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British Journal of Cancer Dec 1999Angiostatin, a potent inhibitor of angiogenesis, tumour growth and metastasis, is a biologically active fragment of plasminogen, containing the kringle domains 1-4. It...
Angiostatin, a potent inhibitor of angiogenesis, tumour growth and metastasis, is a biologically active fragment of plasminogen, containing the kringle domains 1-4. It is generated from plasminogen by limited proteolysis. We show that prostate-specific antigen (PSA), a serine proteinase secreted by human prostate and human prostate cancer cells, is able to convert Lys-plasminogen to biologically active angiostatin-like fragments, containing kringles 1-4, by limited proteolysis of peptide bond Glu439-Ala440 in vitro. In an in vitro morphogenesis assay, the purified angiostatin-like fragments inhibited proliferation and tubular formation of human umbilical vein endothelial cells with the same efficacy as angiostatin. This finding might help to understand growth characteristics of prostate cancer, which usually has low microvessel density and slow proliferation.
Topics: Amino Acid Sequence; Angiostatins; Cells, Cultured; Humans; Male; Peptide Fragments; Plasminogen; Prostate-Specific Antigen
PubMed: 10604721
DOI: 10.1038/sj.bjc.6692167 -
Human Gene Therapy Jan 2017Neovascular age-related macular degeneration (NVAMD) is a prevalent cause of vision loss. Intraocular injections of VEGF-neutralizing proteins provide benefit, but many...
Neovascular age-related macular degeneration (NVAMD) is a prevalent cause of vision loss. Intraocular injections of VEGF-neutralizing proteins provide benefit, but many patients require frequent injections for a prolonged period. Benefits are often lost over time due to lapses in treatment. New treatments that sustain anti-angiogenic activity are needed. This study tested the safety and expression profile of a lentiviral Equine Infectious Anemia Virus (EIAV) vector expressing endostatin and angiostatin (RetinoStat). Patients with advanced NVAMD were enrolled at three centers in the United States, and the study eye received a subretinal injection of 2.4 × 10 (n = 3), 2.4 × 10 (n = 3), or 8.0 × 10 transduction units (TU; n = 15). Each of the doses was well-tolerated with no dose-limiting toxicities. There was little or no ocular inflammation. There was one procedure-related serious adverse event (AE), a macular hole, which was managed without difficulty and resolved. There was a vector dose-related increase in aqueous humor levels of endostatin and angiostatin with high reproducibility among subjects within cohorts. Mean levels of endostatin and angiostatin peaked between 12 and 24 weeks after injection of 2.4 × 10 TU or 8.0 × 10 TU at 57-81 ng/mL for endostatin and 15-27 ng/mL for angiostatin, and remained stable through the last measurement at week 48. Long-term follow-up demonstrated expression was maintained at last measurement (2.5 years in eight subjects and >4 years in two subjects). Despite an apparent reduction in fluorescein angiographic leakage that broadly correlated with the expression levels in the majority of patients, only one subject showed convincing evidence of anti-permeability activity in these late-stage patients. There was no significant change in mean lesion size in subjects injected with 8.0 × 10 TU. These data demonstrate that EIAV vectors provide a safe platform with robust and sustained transgene expression for ocular gene therapy.
Topics: Aged; Aged, 80 and over; Angiostatins; Cohort Studies; Endostatins; Female; Genetic Therapy; Genetic Vectors; Humans; Injections, Intraocular; Lentivirus; Macular Degeneration; Male; Maximum Tolerated Dose
PubMed: 27710144
DOI: 10.1089/hum.2016.117 -
Biotechnology Letters Jan 2003A recombinant strain of Pichia pastoris with a phenotype of MutS was used to produce angiostatin. Due to the low methanol consumption rate of this strain, both methanol...
A recombinant strain of Pichia pastoris with a phenotype of MutS was used to produce angiostatin. Due to the low methanol consumption rate of this strain, both methanol and glycerol feedings, that produced oscillation in dissolved O2 concentration, were used during the expression phase to improve cell growth and angiostatin expression. However, enhanced cell growth led to nitrogen limitation that suppressed further production of angiostatin, but addition of ammonia allowed angiostatin concentration to reach 108 mg l(-1) after an expression period of 96 h. The ratio of consumed glycerol to methanol of 1.5:1 (w/w) in the expression phase suggested that methanol played an important role in the metabolism of carbon sources.
