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Elevated levels of beta-catenin and fibronectin in three-dimensional collagen cultures of Dupuytren's disease cells are regulated by tension in vitro.

Howard JC, Varallo VM, Ross DC, Roth JH, Faber KJ, Alman B, Gan BS - BMC Musculoskelet Disord (2003)

Bottom Line: Dupuytren's contracture or disease (DD) is a fibro-proliferative disease of the hand that results in the development of scar-like, collagen-rich disease cords within specific palmar fascia bands.Immunocytochemistry analysis also revealed extensive filamentous actin networks in disease cells, and enhanced attachment and spreading of disease cell in collagen matrices.The elevated levels of beta-catenin and Fn seen in collagen matrix cultures of disease fibroblasts can be regulated by changes in isometric tension.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Surgery, University of Western Ontario, London, Ontario, Canada. jhoward@lri.sjhc.london.on.ca

ABSTRACT

Background: Dupuytren's contracture or disease (DD) is a fibro-proliferative disease of the hand that results in the development of scar-like, collagen-rich disease cords within specific palmar fascia bands. Although the molecular pathology of DD is unknown, recent evidence suggests that beta-catenin may play a role. In this study, collagen matrix cultures of primary disease fibroblasts show enhanced contraction and isometric tension-dependent changes in beta-catenin and fibronectin levels.

Methods: Western blots of beta-catenin and fibronectin levels were determined for control and disease primary cell cultures grown within stressed- and attached-collagen matrices. Collagen contraction was quantified, and immunocytochemistry analysis of filamentous actin performed.

Results: Disease cells exhibited enhanced collagen contraction activity compared to control cells. Alterations in isometric tension of collagen matrices triggered dramatic changes in beta-catenin and fibronectin levels, including a transient increase in beta-catenin levels within disease cells, while fibronectin levels steadily decreased to levels below those seen in normal cell cultures. In contrast, both fibronectin and beta-catenin levels increased in attached collagen-matrix cultures of disease cells, while control cultures showed only increases in fibronectin levels. Immunocytochemistry analysis also revealed extensive filamentous actin networks in disease cells, and enhanced attachment and spreading of disease cell in collagen matrices.

Conclusion: Three-dimensional collagen matrix cultures of primary disease cell lines are more contractile and express a more extensive filamentous actin network than patient-matched control cultures. The elevated levels of beta-catenin and Fn seen in collagen matrix cultures of disease fibroblasts can be regulated by changes in isometric tension.

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Fibroblast Populated Collagen Lattice contraction assays. Fibroblast populated collagen lattice (FPCL) cultures have been used by many researchers to model cell-ECM interactions associated with wound contraction and DD [58]. As illustrated here three types of FPCLs have been used by investigators, including (A) Floating- (B) Attached- and (C) Stressed-matrices. Floating-matrices are mechanically released from the sides of the dishes immediately after gel polymerization (1 hour). In contrast, isometric tension is allowed to build up in cells placed in attached- and stressed-matrix cultures. In attached-matrices the contractile forces exerted by cells encounter mechanical resistance, which is thought to mimic 'granulation tissue contraction' (D, in vivo-like phenotypes), while cells seeded in stressed-matrices develop isometric tension during an initial attached period (1 to 2 days) that dissipates when the lattices are mechanically released.
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Figure 3: Fibroblast Populated Collagen Lattice contraction assays. Fibroblast populated collagen lattice (FPCL) cultures have been used by many researchers to model cell-ECM interactions associated with wound contraction and DD [58]. As illustrated here three types of FPCLs have been used by investigators, including (A) Floating- (B) Attached- and (C) Stressed-matrices. Floating-matrices are mechanically released from the sides of the dishes immediately after gel polymerization (1 hour). In contrast, isometric tension is allowed to build up in cells placed in attached- and stressed-matrix cultures. In attached-matrices the contractile forces exerted by cells encounter mechanical resistance, which is thought to mimic 'granulation tissue contraction' (D, in vivo-like phenotypes), while cells seeded in stressed-matrices develop isometric tension during an initial attached period (1 to 2 days) that dissipates when the lattices are mechanically released.

Mentions: Alterations in the extracellular matrix (ECM) are another important clinical feature of DD. Disease cords are largely composed of collagen type I, and have elevated levels of collagen type III compared to normal palmar fascia tissue [35-38]. Fibronectin (Fn), a well known extracellular glycoprotein that plays a vital role in numerous cell functions, including adhesion, proliferation, migration, and differentiation [39], is also prominently expressed in DD lesions, most notably within extracellular plaques, termed fibronexus, that are closely associated with DD myofibroblasts [40]. To-date, various Fn isoforms and their post-translational modified forms (ED-A, ED-B, oncofetal Fn), which are typically associated with tissues undergoing extensive proliferation and remodelling, have been documented in DD [41,42]. Recent studies in our lab have also documented aberrant expression of oncofetal Fn in primary cultures of DD lesions compared to patient-matched control fascia derived primary cultures (Varallo et al. unpublished). Although the importance of oncofetal production by DD cells are not clear the ability of these cells to actively modify the composition of the ECM appears to facilitate collagen contraction in vitro (Fig. 3) [43]. Nevertheless, the exact roles that these Fn isoforms, or indeed other ECM components play in the pathogenesis of DD is not known.


