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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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Related in: MedlinePlus

Exposure of the HUIV26 cryptic site within human tissue by proteolysis. Frozen sections of human tissue were mounted on glass slides. (A) Nonfixed normal human skin was incubated for 2 h with either control buffer, pro or activated MMP-2 (1.0 μg/ml), or HT1080 tumor–conditioned medium. The sections were costained with Mab HUIV26 and a polyclonal antibody directed to factor VIII–related antigen. Red indicates factor VIII staining of blood vessels, green indicates exposure of the HUIV26 cryptic epitope, and yellow indicates colocalization. Photo were taken at 630× under oil immersion (B) Normal human skin or malignant melanoma were costained with Mab HUIV26 and a polyclonal antibody directed to factor VIII–related antigen. Red indicates exposure of the HUIV26 cryptic site, green indicates human blood vessels, and yellow indicates colocalization. Photographs were taken at 200× magnification. (C) Representative example of human melanoma tumor tissue costained with Mab HUIV26 (green) and polyclonal antibody directed to factor VIII–related antigen (red), indicating that not all tumor vessels stain positive for HUIV26 epitope. Photos were taken at low power (200×). (D) Normal human retinal tissue or retina from subjects with diabetic retinopathy were costained with Mab HUIV26 and a polyclonal antibody to factor VIII–related antigen. Green indicates human blood vessels and red indicates HUIV26 cryptic sites. Arrows indicate nonspecific fluorescence of retinal pigmented epithelium due to auto-fluorescence of lipofusion. Photomicrographs were taken at 200× magnification. Bars, 50.0 μm.
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fig2: Exposure of the HUIV26 cryptic site within human tissue by proteolysis. Frozen sections of human tissue were mounted on glass slides. (A) Nonfixed normal human skin was incubated for 2 h with either control buffer, pro or activated MMP-2 (1.0 μg/ml), or HT1080 tumor–conditioned medium. The sections were costained with Mab HUIV26 and a polyclonal antibody directed to factor VIII–related antigen. Red indicates factor VIII staining of blood vessels, green indicates exposure of the HUIV26 cryptic epitope, and yellow indicates colocalization. Photo were taken at 630× under oil immersion (B) Normal human skin or malignant melanoma were costained with Mab HUIV26 and a polyclonal antibody directed to factor VIII–related antigen. Red indicates exposure of the HUIV26 cryptic site, green indicates human blood vessels, and yellow indicates colocalization. Photographs were taken at 200× magnification. (C) Representative example of human melanoma tumor tissue costained with Mab HUIV26 (green) and polyclonal antibody directed to factor VIII–related antigen (red), indicating that not all tumor vessels stain positive for HUIV26 epitope. Photos were taken at low power (200×). (D) Normal human retinal tissue or retina from subjects with diabetic retinopathy were costained with Mab HUIV26 and a polyclonal antibody to factor VIII–related antigen. Green indicates human blood vessels and red indicates HUIV26 cryptic sites. Arrows indicate nonspecific fluorescence of retinal pigmented epithelium due to auto-fluorescence of lipofusion. Photomicrographs were taken at 200× magnification. Bars, 50.0 μm.

Mentions: We assessed whether the HUIV26 cryptic epitope could be exposed within the basal lamina of blood vessels in vivo. Unfixed biopsy sections from normal human skin were incubated with either activated or proMMP-2, HT1080 tumor–conditioned medium, or control buffer. The tissues were costained with Mab HUIV26 (green) and a polyclonal antibody directed to factor VIII–related antigen (red), a known marker of blood vessels. As shown in Fig. 2 A, blood vessels (red) from normal human skin were readily detected. Little if any of the cryptic HUIV26 epitope (green) was detected within the vascular basement membranes or the surrounding interstitial matrix from tissues treated with either inactive proMMP-2 or control buffer (Fig. 2 A, top). In contrast, tissues treated with either proteolytically active MMP-2 (Fig. 2 A, bottom left) or HT1080 tumor–conditioned medium (Fig. 2 A, bottom right) demonstrated exposure of the HUIV26 cryptic epitope, as indicated by colocalization (yellow) due to overlap of the exposed HUIV26 epitope (green) and factor VIII–related antigen (red). Together, these findings provide further evidence that the HUIV26 cryptic sites could be exposed by proteolytic activity in a physiological tissue.


