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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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Stressed-matrix contraction of primary DD cultures. The graph displays the percent FPCL contraction of five patient normal/control (C) and diseased (D) matched primary cell lines over an incubation period of 5 days. Data plotted represents the mean ± SEM (standard error of the mean) values of all five patient-matched primary cell lines. Representative images of contracting FPCLs are shown in the lower panel for the indicated time points after mechanical release of the FPCLs. Digital images of the contracting FPCLs were analysed using NIH Imaging software to determine lattice areas for the indicated time points. Statistical pair-wise analysis was performed using a student t-test analysis. *Statistical significance (P < 0.05) between disease and control FPCLs.
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Figure 4: Stressed-matrix contraction of primary DD cultures. The graph displays the percent FPCL contraction of five patient normal/control (C) and diseased (D) matched primary cell lines over an incubation period of 5 days. Data plotted represents the mean ± SEM (standard error of the mean) values of all five patient-matched primary cell lines. Representative images of contracting FPCLs are shown in the lower panel for the indicated time points after mechanical release of the FPCLs. Digital images of the contracting FPCLs were analysed using NIH Imaging software to determine lattice areas for the indicated time points. Statistical pair-wise analysis was performed using a student t-test analysis. *Statistical significance (P < 0.05) between disease and control FPCLs.

Mentions: FPCL cultures of DD primary cell lines not only provides a functional assay to quantify cell-mediated collagen contraction events, but also an 'in vivo-like' environment (i.e. three-dimensional collagen-rich matrix environment) to study cell-matrix interactions. Primary cell lines established from patient-matched disease fascia and adjacent uninvolved normal fascia tissue (n = 5 patient) were cultured as 'stressed-matrices' (Fig. 3). Following 2 days of attached-culture FPCLs were mechanically released from the sides of the culture dishes and digital images of the contracting gels collected over a 5 day incubation period. As shown in Fig. 4, area measurements of the contracting FPCL for all five patient-matched primary cell lines show that the disease cell cultures contracted collagen significantly more between 30 min. and 5 days compared to control FPCLs (*P < 0.05, student T-test). The differences in contraction rates is consistent with a number of earlier FPCL studies that demonstrated enhanced contractile activity of fibroblasts derived from DD lesions [47-49]. However, these studies utilized carpal tunnel fibroblasts as control cells and often used a floating-matrix rather than stressed-matrix contraction model to quantify cell-mediated collagen contraction, thus making it difficult to directly compare results.


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)

Stressed-matrix contraction of primary DD cultures. The graph displays the percent FPCL contraction of five patient normal/control (C) and diseased (D) matched primary cell lines over an incubation period of 5 days. Data plotted represents the mean ± SEM (standard error of the mean) values of all five patient-matched primary cell lines. Representative images of contracting FPCLs are shown in the lower panel for the indicated time points after mechanical release of the FPCLs. Digital images of the contracting FPCLs were analysed using NIH Imaging software to determine lattice areas for the indicated time points. Statistical pair-wise analysis was performed using a student t-test analysis. *Statistical significance (P < 0.05) between disease and control FPCLs.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Stressed-matrix contraction of primary DD cultures. The graph displays the percent FPCL contraction of five patient normal/control (C) and diseased (D) matched primary cell lines over an incubation period of 5 days. Data plotted represents the mean ± SEM (standard error of the mean) values of all five patient-matched primary cell lines. Representative images of contracting FPCLs are shown in the lower panel for the indicated time points after mechanical release of the FPCLs. Digital images of the contracting FPCLs were analysed using NIH Imaging software to determine lattice areas for the indicated time points. Statistical pair-wise analysis was performed using a student t-test analysis. *Statistical significance (P < 0.05) between disease and control FPCLs.
Mentions: FPCL cultures of DD primary cell lines not only provides a functional assay to quantify cell-mediated collagen contraction events, but also an 'in vivo-like' environment (i.e. three-dimensional collagen-rich matrix environment) to study cell-matrix interactions. Primary cell lines established from patient-matched disease fascia and adjacent uninvolved normal fascia tissue (n = 5 patient) were cultured as 'stressed-matrices' (Fig. 3). Following 2 days of attached-culture FPCLs were mechanically released from the sides of the culture dishes and digital images of the contracting gels collected over a 5 day incubation period. As shown in Fig. 4, area measurements of the contracting FPCL for all five patient-matched primary cell lines show that the disease cell cultures contracted collagen significantly more between 30 min. and 5 days compared to control FPCLs (*P < 0.05, student T-test). The differences in contraction rates is consistent with a number of earlier FPCL studies that demonstrated enhanced contractile activity of fibroblasts derived from DD lesions [47-49]. However, these studies utilized carpal tunnel fibroblasts as control cells and often used a floating-matrix rather than stressed-matrix contraction model to quantify cell-mediated collagen contraction, thus making it difficult to directly compare results.

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