Topics: Ammonia; Angiostatins; Carbon; Cloning, Molecular; Glycerol; Humans; Methanol; Oxygen; Peptide Fragments; Pichia; Plasminogen; Quality Control; Recombinant Proteins; Species Specificity
PubMed: 12882295
DOI: 10.1023/a:1021905010021 -
Molecular Therapy : the Journal of the... Jan 2004Angiostatin is a potent endogenous inhibitor of angiogenesis and tumor growth in vivo. The therapeutic potential of adeno-associated viral (AAV) gene delivery of...
Angiostatin is a potent endogenous inhibitor of angiogenesis and tumor growth in vivo. The therapeutic potential of adeno-associated viral (AAV) gene delivery of angiostatin in modulating tumor growth in vivo was evaluated. Sustained levels of angiostatin were detected in the sera of mice for up to 6 months after they received a single injection of AAV-angiostatin. AAV-mediated stable expression of angiostatin inhibited tumor burden in the highly aggressive B16F10 melanoma and Lewis lung carcinoma (LLC) models of experimental metastasis. Moreover, AAV-angiostatin prolonged survival in B16F10 and LLC tumor-bearing mice compared to control groups. Anti-tumor efficacy was consistently observed when angiostatin serum levels of 15-50 ng/ml were detected following gene transfer, but the effect was minimal when the levels were lower or higher than this range. The combination of AAV-angiostatin gene therapy with chemotherapy was also shown to extend marginally the survival of mice bearing preestablished human tumors; however, the effect was evident only within a narrow dose of circulating angiostatin. These studies demonstrate the feasibility of using AAV anti-angiogenic gene therapy as a cancer treatment modality and suggest that the optimal anti-tumor efficacy of angiostatin following gene transfer may be limited to a narrow dose range.
Topics: Angiogenesis Inhibitors; Angiostatins; Animals; Carcinoma, Lewis Lung; Cell Line; Chick Embryo; Combined Modality Therapy; Dependovirus; Female; Gene Expression; Genetic Therapy; Genetic Vectors; Humans; Liver; Melanoma, Experimental; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasms, Experimental; Neovascularization, Pathologic; Transduction, Genetic
PubMed: 14741778
DOI: 10.1016/j.ymthe.2003.10.001 -
Cancer Treatment and Research 2004Angiogenesis and the development of metastases are intrinsically connected. Experimental data suggest that establishment and growth of metastases are influenced by... (Review)
Review
Angiogenesis and the development of metastases are intrinsically connected. Experimental data suggest that establishment and growth of metastases are influenced by soluble factors secreted from the originating solid tumor. Among these factors are so-called endogenous inhibitors of angiogenesis which keep metastasis in a non-proliferating quiescent state. For a number of tumors it has been shown that this dormant state is mediated through inhibition of angiogenesis. This dormant state is characterized by normal proliferation, increased apoptosis, and insufficient neovascularization. Removal of inhibiting antiangiogenic factors leads to growth of dormant metastases. Several endogenous inhibitors have been identified so far and some of them have already been successfully applied in experimental therapeutic trials. This might be of special interest for the treatment of cerebral metastases which are the most common type of malignant brain tumors. Similar to the spread of metastases, it is known that single glioma cells can be found in distant parts of the brain. While local recurrence is a common phenomenon in glioma, formation of clinical apparent distant metastasis occurs rarely. Several lines of evidence suggest that growth inhibition of remote glioma cells may be mediated by an endogenous inhibitory mechanism.
Topics: Angiogenesis Inhibitors; Angiostatins; Animals; Brain; Brain Neoplasms; Cell Division; Endostatins; Humans; Neoplasm Metastasis; Neovascularization, Pathologic
PubMed: 15015566
DOI: 10.1007/978-1-4419-8871-3_17 -
Ophthalmic Research 2005Clinically, preretinal neovascularization (PNV) induced by vessel occlusion is one of the leading causes to induce blindness. The present study was designed to determine...
Clinically, preretinal neovascularization (PNV) induced by vessel occlusion is one of the leading causes to induce blindness. The present study was designed to determine if a recombinant adeno-associated viral vector expressing mouse angiostatin (rAAV-angiostatin) can inhibit experimental PNV in an adult Sprague-Dawley rat model. rAAV-angiostatin and rAAV-lacZ were delivered by intravitreal injections to the right and left eyes of rats. Transgenetic expression of angiostatin in the retina was determined by reverse-transcriptase polymerase chain reaction (RT-PCR). PNV was established by rose-bengal-assisted laser-induced retinal vein occlusion 21 days after the viral injections. The total number and sizes of the neovascular tufts were analyzed 14 days after venous occlusion using retinal flat mount by fluorescein-isothiocyanate-dextran angiography. Electroretinograms (ERGs) were recorded to study any possibility of retinal toxicity of rAAV-angiostatin 3 months after the injections. Angiostatin gene expression in the retina was detectable by RT-PCR, and ERG analysis showed no reduction of b-waves in the rAAV-angiostatin-injected eyes. The number and size of neovascular tufts were significantly lower in rAAV-angiostatin-injected eyes (p = 0.001) than controls. These findings indicated that rAAV-angiostatin successfully suppressed experimental PNV, and no retinal toxicity of the rAAV-angiostatin injection was observed according to ERG recordings.
