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Proteolytic exposure of a cryptic site within collagen type IV is required for angiogenesis and tumor growth in vivo.

Xu J, Rodriguez D, Petitclerc E, Kim JJ, Hangai M, Moon YS, Davis GE, Brooks PC, Yuen SM - J. Cell Biol. (2001)

Bottom Line: Exposure of this cryptic site was associated with angiogenic, but not quiescent, blood vessels and was required for angiogenesis in vivo.A monoclonal antibody (HUIV26) directed to this site disrupts integrin-dependent endothelial cell interactions and potently inhibits angiogenesis and tumor growth.Together, these studies suggest a novel mechanism by which proteolysis contributes to angiogenesis by exposing hidden regulatory elements within matrix-immobilized collagen type IV.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Kaplan Cancer Center, New York University School of Medicine, New York, NY 10016, USA.

ABSTRACT
Evidence is provided that proteolytic cleavage of collagen type IV results in the exposure of a functionally important cryptic site hidden within its triple helical structure. Exposure of this cryptic site was associated with angiogenic, but not quiescent, blood vessels and was required for angiogenesis in vivo. Exposure of the HUIV26 epitope was associated with a loss of alpha1beta1 integrin binding and the gain of alphavbeta3 binding. A monoclonal antibody (HUIV26) directed to this site disrupts integrin-dependent endothelial cell interactions and potently inhibits angiogenesis and tumor growth. Together, these studies suggest a novel mechanism by which proteolysis contributes to angiogenesis by exposing hidden regulatory elements within matrix-immobilized collagen type IV.

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Effects of Mab HUIV26 on human endothelial cell adhesion and migration. Microtiter plates (96 wells) and Transwell membranes were coated with triple helical or denatured colla- gen type IV (25 μg/ml). (A) Subconfluent HUVECs (105) were resuspended in adhesion buffer and allowed to attach in the presence or absence of Mab HUIV26 or an isotype-matched control antibody for 30 min. Nonattached cells were removed by washing and the attached cells were stained with crystal violet. (B) Subconfluent HUVECs (105) were resuspended in migration buffer and allowed to migrate in the presence or absence of Mab HUIV26 or an isotype-matched control antibody. Cells remaining on the top side of the membrane were removed and cells that had migrated to the under side were stained with crystal violet. Cell adhesion and migration was quantified by measuring the OD of eluted dye at 600 nm. Data bars represent the mean OD ± standard deviation from triplicate wells expressed as a percentage of control.
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fig6: Effects of Mab HUIV26 on human endothelial cell adhesion and migration. Microtiter plates (96 wells) and Transwell membranes were coated with triple helical or denatured colla- gen type IV (25 μg/ml). (A) Subconfluent HUVECs (105) were resuspended in adhesion buffer and allowed to attach in the presence or absence of Mab HUIV26 or an isotype-matched control antibody for 30 min. Nonattached cells were removed by washing and the attached cells were stained with crystal violet. (B) Subconfluent HUVECs (105) were resuspended in migration buffer and allowed to migrate in the presence or absence of Mab HUIV26 or an isotype-matched control antibody. Cells remaining on the top side of the membrane were removed and cells that had migrated to the under side were stained with crystal violet. Cell adhesion and migration was quantified by measuring the OD of eluted dye at 600 nm. Data bars represent the mean OD ± standard deviation from triplicate wells expressed as a percentage of control.

Mentions: It is possible that exposure of the HUIV26 cryptic epitope may contribute to angiogenesis in part by regulating endothelial cell–integrin interactions. To examine this possibility, we evaluated the effects of Mab HUIV26 on human endothelial cell adhesion to either triple helical or denatured human collagen IV. HUVECs were allowed to attach to immobilized triple helical or denatured collagen IV in the presence or absence of Mab HUIV26 or isotype-matched control antibody (50 μg/ml). As shown if Fig. 6 A, HUVECs readily attached to both triple helical and denatured collagen IV. In contrast, HUVEC adhesion to denatured collagen IV was inhibited by ∼60% in the presence of Mab HUIV26, while having little if any effect on adhesion to triple helical collagen IV. An isotype-matched control antibody had no effect of cell adhesion to either triple helical or denatured collagen IV.


Proteolytic exposure of a cryptic site within collagen type IV is required for angiogenesis and tumor growth in vivo.

