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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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Mab HUIV26 reactivity with denatured/proteolyzed collagen IV in solid phase ELISA. Microtiter plates were coated with ECM components at a concentration of 25 μg/ml. (A) Mab HUIV26 was added (1 μg/ml), followed 1 h later with goat anti–mouse peroxidase–labeled IgG. All data was corrected for nonspecific binding of secondary antibody. Data bars represent the mean OD ± standard deviations from triplicate wells. (B) Microtiter wells were coated with triple helical collagen IV at 25 μg/ml. Concentrated (20×) HUVEC serum–free–conditioned media was added to the wells in the presence or absence of EDTA, aprotinin, or both and allowed to incubate for 1, 6, and 24 h. The plates were next washed, blocked, and incubated with Mab HUIV26 or control antibody. All data was corrected for nonspecific secondary antibody binding. Data bars represent the mean OD ± standard deviations from triplicate wells.
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fig1: Mab HUIV26 reactivity with denatured/proteolyzed collagen IV in solid phase ELISA. Microtiter plates were coated with ECM components at a concentration of 25 μg/ml. (A) Mab HUIV26 was added (1 μg/ml), followed 1 h later with goat anti–mouse peroxidase–labeled IgG. All data was corrected for nonspecific binding of secondary antibody. Data bars represent the mean OD ± standard deviations from triplicate wells. (B) Microtiter wells were coated with triple helical collagen IV at 25 μg/ml. Concentrated (20×) HUVEC serum–free–conditioned media was added to the wells in the presence or absence of EDTA, aprotinin, or both and allowed to incubate for 1, 6, and 24 h. The plates were next washed, blocked, and incubated with Mab HUIV26 or control antibody. All data was corrected for nonspecific secondary antibody binding. Data bars represent the mean OD ± standard deviations from triplicate wells.

Mentions: We sought to generate a Mab that would specifically recognize proteolyzed or denatured collagen IV, but would not recognize triple helical collagen IV. To produce this antibody, we used the technique of subtractive immunization in conjunction with pepsin-solubilized human collagen IV (Xu et al., 2000). Although pepsin-solubilized collagen IV is not completely representative of native collagen IV found in vivo, it does retain most of its triple helical structure. As shown in Fig. 1 A, Mab HUIV26 specifically reacts with thermally denatured collagen IV, while showing little if any reactivity to triple helical collagen IV. Mab HUIV26 showed no reactivity to other ECM components, including fibronectin, laminin, vitronectin, or fibrinogen. Importantly, Mab HUIV26 was shown to react with a cryptic site within collagen type IV from a variety of species, including human, mouse, chick, and rat (Xu et al., 2000). Moreover, Mab HUIV26 did not react with denatured forms of collagen I or the triple helical or denatured forms of other collagens, including types II, III, or V (Xu et al., 2000).


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)

Mab HUIV26 reactivity with denatured/proteolyzed collagen IV in solid phase ELISA. Microtiter plates were coated with ECM components at a concentration of 25 μg/ml. (A) Mab HUIV26 was added (1 μg/ml), followed 1 h later with goat anti–mouse peroxidase–labeled IgG. All data was corrected for nonspecific binding of secondary antibody. Data bars represent the mean OD ± standard deviations from triplicate wells. (B) Microtiter wells were coated with triple helical collagen IV at 25 μg/ml. Concentrated (20×) HUVEC serum–free–conditioned media was added to the wells in the presence or absence of EDTA, aprotinin, or both and allowed to incubate for 1, 6, and 24 h. The plates were next washed, blocked, and incubated with Mab HUIV26 or control antibody. All data was corrected for nonspecific secondary antibody binding. Data bars represent the mean OD ± standard deviations from triplicate wells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196184&req=5

fig1: Mab HUIV26 reactivity with denatured/proteolyzed collagen IV in solid phase ELISA. Microtiter plates were coated with ECM components at a concentration of 25 μg/ml. (A) Mab HUIV26 was added (1 μg/ml), followed 1 h later with goat anti–mouse peroxidase–labeled IgG. All data was corrected for nonspecific binding of secondary antibody. Data bars represent the mean OD ± standard deviations from triplicate wells. (B) Microtiter wells were coated with triple helical collagen IV at 25 μg/ml. Concentrated (20×) HUVEC serum–free–conditioned media was added to the wells in the presence or absence of EDTA, aprotinin, or both and allowed to incubate for 1, 6, and 24 h. The plates were next washed, blocked, and incubated with Mab HUIV26 or control antibody. All data was corrected for nonspecific secondary antibody binding. Data bars represent the mean OD ± standard deviations from triplicate wells.
Mentions: We sought to generate a Mab that would specifically recognize proteolyzed or denatured collagen IV, but would not recognize triple helical collagen IV. To produce this antibody, we used the technique of subtractive immunization in conjunction with pepsin-solubilized human collagen IV (Xu et al., 2000). Although pepsin-solubilized collagen IV is not completely representative of native collagen IV found in vivo, it does retain most of its triple helical structure. As shown in Fig. 1 A, Mab HUIV26 specifically reacts with thermally denatured collagen IV, while showing little if any reactivity to triple helical collagen IV. Mab HUIV26 showed no reactivity to other ECM components, including fibronectin, laminin, vitronectin, or fibrinogen. Importantly, Mab HUIV26 was shown to react with a cryptic site within collagen type IV from a variety of species, including human, mouse, chick, and rat (Xu et al., 2000). Moreover, Mab HUIV26 did not react with denatured forms of collagen I or the triple helical or denatured forms of other collagens, including types II, III, or V (Xu et al., 2000).

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