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Angiomotin: an angiostatin binding protein that regulates endothelial cell migration and tube formation.

Troyanovsky B, Levchenko T, Månsson G, Matvijenko O, Holmgren L - J. Cell Biol. (2001)

Bottom Line: Transfected angiomotin as well as endogenous angiomotin protein were localized to the leading edge of migrating endothelial cells.Expression of angiomotin in endothelial cells resulted in increased cell migration, suggesting a stimulatory role of angiomotin in cell motility.These findings indicate that angiostatin inhibits cell migration by interfering with angiomotin activity in endothelial cells.

View Article: PubMed Central - PubMed

Affiliation: Center for Genomics Research and Microbiology and Tumor Biology Center, Karolinska Institutet, S-171 76 Stockholm, Sweden.

ABSTRACT
Angiostatin, a circulating inhibitor of angiogenesis, was identified by its ability to maintain dormancy of established metastases in vivo. In vitro, angiostatin inhibits endothelial cell migration, proliferation, and tube formation, and induces apoptosis in a cell type-specific manner. We have used a construct encoding the kringle domains 1--4 of angiostatin to screen a placenta yeast two-hybrid cDNA library for angiostatin-binding peptides. Here we report the identification of angiomotin, a novel protein that mediates angiostatin inhibition of migration and tube formation of endothelial cells. In vivo, angiomotin is expressed in the endothelial cells of capillaries as well as larger vessels of the human placenta. Upon expression of angiomotin in HeLa cells, angiomotin bound and internalized fluorescein-labeled angiostatin. Transfected angiomotin as well as endogenous angiomotin protein were localized to the leading edge of migrating endothelial cells. Expression of angiomotin in endothelial cells resulted in increased cell migration, suggesting a stimulatory role of angiomotin in cell motility. However, treatment with angiostatin inhibited migration and tube formation in angiomotin-expressing cells but not in control cells. These findings indicate that angiostatin inhibits cell migration by interfering with angiomotin activity in endothelial cells.

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Migration of MAE cells transfected with angiomotin or with the vector alone was studied in the modified Boyden chamber assay. (A) Analysis of spontaneous migration in the absence of chemotactic factor at different time points after start of the experiment. A higher number of angiomotin-expressing cells are migrating through the filter at all time points. Vertical axis, cells migrated per high power field (HPF). (B) Effect of angiostatin on migration of MAE-angiomotin and MAE vector cells with or without stimulation with bFGF. Cells were pretreated for 1 h with angiostatin as indicated. (C) Dose response of angiostatin-mediated inhibition of migration of angiomotin-transfected MAE cells. All samples were performed in quadruplicates (error bars = SD).
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Figure 7: Migration of MAE cells transfected with angiomotin or with the vector alone was studied in the modified Boyden chamber assay. (A) Analysis of spontaneous migration in the absence of chemotactic factor at different time points after start of the experiment. A higher number of angiomotin-expressing cells are migrating through the filter at all time points. Vertical axis, cells migrated per high power field (HPF). (B) Effect of angiostatin on migration of MAE-angiomotin and MAE vector cells with or without stimulation with bFGF. Cells were pretreated for 1 h with angiostatin as indicated. (C) Dose response of angiostatin-mediated inhibition of migration of angiomotin-transfected MAE cells. All samples were performed in quadruplicates (error bars = SD).

Mentions: The localization of angiomotin in the leading edge of migrating cells and the effect of angiostatin on FAK kinase activity in angiomotin-transfected cells, suggested that angiomotin might play a role in endothelial cell migration. To test this hypothesis, we studied the effect of angiomotin expression in an angiomotin-negative immortalized MAE cell line. MAE cells were infected with ecotropic retrovirus expressing full-length angiomotin or vector alone. Polyclonal cells were selected by puromycin treatment in order to remove noninfected cells. The effect of angiomotin on cell migration was tested in a modified Boyden chamber. Analysis of spontaneous migration in the absence of chemoattractant showed that angiomotin cells consistently exhibited a higher random migration than the vector control cells (Fig. 7 A). We then tested whether angiostatin had any effect on the increased migratory rate of the MAE-angiomotin cells. Cells were preincubated with 5 μg/ml of angiostatin for 1 h before being added to the migration chamber. Angiostatin did not affect the random or bFGF-induced migration of the control cells. In contrast, angiostatin reduced the motility of MAE-angiomotin cells to background levels (Fig. 7 B) The inhibition of cell migration was dose dependent with maximal inhibition estimated at 500 ng/ml angiostatin (Fig. 7 C).