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)

Exposure of the HUIV26 cryptic site within human tissue by proteolysis. Frozen sections of human tissue were mounted on glass slides. (A) Nonfixed normal human skin was incubated for 2 h with either control buffer, pro or activated MMP-2 (1.0 μg/ml), or HT1080 tumor–conditioned medium. The sections were costained with Mab HUIV26 and a polyclonal antibody directed to factor VIII–related antigen. Red indicates factor VIII staining of blood vessels, green indicates exposure of the HUIV26 cryptic epitope, and yellow indicates colocalization. Photo were taken at 630× under oil immersion (B) Normal human skin or malignant melanoma were costained with Mab HUIV26 and a polyclonal antibody directed to factor VIII–related antigen. Red indicates exposure of the HUIV26 cryptic site, green indicates human blood vessels, and yellow indicates colocalization. Photographs were taken at 200× magnification. (C) Representative example of human melanoma tumor tissue costained with Mab HUIV26 (green) and polyclonal antibody directed to factor VIII–related antigen (red), indicating that not all tumor vessels stain positive for HUIV26 epitope. Photos were taken at low power (200×). (D) Normal human retinal tissue or retina from subjects with diabetic retinopathy were costained with Mab HUIV26 and a polyclonal antibody to factor VIII–related antigen. Green indicates human blood vessels and red indicates HUIV26 cryptic sites. Arrows indicate nonspecific fluorescence of retinal pigmented epithelium due to auto-fluorescence of lipofusion. Photomicrographs were taken at 200× magnification. Bars, 50.0 μm.
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Related In: Results  -  Collection

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fig2: Exposure of the HUIV26 cryptic site within human tissue by proteolysis. Frozen sections of human tissue were mounted on glass slides. (A) Nonfixed normal human skin was incubated for 2 h with either control buffer, pro or activated MMP-2 (1.0 μg/ml), or HT1080 tumor–conditioned medium. The sections were costained with Mab HUIV26 and a polyclonal antibody directed to factor VIII–related antigen. Red indicates factor VIII staining of blood vessels, green indicates exposure of the HUIV26 cryptic epitope, and yellow indicates colocalization. Photo were taken at 630× under oil immersion (B) Normal human skin or malignant melanoma were costained with Mab HUIV26 and a polyclonal antibody directed to factor VIII–related antigen. Red indicates exposure of the HUIV26 cryptic site, green indicates human blood vessels, and yellow indicates colocalization. Photographs were taken at 200× magnification. (C) Representative example of human melanoma tumor tissue costained with Mab HUIV26 (green) and polyclonal antibody directed to factor VIII–related antigen (red), indicating that not all tumor vessels stain positive for HUIV26 epitope. Photos were taken at low power (200×). (D) Normal human retinal tissue or retina from subjects with diabetic retinopathy were costained with Mab HUIV26 and a polyclonal antibody to factor VIII–related antigen. Green indicates human blood vessels and red indicates HUIV26 cryptic sites. Arrows indicate nonspecific fluorescence of retinal pigmented epithelium due to auto-fluorescence of lipofusion. Photomicrographs were taken at 200× magnification. Bars, 50.0 μm.
Mentions: We assessed whether the HUIV26 cryptic epitope could be exposed within the basal lamina of blood vessels in vivo. Unfixed biopsy sections from normal human skin were incubated with either activated or proMMP-2, HT1080 tumor–conditioned medium, or control buffer. The tissues were costained with Mab HUIV26 (green) and a polyclonal antibody directed to factor VIII–related antigen (red), a known marker of blood vessels. As shown in Fig. 2 A, blood vessels (red) from normal human skin were readily detected. Little if any of the cryptic HUIV26 epitope (green) was detected within the vascular basement membranes or the surrounding interstitial matrix from tissues treated with either inactive proMMP-2 or control buffer (Fig. 2 A, top). In contrast, tissues treated with either proteolytically active MMP-2 (Fig. 2 A, bottom left) or HT1080 tumor–conditioned medium (Fig. 2 A, bottom right) demonstrated exposure of the HUIV26 cryptic epitope, as indicated by colocalization (yellow) due to overlap of the exposed HUIV26 epitope (green) and factor VIII–related antigen (red). Together, these findings provide further evidence that the HUIV26 cryptic sites could be exposed by proteolytic activity in a physiological tissue.

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