Expression and significance of angiostatin, vascular endothelial growth factor and matrix metalloproteinase-9 in brain tissue of diabetic rats with ischemia reperfusion

OBJECTIVE@#To discuss the expression and significance of angiostatin, vascular endothelial growth factor and matrix metalloproteinase-9 in the brain tissue of diabetic rats with ischemia reperfusion.@*METHODS@#A total of 60 male Wistar rats were randomly divided into the normal group, sham group, diabetic cerebral infarction group and single cerebral infarction group according to the random number table, with 15 rats in each group. The high sucrose diet and intraperitoneal injection of streptozotocin were performed for the modeling of diabetic rats, while the thread-occlusion method was employed to build the model of cerebral ischemia reperfusion. The immunohistochemical staining was performed to detect the expression of angiostatin, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) in the brain tissue.@*RESULTS@#The expression of angiostatin after the reperfusion in the brain tissue of rats in the single cerebral infarction group and diabetic cerebral infarction group was increased 6 h after the reperfusion, reached to the peak on 1 d and then decreased gradually. The expression of angiostatin in the diabetic cerebral infarction group 6 h, 1 d, 3 d and 7 d after the reperfusion was significantly higher than that in the single cerebral infarction group (P < 0.05). VEGF began to be increased 1 h after the reperfusion in the single cerebral infarction group and diabetic cerebral infarction group, reached to the peak at 6 h and then decreased gradually. The expression of VEGF in the diabetic cerebral infarction group at each time point after the reperfusion was significantly lower than that in the single cerebral infarction group (P < 0.05). MMP-9 began to be increased 1 h after the reperfusion in the single cerebral infarction group and diabetic cerebral infarction group, reached to the peak on 1 d and then decreased gradually. The expression of MMP-9 in the diabetic cerebral infarction group at each time point after the reperfusion was significantly higher than that in the single cerebral infarction group (P < 0.05).@*CONCLUSIONS@#The high glucose environment in which the diabetic cerebral infarction is occurred is to induce the formation of MMP-9 at first and then activate and increase the expression of angiostatin. Afterwards, the expression of VEGF is inhibited, resulting in the poor angiogenesis after cerebral infarction, which thus makes the injury of brain tissue after cerebral infarction even worse than the non-diabetes mellitus.

Anti-tumor activity of tanshinone IIA in combined with cyclophosphamide against Lewis mice with lung cancer

OBJECTIVE@#To explore the anti-tumor activity of tanshinone IIA in combined with cyclophosphamide against Lewis mice with lung cancer and the effect on cellular immune function.@*METHODS@#Lewis tumor cells were inoculated subcutaneously into the right armpit of mice in each group (n = 20) to establish Lewis lung cancer mice model. After model establishment, mice in the model group were given normal saline by lavage, qd. Mice in treatment I group were given intraperitoneal injection of Tan IIA, 15 mg/kg, qd. Mice in treatment II group were given intraperitoneal injection of CTX, 25 mg/kg, qd. Mice in treatment III group were given intraperitoneal injections of Tan IIA and CTX, in which the administration method of Tan IIA was the same as in treatment I group, continuously for 2 weeks, and the dosage of CTX was the same as in treatment II group, 24 h after model establishment, every other day. Mice were sacrificed 2 weeks after establishment. The tumor tissues were collected to calculate the anti-tumor rate. Immunohistochemistry was used to detect the expressions of Bcl-2, Bax, VEGF, Angiostatin, and Endostatin. FCM was used to detect T lymphocyte subsets in spleen and liver of mice.@*RESULTS@#The tumor weight in treatment I, II, and III groups was significantly lower than that in the model group (P  0.05). NK cell activity in treatment I, II, and III groups was significantly higher than that in the model group (P < 0.05). NK cell activity in treatment III group was significantly higher than that in treatment I and II groups (P < 0.05).@*CONCLUSIONS@#Tan IIA in combined with CTX can down regulate Bcl-2 expression in lung cancer tissues, up regulate Bax expression, inhibit the neovascularization of tumor tissues, and enhance the immunological function, with a significant anti-tumor activity.