Angiomotin: an angiostatin binding protein that regulates endothelial cell migration and tube formation.

Troyanovsky B, Levchenko T, Månsson G, Matvijenko O, Holmgren L - J. Cell Biol. (2001)

Migration of MAE cells transfected with angiomotin or with the vector alone was studied in the modified Boyden chamber assay. (A) Analysis of spontaneous migration in the absence of chemotactic factor at different time points after start of the experiment. A higher number of angiomotin-expressing cells are migrating through the filter at all time points. Vertical axis, cells migrated per high power field (HPF). (B) Effect of angiostatin on migration of MAE-angiomotin and MAE vector cells with or without stimulation with bFGF. Cells were pretreated for 1 h with angiostatin as indicated. (C) Dose response of angiostatin-mediated inhibition of migration of angiomotin-transfected MAE cells. All samples were performed in quadruplicates (error bars = SD).
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Related In: Results  -  Collection

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Figure 7: Migration of MAE cells transfected with angiomotin or with the vector alone was studied in the modified Boyden chamber assay. (A) Analysis of spontaneous migration in the absence of chemotactic factor at different time points after start of the experiment. A higher number of angiomotin-expressing cells are migrating through the filter at all time points. Vertical axis, cells migrated per high power field (HPF). (B) Effect of angiostatin on migration of MAE-angiomotin and MAE vector cells with or without stimulation with bFGF. Cells were pretreated for 1 h with angiostatin as indicated. (C) Dose response of angiostatin-mediated inhibition of migration of angiomotin-transfected MAE cells. All samples were performed in quadruplicates (error bars = SD).
Mentions: The localization of angiomotin in the leading edge of migrating cells and the effect of angiostatin on FAK kinase activity in angiomotin-transfected cells, suggested that angiomotin might play a role in endothelial cell migration. To test this hypothesis, we studied the effect of angiomotin expression in an angiomotin-negative immortalized MAE cell line. MAE cells were infected with ecotropic retrovirus expressing full-length angiomotin or vector alone. Polyclonal cells were selected by puromycin treatment in order to remove noninfected cells. The effect of angiomotin on cell migration was tested in a modified Boyden chamber. Analysis of spontaneous migration in the absence of chemoattractant showed that angiomotin cells consistently exhibited a higher random migration than the vector control cells (Fig. 7 A). We then tested whether angiostatin had any effect on the increased migratory rate of the MAE-angiomotin cells. Cells were preincubated with 5 μg/ml of angiostatin for 1 h before being added to the migration chamber. Angiostatin did not affect the random or bFGF-induced migration of the control cells. In contrast, angiostatin reduced the motility of MAE-angiomotin cells to background levels (Fig. 7 B) The inhibition of cell migration was dose dependent with maximal inhibition estimated at 500 ng/ml angiostatin (Fig. 7 C).

Bottom Line: Transfected angiomotin as well as endogenous angiomotin protein were localized to the leading edge of migrating endothelial cells.Expression of angiomotin in endothelial cells resulted in increased cell migration, suggesting a stimulatory role of angiomotin in cell motility.These findings indicate that angiostatin inhibits cell migration by interfering with angiomotin activity in endothelial cells.

View Article: PubMed Central - PubMed

Affiliation: Center for Genomics Research and Microbiology and Tumor Biology Center, Karolinska Institutet, S-171 76 Stockholm, Sweden.

ABSTRACT
Angiostatin, a circulating inhibitor of angiogenesis, was identified by its ability to maintain dormancy of established metastases in vivo. In vitro, angiostatin inhibits endothelial cell migration, proliferation, and tube formation, and induces apoptosis in a cell type-specific manner. We have used a construct encoding the kringle domains 1--4 of angiostatin to screen a placenta yeast two-hybrid cDNA library for angiostatin-binding peptides. Here we report the identification of angiomotin, a novel protein that mediates angiostatin inhibition of migration and tube formation of endothelial cells. In vivo, angiomotin is expressed in the endothelial cells of capillaries as well as larger vessels of the human placenta. Upon expression of angiomotin in HeLa cells, angiomotin bound and internalized fluorescein-labeled angiostatin. Transfected angiomotin as well as endogenous angiomotin protein were localized to the leading edge of migrating endothelial cells. Expression of angiomotin in endothelial cells resulted in increased cell migration, suggesting a stimulatory role of angiomotin in cell motility. However, treatment with angiostatin inhibited migration and tube formation in angiomotin-expressing cells but not in control cells. These findings indicate that angiostatin inhibits cell migration by interfering with angiomotin activity in endothelial cells.

Show MeSH
Related in: MedlinePlus