Validation of Collagenous Protein Synthesis as an Index for Angiogenesis with the Use of Morphological Methods

. On the mechanism of thrombin-induced angiogenesis: involvement of ␣v␤3-integrin. Am J Physiol Cell Physiol 283: C1501-C1510, 2002. First published July 17, 2002 10.1152/ajpcell.00162.2002Thrombin has been reported to be a potent angiogenic factor both in vitro and in vivo, and many of the cellular effects of thrombin may contribute to activation of angiogenesis. In this report we show that thrombin-treatment of human endothelial cells increases mRNA and protein levels of ␣v␤3-integrin. This thrombin-mediated effect is specific, dose dependent, and requires the catalytic site of thrombin. In addition, thrombin interacts with ␣v␤3 as demonstrated by direct binding of ␣v␤3 protein to immobilized thrombin. This interaction of thrombin with ␣v␤3-integrin, which is an angiogenic marker in vascular tissue, is of functional significance. Immobilized thrombin promotes endothelial cells attachment, migration, and survival. Antibody to ␣v␤3 or a specific peptide antagonist to ␣v␤3 can abolish all these ␣v␤3-mediated effects. Furthermore, in the chick chorioallantoic membrane system, the antagonist peptide to ␣v␤3 diminishes both basal and the thrombin-induced angiogenesis. These results support the pivotal role of thrombin in activation of endothelial cells and angiogenesis and may be related to the clinical observation of neovascularization within thrombi. attachment; migration; apoptosis; reverse transcription-polymerase chain reaction THE FREQUENCY OF BLOOD COAGULATION in cancer patients, known for more than 130 years, is supported by clinical, laboratory, and histopathological evidence. This is explained at the molecular and cellular level by the thromboplastic activity of circulating tumor cells, the existence of "a cancer coagulative factor," the activation of factor X, the generation of prothrombinase by tumor cells, and the encircling of cancerous tissue by fibrin deposits (38,50). In addition, the possibility of a relation between blood clotting mechanisms and tumor progression and development of metastases was postulated as early as 1878 by Billroth (7) on the basis of the observation that cancer cells exist within thrombi. This finding was interpreted as evidence that tumor cells spread by thromboembolism. More recently, large epidemiological studies have provided evidence that the standardized incidence ratio for certain types of cancer is as high as 6.7 within a year following a thromboembolic episode (3, 41). These clinical data are in line with animal experiments where thrombin-treated B16 melanoma cells show a dramatic increase in their metastatic potential in the lung of rats (37). These observations have led to experimental use of heparin, aspirin, and warfarin for the prevention and treatment of tumors in animal models and humans (23, 50).We proposed earlier (33, 47) that the tumor-promoting effect of thrombin/thrombosis may be related to our finding that thrombin is a potent promoter of angiogenesis, a process essential for tumor growth and metastasis. The angiogenic action of thrombin was shown to b...

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“…We have used collagenous protein biosynthesis (CPB) as a biochemical index of angiogenesis (31). As shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The in vivo chick chorioallantoic membrane (CAM) angiogenesis model was used as described previously (31). Briefly, biochemical evaluation of angiogenesis was performed by determining the extent of collagenous protein biosynthesis in the CAM lying directly under the disks applied at day 9 of chick embryo development.…”

Section: Methodsmentioning

confidence: 99%

On the mechanism of thrombin-induced angiogenesis: involvement of α_vβ₃-integrin

Tsopanoglou

Andriopoulou

Maragoudakis

2002

American Journal of Physiology-Cell Physiology

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“…Carrier disks alone and human serum albumin were used as negative controls. Quantitation of angiogenesis in the transparent CAM was based on the optical density of the digitized microphotographs using computer-assisted image analysis (22). Mean relative optical density was measured from vessels with a diameter Ͻ0.1 mm using the program ImageJ (23).…”

Section: Methodsmentioning

confidence: 99%

Vascular Endothelial Growth Factor (VEGF)/VEGF-C Mosaic Molecules Reveal Specificity Determinants and Feature Novel Receptor Binding Patterns