Expression and significance of angiostatin, vascular endothelial growth factor and matrix metalloproteinase-9 in brain tissue of diabetic rats with ischemia reperfusion

Objective To discuss the expression and significance of angiostatin, vascular endothelial growth factor and matrix metalloproteinase-9 in the brain tissue of diabetic rats with ischemia reperfusion. Methods A total of 60 male Wistar rats were randomly divided into the normal group, sham group, diabetic cerebral infarction group and single cerebral infarction group according to the random number table, with 15 rats in each group. The high sucrose diet and intraperitoneal injection of streptozotocin were performed for the modeling of diabetic rats, while the thread-occlusion method was employed to build the model of cerebral ischemia reperfusion. The immunohistochemical staining was performed to detect the expression of angiostatin, vascular endothelial growth factor (VEGF) and matrix metalloproteinase-9 (MMP-9) in the brain tissue. Results The expression of angiostatin after the reperfusion in the brain tissue of rats in the single cerebral infarction group and diabetic cerebral infarction group was increased 6 h after the reperfusion, reached to the peak on 1 d and then decreased gradually. The expression of angiostatin in the diabetic cerebral infarction group 6 h, 1 d, 3 d and 7 d after the reperfusion was significantly higher than that in the single cerebral infarction group (P < 0.05). VEGF began to be increased 1 h after the reperfusion in the single cerebral infarction group and diabetic cerebral infarction group, reached to the peak at 6 h and then decreased gradually. The expression of VEGF in the diabetic cerebral infarction group at each time point after the reperfusion was significantly lower than that in the single cerebral infarction group (P < 0.05). MMP-9 began to be increased 1 h after the reperfusion in the single cerebral infarction group and diabetic cerebral infarction group, reached to the peak on 1 d and then decreased gradually. The expression of MMP-9 in the diabetic cerebral infarction group at each time point after the reperfusion was significantly higher than that in the single cerebral infarction group (P < 0.05). Conclusions The high glucose environment in which the diabetic cerebral infarction is occurred is to induce the formation of MMP-9 at first and then activate and increase the expression of angiostatin. Afterwards, the expression of VEGF is inhibited, resulting in the poor angiogenesis after cerebral infarction, which thus makes the injury of brain tissue after cerebral infarction even worse than the non-diabetes mellitus.

Anti-tumor activity of tanshinone IIA in combined with cyclophosphamide against Lewis mice with lung cancer

Objective To explore the anti-tumor activity of tanshinone IIA in combined with cyclophosphamide against Lewis mice with lung cancer and the effect on cellular immune function. Methods Lewis tumor cells were inoculated subcutaneously into the right armpit of mice in each group (n = 20) to establish Lewis lung cancer mice model. After model establishment, mice in the model group were given normal saline by lavage, qd. Mice in treatment I group were given intraperitoneal injection of Tan IIA, 15 mg/kg, qd. Mice in treatment II group were given intraperitoneal injection of CTX, 25 mg/kg, qd. Mice in treatment III group were given intraperitoneal injections of Tan IIA and CTX, in which the administration method of Tan IIA was the same as in treatment I group, continuously for 2 weeks, and the dosage of CTX was the same as in treatment II group, 24 h after model establishment, every other day. Mice were sacrificed 2 weeks after establishment. The tumor tissues were collected to calculate the anti-tumor rate. Immunohistochemistry was used to detect the expressions of Bcl-2, Bax, VEGF, Angiostatin, and Endostatin. FCM was used to detect T lymphocyte subsets in spleen and liver of mice. Results The tumor weight in treatment I, II, and III groups was significantly lower than that in the model group (P < 0.05). The tumor weight in treatment III group was significantly lower than that in treatment I and II groups (P < 0.05). The anti-tumor rate in treatment II and III groups was significantly higher than that in treatment I group (P < 0.05). Bcl-2 expression in the tumor tissues of treatment I, II, and III groups was significantly lower than that in the model group (P < 0.05), while Bax expression was significantly higher than that in the model group (P < 0.05). Bcl-2 expression in the tumor tissues of treatment I and II groups was significantly higher than that in treatment III group (P < 0.05), while Bax expression was significantly lower than that in treatment III group (P < 0.05). CD4

