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Gene Therapy Apr 2001Retinal neovascularization is central to the pathogenesis of proliferative diabetic retinopathy, the leading cause of blindness among the middle-aged population....
Retinal neovascularization is central to the pathogenesis of proliferative diabetic retinopathy, the leading cause of blindness among the middle-aged population. Angiostatin, a proteolytic fragment of plasminogen is one of the most promising inhibitors of angiogenesis currently in clinical trials. Here we show that recombinant angiostatin can inhibit retinal neovascularization in a mouse model of proliferative retinopathy. Because proliferative diabetic retinopathy is a recurrent disease, effective therapy will need to be sustained. Recombinant adeno-associated viruses permit long-term expression of transfected genes; however, they can only accommodate a small insert sequence. Thus, we engineered and tested a shortened recombinant angiostatin derivative containing a signal sequence to permit secretion. Recombinant protein was purified from the medium of transfected HEK293 cells and injected subcutaneously into treated animals. The retinal vasculature was analyzed in retinal flat mounts and using immunohistochemically stained sections. Both methods demonstrate that this short, secreted form of angiostatin is effective in reducing the development of blood vessels in a nontumor environment and has therapeutic potential for neovascular retinopathies such as diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion and, possibly, age-related macular degeneration.
Topics: Angiostatins; Animals; Diabetic Retinopathy; Disease Models, Animal; Mice; Mice, Inbred C57BL; Peptide Fragments; Plasminogen; Recombinant Proteins; Retinal Neovascularization; Transfection
PubMed: 11320411
DOI: 10.1038/sj.gt.3301423 -
The Journal of Biological Chemistry Nov 2003Bone is a very common metastatic site for breast cancer. In bone metastasis, there is a vicious circle wherein bone-residing metastatic cells stimulate...
Bone is a very common metastatic site for breast cancer. In bone metastasis, there is a vicious circle wherein bone-residing metastatic cells stimulate osteoclast-mediated bone resorption, and bone-derived growth factors released from resorbed bone promote tumor growth. The contribution of tumor angiogenesis in the growth of bone metastases is, however, unknown. By using an experimental model of bone metastasis caused by MDA-MB-231/B02 breast cancer cells that quite closely mimics the conditions likely to occur in naturally arising metastatic human breast cancers, we demonstrate here that when MDA-MB-231/B02 cells were engineered to produce at the bone metastatic site an angiogenesis inhibitor, angiostatin, there was a marked inhibition in the extent of skeletal lesions. Inhibition of skeletal lesions came with a pronounced reduction in tumor burden in bone. However, although angiostatin produced by MDA-MB-231/B02 cells was effective at inhibiting in vitro endothelial cell proliferation and in vivo angiogenesis in a Matrigel implant model, we have shown that it inhibited cancer-induced bone destruction through a direct inhibition of osteoclast activity and generation. Overall, these results indicate that, besides its well known anti-angiogenic activity, angiostatin must also be considered as a very effective inhibitor of bone resorption, broadening its potential clinical use in cancer therapy.
Topics: Angiostatins; Animals; Blotting, Western; Bone Neoplasms; Bone and Bones; Cell Division; Cell Line, Tumor; Collagen; Drug Combinations; Endothelium, Vascular; Female; Humans; Immunohistochemistry; Laminin; Luciferases; Mice; Mice, Inbred BALB C; Mice, Nude; Microcirculation; Neoplasm Metastasis; Neoplasm Transplantation; Neovascularization, Pathologic; Osteoclasts; Proteoglycans; Time Factors; Transfection; Vascular Endothelial Growth Factor A; beta-Galactosidase
PubMed: 12954626
DOI: 10.1074/jbc.M309024200 -
Cancer Research Jul 2003Angiostatin, a proteolytic cleavage product of plasminogen, acts via a selective, yet poorly understood mechanism to potently inhibit angiogenesis (M. S. O'Reilly et...
Angiostatin, a proteolytic cleavage product of plasminogen, acts via a selective, yet poorly understood mechanism to potently inhibit angiogenesis (M. S. O'Reilly et al., Cell, 79: 315-328, 1994). Vascular endothelial cell proliferation assays revealed that angiostatin(4.5), a naturally occurring human isoform consisting of plasminogen kringle domains 1-4 and most of kringle domain 5 (G. A. Soff, Cancer Metastasis Rev., 19: 97-107, 2000), dose dependently reduces cell number despite the presence of a potent stimulus of proliferation. Flow cytometry using the vital dyes Hoechst 33342 and Pyronin Y revealed that approximately 40% of both control and angiostatin(4.5)-treated cells were in the proliferative phase, indicating that cell cycle progression is not impaired by exposure to angiostatin(4.5). Both bovine aortic endothelial cells and human umbilical endothelial cells were shown to undergo apoptosis in response to angiostatin(4.5). Caspases-3, -8, and -9 activation, specified by cleavage of fluorophore-conjugated specific peptide substrates, revealed a cascade of caspase activation that peaks at 36 h of angiostatin(4.5) treatment. Angiostatin(4.5) exposure induced release of cytochrome c from mitochondria in a caspase-dependent manner, but a pan-caspase inhibitor, zVAD-fmk, blocked cytochrome c release. Overall, these data indicate that human angiostatin(4.5) may function in vivo to block blood vessel formation by specifically inducing vascular endothelial cells to apoptose in a process likely involving both the intrinsic and extrinsic apoptosis pathways.
