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Cell Oct 1994The phenomenon of inhibition of tumor growth by tumor mass has been repeatedly studied, but without elucidation of a satisfactory mechanism. In our animal model, a...
The phenomenon of inhibition of tumor growth by tumor mass has been repeatedly studied, but without elucidation of a satisfactory mechanism. In our animal model, a primary tumor inhibits its remote metastases. After tumor removal, metastases neovascularize and grow. When the primary tumor is present, metastatic growth is suppressed by a circulating angiogenesis inhibitor. Serum and urine from tumor-bearing mice, but not from controls, specifically inhibit endothelial cell proliferation. The activity copurifies with a 38 kDa plasminogen fragment that we have sequenced and named angiostatin. A corresponding fragment of human plasminogen has similar activity. Systemic administration of angiostatin, but not intact plasminogen, potently blocks neovascularization and growth of metastases. We here show that the inhibition of metastases by a primary mouse tumor is mediated, at least in part, by angiostatin.
Topics: Amino Acid Sequence; Angiostatins; Animals; Carcinoma, Lewis Lung; Cattle; Endothelium, Vascular; Growth Inhibitors; In Vitro Techniques; Male; Mice; Mice, Inbred C57BL; Molecular Sequence Data; Neoplasm Metastasis; Neovascularization, Pathologic; Peptide Fragments; Plasminogen
PubMed: 7525077
DOI: 10.1016/0092-8674(94)90200-3 -
Cancer Research Jan 2006Angiostatin, a proteolytic fragment of plasminogen, is a potent angiogenesis inhibitor able to suppress tumor growth and metastasis through inhibition of endothelial...
Angiostatin, a proteolytic fragment of plasminogen, is a potent angiogenesis inhibitor able to suppress tumor growth and metastasis through inhibition of endothelial cell proliferation and migration. Previously, we showed that angiostatin binds and inhibits F(1)F(o) ATP synthase on the endothelial cell surface and that anti-ATP synthase antibodies reduce endothelial cell proliferation. ATP synthase also occurs on the extracellular surface of a variety of cancer cells, where its function is as yet unknown. Here, we report that ATP synthase is present and active on the tumor cell surface, and angiostatin, or antibody directed against the catalytic beta-subunit of ATP synthase, inhibits the activity of the synthase. We show that tumor cell surface ATP synthase is more active at low extracellular pH (pH(e)). Low pH(e) is a unique characteristic of the tumor microenvironment. Although the mechanism of action of angiostatin has not been fully elucidated, angiostatin treatment in combination with acidosis decreases the intracellular pH (pH(i)) of endothelial cells, leading to cell death. We also find that, at low pH(e), angiostatin and anti-beta-subunit antibody induce intracellular acidification of A549 cells, as well as a direct cytotoxicity that is absent in tumor cells with low levels of extracellular ATP synthase. These results establish angiostatin as an antitumorigenic and antiangiogenic agent through a mechanism implicating tumor cell surface ATP synthase. Furthermore, these data provide evidence that extracellular ATP synthase plays a role in regulating pH(i) in cells challenged by acidosis.
Topics: ATP Synthetase Complexes; Adenosine Triphosphate; Angiostatins; Cell Death; Cell Transformation, Neoplastic; Humans; Hydrogen-Ion Concentration; Neovascularization, Pathologic; Tumor Cells, Cultured
PubMed: 16424020
DOI: 10.1158/0008-5472.CAN-05-2806 -
Life Sciences Aug 2015Angiogenesis and chronic inflammation are known to be co-dependent in atherosclerosis and cardiovascular diseases. This study was undertaken to investigate whether... (Clinical Trial)
Clinical Trial
AIM
Angiogenesis and chronic inflammation are known to be co-dependent in atherosclerosis and cardiovascular diseases. This study was undertaken to investigate whether simvastatin could affect serum levels of angiostatin, a potent endogenous inhibitor of neovascularization, in patients with ischemic heart disease (IHD).