Effect of Cluster Needling of Scalp Acupuncture Combined with Rehabilitation on Neurological Function and Expression of Basic Fibroblast Growth Factor and Angiostatin in Rats after Focal Cerebral Ischemia / 中国康复理论与实践

@#Objective To study the effect of cluster needling of scalp acupuncture combined with rehabilitation on neurological function and expression of basic fibroblast growth factor (bFGF) and angiostatin (AS) in rats after focal cerebral ischemia. Methods 90 male Sprague-Dawley rats were randomly divided into sham operation group, model group, acupuncture treatment group, rehabilitation treatment group and acupuncture and rehabilitation treatment group, each group was further divided into 3 days, 7 days and 14 days subgroups (n=6). Permanent cerebral ischemia rat model was established according to Longa's method. The model group and the sham operation group accepted no treatment, the acupuncture treatment group was treated by cluster needling of scalp acupuncture, the rehabilitation treatment group received the treadmill training, the acupuncture and rehabilitation treatment group was treated by acupuncture and rehabilitation therapy. 3 days, 7 days, 14 days after operation, the neural function was evaluated with the modified Neurological Severity Score (mNSS), the expression levels of bFGF and AS proteins were detected by Western blotting. Results Compared with the model group, the acupuncture treatment group and the rehabilitation treatment group, mNSS decreased, the expression of bFGF protein increased, and the expression of AS protein decreased in the acupuncture and rehabilitation treatment group 3 days, 7 days, 14 days after operation (P<0.05). Conclusion Acupuncture and rehabilitation therapy can reduce the neurological function defect in rats with focal cerebral ischemia, which may be related to the upregulation of bFGF protein expression and down-regulation of AS protein expression.

Effect of Cluster Needling of Scalp Acupuncture Combined with Rehabilitation on Neurological Function and Ex-pression of Basic Fibroblast Growth Factor and Angiostatin in Rats after Focal Cerebral Ischemia / 中国康复理论与实践

Objective To study the effect of cluster needling of scalp acupuncture combined with rehabilitation on neurological function and expression of basic fibroblast growth factor (bFGF) and angiostatin (AS) in rats after focal cerebral ischemia. Methods 90 male Sprague-Dawley rats were randomly divided into sham operation group, model group, acupuncture treatment group, rehabilitation treatment group and acupuncture and rehabilitation treatment group, each group was further divided into 3 days, 7 days and 14 days subgroups (n=6). Permanent cerebral ischemia rat model was established according to Longa's method. The model group and the sham operation group accept-ed no treatment, the acupuncture treatment group was treated by cluster needling of scalp acupuncture, the rehabilitation treatment group re-ceived the treadmill training, the acupuncture and rehabilitation treatment group was treated by acupuncture and rehabilitation therapy. 3 days, 7 days, 14 days after operation, the neural function was evaluated with the modified Neurological Severity Score (mNSS), the expres-sion levels of bFGF and AS proteins were detected by Western blotting. Results Compared with the model group, the acupuncture treatment group and the rehabilitation treatment group, mNSS decreased, the expression of bFGF protein increased, and the expression of AS protein decreased in the acupuncture and rehabilitation treatment group 3 days, 7 days, 14 days after operation (P<0.05). Conclusion Acupuncture and rehabilitation therapy can reduce the neurological function defect in rats with focal cerebral ischemia, which may be related to the up-regulation of bFGF protein expression and down-regulation of AS protein expression.

Angiostatin Works as Immune Modulatory Molecules via Inhibition of Neutrophil Activation and Migration

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.