Jeltsch

Kärpänen

Strandin

et al. 2006

Journal of Biological Chemistry

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“…The in vivo chick chorioallantoic membrane (CAM) angiogenesis model was used as described previously (Maragoudakis et al, 1995). In brief, biochemical evaluation of angiogenesis was performed by determining the extent of collagenous protein biosynthesis in the CAM lying directly under the pellets applied at day 9 of chick embryo development.…”

Section: Materials and Cellmentioning

confidence: 99%

“…We have used the in vivo model of chick chorioallantoic membrane system of angiogenesis and collagenous protein biosynthesis as a biochemical index of angiogenesis (Maragoudakis et al, 1995). As shown in Fig.…”

Section: Par-1 Antagonists Inhibit Angiogenesis In Vivomentioning

confidence: 99%

Blockade of Angiogenesis by Small Molecule Antagonists to Protease-Activated Receptor-1: Association with Endothelial Cell Growth Suppression and Induction of Apoptosis

Zania¹,

Kritikou²,

Flordellis³

et al. 2006

J Pharmacol Exp Ther

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Many studies support the notion that protease-activated receptor (PAR)-1 plays a pivotal role in angiogenesis. However, direct evidence and understanding the molecular mechanisms involved were limited because PAR-1-specific antagonists have been developed only recently. In the present study, we evaluated the effects of two well characterized PAR-1 antagonists,, in the angiogenic cascade. These antagonists suppressed both the basic angiogenesis and that stimulated by thrombin in the chick chorioallantoic membrane model in vivo. PAR-1 antagonists also abrogated tube formation in the in vitro Matrigel system. These inhibitory effects were dose-dependent and well correlated with the inhibitory effects of SCH79797 and RWJ56110 on primary endothelial cell proliferation and on the initiation of apoptosis. PAR-1 blockage resulted in inhibition of endothelial cell growth by increasing the sub-G 0 /G 1 fraction and reducing the percentage of cells in the S phase. Consistent with this, PAR-1 antagonists reduced incorporation of [ 3 H]thymidine in endothelial cells and blocked the phosphorylation of extracellular signal-regulated kinases in a fashion depending specifically on PAR-1 activation. Analysis by annexin V/propidium iodide staining and poly(ADP-ribose) polymerase cleavage revealed that PAR-1 blockage increased apoptotic cell death by a mechanism involving caspases. These results provide further evidence that PAR-1 is a key receptor that mediates angiogenesis and suggest PAR-1 as target for developing antiangiogenic agents with potential therapeutic application in cancer and other angiogenesis-related diseases.

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Validation of Collagenous Protein Synthesis as an Index for Angiogenesis with the Use of Morphological Methods

Cited by 31 publications

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On the mechanism of thrombin-induced angiogenesis: involvement of α_vβ₃-integrin

On the mechanism of thrombin-induced angiogenesis: involvement of α_vβ₃-integrin

Vascular Endothelial Growth Factor (VEGF)/VEGF-C Mosaic Molecules Reveal Specificity Determinants and Feature Novel Receptor Binding Patterns

Blockade of Angiogenesis by Small Molecule Antagonists to Protease-Activated Receptor-1: Association with Endothelial Cell Growth Suppression and Induction of Apoptosis

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Validation of Collagenous Protein Synthesis as an Index for Angiogenesis with the Use of Morphological Methods

Cited by 31 publications

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On the mechanism of thrombin-induced angiogenesis: involvement of αvβ3-integrin

On the mechanism of thrombin-induced angiogenesis: involvement of αvβ3-integrin

Vascular Endothelial Growth Factor (VEGF)/VEGF-C Mosaic Molecules Reveal Specificity Determinants and Feature Novel Receptor Binding Patterns

Blockade of Angiogenesis by Small Molecule Antagonists to Protease-Activated Receptor-1: Association with Endothelial Cell Growth Suppression and Induction of Apoptosis

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On the mechanism of thrombin-induced angiogenesis: involvement of α_vβ₃-integrin

On the mechanism of thrombin-induced angiogenesis: involvement of α_vβ₃-integrin