MAIN METHODS
Twenty-six patients with clinically confirmed IHD and hypercholesterolemia were assigned 40 mg/day of simvastatin for 8 weeks. Levels of lipid metabolism, C-reactive protein (C-RP) and other biochemical parameters in serum samples were measured using biochemical analyzer. Serum angiostatin levels were determined by Western blot. Association of serum angiostatin levels with total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and C-RP levels was evaluated.
KEY FINDINGS
Simvastatin therapy improved the main parameters of lipid metabolism, including statistically significant (P < 0.05) reductions in TC (by 46%) and LDL-C (by 42%), and decreased inflammatory marker C-RP (by 32%), as compared with the baseline. Simvastatin treatment resulted in marked reduction of serum angiostatin level (by 80% in comparison with baseline, P < 0.05). Strong positive correlations between serum angiostatin level versus concentrations of TC, LDL-C, and C-RP were demonstrated before onset of the study (r = 0.48311, 0.6252, and 0.653, respectively) and after simvastatin therapy (r = 0.67752, 0.6485, and 0.8244, respectively).
SIGNIFICANCE
We describe for the first time novel pleiotropic effect of statin therapy associated with decrease of serum angiostatin levels. Thus, circulating angiostatin represents an independent additional risk marker for cardiovascular events and could be applied as potential supplementary indicator for evaluation of statin therapy efficacy.
Topics: Aged; Angiostatins; C-Reactive Protein; Cholesterol, LDL; Female; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hypercholesterolemia; Male; Middle Aged; Myocardial Ischemia
PubMed: 26032258
DOI: 10.1016/j.lfs.2015.05.008 -
Blood Nov 2003Platelets regulate new blood vessel growth, because they contain a number of angiogenesis promoters and inhibitors. Additionally, platelets contain matrix...
Platelets regulate new blood vessel growth, because they contain a number of angiogenesis promoters and inhibitors. Additionally, platelets contain matrix metalloproteinases (MMPs), which when released mediate platelet adhesion and aggregation, and plasminogen, a fibrinolytic system enzyme that serves to limit blood clot formation. Enzymatic cleavage of plasminogen by MMPs generates angiostatin, an angiogenesis inhibitor. Therefore, we examined whether platelets generate angiostatin during aggregation in vitro. Platelets were isolated from healthy human donors and then aggregated with collagen, thrombin, or HT-1080 fibrosarcoma cells. Angiostatin was detected by Western blot analysis in the platelet releasates of all blood donors irrespective of the aggregating agent used. Platelet pellet homogenates showed the presence of angiostatin in all donors, which was released upon aggregation. Furthermore, platelet-derived angiostatin was isolated and purified by lysine-Sepharose affinity chromatography from collagen-aggregated platelet releasates. Bioassay of platelet-derived angiostatin showed that it inhibited the formation of capillary structures by human umbilical vein endothelial cells (HUV-EC-Cs) in an in vitro angiogenesis model. Inhibition of angiostatin in platelet releasates promoted the formation of capillary structures by HUV-EC-Cs. We conclude that healthy human platelets contain angiostatin, which is released in active form during platelet aggregation, and platelet-derived angiostatin has the capacity to inhibit angiogenesis.
Topics: Angiogenesis Inhibitors; Angiostatins; Blood Platelets; Blotting, Western; Chromatography, Affinity; Collagen; Endothelium, Vascular; Humans; Neovascularization, Physiologic; Platelet Aggregation; Umbilical Veins
PubMed: 12855585
DOI: 10.1182/blood-2003-02-0378 -
Journal of Thrombosis and Haemostasis :... May 2006Angiogenesis, the growth of new capillaries from pre-existing blood vessels, is regulated by a balance between its promoters and inhibitors. Platelets are an important...
BACKGROUND
Angiogenesis, the growth of new capillaries from pre-existing blood vessels, is regulated by a balance between its promoters and inhibitors. Platelets are an important circulating store of angiogenesis regulators. We have previously identified the angiogenesis inhibitor angiostatin in human platelets.
AIM
To identify the mechanism of platelet angiostatin generation and its pharmacological regulation.
METHODS
Platelet aggregometry, flow cytometry, Western blot, zymography, immunofluorescence microscopy, matrigel-induced angiogenesis of human umbilical vein endothelial cells (HUVECs), and a panel of selective proteinase inhibitors were used to study the mechanism of angiostatin generation by platelets, its pharmacological regulation, and effects on angiogenesis. Release of pro-MMP-2 by HUVECs was also used to quantify angiogenesis.
