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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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Angiomotin is expressed in endothelial cells in vivo. (A) Northern blot analysis of angiomotin expression in fetal and adult human tissues. 2 μg of poly A+ RNA was analyzed for expression of angiomotin using a 1-kb probe from the 5′ region. Two detectable transcripts were detected in both fetal and adult tissues (multitissue blot from CLONTECH Laboratories, Inc.; H, heart; Br, brain; Pl placenta; Lu, lung; Li, liver; Ki, kidney; Pa, pancreas; M, marker). (B) Western blot analysis using immunoaffinity-purified rabbit polyclonal antibodies against angiomotin. The antibodies detected a band with an approximate molecular mass of 75 kD in angiomotin-transfected NIH 3T3 (Am.) cells but not in the vector (vect.) control. (C) Immunohistochemical localization of ABP-1 protein in human placenta. Paraffin-embedded placental tissue from term placenta was stained with immunoaffinity-purified angiomotin polyclonal antibodies. (C, a) Shows angiomotin protein in endothelium of larger vessels in a placental villus. (C, b) Immunostaining using a monoclonal antibody against CD34, an endothelial marker. (C, c) Detection of angiomotin protein in capillaries of placental microvilli. (C, d) IgG negative control. (D) Expression of angiomotin in a dermal Kaposis sarcoma. (D, a) Shows angiomotin staining of a blood vessel within the Kaposis lesion. (D, b) CD34 staining of a consecutive section of D, panel a. (D, c) Angiomotin-negative dermal blood vessel (arrow) in the normal tissue adjacent to the Kaposis lesion. (D, d) Same as D, panel c, but stained with CD34. D, panels e and f, are negative controls stained with nonimmune rabbit IgG as a negative control. Bars, 40 μm.
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Figure 4: Angiomotin is expressed in endothelial cells in vivo. (A) Northern blot analysis of angiomotin expression in fetal and adult human tissues. 2 μg of poly A+ RNA was analyzed for expression of angiomotin using a 1-kb probe from the 5′ region. Two detectable transcripts were detected in both fetal and adult tissues (multitissue blot from CLONTECH Laboratories, Inc.; H, heart; Br, brain; Pl placenta; Lu, lung; Li, liver; Ki, kidney; Pa, pancreas; M, marker). (B) Western blot analysis using immunoaffinity-purified rabbit polyclonal antibodies against angiomotin. The antibodies detected a band with an approximate molecular mass of 75 kD in angiomotin-transfected NIH 3T3 (Am.) cells but not in the vector (vect.) control. (C) Immunohistochemical localization of ABP-1 protein in human placenta. Paraffin-embedded placental tissue from term placenta was stained with immunoaffinity-purified angiomotin polyclonal antibodies. (C, a) Shows angiomotin protein in endothelium of larger vessels in a placental villus. (C, b) Immunostaining using a monoclonal antibody against CD34, an endothelial marker. (C, c) Detection of angiomotin protein in capillaries of placental microvilli. (C, d) IgG negative control. (D) Expression of angiomotin in a dermal Kaposis sarcoma. (D, a) Shows angiomotin staining of a blood vessel within the Kaposis lesion. (D, b) CD34 staining of a consecutive section of D, panel a. (D, c) Angiomotin-negative dermal blood vessel (arrow) in the normal tissue adjacent to the Kaposis lesion. (D, d) Same as D, panel c, but stained with CD34. D, panels e and f, are negative controls stained with nonimmune rabbit IgG as a negative control. Bars, 40 μm.

Mentions: We investigated the expression of angiomotin mRNA by Northern blot analysis of mRNA derived from fetal and adult human tissues. Hybridization with a 1-kb probe from nucleotides 953–2,152 showed positive hybridization in both fetal and adult tissues (Fig. 4 A). Two mRNA bands with approximate sizes of 6.5 and 7.5 kb showed the most abundant expression in placenta and skeletal muscle but were detectable in all analyzed tissues. We also analyzed the expression of Angiomotin mRNA in 15 different cell lines by real time PCR. Highest expression could be detected in human dermal microvascular and HUVECs (Table ). To study angiomotin protein expression in vivo, we raised a polyclonal antiserum directed against the recombinant protein that was derived from the sequence identified from the initial yeast two-hybrid clone. The resulting antiangiomotin serum was subsequently affinity purified against the immunizing peptide over an affinity column. The serum and affinity-purified antibodies detected a protein with a molecular mass of 75 kD in angiomotin-transfected NIH 3T3 cells but not in cells transfected with vector alone (Fig. 4 B).