Topics: Angiostatins; Animals; Annexin A5; Apoptosis; Caspase 3; Caspase 9; Caspases; Cattle; Cell Cycle; Cells, Cultured; Cytochrome c Group; Endothelium, Vascular; Enzyme Activation; Peptide Fragments; Plasminogen; S Phase; Signal Transduction
PubMed: 12874037
DOI: No ID Found -
The Journal of Laboratory and Clinical... May 2002Angiostatin (AST), a polypeptide with potent antiangiogenic properties, is released proteolytically from plasminogen in vivo. Plasminogen exists naturally in plasma as...
Angiostatin (AST), a polypeptide with potent antiangiogenic properties, is released proteolytically from plasminogen in vivo. Plasminogen exists naturally in plasma as two glycoforms (PLGs), I and II. Recently it was shown with the use of a chick-embryo chorioallantoic membrane (CAM) assay that rabbit PLG-I and -II yield distinct ASTs-AST-I and -II, respectively-with different antiangiogenic activities. AST glycoforms were of similar molecular weight, approximately 30 to 32,000 kD, and probably consisted of kringles 1 to 3 only. AST has now been identified in the interpleural effusate released from VX-2 lung tumors in rabbits. Effusate was collected from six rabbits with high tumor burdens and fractionated by means of lysine-Sepharose chromatography. The epsilon-aminohexanoic acid-eluted protein of all effusates contained AST (kringles 1-3) at a mean concentration of 1.2 microg/mL of effusate; with regard to AST content, 97% was AST-II. A CAM assay revealed that the lysine-Sepharose-bound fraction from all interpleural effusates contained potent antiangiogenic activity. Blood and urine from rabbits with high burdens of VX-2 contained essentially only AST-II, at mean concentrations of 145 and 4 ng/mL, respectively. AST was absent from the blood of control rabbits. In an attempt to compare their uptake by VX-2, iodine 125-labeled AST-I and iodine 131-labeled AST-II were injected intravenously into tumor-bearing rabbits. AST-I entered the tumor 1.6 times faster than AST-II. As a means of accounting for the preponderance of AST-II in the interpleural effusate, we postulate that VX-2 cells release proteolytic activity to activate plasminogen but that of the two PLGs, PLG-II may be the preferred substrate for AST formation in vivo.
Topics: Angiostatins; Animals; Blotting, Western; Chickens; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Humans; Iodine Radioisotopes; Lung Neoplasms; Molecular Weight; Neoplasm Transplantation; Peptide Fragments; Plasminogen; Pleural Effusion; Protein Isoforms; Rabbits; Tumor Cells, Cultured
PubMed: 12032493
DOI: 10.1067/mlc.2002.123267 -
The Journal of Biological Chemistry Nov 1998Angiostatin is a potent inhibitor of tumor angiogenesis and the growth of metastatic foci. Recent studies have indicated that neoplastic cells can generate angiostatin...
Angiostatin is a potent inhibitor of tumor angiogenesis and the growth of metastatic foci. Recent studies have indicated that neoplastic cells can generate angiostatin directly or in cooperation with tumor-associated macrophages. In studies reported here, we determined whether angiostatin is generated in mice under non-neoplastic settings. Utilizing murine RAW264.7 macrophages and thioglycollate-elicited peritoneal macrophages, we demonstrate that angiostatin-like fragments are generated as a byproduct of the proteolytic regulation of membrane-bound plasmin. Plasmin proteolysis and subsequent loss in membrane-bound plasmin activity requires active plasmin but was unaffected by inhibitors of metalloproteinases. Lysine binding fragments of plasmin, isolated from macrophage-conditioned media utilizing affinity chromatography, appeared as a major (48 kDa) and two minor bands (42 and 50 kDa) in SDS-polyacrylamide gel electrophoresis and were immunoreactive with anti-kringle 1-3 IgG. Each peptide begins with Lys77 and contains the entire sequence of angiostatin. The affinity isolated plasmin fragments inhibited bFGF-induced endothelial cell proliferation. Lavage fluid recovered from the peritoneal cavities of mice previously injected with thioglycollate contained angiostatin-like plasmin fragments similar to those generated in vitro. This is the first demonstration that angiostatin-like plasmin fragments are generated in a non-neoplastic inflammatory setting. Thus, in addition to regulating pericellular plasmin activity, proteolysis of plasmin generates inactive kringle-containing fragments expressing angiostatic properties.