RESULTS
Platelet membranes were identified as the site of angiostatin generation from plasminogen. Generation of angiostatin by platelet membranes was not affected by a matrix metalloproteinase (MMP) inhibitor, phenanthroline, but was inhibited by serine proteinase inhibitors aprotinin, leupeptin, plasminogen activator inhibitor-1, and selective inhibitor of urokinase plasminogen activator (uPA), uPA-STOP(TM). Angiostatin generation by intact platelets was inhibited by aprotinin, and the resulting incubate promoted angiogenesis to a greater extent than incubate where angiostatin generation occurred. Furthermore, HUVECs incubated with reaction mixture, where angiostatin generation was inhibited, released more pro-MMP-2 than HUVECs incubated with supernatants, where angiostatin generation occurred.
CONCLUSIONS
We conclude that; (i) platelets constitutively generate angiostatin on their membranes; (ii) this mechanism is dependent on uPA, but not, MMPs; and (iii) inhibition of platelet angiostatin generation can further promote angiogenesis.
Topics: Angiostatins; Blood Platelets; Blotting, Western; Cell Line; Flow Cytometry; Humans; Microscopy, Fluorescence; Neovascularization, Physiologic; Platelet Aggregation; Urokinase-Type Plasminogen Activator
PubMed: 16689764
DOI: 10.1111/j.1538-7836.2006.01878.x -
International Journal of Rheumatic... Nov 2017The goal of this study is to investigate how urinary angiostatin, vascular cell adhesion molecule 1 (VCAM-1) and established measures of renal function relate to... (Comparative Study)
Comparative Study
AIM
The goal of this study is to investigate how urinary angiostatin, vascular cell adhesion molecule 1 (VCAM-1) and established measures of renal function relate to specific histologic findings in paired kidney biopsy samples from patients with lupus nephritis (LN).
METHOD
Urine samples were collected from 54 LN patients together with paired kidney biopsy samples and examined for urinary angiostatin and VCAM-1 protein levels. Nonparametric tests were used to examine the association of both urinary biomarkers and established traditional laboratory markers of renal function with nine specific renal histologic features seen in LN, including glomerular leukocyte infiltration, endocapillary proliferation, cellular crescents, fibrinoid necrosis, wire loops, interstitial inflammation, glomerulosclerosis, fibrous crescents, tubular atrophy and interstitial fibrosis.
RESULTS
Compared to traditional renal disease metrics, both urinary angiostatin and VCAM-1 exhibited outstanding potential (area under the curve 0.97, 0.98, respectively) to predict renal biopsy activity index score ≥ 7, which is associated with poor long-term prognosis. Whereas urine VCAM-1 was most significantly associated with fibrous crescents, urine angiostatin was most significantly associated with endocapillary proliferation, cellular crescents, fibrinoid necrosis and fibrous crescents in concurrent renal biopsies.
CONCLUSION
Urinary angiostatin and VCAM-1 are predictive of specific histological changes in concurrent LN renal biopsies. Both urinary biomarkers are good candidates for use as noninvasive measures of renal pathology activity changes in LN.
Topics: Adult; Angiostatins; Area Under Curve; Atrophy; Biomarkers; Biopsy; Cross-Sectional Studies; Female; Fibrosis; Humans; Kidney; Lupus Nephritis; Male; Predictive Value of Tests; ROC Curve; Reproducibility of Results; Severity of Illness Index; Urinalysis; Vascular Cell Adhesion Molecule-1
PubMed: 29076253
DOI: 10.1111/1756-185X.13197 -
Blood Dec 1998Angiostatin is a circulating inhibitor of angiogenesis generated by proteolytic cleavage of plasminogen. In this study we have used recombinant human and murine...
