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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 epitope is associated with the expression and activation of MMP-2 in vivo. bFGF-treated CAMs or CAMs containing CS1 melanoma tumors were costained with either Mab HUIV26 and polyclonal anti–MMP-2 (top), or Mab HUIV26 and polyclonal antifactor VIII–related antigen (bottom). (A) Tissues were visualized by incubation with rhodamine- and FITC-conjugated secondary antibodies. Top, red indicates MMP-2 and green indicates HUIV26 cryptic epitope. Bottom, red indicates factor VIII staining of blood vessels, green indicates HUIV26 cryptic epitope, and yellow indicates colocalization. Photographs were taken at 200× magnification. (B) CAMs of 10-d-old embryos were stimulated with bFGF and total CAM lysates were prepared at 2, 24, 48, and 72 h. Top, gelatin zymogram of total CAM lysates after stimulation with bFGF. Bottom, dot blot of total CAM lysates. Total collagen IV (triple helical and denatured) was detected with a polyclonal antibody to both native and denatured collagen IV. Denatured collagen IV was detected with Mab HUIV26. (C) Microtiter plates were coated with triple helical collagen type IV (25 μg/ml). MMP-2 (500 ng/ml), tPA (6 U/ml, specific activity 700 μg/mg protein), or NT (control buffer) were incubated for 18 h. The wells were washed, blocked with BSA, and the HUIV26 cryptic sites were detected with Mab HUIV26 (1.0 μg/ml). Data bars represent the mean OD ± standard deviations from triplicate wells. Bars, 50.0 μm.
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fig3: Exposure of the HUIV26 cryptic epitope is associated with the expression and activation of MMP-2 in vivo. bFGF-treated CAMs or CAMs containing CS1 melanoma tumors were costained with either Mab HUIV26 and polyclonal anti–MMP-2 (top), or Mab HUIV26 and polyclonal antifactor VIII–related antigen (bottom). (A) Tissues were visualized by incubation with rhodamine- and FITC-conjugated secondary antibodies. Top, red indicates MMP-2 and green indicates HUIV26 cryptic epitope. Bottom, red indicates factor VIII staining of blood vessels, green indicates HUIV26 cryptic epitope, and yellow indicates colocalization. Photographs were taken at 200× magnification. (B) CAMs of 10-d-old embryos were stimulated with bFGF and total CAM lysates were prepared at 2, 24, 48, and 72 h. Top, gelatin zymogram of total CAM lysates after stimulation with bFGF. Bottom, dot blot of total CAM lysates. Total collagen IV (triple helical and denatured) was detected with a polyclonal antibody to both native and denatured collagen IV. Denatured collagen IV was detected with Mab HUIV26. (C) Microtiter plates were coated with triple helical collagen type IV (25 μg/ml). MMP-2 (500 ng/ml), tPA (6 U/ml, specific activity 700 μg/mg protein), or NT (control buffer) were incubated for 18 h. The wells were washed, blocked with BSA, and the HUIV26 cryptic sites were detected with Mab HUIV26 (1.0 μg/ml). Data bars represent the mean OD ± standard deviations from triplicate wells. Bars, 50.0 μm.