Efeito da proteína Kint3-4 do plasminogênio humano no crescimento do tumor de Ehrlich / Kint3-4 protein from human plasminogen delays Ehrlich tumor growth in mice

INTRODUCTION AND OBJECTIVE: Kint3-4 protein, originated from a genetic recombination of K1-3 and K1-4 human plasminogen segments, is recognized for its antiangiogenic and anti-inflammatory potential. This study aimed to evaluate the effect of Kint3-4 protein on tumor development in Swiss mice previously inoculated with Ehrlich tumor cells. METHODS: The protein fragment was obtained from Pichia pastoris cloning and transformation. After tumor cell inoculation three different protocols were used to assess tumor growth: beginning (0-6 days), peak (0-12 days) and after peak (0-18 days). We analyzed tumor growth, histomorphological characteristics and immunohistochemistry by use of CDC47 (cellular proliferation marker) and CD31 (blood vessel marker). RESULTS: Animals treated with Kint3-4 protein (150 µg/kg/48 h) showed lower tumor growth in all protocols. Based on histological assessment, inflammation and tumor areas were also reduced. Moreover, both the lowest rate of tumor cell proliferation and low microvessel density were observed in animals treated with Kint3-4 protein compared with the untreated control group. CONCLUSION: The effect of Kint3-4 recombinant protein on tumor angiogenesis and control of malignant cell proliferation enhances the prospects of its use in clinical and antiangiogenic treatment.

INTRODUÇÃO E OBJETIVO: A proteína Kint3-4 originou-se a partir de uma recombinação genética dos segmentos K1-3 e K1--4 do plasminogênio humano e é reconhecida por seu potencial anti-inflamatório e antiangiogênico. Este estudo teve como objetivo avaliar o efeito da proteína Kint3-4 no desenvolvimento de tumores em camundongos inoculados com células do tumor de Ehrlich. MÉTODOS: O fragmento de proteína foi obtido por uma técnica de clonagem e transformação de Pichia pastoris. Três diferentes protocolos foram avaliados após a inoculação das células tumorais: no início (0-6 dias), no pico (0-12 dias) e após o pico (0-18 dias) de crescimento do tumor. Foram analisados o crescimento do tumor e as características histomorfológica e imuno-histoquímica com CDC47 (marcador de proliferação celular) e CD31 (marcador de vasos sanguíneos). RESULTADOS: Os animais tratados com a proteína Kint3-4 (150 µg/kg/48 h) nos três diferentes protocolos apresentaram menor crescimento do tumor. Áreas de inflamação e tumor também foram reduzidas, avaliadas por exame histológico. Além disso, a menor taxa de proliferação das células tumorais e a baixa densidade de microvasos foram observadas nos animais tratados com proteína Kint3-4 em comparação com o grupocontrole. CONCLUSÃO: A participação da proteína recombinante Kint3-4 na angiogênese tumoral e no controle da proliferação de células malignas abre perspectivas para seu uso no tratamento clínico como antiangiogênico.

Mechanism of angiostatin induced reduction of vascular leakage in retina and iris of rats with retinopathy of prematurity / 国际眼科杂志(Guoji Yanke Zazhi)

·AIM: To study the effect of an intravitreal injection of angiostatin on vascular leakage in the retina and iris of oxygen-induced retinopathy of prematurity (ROP).·METHODS: Brown Norway rats at postnatal day 7 (P7) were exposed to hyperoxia (750mL/L O2 )for 5 days (P7-12) and then returned to normoxia to induce retinopathy. Angiostatin was reconstituted in sterile Phosphate Buffered Saline(PBS) and diluted to desired different concentrations. Angiostatin solution was injected into the vitreous of the right eye of the ROP rats at P14 and the age-matched normal rats through pars plana using a glass capillary, and the left eye received the same volume of sterile PBS as the control. Vascular permeability was quantified at 1, 2 and 3 days after the injection by measuring albumin leakage from blood vessels into the retina and iris using the Evans blue method and normalized by total protein concentrations. The expression of vascular endothelial growth factor (VEGF) in retina was evaluated using the Western Blot analysis and immuno-histochemistry 24 hours following the injection.·RESULTS: ROP rats showed significant increases of vascular permeability in the retina and iris (P<0.01). Angio-statin reduces vascular permeability in a dose-dependent manner in the retina of ROP rats. The reduction showed a time course trend. [Angiostatin injection reduced retinal vascular permeability by approximately 1.5 and 2-fold at P15 (P<0.05) and P16 (P<0.01), respectively.] Angiostatin injection significantly reduced VEGF levels in the retina of ROP rats but did not affect retinal VEGF levels in normal rats.·CONCLUSION: Angiostatin significantly decreases pa-thological vascular permeability in the retina and iris of ROP rats but not in normal rats. Angiostatin down-regulates VEGF expression in retina of ROP rats. These results suggest that angiostatin may have a therapeutic potential in the treatment of ROP and other diseases with vascular leakage.