Elevated levels of beta-catenin and fibronectin in three-dimensional collagen cultures of Dupuytren's disease cells are regulated by tension in vitro.

Howard JC, Varallo VM, Ross DC, Roth JH, Faber KJ, Alman B, Gan BS - BMC Musculoskelet Disord (2003)

Fibroblast Populated Collagen Lattice contraction assays. Fibroblast populated collagen lattice (FPCL) cultures have been used by many researchers to model cell-ECM interactions associated with wound contraction and DD [58]. As illustrated here three types of FPCLs have been used by investigators, including (A) Floating- (B) Attached- and (C) Stressed-matrices. Floating-matrices are mechanically released from the sides of the dishes immediately after gel polymerization (1 hour). In contrast, isometric tension is allowed to build up in cells placed in attached- and stressed-matrix cultures. In attached-matrices the contractile forces exerted by cells encounter mechanical resistance, which is thought to mimic 'granulation tissue contraction' (D, in vivo-like phenotypes), while cells seeded in stressed-matrices develop isometric tension during an initial attached period (1 to 2 days) that dissipates when the lattices are mechanically released.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC183833&req=5

Figure 3: Fibroblast Populated Collagen Lattice contraction assays. Fibroblast populated collagen lattice (FPCL) cultures have been used by many researchers to model cell-ECM interactions associated with wound contraction and DD [58]. As illustrated here three types of FPCLs have been used by investigators, including (A) Floating- (B) Attached- and (C) Stressed-matrices. Floating-matrices are mechanically released from the sides of the dishes immediately after gel polymerization (1 hour). In contrast, isometric tension is allowed to build up in cells placed in attached- and stressed-matrix cultures. In attached-matrices the contractile forces exerted by cells encounter mechanical resistance, which is thought to mimic 'granulation tissue contraction' (D, in vivo-like phenotypes), while cells seeded in stressed-matrices develop isometric tension during an initial attached period (1 to 2 days) that dissipates when the lattices are mechanically released.
Mentions: Alterations in the extracellular matrix (ECM) are another important clinical feature of DD. Disease cords are largely composed of collagen type I, and have elevated levels of collagen type III compared to normal palmar fascia tissue [35-38]. Fibronectin (Fn), a well known extracellular glycoprotein that plays a vital role in numerous cell functions, including adhesion, proliferation, migration, and differentiation [39], is also prominently expressed in DD lesions, most notably within extracellular plaques, termed fibronexus, that are closely associated with DD myofibroblasts [40]. To-date, various Fn isoforms and their post-translational modified forms (ED-A, ED-B, oncofetal Fn), which are typically associated with tissues undergoing extensive proliferation and remodelling, have been documented in DD [41,42]. Recent studies in our lab have also documented aberrant expression of oncofetal Fn in primary cultures of DD lesions compared to patient-matched control fascia derived primary cultures (Varallo et al. unpublished). Although the importance of oncofetal production by DD cells are not clear the ability of these cells to actively modify the composition of the ECM appears to facilitate collagen contraction in vitro (Fig. 3) [43]. Nevertheless, the exact roles that these Fn isoforms, or indeed other ECM components play in the pathogenesis of DD is not known.

Bottom Line: Dupuytren's contracture or disease (DD) is a fibro-proliferative disease of the hand that results in the development of scar-like, collagen-rich disease cords within specific palmar fascia bands.Immunocytochemistry analysis also revealed extensive filamentous actin networks in disease cells, and enhanced attachment and spreading of disease cell in collagen matrices.The elevated levels of beta-catenin and Fn seen in collagen matrix cultures of disease fibroblasts can be regulated by changes in isometric tension.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Surgery, University of Western Ontario, London, Ontario, Canada. jhoward@lri.sjhc.london.on.ca

ABSTRACT

Background: Dupuytren's contracture or disease (DD) is a fibro-proliferative disease of the hand that results in the development of scar-like, collagen-rich disease cords within specific palmar fascia bands. Although the molecular pathology of DD is unknown, recent evidence suggests that beta-catenin may play a role. In this study, collagen matrix cultures of primary disease fibroblasts show enhanced contraction and isometric tension-dependent changes in beta-catenin and fibronectin levels.

Methods: Western blots of beta-catenin and fibronectin levels were determined for control and disease primary cell cultures grown within stressed- and attached-collagen matrices. Collagen contraction was quantified, and immunocytochemistry analysis of filamentous actin performed.

Results: Disease cells exhibited enhanced collagen contraction activity compared to control cells. Alterations in isometric tension of collagen matrices triggered dramatic changes in beta-catenin and fibronectin levels, including a transient increase in beta-catenin levels within disease cells, while fibronectin levels steadily decreased to levels below those seen in normal cell cultures. In contrast, both fibronectin and beta-catenin levels increased in attached collagen-matrix cultures of disease cells, while control cultures showed only increases in fibronectin levels. Immunocytochemistry analysis also revealed extensive filamentous actin networks in disease cells, and enhanced attachment and spreading of disease cell in collagen matrices.

Conclusion: Three-dimensional collagen matrix cultures of primary disease cell lines are more contractile and express a more extensive filamentous actin network than patient-matched control cultures. The elevated levels of beta-catenin and Fn seen in collagen matrix cultures of disease fibroblasts can be regulated by changes in isometric tension.

Show MeSH
Related in: MedlinePlus