Mentions: Our previous studies indicated that incubation of normal human tissue with proteolytically active MMP-2, but not inactive pro-MMP-2, can expose the HUIV26 cryptic site in situ. Therefore, we examined whether the expression of MMP-2 was associated with the exposure of the HUIV26 epitope during angiogenesis in vivo. To facilitate these studies, angiogenesis was induced within the chick chorioallantoic membrane (CAM) by either purified bFGF or a single cell suspension of CS1 melanoma cells. CAM tissues were costained with either Mab HUIV26 or a polyclonal antibody to MMP-2, or a polyclonal antibody directed to factor VIII–related antigen. As shown in Fig. 3 A, top, MMP-2 (red) colocalized (yellow) with the cryptic HUIV26 epitope (green) in blood vessels from either cytokine- or tumor-induced angiogenesis. To confirm the exposure of the HUIV26 cryptic epitope within the basement membrane of blood vessels, similar CAM tissues were costained with polyclonal antibody directed to factor VIII–related antigen and Mab HUIV26. As shown in Fig. 3 A, bottom, the HUIV26 cryptic epitope was associated with both bFGF-induced and tumor-induced angiogenic blood vessels. To determine whether the proteolytically active form of MMP-2 correlated with exposure of the HUIV26 cryptic epitope, tissue lysates were prepared from these CAMs after bFGF stimulation and analyzed by both gelatin zymography and dot blot analysis. As shown in Fig. 3 B, top, bFGF treatment was associated with a time-dependent conversion of the latent 72-kD MMP-2 to its proteolytically active 62-kD species between 24 and 48 h after stimulation. These findings were consistent with our previously published results (Brooks et al., 1996). The gelatinolytic bands were confirmed to be pro (72 kD) and active (62 kD) MMP-2 by Western blot analysis (data not shown). Interestingly, dot blot analysis of these same lysates demonstrated the generation of the HUIV26 epitope between 24 and 48 h which correlated directly with the bFGF-associated activation of MMP-2. To confirm that activated MMP-2 can contribute to the exposure of the HUIV26 epitope, in vitro ELISA assays were conducted. Triple helical collagen type IV was coated on microtiter wells as described above. The wells were next incubated with either activated MMP-2, the serine protease tPA, or control buffer for 18 h. The wells were washed and the HUIV26 cryptic epitope was detected with Mab HUIV26. As shown in Fig. 3 C, incubation of triple helical collagen type IV with activated MMP-2 caused an approximately threefold increase in the exposure of the HUIV26 epitope as compared with either no treatment (control buffer), or the serine protease tPA. These findings provide further evidence that MMPs such as MMP-2 may contribute to the exposure of the HUIV26 epitope. However, these results do not rule out the likely contributions of other MMPs or serine proteases in the exposure of the HUIV26 cryptic site in vivo. Since MMP-9, the second major MMP capable of cleaving triple helical collagen IV, was not detected in the CAM lysates, it is likely, but not direct proof, that MMP-2 is at least one protease that may contribute to the generation of the HUIV26 epitope in the chick embryo model.


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 epitope is associated with the expression and activation of MMP-2 in vivo. bFGF-treated CAMs or CAMs containing CS1 melanoma tumors were costained with either Mab HUIV26 and polyclonal anti–MMP-2 (top), or Mab HUIV26 and polyclonal antifactor VIII–related antigen (bottom). (A) Tissues were visualized by incubation with rhodamine- and FITC-conjugated secondary antibodies. Top, red indicates MMP-2 and green indicates HUIV26 cryptic epitope. Bottom, red indicates factor VIII staining of blood vessels, green indicates HUIV26 cryptic epitope, and yellow indicates colocalization. Photographs were taken at 200× magnification. (B) CAMs of 10-d-old embryos were stimulated with bFGF and total CAM lysates were prepared at 2, 24, 48, and 72 h. Top, gelatin zymogram of total CAM lysates after stimulation with bFGF. Bottom, dot blot of total CAM lysates. Total collagen IV (triple helical and denatured) was detected with a polyclonal antibody to both native and denatured collagen IV. Denatured collagen IV was detected with Mab HUIV26. (C) Microtiter plates were coated with triple helical collagen type IV (25 μg/ml). MMP-2 (500 ng/ml), tPA (6 U/ml, specific activity 700 μg/mg protein), or NT (control buffer) were incubated for 18 h. The wells were washed, blocked with BSA, and the HUIV26 cryptic sites were detected with Mab HUIV26 (1.0 μg/ml). Data bars represent the mean OD ± standard deviations from triplicate wells. Bars, 50.0 μm.