Xu J, Rodriguez D, Petitclerc E, Kim JJ, Hangai M, Moon YS, Davis GE, Brooks PC, Yuen SM - J. Cell Biol. (2001)

Effects of Mab HUIV26 on human endothelial cell adhesion and migration. Microtiter plates (96 wells) and Transwell membranes were coated with triple helical or denatured colla- gen type IV (25 μg/ml). (A) Subconfluent HUVECs (105) were resuspended in adhesion buffer and allowed to attach in the presence or absence of Mab HUIV26 or an isotype-matched control antibody for 30 min. Nonattached cells were removed by washing and the attached cells were stained with crystal violet. (B) Subconfluent HUVECs (105) were resuspended in migration buffer and allowed to migrate in the presence or absence of Mab HUIV26 or an isotype-matched control antibody. Cells remaining on the top side of the membrane were removed and cells that had migrated to the under side were stained with crystal violet. Cell adhesion and migration was quantified by measuring the OD of eluted dye at 600 nm. Data bars represent the mean OD ± standard deviation from triplicate wells expressed as a percentage of control.
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Related In: Results  -  Collection

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fig6: Effects of Mab HUIV26 on human endothelial cell adhesion and migration. Microtiter plates (96 wells) and Transwell membranes were coated with triple helical or denatured colla- gen type IV (25 μg/ml). (A) Subconfluent HUVECs (105) were resuspended in adhesion buffer and allowed to attach in the presence or absence of Mab HUIV26 or an isotype-matched control antibody for 30 min. Nonattached cells were removed by washing and the attached cells were stained with crystal violet. (B) Subconfluent HUVECs (105) were resuspended in migration buffer and allowed to migrate in the presence or absence of Mab HUIV26 or an isotype-matched control antibody. Cells remaining on the top side of the membrane were removed and cells that had migrated to the under side were stained with crystal violet. Cell adhesion and migration was quantified by measuring the OD of eluted dye at 600 nm. Data bars represent the mean OD ± standard deviation from triplicate wells expressed as a percentage of control.
Mentions: It is possible that exposure of the HUIV26 cryptic epitope may contribute to angiogenesis in part by regulating endothelial cell–integrin interactions. To examine this possibility, we evaluated the effects of Mab HUIV26 on human endothelial cell adhesion to either triple helical or denatured human collagen IV. HUVECs were allowed to attach to immobilized triple helical or denatured collagen IV in the presence or absence of Mab HUIV26 or isotype-matched control antibody (50 μg/ml). As shown if Fig. 6 A, HUVECs readily attached to both triple helical and denatured collagen IV. In contrast, HUVEC adhesion to denatured collagen IV was inhibited by ∼60% in the presence of Mab HUIV26, while having little if any effect on adhesion to triple helical collagen IV. An isotype-matched control antibody had no effect of cell adhesion to either triple helical or denatured collagen IV.

Bottom Line: Exposure of this cryptic site was associated with angiogenic, but not quiescent, blood vessels and was required for angiogenesis in vivo.A monoclonal antibody (HUIV26) directed to this site disrupts integrin-dependent endothelial cell interactions and potently inhibits angiogenesis and tumor growth.Together, these studies suggest a novel mechanism by which proteolysis contributes to angiogenesis by exposing hidden regulatory elements within matrix-immobilized collagen type IV.

View Article: PubMed Central - PubMed

Affiliation: Department of Radiation Oncology, Kaplan Cancer Center, New York University School of Medicine, New York, NY 10016, USA.

ABSTRACT
Evidence is provided that proteolytic cleavage of collagen type IV results in the exposure of a functionally important cryptic site hidden within its triple helical structure. Exposure of this cryptic site was associated with angiogenic, but not quiescent, blood vessels and was required for angiogenesis in vivo. Exposure of the HUIV26 epitope was associated with a loss of alpha1beta1 integrin binding and the gain of alphavbeta3 binding. A monoclonal antibody (HUIV26) directed to this site disrupts integrin-dependent endothelial cell interactions and potently inhibits angiogenesis and tumor growth. Together, these studies suggest a novel mechanism by which proteolysis contributes to angiogenesis by exposing hidden regulatory elements within matrix-immobilized collagen type IV.

Show MeSH
Related in: MedlinePlus