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

OBJECTIVETo investigate endostatin gene therapy of rat corneal neovascularization induced by acid cauterization.

METHODSpBlast-hEndostatin and pBlast-Mcs were identified by digestion with Nhe Iand Sal I, by PCR reaction, by sequence, and then by alignment of PCR products with the gene Bank using NCBIBLAST software. They were then purified with QIAGEN Endofree plasmid maxi kit. Rat corneal neovascularization models were made with 75% AgNO(3) and 25% KNO(3) cauterization. The treatment method was subconjunctive injection of the pBlast-hEndostatin with the control of pBlast-Mcs.

RESULTSpBlast-hEndostatin was found to contain the human endostatin gene. The rat corneal neovascularization induced by acid cauterization was significantly suppressed after subconjunctive injection of the pBlast-hEndostatin with inhibition rates of 37%, 40.2%, and 42.8% respectively on the sixth, tenth, and fifteenth day. The inhibition rate for the density of corneal neovascularization was 40%. However, no inhibition effect on the length of the neovascularization and corneal inflammatory cells was observed. Corneal neovascularization areas were positively correlated with edema and corneal opacity.

CONCLUSIONSThe plasmid of pBlast-hEndostatin contained the human endostatin gene. The rat corneal neovascularization induced by acid cauterization can be partially inhibited by subconjunctive injection of the pBlast-hEndostatin mediated by liposomes. Endostatin produced by transfected fibroblast cells directly inhibits corneal neovascularization. This is not caused by inflammatory reaction inhibition.

Animals ; Corneal Neovascularization ; pathology ; therapy ; Endostatins ; genetics ; Genetic Therapy ; methods ; Humans ; Rats ; Rats, Wistar ; Transfection

OBJECTIVETo throw light on the superiority of the anti-angiogenesis activity of endostatin (ES) derivatives by reviewing the recent progress in the field of ES molecular structure modification.

DATA SOURCESThe data used in this article were mainly from PubMed with relevant English articles published from 1971 to May 2008. The search terms were "endostatin" and "angiothesis".

STUDY SELECTIONArticles involved in the ES molecular structure modification and the original milestone articles were selected.

RESULTSA number of ES derivatives were designed and studied to improve its clinical relevance. The modified ES with polyethylene glycol (PEG), low molecular weight heparin (LMWH) and IgG Fc domain extended the circulation half-life. Meanwhile the recombinant ESs showed more potent anti-tumor activity than native ES in mouse xenografts. Mutated ES also changed its anti-angiogenesis activity.

CONCLUSIONSThe anti-angiogenesis treatment remains a promising tumor therapeutic strategy. New ES derivatives would be a good choice to meet the future challenge on clinical application of ES.

Angiogenesis Inhibitors ; pharmacokinetics ; therapeutic use ; Animals ; Endostatins ; pharmacokinetics ; therapeutic use ; Humans ; Neovascularization, Pathologic ; drug therapy

OBJECTIVETo explore the in vitro anti-leukemia effect of endostar (recombinant human endostatin, rhES).

METHODSThe anti-leukemia effect of endostar on fresh bone marrow cell from acute leukemia patients and healthy adult was analyzed by typan-blue exclusion assay, MTT assay, transmission electron microscopy and flow cytometry with Annexin V-FITC/PI staining.

RESULTSTreatment with endostar at concentrations of 50, 100 and 200 µg/ml for 72 hours could inhibit the proliferation of HL-60 cells. The inhibition rates were 11.6%, 30.4% and 33.5%, respectively. For NB4 cells, the inhibitory rates of 100 and 200 µg/ml endostar were 12.4% and 16.4%, respectively. The inhibition of endostar on acute leukemia cells was time and dose dependent, 50 - 200 µg/ml endostar could also inhibit the growth of fresh bone marrow cell from acute leukemia patients. While 0 - 400 µg/ml endostar had no significant effect on fresh primary bone marrow cells from healthy adult. 100 µg/ml endostar could induce apoptosis of HL-60 and NB4 cells after treatment for 72 hours. The apoptotic rates of HL-60 and NB4 cells were 19.6% and 20.8%, respectively.

