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W. J. Foster, J. T. Davis, Q. Wen, P. K. Lam, D. C. Otteson, P. A. Janmey; Substrate Stiffness Influences Müller Cell Morphology, Proliferation, and the Expression of Genes That Are Known to Play a Role in Proliferative Vitreoretinopathy. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6090.
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The mechanical properties of tissue have increasingly become appreciated to be a significant factor in cell behavior. Matrix stiffness, in particular, is known to have marked influence on numerous cell types, including neurons, glia, hepatocytes, and mesenchymal stem cells. To better understand the mechanosensitivity of Müller cells and its association with the development of proliferative vitreoretinopathy (PVR) and related pathologic retinal conditions, we examined cell morphology, proliferation, and expression of extracellular matrix-related (ECM) genes in Müller cells when cultured on substrates of varying elastic modulus.
A conditionally immortalized mouse Müller cell line (ImM10) was cultured on polyacrylamide substrates with a calibrated Young’s modulus of 500 Pa, 1000 Pa, and 5000 Pa with glass as a control. A uniform coating of laminin was cross-linked to the substrates. Fluorescence and atomic force microscopy were utilized to study cell morphology and proliferation. Because of the known time-course of proliferative vitreoretinopathy (PVR), Müller cells were cultured for 21 days, and then harvested, mRNA was extracted, and real-time PCR was performed in triplicate. We then examined genes that demonstrated at least a 4-fold increase or decrease in expression of mRNA between different substrates that continuously increased or decreased with substrate elastic modulus.
Müller cells on firmer substrates were quantitatively found to proliferate more rapidly, to change in morphology, and to have increased cortical actin stiffness. Of the 85 ECM genes tested, two genes met our criteria. Ctgf (connective tissue growth factor, 22-fold change, R2=0.93) was upregulated on softer substrates and Col4a1 (collagen IV, 10-fold change, R2=0.84) expression was downregulated on softer substrates.
There are significant changes in ECM gene regulation in Müller cells as a function of the stiffness of the substrate. The ECM genes found have been previously noted to play a critical role in PVR. Substantial Ctgf expression has been previously found in PVR membranes, and collagen IV is known to play a role in the structure of PVR membranes. Tissue elastic modulus may play a role in the development and progression of retinal disorders.
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