Construction of adenovirus vector with angiostatin K1-5 gene and suppression to proliferation and migration of human vascular endothelial cells / 基础医学与临床

Objective To construct adenovirus vector with agiostatinK1-5 gene and to investigate the function of suppression to proliferation and migration for vascular endothelial cells.Methods With the use of gene recombination and clone technology, we constructed the adenovirus vector with the gene of angiostatin K1-5. In vitro vascular endothelial eclls proliferation assay and migration activity were performed through direct infection,MTT and transwell chemotaxis assay. Results 50% TCID indicated that the condence of resultant viruses was 1.5?109PFU/mL. It was purified by CsCL banding,final yield were generally 1.1?1010 PFU/mL plaquing-forming units. Through indirect infect assay and MTT, we found angiostatin K1-5 inhibited human vascular endothelial cells proliferation. We utilized human vascular endothelial cells to study the effect angiostatin K1-5 on cell migration,the result showed that adenoviruse vector with angiostatin K1-5 significantly inhibited HUVEC migration.Conclusion We successfully constructed adenoviruse vector with angiostatin K1-5 and demonstrated its inhibitory effect on proliferation andmigration of HUVEC.

The effect of co-transfection of p53 and angiostatin gene in SG7901 / 中国医师进修杂志

Objective To investigate the co-transfection of p53 and angiostatin gene in the inhibition of SG7901. Methods Transfected the pVITRO2-hp53-hAS into SG7901 with lipofectamine.After transfection, RT-PCR were used to know whether the aimed gene had been transfected and expressed or not. Cell clones trial, MTT growth curve, cell cycle measuring were used to analyze the differences. Results The cells were suppressed by the two genes and inhibition of the combined genes is more powerful than single one. Conclusion The effection of combined genes pVITRO2-hp53-hAS on SG7901 is stronger than either single one. Combined-gene-therapy is a useful anti-carcinoma method.

Construction and Expression of Hepatocellular Carcinoma-Specific Expressing Eukaryotic Vector for Anti-Angiogenesis Therapy / 中国普外基础与临床杂志

Objective To construct a mammalian vector encoding angiostatin kringle 5 (K5) under the control of ?-fetoprotein (AFP) enhancer and albumin promoter, and to observe the expression of angiostatin by introducting angiostatin gene into hepatocellular carcinoma cells through gene transfection. Methods Angiostatin cDNA was amplified from normal human eukaryotic cells by using RT-PCR. Meanwhile, AFP enhancer and albumin promoter sequences were directed cloned and were inserted into vector pcDNA3.1. The recombinant vector of pcDNA3.1-AFAB-angiostatin K5-His was constructed, which contained the angiostatin K5 cDNA sequence that was under the control of the AFP enhancer and promoter. Angiostatin K5 cDNA was introduced into human AFP positive hepatocellular carcinoma cell lines with the transfected cultured cells that were mediated with Lipofectamine 2000. The expression of angiostatin K5 was analyzed by Western blot and the protein was dectected with anti-His antibody. Results The 500-base pair of angiostatin K5 was in accordance with the expected sequence and the recombinant vector of pcDNA3.1-AFAB-angiostatin K5-His was also confirmed as the anticipated sequence. The expression of angiostatin K5 in AFP positive hepatocellular carcinoma cells was detected both by SDS-PAGE and Western blot. Conclusion Efficient construction and expression of angiostatin K5 to AFP positive cells make it possible for antiangiogenesis therapy of human hepatocellular carcinomas, which may provide a promising approach.