Topics: Angiostatins; Animals; Cell Division; Cells, Cultured; Endothelium, Vascular; Enzyme Activation; Exudates and Transudates; Fibrinolysin; Inflammation; Kringles; Macrophages; Macrophages, Peritoneal; Membrane Proteins; Metalloendopeptidases; Mice; Peptide Fragments; Plasminogen; Thioglycolates
PubMed: 9813061
DOI: 10.1074/jbc.273.47.31480 -
The Journal of Biological Chemistry Nov 1997Angiostatin is one of the most potent inhibitors of angiogenesis. Reports have shown that metalloelastase, pancreas elastase, plasmin reductase, and plasmin convert...
Angiostatin is one of the most potent inhibitors of angiogenesis. Reports have shown that metalloelastase, pancreas elastase, plasmin reductase, and plasmin convert plasminogen to angiostatin. However, the cleavage sites of plasminogen by those enzymes have not been determined. Here we demonstrate that two members of the human matrix metalloproteinase (MMP) family, matrilysin (MMP-7) and gelatinase B/type IV collagenase (MMP-9), hydrolyze human plasminogen to generate angiostatin fragments. The cleavage sites have been determined. The 58-kDa bands derived from plasminogen by MMP-7 and MMP-9 both have the N-terminal sequence KVYLSEXKTG, which corresponds to that of angiostatin. This N terminus is identical to that of the starting plasminogen itself and corresponds to residues 97-106 of prepro-plasminogen. The 42- and 38-kDa bands generated by MMP-7 both have the N-terminal sequence VVLLPNVETP, which corresponds to the amino acid sequence 467-476 of prepro-plasminogen, between kringle domain 4 and 5. MMP-9 cleaves plasminogen to generate a 42-kDa fragment with the N-terminal sequence PVVLLPNVE, 1 residue upstream of the MMP-7 cleavage site. These results indicate that MMP-7 and MMP-9 may regulate new blood vessel formation by cleaving plasminogen and generating angiostatin molecules.
Topics: Amino Acid Sequence; Angiostatins; Collagenases; Humans; Hydrolysis; Kringles; Matrix Metalloproteinase 7; Matrix Metalloproteinase 9; Metalloendopeptidases; Molecular Sequence Data; Peptide Fragments; Plasminogen
PubMed: 9360944
DOI: 10.1074/jbc.272.46.28823 -
Microcirculation (New York, N.Y. : 1994) Feb 2007Ischemic angiogenesis is dependent on vascular nitric oxide (NO) bioavailability, in part by reducing matrix metalloproteinases (MMP)-2 and -9 activity and angiostatin...
OBJECTIVE
Ischemic angiogenesis is dependent on vascular nitric oxide (NO) bioavailability, in part by reducing matrix metalloproteinases (MMP)-2 and -9 activity and angiostatin production. In the metabolic syndrome, the authors have demonstrated that reduced skeletal muscle microvessel density (SKMVD) was correlated with chronic reductions in NO bioavailability, but these relationships are complicated by the presence of the multiple pathological states inherent in the metabolic syndrome. Given this, the authors hypothesized that low NO bioavailability could reduce SKMVD in normal rats, independent of any systemic pathologies associated with the metabolic syndrome, and that this would be correlated with increased angiostatin production.
METHODS
Rats were separated into 3 groups: (1) control, (2) chronic NOS inhibition (10-4 M L-NAME; drinking water), and (3) NOS inhibition/normotensive (combined L-NAME/hydralazine treatment; 50 mg/kg/d; drinking water). Vessel structure, reactivity, and NO bioavailability were assessed in isolated vessels using standard techniques. SKMVD was determined using established immunohistochemical methods. Established protein analyses were performed for MMP-2 and MMP-9 expression/activity and for angiostatin expression. Alterations in vascular endothelial growth factor (VEGF) levels under the conditions of the study were assessed using ELISA.
RESULTS
After 6 weeks, MAP was elevated in L-NAME rats vs. control, although this difference was abolished in L-NAME/hydralazine rats. NOS inhibition abrogated dilation to acetylcholine and arterial eNOS activity. While NOS inhibition reduced SKMVD vs. control, hydralazine treatment did not improve density, suggesting that rarefaction in NOS-inhibited rats was independent of elevated pressure. Skeletal muscle demonstrated reduced active hyperemia and increased minimum vascular resistance in L-NAME rats, which was also associated with increased arteriolar wall stiffness. L-NAME/hydralazine treatment, while still causing an elevated resistance, prevented arteriolar wall stiffening. Protein analysis demonstrated that neither expressions nor activities of MMP-2 or MMP-9 were altered from control in skeletal muscle of rats treated with L-NAME and angiostatin production was not altered in any group. Chronic NOS inhibition was associated with no consistent change in plasma VEGF levels.