Angiostatin is a circulating inhibitor of angiogenesis generated by proteolytic cleavage of plasminogen. In this study we have used recombinant human and murine angiostatins (kringles 1-4) as well as native human angiostatin (prepared by elastase digestion of plasminogen [kringles 1-3] or by plasmin autocatalysis in the presence of a free sulfhydryl donor [kringles 1-4]). We report that angiostatin reduces endothelial cell number in a 4-day proliferation assay without affecting cell cycle progression into S-phase (as determined by bromodeoxyuridine labeling). This suggested that the reduction in cell number in the proliferation assay might in part be due to cytotoxicity. This was confirmed by the observation that ethidium homodimer incorporation (a measure of plasma membrane integrity) into endothelial cells was increased by angiostatin in a manner similar to that seen with tumor necrosis factor- (TNF-) and transforming growth factor-beta1 (TGF-beta1), both of which induce apoptosis in endothelial cells. In contrast to TNF- and TGF-beta1, angiostatin did not induce cytotoxicity in human MRC-5 fibroblast, rat smooth muscle, canine MDCK epithelial, or murine B16-F10 melanoma cell lines. Angiostatin-induced apoptosis was confirmed by endothelial cell nuclear acridine orange incorporation as well as by annexin V and TUNEL staining. These in vitro findings point to endothelial cell apoptosis as a mechanism for the antiangiogenic effect of angiostatin in vivo.
Topics: Angiostatins; Animals; Antibodies, Monoclonal; Apoptosis; Cattle; Cell Count; Cell Cycle; Cell Division; Cells, Cultured; Dactinomycin; Dogs; Dose-Response Relationship, Drug; Endothelium, Vascular; Humans; Kringles; Mice; Organ Specificity; Peptide Fragments; Plasminogen; Rats; Recombinant Proteins; S Phase; Time Factors; Tumor Necrosis Factor-alpha
PubMed: 9845539
DOI: No ID Found -
The Journal of Biological Chemistry Oct 1999We have previously reported the identification of the endogenous angiogenesis inhibitor angiostatin, a specific inhibitor of endothelial cell proliferation in vitro and...
We have previously reported the identification of the endogenous angiogenesis inhibitor angiostatin, a specific inhibitor of endothelial cell proliferation in vitro and angiogenesis in vivo. In our original studies, we demonstrated that a Lewis lung carcinoma (LLC-LM) primary tumor could suppress the growth of its metastases by generating angiostatin. Angiostatin, a 38-kDa internal fragment of plasminogen, was purified from the serum and urine of mice bearing LLC-LM, and its discovery provides the first proven mechanism for concomitant resistance (O'Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R. A., Moses, M. A., Lane, W. S., Cao, Y., Sage, E. H., and Folkman, J. (1994) Cell 79, 315-328). Subsequently, we have shown that systemic administration of angiostatin can regress a wide variety of malignant tumors in vivo. However, at the time of our initial discovery of angiostatin, the source of the protein was unclear. We hypothesized that the tumor or stromal cells might produce an enzyme that could cleave plasminogen sequestered by the primary tumor into angiostatin. Alternatively, we speculated that the tumor cells might express angiostatin. By Northern analysis, however, we have found no evidence that the tumor cells express angiostatin or other fragments of plasminogen (data not shown). We now report that gelatinase A (matrix metalloproteinase-2), produced directly by the LLC-LM cells, is responsible for the production of angiostatin, which suppresses the growth of metastases in our original model.
Topics: Angiostatins; Animals; Antineoplastic Agents; Cell Division; Culture Media, Conditioned; Endothelium, Vascular; Gelatin; Lung Neoplasms; Matrix Metalloproteinase 2; Mice; Neovascularization, Pathologic; Peptide Fragments; Plasminogen; RNA, Messenger; Sequence Analysis; Tumor Cells, Cultured
PubMed: 10506224
DOI: 10.1074/jbc.274.41.29568 -
Molecular and Biochemical Parasitology Jan 2014Malaria is a disease caused by Plasmodium parasites and remains one of the most prevalent and persistent maladies, affecting hundreds of millions of people. In the...