Angiostatin down-regulates vascular endothelial growth factor expression in human tongue squamous cell carcinoma cells / 第三军医大学学报

Objective To evaluate the effects of angiostatin on vascular endothelial growth factor expression in human tongue squamous cell carcinoma cells.Methods Subcutaneous xenografts of human tongue squamous cell carcinoma(Tca8113)cell line in BALB/c nude mice were employed and eighteen mice were randomly divided into three groups(6 mice each):the PBS group injected with PBS,the AS group injected with 250 ng purified angiostatin,and the AS group injected with 30 ?g purified angiostatin.Reagents were injected intraperitoneally twice daily and tumor volume was calculated twice weekly for 21 d,then animals were sacrificed and each tumor specimen was divided into two parts after surgical resection.The first part was submitted to both immunocytochemistry for VEGF,VEGFR1,VEGFR2 and CD34,and TUNEL apoptosis assay.The second part was prepared for semi-quantitative RT-PCR analysis of VEGF,VEGFR1 and VEGFR2 mRNA abundance.Results There was an increase of apoptotic index(AI)(P

Target of Angiostatin / 中国药理学通报

Aim To investigate the specificity of angiostatin to vascular endothelial cells. Methods S-180 tumor imaging and autoradiography of angiogenesis on Matrigel model was developed with 99 Tcm-recombinant human angiostatin ( 99 Tcm-rhAS) as a tracer, and FCM on human microvascular endothelial cell-1 (HMEC-1) with FITC-rhAS or rhAS antibody. The binding protein of rhAS on HMEC-1 was isolated by af-finity chromatography, and the proteins was sequenced with MS. Results 99 Tcm-rhAS was concentrated on the tumor site in vivo, and the rhAS was specific to angiogenesis of tumor. There were some binding sites on the surface of HMEC-1. Three proteins which are able to bind rhAS were obtained by affinity chromatography, among which tubulin sequenced was an important target for tumor. Conclusion The angiostatin is specific to novel vascular endothelial cell, and its mechanism targeting tumor is complicated.

Cirurgião diante das novas terapias para o tratamento do câncer / The surgeon facing new therapies for cancer treatment

The cancer treatement has been subjected to substantial changes, mainly concerning clinical specialities. The advances on tumours study, especially related to genetics and molecular biology, greatly increased our understanding about many aspects of carcinogenesis, neoplasic growth and metastasizing process, untill now obscure. In this context, surgery seems to be attained its limits in trying to erradicate completely the disease, and although the great resections made, this aim has not been succeeded in many cases. New treatments are emerging each year and between the most promising we can highlight tumour angiogenesis chemical blocking agents, with highly promising experimental results. At the same time of the beginning of clinical researchs about these drugs, the authors present a review work, with the objective of presenting a general survey of the knowledge achieved about these recently discovered drugs in tumours control.

Inhibitory effect of mouse angiostatin cDNA gene transduction on human liver cancer cell / 中华消化杂志

Objective To study the effect of the mouse angiostatin cDNA gene transfecting into human liver cancer cell line HCC7721 on cell growth, cell cycle phase distribution, cell morphology in vitro and tumorigenesis in vivo , and the mechanisms. Methods The gene fragment of mouse angiostatin cDNA was directly cloned into an eukaryotic expression plasmid pcDNA3.1(+) of the promoter CMV between the multicloning sites HindⅢ and XbaⅠ and confirmed the correct recombinant plasmid pcDNA3.1(+) angio through enzymatic digestion and gene sequencing. Then it was transfected into human liver cancer cell line HCC7721 with liposome, pcDNA3.1(+) as vecter control and liposome as mock control. After 30 day selection by neomycin G418, angiostatin expression at the levels of mRNA and protein in vitro were tested by RNA dot blot and FACS respectively. Cell morphology under was observed light microscope, made growth curves were made, and cell cycle distribution checked in FACS. Animal model was set up to study tumorigenesis in vivo of those cells transplanted subcutaneously in the right hind legs of nude mice BALBc. Microvessel density (MVD) was analyzed and angiostatin expression in the tumor tissues by in situ immunohistochemistry. Results The recombinant eukaryotic expression plasmid pcDNA3.1(+) angio was constructed Auccessfully. After selection of G418 for about 4 weeks there were macroscopic cell clones in the experimental group and vector control group, but no survival cells in the mock control. In vitro angiostatin were expressed in the experimental group, but not in the vector control. There weren't significant changes in cell morphology, cell growth curves and cell cycle distribution between the experimental and the control group. However, the nude mice experiment showed that tumorigenic capability of the experimental cells had been reduced greatly. Immunochemistry study showed that there were much less microvessels and conversely stronger angiostatin positive staining in the tumor tissues than in the control group. Conclusions Mouse angiostatin has no direct inhibition on human liver cancer cell line HCC7721 in vitro , but it inhibits effectively the tumorigenesis of HCC7721 in vivo , which is probably due to its inhibition on tumor angiogenesis in a paracrine path way.