CONCLUSIONS
These results suggest that a reduced SKMVD develops with low NO bioavailability, although this process was not associated with significant alterations to either VEGF or angiostatin production.
Topics: Angiostatins; Animals; Arterioles; Enzyme Inhibitors; Hydralazine; Male; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Muscle, Skeletal; NG-Nitroarginine Methyl Ester; Nitric Oxide; Rats; Rats, Zucker; Regional Blood Flow; Vascular Endothelial Growth Factor A; Vascular Resistance; Vasodilator Agents
PubMed: 17365669
DOI: 10.1080/10739680601131242 -
Proceedings of the National Academy of... May 1998Angiostatin, a fragment of plasminogen, has been identified and characterized as an endogenous inhibitor of neovascularization. We show that angiostatin treatment of...
Angiostatin, a fragment of plasminogen, has been identified and characterized as an endogenous inhibitor of neovascularization. We show that angiostatin treatment of endothelial cells in the absence of growth factors results in an increased apoptotic index whereas the proliferation index is unchanged. Angiostatin also inhibits migration and tube formation of endothelial cells. Angiostatin treatment has no effect on growth factor-induced signal transduction but leads to an RGD-independent induction of the kinase activity of focal adhesion kinase, suggesting that the biological effects of angiostatin relate to subversion of adhesion plaque formation in endothelial cells.
Topics: Angiostatins; Antineoplastic Agents; Apoptosis; Cell Adhesion Molecules; Cells, Cultured; Endothelium, Vascular; Enzyme Activation; Focal Adhesion Kinase 1; Focal Adhesion Protein-Tyrosine Kinases; Humans; Oligopeptides; Peptide Fragments; Phosphorylation; Plasminogen; Protein-Tyrosine Kinases; Receptor, Insulin; Signal Transduction; Tyrosine
PubMed: 9576925
DOI: 10.1073/pnas.95.10.5579 -
Molecular Vision Dec 2004To investigate the effect of different doses of adjuvant angiostatin affecting hepatic micrometastasis in a murine model of metastatic ocular melanoma.
PURPOSE
To investigate the effect of different doses of adjuvant angiostatin affecting hepatic micrometastasis in a murine model of metastatic ocular melanoma.
METHODS
Angiostatin and plasminogen expression was detected in three murine melanoma cell lines (Queens, B16F10, and B16LS9). The three cell lines were heterotopically inoculated into the posterior compartment (PC) of the right eyes of C57BL/6 mice. After enucleation, the mice were given injections of 100 microl PBS and low dose (0.1 microg/microl) or high dose (0.3 microg/microl) murine recombinant angiostatin every day for 14 days after enucleation. The mice were sacrificed at 21 days post-enucleation and hepatic micrometastases were counted. In vitro migration/invasion assays were performed with low (0.1 microg/microl) and high (50 microg/microl) concentration angiostatin supplementation. Quantitative RT-PCR detected mRNA and Western analysis determined protein expression of VEGF for all cell lines. Evaluation of TdT mediated dUTP nick end labeling (TUNEL) and MIB1 immunostaining of the micrometastases determined apoptosis and proliferation ratios.
RESULTS
There was a decrease in micrometastasis in the low dose group for Queens (p<0.05), B16F10 (p<0.05), and B16LS9 melanoma (p<0.01) cell lines. Two of the cell lines (B16F10 and B16LS9) elaborated plasminogen and were able to cleave plasminogen into K1-K4 (angiostatin). There was a decrease in the in vitro migration and invasion after supplementation with low concentration compared to high concentration angiostatin (p<0.01). VEGF mRNA and protein expression decreased in all cells lines in low concentration angiostatin, with the greatest decrease in B16LS9 cells (p<0.05). Apoptosis ratios were increased (p<0.01) and proliferation ratios were decreased (p<0.01) in hepatic micrometastases after treatment with low dose angiostatin.
CONCLUSIONS
There were significantly fewer micrometastases in treated compared to controls with low dose compared to high dose angiostatin. This treatment results in apoptosis in the micrometastases. The mechanism appears to be related an anti-migratory effect and altered VEGF expression by melanoma cells.
Topics: Angiogenesis Inhibitors; Angiostatins; Animals; Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Proliferation; Disease Models, Animal; Eye Enucleation; Eye Neoplasms; Female; In Situ Nick-End Labeling; Liver Neoplasms; Melanoma, Experimental; Mice; Mice, Inbred C57BL; Neoplasm Invasiveness; Plasminogen; RNA, Messenger; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Vascular Endothelial Growth Factor A
PubMed: 15623988
DOI: No ID Found -
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