CONCLUSIONEndostar inhibits proliferation of leukemia cells by inducing apoptosis. It might be a potential medication for acute leukemia.

Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Endostatins ; HL-60 Cells ; Humans ; Leukemia

Animals ; Endostatins ; blood ; Estrogens ; therapeutic use ; Liver Cirrhosis, Experimental ; drug therapy ; metabolism ; Male ; Rats ; Rats, Wistar

OBJECTIVETo construct and express secretive endostatin eukaryotic plasmid for treatment of hepatoma.

METHODSMouse Igk signal peptide sequence was synthesized and cloned into pcDNA3.1 with endostatin gene. The supernant of BHK-21 transfected with recombinant was used to culture ECV304. The proliferation of latter was evaluated by MTT assay. H22 was inoculated intramusclely, then naked DNA of endostatin plasmid was injected into the inoculation site. Tumors were dissected and weighted after treatments. All data was analyzed by SPSS10.0.

RESULTSThe supernant of BHK-21 transfected with recombinant can inhibit the proliferation of ECV304 by 29.2%. Tumor weight lighter after injected with naked pSecES (1.34 g+/-0.96g) compared with naked pcDNA3.1 (2.70g+/-0.82g) and saline (3.73g+/-1.41g).

CONCLUSIONThe endostatin eukaryotic plasmid was constructed and it can be used for gene therapy on hepatoma.

Animals ; Endostatins ; genetics ; secretion ; Genetic Therapy ; Liver Neoplasms, Experimental ; therapy ; Male ; Mice ; Plasmids

BACKGROUND: The purpose of this study was to investigate plasma levels of endostatin and vascular endothelial growth factor (VEGF) in normal subjects and in patients with pituitary adenoma and to evaluate change in these levels following stereotactic radiosurgery (SRS) for pituitary adenoma. METHODS: Peripheral venous blood was collected from five patients with pituitary adenoma before SRS using Gamma Knife and at the 1 week and 1 month follow-up visits. Plasma endostatin and VEGF levels were measured using commercially available enzyme-linked immunosorbent assay kits. Peripheral blood samples were obtained from 10 healthy volunteers as controls. RESULTS: Mean baseline plasma endostatin level (105.3 ng/mL, range, 97.0-120.2 ng/mL) in patients with pituitary adenoma was higher than that of the healthy controls (86.6 ng/mL, range, 71.3-98.2 ng/mL) (p=0.001). Mean plasma VEGF level was 89.5 pg/mL (range, 24.1-171.8 pg/mL) in patients with pituitary adenoma at baseline and 29.3 pg/mL (range, 9.2-64.3 pg/mL) in the control group (p=0.050). Plasma endostatin level changed to 106.6 ng/mL 1 week after SRS and decreased to 95.9 ng/mL after 1 month. Plasma VEGF level following SRS decreased to 74.1 pg/mL after 1 week and 79.0 pg/mL after 1 month. There was a trend toward decreased plasma endostatin and VEGF concentrations 1 month after SRS compared to baseline levels (p=0.195, p=0.812, respectively). CONCLUSION: Plasma endostatin and VEGF levels in patients with pituitary adenoma were significantly elevated over controls at baseline, which decreased from baseline to 1 month after SRS for pituitary adenomas.
Endostatins* ; Enzyme-Linked Immunosorbent Assay ; Follow-Up Studies ; Healthy Volunteers ; Humans ; Pituitary Neoplasms* ; Plasma ; Radiosurgery* ; Vascular Endothelial Growth Factor A*