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Related In: Results  -  Collection

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fig3: Exposure of the HUIV26 cryptic epitope is associated with the expression and activation of MMP-2 in vivo. bFGF-treated CAMs or CAMs containing CS1 melanoma tumors were costained with either Mab HUIV26 and polyclonal anti–MMP-2 (top), or Mab HUIV26 and polyclonal antifactor VIII–related antigen (bottom). (A) Tissues were visualized by incubation with rhodamine- and FITC-conjugated secondary antibodies. Top, red indicates MMP-2 and green indicates HUIV26 cryptic epitope. Bottom, red indicates factor VIII staining of blood vessels, green indicates HUIV26 cryptic epitope, and yellow indicates colocalization. Photographs were taken at 200× magnification. (B) CAMs of 10-d-old embryos were stimulated with bFGF and total CAM lysates were prepared at 2, 24, 48, and 72 h. Top, gelatin zymogram of total CAM lysates after stimulation with bFGF. Bottom, dot blot of total CAM lysates. Total collagen IV (triple helical and denatured) was detected with a polyclonal antibody to both native and denatured collagen IV. Denatured collagen IV was detected with Mab HUIV26. (C) Microtiter plates were coated with triple helical collagen type IV (25 μg/ml). MMP-2 (500 ng/ml), tPA (6 U/ml, specific activity 700 μg/mg protein), or NT (control buffer) were incubated for 18 h. The wells were washed, blocked with BSA, and the HUIV26 cryptic sites were detected with Mab HUIV26 (1.0 μg/ml). Data bars represent the mean OD ± standard deviations from triplicate wells. Bars, 50.0 μm.
Mentions: Our previous studies indicated that incubation of normal human tissue with proteolytically active MMP-2, but not inactive pro-MMP-2, can expose the HUIV26 cryptic site in situ. Therefore, we examined whether the expression of MMP-2 was associated with the exposure of the HUIV26 epitope during angiogenesis in vivo. To facilitate these studies, angiogenesis was induced within the chick chorioallantoic membrane (CAM) by either purified bFGF or a single cell suspension of CS1 melanoma cells. CAM tissues were costained with either Mab HUIV26 or a polyclonal antibody to MMP-2, or a polyclonal antibody directed to factor VIII–related antigen. As shown in Fig. 3 A, top, MMP-2 (red) colocalized (yellow) with the cryptic HUIV26 epitope (green) in blood vessels from either cytokine- or tumor-induced angiogenesis. To confirm the exposure of the HUIV26 cryptic epitope within the basement membrane of blood vessels, similar CAM tissues were costained with polyclonal antibody directed to factor VIII–related antigen and Mab HUIV26. As shown in Fig. 3 A, bottom, the HUIV26 cryptic epitope was associated with both bFGF-induced and tumor-induced angiogenic blood vessels. To determine whether the proteolytically active form of MMP-2 correlated with exposure of the HUIV26 cryptic epitope, tissue lysates were prepared from these CAMs after bFGF stimulation and analyzed by both gelatin zymography and dot blot analysis. As shown in Fig. 3 B, top, bFGF treatment was associated with a time-dependent conversion of the latent 72-kD MMP-2 to its proteolytically active 62-kD species between 24 and 48 h after stimulation. These findings were consistent with our previously published results (Brooks et al., 1996). The gelatinolytic bands were confirmed to be pro (72 kD) and active (62 kD) MMP-2 by Western blot analysis (data not shown). Interestingly, dot blot analysis of these same lysates demonstrated the generation of the HUIV26 epitope between 24 and 48 h which correlated directly with the bFGF-associated activation of MMP-2. To confirm that activated MMP-2 can contribute to the exposure of the HUIV26 epitope, in vitro ELISA assays were conducted. Triple helical collagen type IV was coated on microtiter wells as described above. The wells were next incubated with either activated MMP-2, the serine protease tPA, or control buffer for 18 h. The wells were washed and the HUIV26 cryptic epitope was detected with Mab HUIV26. As shown in Fig. 3 C, incubation of triple helical collagen type IV with activated MMP-2 caused an approximately threefold increase in the exposure of the HUIV26 epitope as compared with either no treatment (control buffer), or the serine protease tPA. These findings provide further evidence that MMPs such as MMP-2 may contribute to the exposure of the HUIV26 epitope. However, these results do not rule out the likely contributions of other MMPs or serine proteases in the exposure of the HUIV26 cryptic site in vivo. Since MMP-9, the second major MMP capable of cleaving triple helical collagen IV, was not detected in the CAM lysates, it is likely, but not direct proof, that MMP-2 is at least one protease that may contribute to the generation of the HUIV26 epitope in the chick embryo model.

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