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Fibrin binds to collagen and provides a bridge for αVβ3 integrin-dependent contraction of collagen gels.

Reyhani V, Seddigh P, Guss B, Gustafsson R, Rask L, Rubin K - Biochem. J. (2014)

Bottom Line: This allowed murine myoblast C2C12 cells to contract the collagenous composite gel via αVβ3 integrin.A specific competitive inhibitor blocking the Col-I-binding site for fibrinogen abolished the organization of fibrin into discernable fibrils, as well as the C2C12-mediated contraction of Col I gels.Our data show that fibrin can function as a linkage protein between Col I fibres and cells, and suggest that fibrin at inflammatory sites indirectly connects αVβ3 integrins to Col I fibres and thereby promotes cell-mediated contraction of collagenous tissue structures.

View Article: PubMed Central - PubMed

Affiliation: *Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, BMC Box 582, SE-751 23 Uppsala, Sweden.

ABSTRACT
The functional significance of fibrin deposits typically seen in inflammatory lesions, carcinomas and in healing wounds is not fully understood. In the present study, we demonstrate that fibrinogen/fibrin specifically bound to native Col I (collagen type I) and used the Col I fibre network as a base to provide a functional interface matrix that connects cells to the Col I fibres through αVβ3 integrins. This allowed murine myoblast C2C12 cells to contract the collagenous composite gel via αVβ3 integrin. We show that fibrinogen specifically bound to immobilized native Col I at the site known to bind matrix metalloproteinase-1, discoidin domain receptor-2 and fibronectin, and that binding had no effect on Col I fibrillation. A specific competitive inhibitor blocking the Col-I-binding site for fibrinogen abolished the organization of fibrin into discernable fibrils, as well as the C2C12-mediated contraction of Col I gels. Our data show that fibrin can function as a linkage protein between Col I fibres and cells, and suggest that fibrin at inflammatory sites indirectly connects αVβ3 integrins to Col I fibres and thereby promotes cell-mediated contraction of collagenous tissue structures.

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Related in: MedlinePlus

Fibrin triggers αVβ3 integrin-dependent cell-mediated contraction of collagen gels(A) Murine myoblast C2C12 cells that lack collagen-binding β1 integrins effectively contracted fibrin/Col I gels (Fib/Col I gel), but only marginally contracted pure Col I gels. Two selective αVβ3 integrin inhibitors, c-RGD (20 nM) and the anti-(mouse integrin-β3) antibody HMβ3 (10 μg/ml), abolished contraction. (B) C2C12 cells with forced expression of the α2β1 integrin (C2C12-α2β1 cells) contracted pure Col I gels to the same extent regardless of addition of 20 nM c-RGD or thrombin. (C) PAE cells, which express collagen-binding β1 integrins and αVβ3, showed additional contraction in response to the presence of fibrin in Col I gels. c-RGD at 20 nM repressed the fibrin-induced enhanced contraction. (D) C2C12 cells contracted fibrin/Col I gels in the presence of 50 and 100 μg/ml fibrin, but not at higher concentrations such as 500 and 1000 μg/ml fibrin. The values are after 4 h of contraction. (E) Thrombin did not enhance contraction of fibronectin/Col I (Fn/Col I) gels by C2C12 cells, whereas addition of 20 nM cRGD abolished contraction. (F) Similar fibrillation rates of Col I were observed in the absence or presence of fibrinogen (at three different concentrations). Values are means of at least three independent experiments performed in tetraplicates and error bars are S.E.M. Abs, absorbance; AU, arbitrary unit.
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Figure 1: Fibrin triggers αVβ3 integrin-dependent cell-mediated contraction of collagen gels(A) Murine myoblast C2C12 cells that lack collagen-binding β1 integrins effectively contracted fibrin/Col I gels (Fib/Col I gel), but only marginally contracted pure Col I gels. Two selective αVβ3 integrin inhibitors, c-RGD (20 nM) and the anti-(mouse integrin-β3) antibody HMβ3 (10 μg/ml), abolished contraction. (B) C2C12 cells with forced expression of the α2β1 integrin (C2C12-α2β1 cells) contracted pure Col I gels to the same extent regardless of addition of 20 nM c-RGD or thrombin. (C) PAE cells, which express collagen-binding β1 integrins and αVβ3, showed additional contraction in response to the presence of fibrin in Col I gels. c-RGD at 20 nM repressed the fibrin-induced enhanced contraction. (D) C2C12 cells contracted fibrin/Col I gels in the presence of 50 and 100 μg/ml fibrin, but not at higher concentrations such as 500 and 1000 μg/ml fibrin. The values are after 4 h of contraction. (E) Thrombin did not enhance contraction of fibronectin/Col I (Fn/Col I) gels by C2C12 cells, whereas addition of 20 nM cRGD abolished contraction. (F) Similar fibrillation rates of Col I were observed in the absence or presence of fibrinogen (at three different concentrations). Values are means of at least three independent experiments performed in tetraplicates and error bars are S.E.M. Abs, absorbance; AU, arbitrary unit.