Topics: Angiostatins; Animals; Defective Viruses; Dependovirus; Disease Models, Animal; Electroretinography; Fluorescein Angiography; Gene Expression; Genetic Therapy; Genetic Vectors; RNA, Messenger; Rats; Rats, Sprague-Dawley; Retinal Neovascularization; Retinal Vein Occlusion; Reverse Transcriptase Polymerase Chain Reaction; Transgenes
PubMed: 15637422
DOI: 10.1159/000083040 -
The Journal of Biological Chemistry Dec 2000Angiostatin, a potent endogenous inhibitor of angiogenesis, is generated by cancer-mediated proteolysis of plasminogen. The culture medium of human prostate carcinoma...
Angiostatin, a potent endogenous inhibitor of angiogenesis, is generated by cancer-mediated proteolysis of plasminogen. The culture medium of human prostate carcinoma cells, when incubated with plasminogen at a variety of pH values, generated angiostatic peptides and miniplasminogen. The enzyme(s) responsible for this reaction was purified and identified as procathepsin D. The purified procathepsin D, as well as cathepsin D, generated two angiostatic peptides having the same NH(2)-terminal amino acid sequences and comprising kringles 1-4 of plasminogen in the pH range of 3.0-6.8, most strongly at pH 4.0 in vitro. This reaction required the concomitant conversion of procathepsin D to catalytically active pseudocathepsin D. The conversion of pseudocathepsin D to the mature cathepsin D was not observed by the prolonged incubation. The affinity-purified angiostatic peptides inhibited angiogenesis both in vitro and in vivo. Importantly, procathepsin D secreted by human breast carcinoma cells showed a significantly lower angiostatin-generating activity than that by human prostate carcinoma cells. Since deglycosylated procathepsin D from both prostate and breast carcinoma cells exhibited a similar low angiostatin-generating activity, this discrepancy appeared to be attributed to the difference in carbohydrate structures of procathepsin D molecules between the two cell types. The seminal vesicle fluid from patients with prostate carcinoma contained the mature cathepsin D and procathepsin D, but not pseudocathepsin D, suggesting that pseudocathepsin D is not a normal intermediate of procathepsin D processing in vivo. The present study provides evidence for the first time that cathepsin D secreted by human prostate carcinoma cells is responsible for angiostatin generation, thereby causing the prevention of tumor growth and angiogenesis-dependent growth of metastases.
Topics: Angiostatins; Antineoplastic Agents; Breast Neoplasms; Cathepsin D; Female; Humans; Hydrogen-Ion Concentration; Kinetics; Male; Models, Molecular; Peptide Fragments; Plasminogen; Prostatic Neoplasms; Protease Inhibitors; Protein Structure, Secondary; Serine Endopeptidases; Tumor Cells, Cultured
PubMed: 10986284
DOI: 10.1074/jbc.M005402200 -
The Journal of Experimental Medicine Aug 1998We determined whether tumor cells consistently generating granulocyte/macrophage colony- stimulating factor (GM-CSF) can recruit and activate macrophages to generate...
We determined whether tumor cells consistently generating granulocyte/macrophage colony- stimulating factor (GM-CSF) can recruit and activate macrophages to generate angiostatin and, hence, inhibit the growth of distant metastasis. Two murine melanoma lines, B16-F10 (syngeneic to C57BL/6 mice) and K-1735 (syngeneic to C3H/HeN mice), were engineered to produce GM-CSF. High GM-CSF (>1 ng/10(6) cells)- and low GM-CSF (<10 pg/10(6) cells)-producing clones were identified. Parental, low, and high GM-CSF-producing cells were injected subcutaneously into syngeneic and into nude mice. Parental and low-producing cells produced rapidly growing tumors, whereas the high-producing cells produced slow-growing tumors. Macrophage density inversely correlated with tumorigenicity and directly correlated with steady state levels of macrophage metalloelastase (MME) mRNA. B16 and K-1735 subcutaneous (s.c.) tumors producing high levels of GM-CSF significantly suppressed lung metastasis of 3LL, UV-2237 fibrosarcoma, K-1735 M2, and B16-F10 cells, but parental or low-producing tumors did not. The level of angiostatin in the serum directly correlated with the production of GM-CSF by the s.c. tumors. Macrophages incubated with medium conditioned by GM-CSF- producing B16 or K-1735 cells had higher MME activity and generated fourfold more angiostatin than control counterparts. These data provide direct evidence that GM-CSF released from a primary tumor can upregulate angiostatin production and suppress growth of metastases.
