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J. Chua, L. F. Seet, R. Su, M. S. Weaver, T. T. Wong; Increased SPARC Expression in 3D Collagen Gel Stressed Fibroblasts Is Required for the Development of the Myofibroblast Phenotype. Invest. Ophthalmol. Vis. Sci. 2009;50(13):467.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate SPARC (secreted protein acidic and rich in cysteine) expression in Tenons fibroblasts following stress induction using an in vitro collagen 3D stress model and its requirement in developing the myofibroblast phenotype.
Human Tenons fibroblasts were cultured in relaxed (control) and stressed 3D collagen I gels for 5 days. mRNA and protein expression for SPARC and alpha smooth muscle actin (ASMA) were determined for relaxed and stressed fibroblasts by RTPCR and western blotting respectively. Since SPARC signals through integrin-β1, we also determined integrin-β1 expression by western blotting. In addition, the culture medium was analyzed for collagen I secretion using enzyme-linked immunosorbent assay (ELISA). The experiments were replicated using SPARC knockout mouse fibroblasts.
SPARC expression was increased in the stressed Tenons fibroblasts relative to the relaxed ones. Corresponding increases in ASMA and integrin-β1 expression as well as collagen I secretion were also detected. There was no increase in ASMA, integrin-β1 and collagen I expression in the SPARC knockout mouse fibroblasts under stress, whereas the wild type fibroblasts responded in a similar manner to the human Tenons fibroblasts under both relaxed and stressed conditions.
SPARC is elevated under stress conditions generated in the 3D collagen stress model. Induction of SPARC is necessary for the expression of ASMA and collagen I, both of which are markers for the myofibroblast phenotype. Increased integrin-β1 expression under stress conditions corroborates the role of SPARC in the development of the myofibroblast phenotype.
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