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Biochemistry and Cell Biology =... Feb 2005Previous studies from this laboratory have demonstrated that plasminogen binds to endothelial cell surface-associated actin via its kringles in a dose-dependent and...
Previous studies from this laboratory have demonstrated that plasminogen binds to endothelial cell surface-associated actin via its kringles in a dose-dependent and specific manner. The purpose of this study was to determine whether angiostatin, a proteolytic fragment of plasminogen, shares binding properties with plasminogen. Our results indicated that like plasminogen, angiostatin bound to actin in a time-, concentration-, and kringle-dependent manner. Furthermore, this binding was significantly inhibited by excess plasminogen, suggesting that both proteins shared binding motifs on the actin molecule. Fluorescence studies demonstrated that angiostatin bound to intact endothelial cells through its kringles, and this binding was also inhibited by plasminogen but not by unrelated proteins. Ligand blot analyses on endothelial cell lysates indicated that angiostatin interacted with a 42 kDa protein, which was identified as actin. Furthermore, an anti-actin antibody inhibited binding of angiostatin to endothelial cells by approximately 25%. These results suggest that angiostatin and plasminogen share binding to endothelial cell surface actin and, therefore, that angiostatin has the potential to inhibit plasmin-dependent processes such as cell migration-movement.
Topics: Actins; Angiostatins; Animals; Cell Membrane; Cells, Cultured; Endothelial Cells; Humans; Kringles; Microscopy, Fluorescence; Plasminogen; Protein Binding; Umbilical Veins
PubMed: 15746964
DOI: 10.1139/o04-109 -
Molecular Vision Jan 2007To evaluate the efficacy of recombinant adeno-associated virus (rAAV) vector expressing mouse angiostatin (Kringle domains 1 to 4) in reducing retinal vascular leakage...
PURPOSE
To evaluate the efficacy of recombinant adeno-associated virus (rAAV) vector expressing mouse angiostatin (Kringle domains 1 to 4) in reducing retinal vascular leakage in an experimental diabetic rat model.
METHODS
rAAV-angiostatin was delivered by intravitreal injection to the right eyes of Sprague-Dawley rats. As a control, the contralateral eye received an intravitreal injection of rAAV-lacZ. Gene delivery was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR). Diabetes was induced by intravenous injection of streptozotocin (STZ). Vascular permeability changes were evaluated by extravascular albumin accumulation and leakage of intravenous-injected fluorescein isothiocynate-bovine serum albumin (FITC-BSA). Effects of rAAV-angiostatin on expression of vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), occludin, and phospho-p42/p44 MAP kinase in retina tissue were analyzed by western blotting.
RESULTS
The rAAV-angiostatin injections led to sustained angiostatin gene expression in retina as confirmed by RT-PCR, and reduced extravascular albumin accumulation in STZ-induced diabetic retina. Further, rAAV-angiostatin significantly decreased intravascularly injected FITC-BSA leakage at 5 days (p=0.001), 10 days (p<0.001), and 15 days (p=0.001) after STZ-induced diabetes, as compared to the control eyes receiving rAAV-lacZ. Expression of VEGF and phosphorylation of p42/p44 MAP kinase in retina was reduced by rAAV-angiostatin at day 1 (p=0.043 for both VEGF and phospho-p42/p44 MAP kinase) after STZ-induced diabetes compared with rAAV-lacZ eyes. rAAV-angiostatin reduced retinal occludin loss at 10 days after STZ-induced diabetes (n=5, p=0.041). There was no significant difference in retinal PEDF expression between eyes injected with rAAV-angiostatin and rAAV-lacZ.
CONCLUSIONS
Intravitreal delivery of rAAV-angiostatin reduces vascular leakage in an STZ-induced diabetic model. This effect is associated with a reduction in the retinal occludin loss induced by diabetes and downregulation of retinal VEGF and phosphor-p42/p44 MAP kinase expression. This gene transfer approach may reduce diabetic macular edema, providing protection in diabetic patients at risk for macular edema.