PURPOSE: The purpose of this study was to examine whether recombinant human endostatin (rhEndostatin), an antiangiogenic agent, is effective against a human neuroblastoma cell line (designated TNB9). We employed a human neuroblastoma xenograft model, and we investigated whether continuous infusion is more effective than an intermittent administration. METHODS: In the first experiment, when the tumors on the backs of nude mice reached a weight of 90 mg, rhEndostatin was administered subcutaneously to the mice (n=5) every day for 10 consecutive days. In the second experiment, the same daily dose of rhEndostatin was administered continuously to the TNB9- bearing mice (n=6) via subcutaneous infusion pumps for 3 consecutive days with the total dose being 30% of the dose given in the first experiment. Nestin and factor VIII expression levels were assessed immunohistochemically to elucidate whether the effects of rhEndostatin was present according to the histologic evidence at day 4 in the second experiment. RESULTS: In the first experiment, the relative tumor weight in treated mice (n=5) was significantly less than that in the controls (n=12) on day 2 after treatment initiation only (P<.05). The maximum inhibition rate (MIR) of TNB9 xenograft growth by rhEndostatin was 46.4%, indicating the lack of efficacy. In the second experiment, the effects of rhEndostatin were much more marked than those noted in the first experiment, with the MIR being 60.7%. The mean relative tumor weight in the treated group (n=6) in the second experiment was significantly less than that in the controls (n=10) on days 2, 4 and 6 (P<.01), as well as on days 8 and 10 (P<.05). The nestin staining in the endothelium of the control tumors (n=2) was remarkable, whereas the nestin staining showed as a loss of fibrillar structure in the rhEndostatin-treated tumors (n=2). The number of vessels immunostained with antifactor VIII antibody was markedly reduced in the tumors (n=2) from the rhEndostatin-treated mice compared with that from the control mice (n=2). CONCLUSION: Continuous administration of rhEndostatin resulted in more significant tumor regression than an intermittent administration of the agent. This result suggests that the continuous infusion of rhEndostatin is an effective agent and administration method for treating patients with neuroblastoma in the future.
Animals ; Cell Line ; Endostatins* ; Endothelium ; Factor VIII ; Heterografts* ; Humans* ; Infusions, Subcutaneous ; Mice ; Mice, Nude ; Nestin ; Neuroblastoma* ; Tumor Burden

OBJECTIVETo investigate the therapeutic effect of retroviral endostatin gene transfer on the human colon cancer cell line, LoVo.

METHODSA retroviral vector pLESSN expressing secretable endostatin was constructed and packaged with a titer of 8.2 x 10(5) CFU/ml. A LoVo cell line was subjected to retrovirus-mediated endostatin gene transfer. The proviral integration of endostatin was analyzed with PCR. The function of endostatin was tested by MTT assay in vitro and a mouse xenograft model in vivo.

RESULTSAfter transfection and superinfection, amphotropic retrovirus was collected, and transduction with amphotropic retroviruses resulted in endostatin proviral integration. The endostatin secreted by transduced LoVo cells markedly inhibited cell growth up to 67% (P<0.001), compared with the control cells. The gene expression of endostatin in LoVo colon tumor cells significantly inhibited tumor growth in vivo. There was an 86% reduction in tumor size in the endostatin-transduced group, accompanied by a reduction in vessels, compared with the control group (P<0.01).

CONCLUSIONRetroviruses can allow functional expression of the endostatin gene in human colon tumors, showing promise for an antitumor strategy using antiangiogenesis.

Cell Division ; Cell Line, Tumor ; Colonic Neoplasms ; pathology ; therapy ; Endostatins ; genetics ; Gene Transfer Techniques ; Genetic Vectors ; Humans ; Retroviridae

AIMTo establish the quality control methods for recombinant human endostatin.

METHODSBiological activity was determined by endothelial cell migration assays. Peptide mapping was tested by trypsin digestion and RP-HPLC. Purity was determined by non-reduced SDS-PAGE and RP-HPLC. Other tests including molecular weight, isoelectrical point, etc. were done according to the National Requirements for Biological Products (2000).

RESULTSThe method of bioassay was established and used for determining activity of endostatin. Specific activity of the three batchs of drug substance was 1.45 x 10(6), 1.57 x 10(6) and 2.73 x 10(6) u.mg-1 proteins. Peptide mappings of the three batches of drug substance were completely identified. Both purity results of the products tested by SDS-PAGE and RP-HPLC were more than 99%.

CONCLUSIONThe established methods can effectively control the quality of recombinant human endostatin.

Cell Movement ; drug effects ; Cells, Cultured ; Endostatins ; pharmacology ; Endothelium, Vascular ; cytology ; Humans ; Quality Control ; Recombinant Proteins ; pharmacology ; Technology, Pharmaceutical ; methods