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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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Subcellular localization of transfected and endogenous angiomotin. GFP-tagged angiomotin was expressed in NIH 3T3 cells using a retroviral expression system. Colocalization of GFP-angiomotin (a) with FAK (b) in the extending lamellipodium. (c) Immunofluorescence staining with a polyclonal antibody against angiomotin shows similar localization of endogenous angiomotin in a migrating HUVEC. (d) Control IgG from the same immunized animal. e and f shows the localization of angiomotin (e) to actin ruffles (f) and focal complexes in spreading HUVECs as visualized by phalloidin staining (arrows). Bars, 10 μm.
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Figure 5: Subcellular localization of transfected and endogenous angiomotin. GFP-tagged angiomotin was expressed in NIH 3T3 cells using a retroviral expression system. Colocalization of GFP-angiomotin (a) with FAK (b) in the extending lamellipodium. (c) Immunofluorescence staining with a polyclonal antibody against angiomotin shows similar localization of endogenous angiomotin in a migrating HUVEC. (d) Control IgG from the same immunized animal. e and f shows the localization of angiomotin (e) to actin ruffles (f) and focal complexes in spreading HUVECs as visualized by phalloidin staining (arrows). Bars, 10 μm.

Mentions: Next we assessed the subcellular localization of Angiomotin in transfected cells. The angiomotin open reading frame was fused to GFP in the COOH terminus. Angiomotin was expressed in NIH 3T3 cells using a retroviral expression vector. The cellular localization differed depending on whether cells were migrating or not. In migrating cells, angiomotin localized to the leading edge of the lamellipodia of the migrating cell. Double staining with antibodies against FAK showed overlapping localization of these proteins in migratory cells (Fig. 5, a and b). We also examined the distribution of endogenous angiomotin in human umbilical cord endothelial cells. Cells were incubated with angiomotin serum and rhodamine-labeled phalloidin in order to visualize F-actin. The angiomotin antibodies stained the leading edge of migrating cells (Fig. 5 c) and peripheral as well as circular ruffles in spreading cells. Angiomotin overlaps with F-actin staining in these structures, whereas no positive angiomotin staining was detected in stress fibers (Fig. 5e and Fig. f).


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)

Subcellular localization of transfected and endogenous angiomotin. GFP-tagged angiomotin was expressed in NIH 3T3 cells using a retroviral expression system. Colocalization of GFP-angiomotin (a) with FAK (b) in the extending lamellipodium. (c) Immunofluorescence staining with a polyclonal antibody against angiomotin shows similar localization of endogenous angiomotin in a migrating HUVEC. (d) Control IgG from the same immunized animal. e and f shows the localization of angiomotin (e) to actin ruffles (f) and focal complexes in spreading HUVECs as visualized by phalloidin staining (arrows). Bars, 10 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199208&req=5

Figure 5: Subcellular localization of transfected and endogenous angiomotin. GFP-tagged angiomotin was expressed in NIH 3T3 cells using a retroviral expression system. Colocalization of GFP-angiomotin (a) with FAK (b) in the extending lamellipodium. (c) Immunofluorescence staining with a polyclonal antibody against angiomotin shows similar localization of endogenous angiomotin in a migrating HUVEC. (d) Control IgG from the same immunized animal. e and f shows the localization of angiomotin (e) to actin ruffles (f) and focal complexes in spreading HUVECs as visualized by phalloidin staining (arrows). Bars, 10 μm.
Mentions: Next we assessed the subcellular localization of Angiomotin in transfected cells. The angiomotin open reading frame was fused to GFP in the COOH terminus. Angiomotin was expressed in NIH 3T3 cells using a retroviral expression vector. The cellular localization differed depending on whether cells were migrating or not. In migrating cells, angiomotin localized to the leading edge of the lamellipodia of the migrating cell. Double staining with antibodies against FAK showed overlapping localization of these proteins in migratory cells (Fig. 5, a and b). We also examined the distribution of endogenous angiomotin in human umbilical cord endothelial cells. Cells were incubated with angiomotin serum and rhodamine-labeled phalloidin in order to visualize F-actin. The angiomotin antibodies stained the leading edge of migrating cells (Fig. 5 c) and peripheral as well as circular ruffles in spreading cells. Angiomotin overlaps with F-actin staining in these structures, whereas no positive angiomotin staining was detected in stress fibers (Fig. 5e and Fig. f).

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