Malaria is a disease caused by Plasmodium parasites and remains one of the most prevalent and persistent maladies, affecting hundreds of millions of people. In the present work, we evaluated the capability of Plasmodium falciparum proteases to hydrolyze the multifunctional protein plasminogen, which is implicated in angiogenesis and coagulation processes by the generation of angiostatin and plasmin, respectively. Using fluorescence microscopy, we visualized the internalization of FITC-labeled plasminogen in erythrocytes infected by P. falciparum and showed that the parasites are able to hydrolyze the protein. The cleavage of plasminogen by the P. falciparum proteases was also observed by SDS-PAGE, followed by immunoblotting with anti-angiostatin antibody. N-terminal sequencing of the main generated fragments indicated that they are comprised in the five plasminogen kringle domains, suggesting as being angiostatin-like peptides. This assumption was reinforced by the demonstration that the products of plasminogen processing mimic angiostatin functions, including the capability to inhibit angiogenesis and to stimulate calcium response in endothelial cells in vitro. However, no plasmin activity was detected after plasminogen hydrolysis by P. falciparum. Nonetheless, exogenous tissue plasminogen activator (tPA) activated plasmin in infected erythrocytes, suggesting that the uptake of plasminogen by P. falciparum may be modulated by the vertebrate host. Taken together, the data presented here provide evidence for the processing of host plasminogen by malaria parasites to generate active fragments that may modulate host physiology events during malaria infection.
Topics: Angiostatins; Electrophoresis, Polyacrylamide Gel; Erythrocytes; Fibrinolysin; Fluorescein-5-isothiocyanate; Fluorescent Dyes; Host-Pathogen Interactions; Humans; Hydrolysis; Immunoblotting; Peptide Hydrolases; Plasminogen; Plasmodium falciparum; Staining and Labeling
PubMed: 24503144
DOI: 10.1016/j.molbiopara.2014.01.004 -
Angiostatin K1-3 induces E-selectin via AP1 and Ets1: a mediator for anti-angiogenic action of K1-3.Journal of Thrombosis and Haemostasis :... Nov 2008Angiostatin, a circulating angiogenic inhibitor, is an internal fragment of plasminogen and consists of several isoforms, K1-3 included. We previously showed that K1-3...
BACKGROUND
Angiostatin, a circulating angiogenic inhibitor, is an internal fragment of plasminogen and consists of several isoforms, K1-3 included. We previously showed that K1-3 was the most potent angiostatin to induce E-selectin mRNA expression. The purpose of this study was to identify the mechanism responsible for K1-3-induced E-selectin expression and investigate the role of E-selectin in the anti-angiogenic action of K1-3.
METHODS AND RESULTS
Quantitative real time RT-PCR and Western blotting analyses confirmed a time-dependent increase of E-selectin mRNA and protein induced by K1-3. Subcellular fractionation and immunofluorescence microscopy showed the co-localization of K1-3-induced E-selectin with caveolin 1 (Cav1) in lipid rafts in which E-selectin may behave as a signaling receptor. Promoter-driven reporter assays and site-directed mutagenesis showed that K1-3 induced E-selectin expression via promoter activation and AP1 and Ets-1 binding sites in the proximal E-selectin promoter were required for E-selectin induction. The in vivo binding of both protein complexes to the proximal promoter was confirmed by chromatin immunoprecipitation (ChIP). Although K1-3 induced the activation of ERK1/2 and JNK, only repression of JNK activation attenuated the induction of E-selectin by K1-3. A modulatory role of E-selectin in the anti-angiogenic action of K1-3 was manifested by both overexpression and knockdown of E-selectin followed by cell proliferation assay.
CONCLUSIONS
We show that K1-3 induced E-selectin expression via AP1 and Ets-1 binding to the proximal E-selectin promoter (-356/+1), which was positively mediated by JNK activation. Our findings also demonstrate E-selectin as a novel target for the anti-angiogenic therapy.
Topics: Angiostatins; Binding Sites; Caveolin 1; Cell Line; Cell Line, Tumor; E-Selectin; Humans; JNK Mitogen-Activated Protein Kinases; Kinetics; Membrane Microdomains; Promoter Regions, Genetic; Protein Binding; Protein Isoforms; Proto-Oncogene Protein c-ets-1; RNA, Messenger; Transcription Factor AP-1; Transcriptional Activation
PubMed: 18761727
DOI: 10.1111/j.1538-7836.2008.03139.x