broken. There is a magnificent predominant expression of Th2 cytokines in human gliomas and human glioma cells, the switching of Th2 to Th1 can inhibit the proliferation of glioma cells. The dosage of 100 micrograms/ml of erythromycin is the best of therapeutic effect. Angiostatin can not only inhibit the vascular endothelial growth, but also have the inhibitory role on the growth of glioma cells in vivo. The above studies have provided some new ideas and will be very helpful for the treatment of glioma patients.
Angiostatins ; pharmacology ; Animals ; Biological Therapy ; Brain Neoplasms ; secretion ; therapy ; Drug Resistance, Neoplasm ; Glioma ; secretion ; therapy ; Humans ; Interleukin-6 ; genetics ; secretion ; RNA, Messenger ; genetics ; secretion ; Th1 Cells ; metabolism ; Th2 Cells ; metabolism

OBJECTIVETo study the effects of murine angiostatin, which was transfected into the human hepatocellular cancer cell line SMMC-7721, on the implant carcinoma of nude mouse.

METHODSThe human hepatocellular cancer cell line SMMC-7721, which could express murine angiostatin gene stably, was constructed. The animals were divided into three groups: SMMC-7721 cell was implanted into control group, SMMC-7721/pcDNA3.1 (+) cell was implanted into vector group, and SMMC-7721/pcDNA3.1-mAST cell was implanted into angiostatin group. The carcinoma volume, weight, and microvessel density (MVD) of each group were compared.

RESULTSThe implant carcinoma volume in 35 days was (3 538.1 +/- 643.3) mm(3), (3 128.5 +/- 546.6) mm(3), and (755.8 +/- 198.2) mm(3) in the control group, vector group, and angiostatin group. The carcinoma weight of the control group, vector group, and angiostatin group was (6.0 +/- 0.7) g, (5.9 +/- 0.5) g, (2.1 +/- 0.5) g, respectively. The carcinoma MVD was 52.2 +/- 6.6, 49.4 +/- 7.0, and 25.5 +/- 4.1 accordingly. The carcinoma volume, weight, and MVD of the angiostatin group were significantly smaller than those of the control group and vector group (P < 0.01). The inhibitory rate of carcinoma reached 78.6%.

CONCLUSIONSNude mouse experiments showed that the tumorigenic capacity of cells transfected had been reduced greatly, and that the carcinoma volume, weight and MVD were significantly lower than those of the control group. We conclude that angiostatin inhibits the growth of carcinoma by its inhibition of carcinoma angiogenesis.

Angiostatins ; Animals ; Genetic Therapy ; methods ; Liver Neoplasms, Experimental ; blood supply ; prevention & control ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neovascularization, Pathologic ; prevention & control ; Peptide Fragments ; genetics ; Plasminogen ; genetics ; Tumor Cells, Cultured

OBJECTIVETo observe the effects of angiostatin gene combined with chemotherapy on implanted human ovarian carcinoma of nude mouse.

METHODSThe mice were randomly divided into four groups after 7 days of the intraperitoneal injection of tumor cells (4 x 10(6)), and injected respectively with empty plasmid pcDNA3.0, angiostatin plasmid, cisplatin, and angiostatin plasmid + cisplatin. For combinational treatment, reagents were delivered in a timed fashion, where angiostatin plasmid was injected first, followed by cisplatin 24h later. The tumor samples were prepared to be used in the examinations of the expression of angiostatin with immunohistochemistry, of MVD in the tumor with immunohistochemistry, and of cell apoptosis with TUNEL staining.

RESULTSTumor growth and ascites formation were inhibited in all 3 groups except for the control group. The therapeutic effectiveness in the combined group was more significant than in the other two groups. In this group, MVD (32.5 +/- 4.3) was the lowest and apoptosis index (5.12 +/- 0.63) was the highest (P < 0.01).

CONCLUSIONSAngiostatin gene therapy combined with chemotherapy has a synergistic effect on the inhibition of ovarian cancer angiogenesis and ascites formation. Combining multiple therapies to treat ovarian cancer is an effective strategy.

Angiostatins ; biosynthesis ; genetics ; Animals ; Antineoplastic Agents ; therapeutic use ; Cisplatin ; therapeutic use ; Combined Modality Therapy ; Female ; Humans ; Injections, Intraperitoneal ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Ovarian Neoplasms ; blood supply ; pathology ; therapy ; Peritoneum ; Random Allocation ; Transplantation, Heterologous

Carbon source plays an important role in the constitutive expression of foreign proteins in Pichia pastoris. In present study, glucose , glycerol , methanol and oil acid, was used respectively as the only carbon source to constitutively express hAS in Pichia pastoris GS115 (pGAP9K-AS)in shaking flask. The result shows that oleic acid is the best (163 mg/L) compared with glycerol (83mg/L), glucose (76 mg/L)and methanol (57 mg/L). Since oleic acid is insoluble in water, glycerol was used as the carbon source in the high-density cell culture of GS115 (pGAP9K-AS) in a 30 liter bioreactor and 169 mg/L of angiostatin was obtained after 48h of culture. The expressed angiostatin is immunologically active as shown by Western blotting. The recombinant hAS inhibits bFGF induced CAM angiogenesis and suppresses the growth of B16 melanoma in C57BL/6J mice. The tumor inhibition rate is 90% after 12 days of treatment. Statistics analysis revealed that the tumor volume difference of mice between the hAS group and PBS group is prominent (P < 0.01).
Angiogenesis Inhibitors ; biosynthesis ; genetics ; therapeutic use ; Angiostatins ; biosynthesis ; genetics ; therapeutic use ; Animals ; Bioreactors ; microbiology ; Culture Media ; pharmacology ; Fermentation ; Glycerol ; pharmacology ; Humans ; Melanoma, Experimental ; drug therapy ; Mice ; Mice, Inbred C57BL ; Pichia ; genetics ; growth & development ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics ; therapeutic use