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)

Angiomotin is expressed in endothelial cells in vivo. (A) Northern blot analysis of angiomotin expression in fetal and adult human tissues. 2 μg of poly A+ RNA was analyzed for expression of angiomotin using a 1-kb probe from the 5′ region. Two detectable transcripts were detected in both fetal and adult tissues (multitissue blot from CLONTECH Laboratories, Inc.; H, heart; Br, brain; Pl placenta; Lu, lung; Li, liver; Ki, kidney; Pa, pancreas; M, marker). (B) Western blot analysis using immunoaffinity-purified rabbit polyclonal antibodies against angiomotin. The antibodies detected a band with an approximate molecular mass of 75 kD in angiomotin-transfected NIH 3T3 (Am.) cells but not in the vector (vect.) control. (C) Immunohistochemical localization of ABP-1 protein in human placenta. Paraffin-embedded placental tissue from term placenta was stained with immunoaffinity-purified angiomotin polyclonal antibodies. (C, a) Shows angiomotin protein in endothelium of larger vessels in a placental villus. (C, b) Immunostaining using a monoclonal antibody against CD34, an endothelial marker. (C, c) Detection of angiomotin protein in capillaries of placental microvilli. (C, d) IgG negative control. (D) Expression of angiomotin in a dermal Kaposis sarcoma. (D, a) Shows angiomotin staining of a blood vessel within the Kaposis lesion. (D, b) CD34 staining of a consecutive section of D, panel a. (D, c) Angiomotin-negative dermal blood vessel (arrow) in the normal tissue adjacent to the Kaposis lesion. (D, d) Same as D, panel c, but stained with CD34. D, panels e and f, are negative controls stained with nonimmune rabbit IgG as a negative control. Bars, 40 μm.
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Figure 4: Angiomotin is expressed in endothelial cells in vivo. (A) Northern blot analysis of angiomotin expression in fetal and adult human tissues. 2 μg of poly A+ RNA was analyzed for expression of angiomotin using a 1-kb probe from the 5′ region. Two detectable transcripts were detected in both fetal and adult tissues (multitissue blot from CLONTECH Laboratories, Inc.; H, heart; Br, brain; Pl placenta; Lu, lung; Li, liver; Ki, kidney; Pa, pancreas; M, marker). (B) Western blot analysis using immunoaffinity-purified rabbit polyclonal antibodies against angiomotin. The antibodies detected a band with an approximate molecular mass of 75 kD in angiomotin-transfected NIH 3T3 (Am.) cells but not in the vector (vect.) control. (C) Immunohistochemical localization of ABP-1 protein in human placenta. Paraffin-embedded placental tissue from term placenta was stained with immunoaffinity-purified angiomotin polyclonal antibodies. (C, a) Shows angiomotin protein in endothelium of larger vessels in a placental villus. (C, b) Immunostaining using a monoclonal antibody against CD34, an endothelial marker. (C, c) Detection of angiomotin protein in capillaries of placental microvilli. (C, d) IgG negative control. (D) Expression of angiomotin in a dermal Kaposis sarcoma. (D, a) Shows angiomotin staining of a blood vessel within the Kaposis lesion. (D, b) CD34 staining of a consecutive section of D, panel a. (D, c) Angiomotin-negative dermal blood vessel (arrow) in the normal tissue adjacent to the Kaposis lesion. (D, d) Same as D, panel c, but stained with CD34. D, panels e and f, are negative controls stained with nonimmune rabbit IgG as a negative control. Bars, 40 μm.
Mentions: We investigated the expression of angiomotin mRNA by Northern blot analysis of mRNA derived from fetal and adult human tissues. Hybridization with a 1-kb probe from nucleotides 953–2,152 showed positive hybridization in both fetal and adult tissues (Fig. 4 A). Two mRNA bands with approximate sizes of 6.5 and 7.5 kb showed the most abundant expression in placenta and skeletal muscle but were detectable in all analyzed tissues. We also analyzed the expression of Angiomotin mRNA in 15 different cell lines by real time PCR. Highest expression could be detected in human dermal microvascular and HUVECs (Table ). To study angiomotin protein expression in vivo, we raised a polyclonal antiserum directed against the recombinant protein that was derived from the sequence identified from the initial yeast two-hybrid clone. The resulting antiangiomotin serum was subsequently affinity purified against the immunizing peptide over an affinity column. The serum and affinity-purified antibodies detected a protein with a molecular mass of 75 kD in angiomotin-transfected NIH 3T3 cells but not in cells transfected with vector alone (Fig. 4 B).

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