Topics: Angiostatins; Animals; Blood-Retinal Barrier; Capillary Permeability; Dependovirus; Diabetes Mellitus, Experimental; Diabetic Retinopathy; Down-Regulation; Eye Proteins; Gene Transfer Techniques; Male; Membrane Proteins; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nerve Growth Factors; Occludin; Phosphorylation; Rats; Rats, Sprague-Dawley; Recombination, Genetic; Retina; Retinal Vessels; Serpins; Vascular Endothelial Growth Factor A; Vitreous Body
PubMed: 17293777
DOI: No ID Found -
Mathematical Biosciences and... Jan 2023Anti-angiogenesis as a treatment strategy for normalizing the microvascular network of tumors is of great interest among researchers, especially in combination with...
Anti-angiogenesis as a treatment strategy for normalizing the microvascular network of tumors is of great interest among researchers, especially in combination with chemotherapy or radiotherapy. According to the vital role that angiogenesis plays in tumor growth and in exposing the tumor to therapeutic agents, this work develops a mathematical framework to study the influence of angiostatin, a plasminogen fragment that shows the anti-angiogenic function, in the evolutionary behavior of tumor-induced angiogenesis. Angiostatin-induced microvascular network reformation is investigated in a two-dimensional space by considering two parent vessels around a circular tumor by a modified discrete angiogenesis model in different tumor sizes. The effects of imposing modifications on the existing model, i.e., the matrix-degrading enzyme effect, proliferation and death of endothelial cells, matrix density function, and a more realistic chemotactic function, are investigated in this study. Results show a decrease in microvascular density in response to the angiostatin. A functional relationship exists between angiostatin's ability to normalize the capillary network and tumor size or progression stage, such that capillary density decreases by 55%, 41%, 24%, and 13% in tumors with a non-dimensional radius of 0.4, 0.3, 0.2, and 0.1, respectively, after angiostatin administration.
Topics: Humans; Angiostatins; Angiogenesis Inhibitors; Endothelial Cells; Neoplasms; Neovascularization, Pathologic; Microvessels
PubMed: 36896553
DOI: 10.3934/mbe.2023252 -
Acta Clinica Belgica Aug 2014Angiogenesis plays an important role in the pathogenesis of inflammatory diseases, but the possible role of angiogenesis in Behçet's disease (BD) has not yet been...
Angiogenesis plays an important role in the pathogenesis of inflammatory diseases, but the possible role of angiogenesis in Behçet's disease (BD) has not yet been studied. The aim of this study was to determine angiostatin levels in patients with BD and the role of angiogenesis in the pathogenesis of the disease. Thirty-seven patients with BD (mean age: 28·6±5·4 years, mean disease duration: 9·3±3·7 years) and 18 healthy controls were enrolled to the study. Twenty-four patients were in active and 13 patients were in inactive stage of the disease. The mean serum angiostatin level of patients with BD was 113·9±53·2 and 60·7±20·1 ng/ml in healthy controls. The mean serum angiostatin level was 142·7±43·1 ng/ml in active and 86·9±15·5 ng/ml in inactive patients with BD. Serum angiostatin levels were significantly high in patients with BD compared with healthy controls (P<0·001) and it was significantly high in active patients compared with inactive patients with BD (P<0·001). In inactive patients with BD, serum angiostatin concentrations were found to be higher compared with healthy controls (P<0·01). In active BD patients, the mean serum angiostatin level was correlated with the deep vein thrombosis (r = 0·482, P = 0·05), uveitis (r = 0·582, P = 0·01), and arthritis (r = 0·492, P = 0·05). According to these results; elevated serum angiostatin levels in patients with BD suggest the possible role of angiogenesis in the pathogenesis of the disease and its high levels in inactive Behçet's patients is related with the continuous activation of the disease even in the subclinical period.
Topics: Adult; Angiostatins; Behcet Syndrome; Case-Control Studies; Female; Humans; Male; Middle Aged; Neovascularization, Pathologic
PubMed: 25012748
DOI: 10.1179/2295333714Y.0000000030 -
Biochemical and Biophysical Research... Oct 2003Angiostatin consisting of the first four-kringle domains of the plasminogen potently inhibits angiogenesis in vitro and in vivo. However, the molecular mechanism of...
