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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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Binding of FITC-labeled angiostatin to living cells in culture. Cells were incubated with 10 μg/ml FITC-labeled angiostatin at 4°C for 60 min. The cells were placed in 37°C incubator 15 min before washing in PBS and fixation in 3.7% formaldehyde. Angiostatin binds to and becomes internalized in bovine microcapillary endothelial (BME) cells resulting in a patchy staining of endosomes (top left). In contrast, angiostatin binds to the matrix of human fetal fibroblasts but is not internalized (top right). HeLa cells transfected with angiomotin bind and internalize angiostatin in a similar pattern to that of endothelial cells (middle left). HeLa-Ctrl cells are transfected with an empty vector and were negative for angiostatin binding (middle right). The bottom panel shows colocalization of FITC-angiostatin (ang.) with angiomotin during internalization after surface binding to HeLa cells transfected with angiomotin. Angiomotin was visualized by immunofluorescent staining using immunoaffinity-purified rabbit polyclonal antibodies against angiomotin as described in Materials and Methods. Bars, 10 μm.
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Figure 3: Binding of FITC-labeled angiostatin to living cells in culture. Cells were incubated with 10 μg/ml FITC-labeled angiostatin at 4°C for 60 min. The cells were placed in 37°C incubator 15 min before washing in PBS and fixation in 3.7% formaldehyde. Angiostatin binds to and becomes internalized in bovine microcapillary endothelial (BME) cells resulting in a patchy staining of endosomes (top left). In contrast, angiostatin binds to the matrix of human fetal fibroblasts but is not internalized (top right). HeLa cells transfected with angiomotin bind and internalize angiostatin in a similar pattern to that of endothelial cells (middle left). HeLa-Ctrl cells are transfected with an empty vector and were negative for angiostatin binding (middle right). The bottom panel shows colocalization of FITC-angiostatin (ang.) with angiomotin during internalization after surface binding to HeLa cells transfected with angiomotin. Angiomotin was visualized by immunofluorescent staining using immunoaffinity-purified rabbit polyclonal antibodies against angiomotin as described in Materials and Methods. Bars, 10 μm.

Mentions: Next we assessed whether the expression of angiomotin in cells in vitro would confer binding of fluorescein-labeled angiostatin. In our experience, iodination of angiostatin results in loss of activity whereas activity can be retained when fluorescein labeling (FITC) is used (data not shown). Primary bovine endothelial cells were incubated with FITC-angiostatin for 60 min at 4°C before incubation at 37°C for 15 min. Incubation at 37°C resulted in aggregation and internalization of angiostatin (Fig. 3). This pattern of internalization is not detected in fibroblasts where angiostatin only binds to the extracellular matrix. HeLa cells were used for binding experiments, as they do not express angiomotin and exhibit little or no background angiostatin binding (Fig. 3). HeLa cells transfected with angiomotin bound and internalized angiostatin in a similar pattern to that of endothelial cells whereas vector control cells were negative. In addition, internalized FITC-angiostatin colocalized with angiomotin in intracellular vesicles (Fig. 3, bottom).


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)

Binding of FITC-labeled angiostatin to living cells in culture. Cells were incubated with 10 μg/ml FITC-labeled angiostatin at 4°C for 60 min. The cells were placed in 37°C incubator 15 min before washing in PBS and fixation in 3.7% formaldehyde. Angiostatin binds to and becomes internalized in bovine microcapillary endothelial (BME) cells resulting in a patchy staining of endosomes (top left). In contrast, angiostatin binds to the matrix of human fetal fibroblasts but is not internalized (top right). HeLa cells transfected with angiomotin bind and internalize angiostatin in a similar pattern to that of endothelial cells (middle left). HeLa-Ctrl cells are transfected with an empty vector and were negative for angiostatin binding (middle right). The bottom panel shows colocalization of FITC-angiostatin (ang.) with angiomotin during internalization after surface binding to HeLa cells transfected with angiomotin. Angiomotin was visualized by immunofluorescent staining using immunoaffinity-purified rabbit polyclonal antibodies against angiomotin as described in Materials and Methods. Bars, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2199208&req=5

Figure 3: Binding of FITC-labeled angiostatin to living cells in culture. Cells were incubated with 10 μg/ml FITC-labeled angiostatin at 4°C for 60 min. The cells were placed in 37°C incubator 15 min before washing in PBS and fixation in 3.7% formaldehyde. Angiostatin binds to and becomes internalized in bovine microcapillary endothelial (BME) cells resulting in a patchy staining of endosomes (top left). In contrast, angiostatin binds to the matrix of human fetal fibroblasts but is not internalized (top right). HeLa cells transfected with angiomotin bind and internalize angiostatin in a similar pattern to that of endothelial cells (middle left). HeLa-Ctrl cells are transfected with an empty vector and were negative for angiostatin binding (middle right). The bottom panel shows colocalization of FITC-angiostatin (ang.) with angiomotin during internalization after surface binding to HeLa cells transfected with angiomotin. Angiomotin was visualized by immunofluorescent staining using immunoaffinity-purified rabbit polyclonal antibodies against angiomotin as described in Materials and Methods. Bars, 10 μm.
Mentions: Next we assessed whether the expression of angiomotin in cells in vitro would confer binding of fluorescein-labeled angiostatin. In our experience, iodination of angiostatin results in loss of activity whereas activity can be retained when fluorescein labeling (FITC) is used (data not shown). Primary bovine endothelial cells were incubated with FITC-angiostatin for 60 min at 4°C before incubation at 37°C for 15 min. Incubation at 37°C resulted in aggregation and internalization of angiostatin (Fig. 3). This pattern of internalization is not detected in fibroblasts where angiostatin only binds to the extracellular matrix. HeLa cells were used for binding experiments, as they do not express angiomotin and exhibit little or no background angiostatin binding (Fig. 3). HeLa cells transfected with angiomotin bound and internalized angiostatin in a similar pattern to that of endothelial cells whereas vector control cells were negative. In addition, internalized FITC-angiostatin colocalized with angiomotin in intracellular vesicles (Fig. 3, bottom).

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