Mentions: To investigate the effect of fibrin on the ability of cells to contract Col I gels in an αVβ3 integrin-dependent fashion, we used the mouse muscle satellite C2C12 cell line. C2C12 cells lack functional binding β1 integrins but express the αVβ3 integrin. In the absence of exogenously added stimulators, such as PDGF-BB, C2C12 cells are not capable of adhesion to native Col I or contraction of reconstituted Col I gels [36]. Therefore C2C12-mediated Col I gel contraction can serve as an in vitro model to investigate integrin αVβ3-mediated Col I gel contraction. In response to the addition of 50 μg/ml fibrinogen (equal to ~150 nM) into Col I gels (1 mg/ml collagen; equal to 3.3 μM), the C2C12 cells significantly contracted the resulting fibrin/Col I composite gels compared with pure Col I gels (Figure 1A). The selective inhibitor of the αVβ3 integrin, c-RGD peptide, at a concentration of 20 nM, which specifically inhibits αVβ3 [31], completely blocked the effective contraction of fibrin/Col I composite gels (Figure 1A). Similar to c-RGD, addition of 10 μg/ml HMβ3 antibody (inhibitor of the β3 integrin subunit) also completely inhibited the contraction (Figure 1A). As a control for potential toxicity of the c-RGD, C2C12-α2β1 cells were treated with c-RGD in the collagen gel contraction assay. C2C12-α2β1 cells express functional α2β1 and hence they can effectively contract pure Col I gels in the absence of exogenous stimulators. Addition of the same concentration of c-RGD had no effect on C2C12-α2β1-mediated Col I gel contraction, excluding the cytotoxicity of the peptide in the applied concentration (Figure 1B). In order to investigate whether fibrin-induced composite gel contraction could be observed with other cell lines, PAE cells were used both in composite gels and pure Col I gels. These cells express αVβ3 integrins, as well as collagen-binding β1 integrins. In presence of fibrin (50 μg/ml), PAE cells showed an enhanced gel contraction compared with contraction of pure Col I gels. Treatment of these cells with 20 nM c-RGD repressed the observed fibrin-enhanced gel contraction, emphasizing the dependency of fibrin-enhanced gel contraction on αVβ3 (Figure 1C).


Fibrin binds to collagen and provides a bridge for αVβ3 integrin-dependent contraction of collagen gels.

Reyhani V, Seddigh P, Guss B, Gustafsson R, Rask L, Rubin K - Biochem. J. (2014)