Angiostatin consisting of the first four-kringle domains of the plasminogen potently inhibits angiogenesis in vitro and in vivo. However, the molecular mechanism of action whereby angiostatin mediates its inhibitory effect on proliferating endothelial cells remains elusive. We therefore used the proliferating cultured human umbilical vein endothelial cells (HUVECs) promoted by vascular endothelial growth factor A to identify the endogenous signaling elements that mediate the antiangiogenic effect of angiostatin. Treatment of HUVEC with angiostatin at a concentration known to inhibit cell proliferation and induce apoptosis resulted in induction of p53-, Bax-, and tBid-mediated release of cytochrome c into the cytosol. In addition, angiostatin also activated the Fas-mediated apoptotic pathway in part via up-regulation of FasL mRNA, down-regulation of c-Flip, and activation of caspase 3. These results suggest that the anti-angiogenic action of angiostatin is likely mediated by two distinct signaling pathways, one intrinsic mediated by p53 while the other extrinsic involved in FasL engagement and mitochondria dysfunction.
Topics: Angiostatins; Apoptosis; BH3 Interacting Domain Death Agonist Protein; Blotting, Southern; Blotting, Western; Carrier Proteins; Cell Division; Cells, Cultured; Cytochromes c; Cytosol; Dose-Response Relationship, Drug; Down-Regulation; Endothelium, Vascular; Fas Ligand Protein; Humans; Membrane Glycoproteins; Mitochondria; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; RNA, Messenger; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Time Factors; Tumor Suppressor Protein p53; Umbilical Veins; Up-Regulation; Vascular Endothelial Growth Factor A; bcl-2-Associated X Protein
PubMed: 14550275
DOI: 10.1016/j.bbrc.2003.09.081 -
Experimental Eye Research Mar 2004The purpose of this study is to determine whether angiostatin is involved in maintaining corneal avascularity after wounding. We generated polyclonal rabbit anti-mouse...
The purpose of this study is to determine whether angiostatin is involved in maintaining corneal avascularity after wounding. We generated polyclonal rabbit anti-mouse angiostatin antibodies directed against each of the five kringle domains, (K1-5) and anti-mouse plasmin B chain antibodies. Mouse corneas were immunostained with anti-K1 angiostatin antibody after excimer laser keratectomy. Corneal epithelial cell lysate was harvested and angiostatin was isolated using lysine sepharose. Purified plasminogen was incubated with lysate of mouse corneal epithelial cells from wild type mice in the presence or absence of MMP inhibitors. Angiostatin activity was determined using calf pulmonary artery endothelial (CPAE) cell proliferation assay with and without angiostatin immunoprecipitation; and corneal neovascularization was assayed by intrastromal injection of anti-plasminogen, anti-K1-3 or anti-B chain antibodies after corneal wounding. Using the anti-mouse angiostatin antibodies that we generated, we confirmed that angiostatin-like molecules were expressed in the corneal epithelium and in cultured corneal epithelial cells. Western blotting after incubation of scraped corneal epithelial cell lysate with purified plasminogen showed reduction of the plasminogen bands at 6, 12, and 24 hr, respectively. Complete cleavage of plasminogen occurred by 48 hr. Functional assays in which corneal epithelial cell extracts were incubated with CPAE cells resulted in inhibition of vascular endothelial cell proliferation. Depletion experiments using anti-angiostatin (K1) antibodies resulted in a 25 +/- 1.2% increase in vascular endothelial cell proliferation as compared to 12 +/- 1.8% using the protein A control (p < 0.05). Corneal neovascularization was observed after excimer laser keratectomy when anti-angiostatin antibodies were injected into the cornea (65 +/- 13%) which was significantly higher than when plasmin B chain antibodies were injected (10 +/- 2.6%; p < 0.05). Plasminogen and angiostatin are produced in the cornea. They may play a role in preventing vascularization and may contribute to the maintenance of corneal avascularity after excimer laser keratectomy.