Topics: Angiostatins; Animals; Antineoplastic Agents; Cells, Cultured; Granulocyte-Macrophage Colony-Stimulating Factor; Injections, Subcutaneous; Lung Neoplasms; Macrophages; Male; Melanoma; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Nude; Peptide Fragments; Plasminogen; Tumor Cells, Cultured
PubMed: 9705957
DOI: 10.1084/jem.188.4.755 -
Gene Therapy Feb 2003Ischemic retinal diseases, such as diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration, are a major cause of blindness worldwide....
Ischemic retinal diseases, such as diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration, are a major cause of blindness worldwide. Angiostatin is an internal peptide fragment of plasminogen that inhibits endothelial proliferation in vitro and tumor growth in vivo. We now demonstrate that HIV vector encoding angiostatin (HIV-angiostatin) can inhibit retinal neovascularization in a mouse model of proliferative retinopathy. Intravitreal injections of HIV-angiostatin led to stable expression of the angiostatin gene in retinal tissue. Retinal neovascularization was histologically quantitated by a masked protocol. Retinal neovascularization in the eye injected with HIV-angiostatin was reduced in 90% (9/10; P=0.025) of animals, compared with the eye injected with phosphate-buffered saline. Reduction of histologically evident neovascular nuclei per 6-microm section averaged 68%, with maximal inhibitory effects of 87%. Neovascularization was not reduced in the eyes injected with HIV vector encoding enhanced green fluorescent protein. This is the first report that HIV-angiostatin can reduce neovascular cell nuclei in a murine proliferative retinopathy model. These data suggest that the anti-angiogenic activity of angiostatin has therapeutic potential for the treatment of retinal neovascularization.
Topics: Angiostatins; Animals; Arthritis, Rheumatoid; Diabetic Retinopathy; Female; Gene Expression; Genetic Therapy; Genetic Vectors; HIV-1; Humans; Mice; Mice, Inbred C57BL; Models, Animal; Peptide Fragments; Plasminogen; Retina; Retinal Neovascularization; Transduction, Genetic
PubMed: 12571629
DOI: 10.1038/sj.gt.3301878 -
International Journal of Colorectal... Jul 2004Liver fibrosis is a response to chronic hepatic damage, which ultimately leads to liver failure and necessitates liver transplantation. A characteristic of fibrosis is...
BACKGROUND AND AIMS
Liver fibrosis is a response to chronic hepatic damage, which ultimately leads to liver failure and necessitates liver transplantation. A characteristic of fibrosis is pathological vessel growth. This type of angiogenesis may contribute to the disturbance of hepatocyte perfusion dynamics and lead to aggravation of disease. We hypothesized that angiostatin can inhibit pathological vessel growth and, consequently, the development of hepatic fibrosis.
METHODS
Hepatic fibrosis was induced by injection of carbon tetrachloride for 5 weeks. Angiostatin mice received carbon tetrachloride for 5 weeks and angiostatin during weeks 4 and 5. After 5 weeks, immunohistochemistry for endothelial cell marker von Willebrand factor and for cell proliferation was performed. Angiogenesis was quantified by counting the number of immunopositive microvessels. Also, the relative fibrotic surface was determined using Sirius Red histostaining and computer image analysis.
RESULTS
Immunohistochemistry revealed increased expression for von Willebrand factor in fibrotic livers. Immunopositive microvessels were localized in fibrotic areas surrounding larger vessels and in emerging fibrotic septa. Angiostatin reduced the number of immunopositive microvessels by 69% (p<0.001). In addition, angiostatin reduced the relative fibrotic area in the liver by 63+/-0.1% (p<0.001). Finally, angiostatin treatment was not associated with differences in cell proliferation.
CONCLUSIONS
Angiostatin inhibits the development of pathological angiogenesis and liver fibrosis in mice. These results warrant further evaluation of angiostatin as an antifibrotic agent, potentially contributing to the deferment of liver transplantation and reduced recurrence of fibrotic disease in the transplanted liver.
Topics: Angiogenesis Inhibitors; Angiostatins; Animals; Cell Differentiation; Female; Liver; Liver Cirrhosis; Mice; Mice, Inbred BALB C; Neovascularization, Pathologic; von Willebrand Factor
PubMed: 14716496
DOI: 10.1007/s00384-003-0562-4