Fibrin triggers αVβ3 integrin-dependent cell-mediated contraction of collagen gels(A) Murine myoblast C2C12 cells that lack collagen-binding β1 integrins effectively contracted fibrin/Col I gels (Fib/Col I gel), but only marginally contracted pure Col I gels. Two selective αVβ3 integrin inhibitors, c-RGD (20 nM) and the anti-(mouse integrin-β3) antibody HMβ3 (10 μg/ml), abolished contraction. (B) C2C12 cells with forced expression of the α2β1 integrin (C2C12-α2β1 cells) contracted pure Col I gels to the same extent regardless of addition of 20 nM c-RGD or thrombin. (C) PAE cells, which express collagen-binding β1 integrins and αVβ3, showed additional contraction in response to the presence of fibrin in Col I gels. c-RGD at 20 nM repressed the fibrin-induced enhanced contraction. (D) C2C12 cells contracted fibrin/Col I gels in the presence of 50 and 100 μg/ml fibrin, but not at higher concentrations such as 500 and 1000 μg/ml fibrin. The values are after 4 h of contraction. (E) Thrombin did not enhance contraction of fibronectin/Col I (Fn/Col I) gels by C2C12 cells, whereas addition of 20 nM cRGD abolished contraction. (F) Similar fibrillation rates of Col I were observed in the absence or presence of fibrinogen (at three different concentrations). Values are means of at least three independent experiments performed in tetraplicates and error bars are S.E.M. Abs, absorbance; AU, arbitrary unit.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Fibrin triggers αVβ3 integrin-dependent cell-mediated contraction of collagen gels(A) Murine myoblast C2C12 cells that lack collagen-binding β1 integrins effectively contracted fibrin/Col I gels (Fib/Col I gel), but only marginally contracted pure Col I gels. Two selective αVβ3 integrin inhibitors, c-RGD (20 nM) and the anti-(mouse integrin-β3) antibody HMβ3 (10 μg/ml), abolished contraction. (B) C2C12 cells with forced expression of the α2β1 integrin (C2C12-α2β1 cells) contracted pure Col I gels to the same extent regardless of addition of 20 nM c-RGD or thrombin. (C) PAE cells, which express collagen-binding β1 integrins and αVβ3, showed additional contraction in response to the presence of fibrin in Col I gels. c-RGD at 20 nM repressed the fibrin-induced enhanced contraction. (D) C2C12 cells contracted fibrin/Col I gels in the presence of 50 and 100 μg/ml fibrin, but not at higher concentrations such as 500 and 1000 μg/ml fibrin. The values are after 4 h of contraction. (E) Thrombin did not enhance contraction of fibronectin/Col I (Fn/Col I) gels by C2C12 cells, whereas addition of 20 nM cRGD abolished contraction. (F) Similar fibrillation rates of Col I were observed in the absence or presence of fibrinogen (at three different concentrations). Values are means of at least three independent experiments performed in tetraplicates and error bars are S.E.M. Abs, absorbance; AU, arbitrary unit.
Mentions: To investigate the effect of fibrin on the ability of cells to contract Col I gels in an αVβ3 integrin-dependent fashion, we used the mouse muscle satellite C2C12 cell line. C2C12 cells lack functional binding β1 integrins but express the αVβ3 integrin. In the absence of exogenously added stimulators, such as PDGF-BB, C2C12 cells are not capable of adhesion to native Col I or contraction of reconstituted Col I gels [36]. Therefore C2C12-mediated Col I gel contraction can serve as an in vitro model to investigate integrin αVβ3-mediated Col I gel contraction. In response to the addition of 50 μg/ml fibrinogen (equal to ~150 nM) into Col I gels (1 mg/ml collagen; equal to 3.3 μM), the C2C12 cells significantly contracted the resulting fibrin/Col I composite gels compared with pure Col I gels (Figure 1A). The selective inhibitor of the αVβ3 integrin, c-RGD peptide, at a concentration of 20 nM, which specifically inhibits αVβ3 [31], completely blocked the effective contraction of fibrin/Col I composite gels (Figure 1A). Similar to c-RGD, addition of 10 μg/ml HMβ3 antibody (inhibitor of the β3 integrin subunit) also completely inhibited the contraction (Figure 1A). As a control for potential toxicity of the c-RGD, C2C12-α2β1 cells were treated with c-RGD in the collagen gel contraction assay. C2C12-α2β1 cells express functional α2β1 and hence they can effectively contract pure Col I gels in the absence of exogenous stimulators. Addition of the same concentration of c-RGD had no effect on C2C12-α2β1-mediated Col I gel contraction, excluding the cytotoxicity of the peptide in the applied concentration (Figure 1B). In order to investigate whether fibrin-induced composite gel contraction could be observed with other cell lines, PAE cells were used both in composite gels and pure Col I gels. These cells express αVβ3 integrins, as well as collagen-binding β1 integrins. In presence of fibrin (50 μg/ml), PAE cells showed an enhanced gel contraction compared with contraction of pure Col I gels. Treatment of these cells with 20 nM c-RGD repressed the observed fibrin-enhanced gel contraction, emphasizing the dependency of fibrin-enhanced gel contraction on αVβ3 (Figure 1C).

Bottom Line: This allowed murine myoblast C2C12 cells to contract the collagenous composite gel via αVβ3 integrin.A specific competitive inhibitor blocking the Col-I-binding site for fibrinogen abolished the organization of fibrin into discernable fibrils, as well as the C2C12-mediated contraction of Col I gels.Our data show that fibrin can function as a linkage protein between Col I fibres and cells, and suggest that fibrin at inflammatory sites indirectly connects αVβ3 integrins to Col I fibres and thereby promotes cell-mediated contraction of collagenous tissue structures.

View Article: PubMed Central - PubMed

Affiliation: *Department of Medical Biochemistry and Microbiology, Science for Life Laboratory, Uppsala University, BMC Box 582, SE-751 23 Uppsala, Sweden.

ABSTRACT
The functional significance of fibrin deposits typically seen in inflammatory lesions, carcinomas and in healing wounds is not fully understood. In the present study, we demonstrate that fibrinogen/fibrin specifically bound to native Col I (collagen type I) and used the Col I fibre network as a base to provide a functional interface matrix that connects cells to the Col I fibres through αVβ3 integrins. This allowed murine myoblast C2C12 cells to contract the collagenous composite gel via αVβ3 integrin. We show that fibrinogen specifically bound to immobilized native Col I at the site known to bind matrix metalloproteinase-1, discoidin domain receptor-2 and fibronectin, and that binding had no effect on Col I fibrillation. A specific competitive inhibitor blocking the Col-I-binding site for fibrinogen abolished the organization of fibrin into discernable fibrils, as well as the C2C12-mediated contraction of Col I gels. Our data show that fibrin can function as a linkage protein between Col I fibres and cells, and suggest that fibrin at inflammatory sites indirectly connects αVβ3 integrins to Col I fibres and thereby promotes cell-mediated contraction of collagenous tissue structures.

Show MeSH
Related in: MedlinePlus