Topics: Angiostatins; Animals; Cells, Cultured; Corneal Neovascularization; Endothelium, Vascular; Epithelium, Corneal; Female; Lasers, Excimer; Mice; Mice, Inbred C57BL; Photorefractive Keratectomy; Plasminogen; Rabbits; Rats; Rats, Sprague-Dawley; Wound Healing
PubMed: 15106938
DOI: 10.1016/j.exer.2003.09.005 -
Deutsche Medizinische Wochenschrift... Mar 1997
Topics: Angiostatins; Animals; Antineoplastic Agents; Humans; Mice; Neoplasm Metastasis; Neoplasms; Neoplasms, Experimental; Neovascularization, Pathologic; Peptide Fragments; Plasminogen
PubMed: 9138915
DOI: 10.1055/s-0029-1233709 -
Oncology Reports Feb 2004The aim of this study was to determine the presence of angiostatin in ascitic and pleural effusions from cancer patients, as well as of metalloproteinases (MMPs) and... (Comparative Study)
Comparative Study
The aim of this study was to determine the presence of angiostatin in ascitic and pleural effusions from cancer patients, as well as of metalloproteinases (MMPs) and urokinase-type plasminogen activator (uPA), both involved in angiostatin generation in in vitro models. Ascitic fluids, pleural exudates, and sera from 21 cancer patients were analyzed for the presence of angiostatin by western blot, whereas gelatinases MMP-2 and MMP-9, and uPA were evaluated by zymography. Our study revealed elevated levels of angiostatin in effusions of cancer patients, contrasting with mostly intermediate levels in less than half of their sera, and undetectable levels in normal sera. Despite the observation of enhanced levels of HMW-uPA and MMP-2 in malignant effusions from cancer patients, their analysis in individual samples showed no association between angiostatin presence and the enzymes, suggesting that the latter would not play an unimportant role, if any, in in vivo generation of angiostatin.
Topics: Aged; Aged, 80 and over; Angiogenesis Inhibitors; Angiostatins; Ascites; Female; Humans; Male; Matrix Metalloproteinase 2; Middle Aged; Neoplasms; Pleural Effusion; Reference Values; Urokinase-Type Plasminogen Activator
PubMed: 14719094
DOI: No ID Found -
Clinical and Experimental Rheumatology 2016To determine the concentrations of circulating endostatin and angiostatin in patients with systemic sclerosis (SSc) and to assess its relationship to disease subsets,... (Comparative Study)
Comparative Study
OBJECTIVES
To determine the concentrations of circulating endostatin and angiostatin in patients with systemic sclerosis (SSc) and to assess its relationship to disease subsets, evolution phase, organ involvement and nailfold capillaroscopic changes.
METHODS
Endostatin and angiostatin serum levels were measured by ELISA in a cohort of 57 patients with SSc, and correlated with disease subsets, evolution phase, organ involvement and nailfold capillaroscopic changes.
RESULTS
Endostatin and angiostatin serum levels were significantly higher in patients with SSc than in healthy controls. Also, angiostatin was elevated in diffuse cutaneous SSc (dcSSc) and limited cutaneous SSc (lcSSc), but not in pre-SSc, while endostatin was increased in all SSc subsets. Moreover, endostatin was augmented in lcSSc, with or without CREST syndrome, whereas angiostatin was increased exclusively in patients with CREST. Analysis according to disease evolution phase found that endostatin was elevated in all phases while angiostatin was only significantly higher in intermediate and late phases of disease. Analysis regarding organ involvement revealed that angiostatin was significantly higher in patients with osteoarticular involvement and with more serious lung affection; no significant differences were found for endostatin. Finally, endostatin was significantly increased in all nailfold capillaroscopy stages, while angiostatin was only elevated in active and late phases.
CONCLUSIONS
In accordance with previous studies, we found that endostatin and angiostatin concentrations are elevated in SSc patients. Additionally, we recognised the important role that endostatin might play as an early disease marker and realized that angiostatin is a marker of late disease and relates to lung disease severity.
Topics: Adolescent; Adult; Aged; Aged, 80 and over; Angiostatins; Biomarkers; CREST Syndrome; Case-Control Studies; Cohort Studies; Disease Progression; Early Diagnosis; Endostatins; Enzyme-Linked Immunosorbent Assay; Female; Humans; Male; Microscopic Angioscopy; Middle Aged; Neovascularization, Pathologic; Predictive Value of Tests; Scleroderma, Diffuse; Scleroderma, Limited; Severity of Illness Index; Signal Transduction; Skin; Up-Regulation; Young Adult
PubMed: 26885625
DOI: No ID Found -
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