Effects of angiostatin on the activities of extra-cellular signal-regulated protein kinase in microvascular endothelial cells of rat′s retina / 中华眼底病杂志

Objective To observe the effects of angiostatin on the activity of extra-cellular signal-regulated protein kinase (ERK) of retinal microvascular endothelial cells of mice. Methods Angiostatin was separated and purified by l-lysine sepharose 4B from human plasma. The primary retinal microvascular endothelial cells were divided into 4 groups: the control group, vascular endothelial growth factor (VEGF) 10 ng/ml group, angiostatin 130 ?g/ml group, and VEGF (10 ng/ml) + angiostatin (130 ?g/ml) group. The expression of ERK-1 was assayed by Western-blotting method 1, 2, 5, 10, 15, and 30 minutes after the treatment of angiostatin. Results Compared with the control group, the expression of ERK-1 reduced 1 minute after treatment, reduced markedly after 10 minutes. After 30 minutes, no differences of the expression of ERK were seen between the control group and angiostatin group. The activation of ERK-1 of retinal microvascular endothelial cells occurred after stimulated by VEGF, and at the pitch at the peak after 5 minutes. The level of ERK in VEGF group increased 210% than that in the control ( P

Inhibition Effect of Angiostatin and Endostatin on Human Angiogenesis

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.

The Inhibition of Endostatin on Tumor Growth and Metastasis of Lung Adenocarcinoma LA795 in Mice / 中国肿瘤生物治疗杂志

Objective: To evaluate the inhibition of human endostatin on tumor growth and metastasis of lung adenocarcinoma LA795 in mice. Methods: Recombinant human endostatin was purified from pCX expressed endostatin clones. Plasminogen was purified from outdated human plasma by affinity chromatography, and human angiostatin was produced from human plasminogen digested by elastase and purified by affinity chromatography. LA795 cells were inoculated subcutaneously into the dorsa of T739 mice, and the mice were randomized into 3 groups. From the 1Oth day, the first group was given 20 mg/kg of recombinant human endostatin s.c. qd, the second was treated daily s. c. of 7.5 mg/kg of human angiostatin, and the third group received daily s.c. with equal volumes of PBS for 14 days. Volumes of the subcutaneous tumors, lung weights, the number of lung surface metastases and mice life span were observed. The results were analyzed by q-test. Results: The tumor volumes of both the 1 st and the 2nd groups increased slowly. From the 8th day after being treated, the tumor volumes were decreasing. However, in the 3rd group, the tumor volumes increased continuously. The lung weight and the number of lung surface metastases of the 1st and 2nd groups were less than that of the 3rd group. The average survival periods of the 1st and 2nd groups were longer than that of the 3rd group. Conclusion: Human endostatin and angiostatin have strong inhibitory effects both on growth of primary tumor and metastasis of lung adenocarcinoma LA795, and prolongs the survival period of the tumor-bearing mice.

Cloning, Expression and Activity of Recombinant Human Angiostatin / 中国肿瘤生物治疗杂志

Objective: To express recombinant human angiostatin for further application in clinic. Methods: The complete encoding eDNA of human angiostatin was isolated from human embryo liver with RT-PCR and expressed in secretory Pichia expression system. Recombinant human angiostatin was purified with heparin sepharose chromatography and its activity was determined in chick embryo chorioallantoic membrane (CAM) and wound healing assays. Results: Expressed in large quantity (yield=5 mg/L) and purified with heparin sepharose, recombinant angiostatin was showed to have a molecular weight of 43 kD in SDS-PAGE and potently inhibit angiogenesis and wound healing. Conclusion: Recombinant human angiostatin was expressed efficiently in a biologically active form.