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D. Karamichos, A. E. K. Hutcheon, X. Q. Guo, J. D. Zieske; A Novel 3D Scar Model for Assessing the Effect of TGF-β Isoforms in Corneal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2009;50(13):4538.
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© ARVO (1962-2015); The Authors (2016-present)
One of the most devastating outcomes of a corneal wound is the onset of corneal fibrosis leading to a scar. Corneal fibrosis is characterized by the presence of myofibroblasts and improper deposition of extracellular matrix components (ECM). TGF-β1 (T1) is known to stimulate overproduction and deposition of ECM components; however, two more isoforms are present: TGF-β2 and -β3 (T2 and T3). We have developed an in-vitro 3D scar model using human corneal fibroblasts (HCF) in order to investigate the effect of these three isoforms.
HCF were stimulated by a stable Vitamin C (VitC) derivative ± 0.1ng/ml of either T1, T2, or T3. HCF were grown for 4 and 8 weeks and analyzed by indirect-immunofluorescence microscopy.
All three TGF-β isoforms stimulated matrix production by ~3 times compared to controls. High levels of collagen III with T1, T2 and T3 were seen; however, the timing and the localization varied: While T1 did not show expression of collagen III until week 8, T3 lost the expression by that time. T2 showed expression in the top-most layers at both time points. Alpha-smooth muscle-actin (SMA) expression was seen in all constructs, with the number of myofibroblasts being far greater in T1 and T2. At week 4, Thrombospondin-1 (TSP-1) was found in the middle of the constructs; in the top layers for T3, in the bottom layers for T2, and was absent in T1. By week 8, the control and T3 samples showed similar expression to week 4; however, T1 showed an increase. Finally, fibronectin (cFN) was absent from the controls at week 4; however, intense cFN expression appeared in T1 and T2 in the top layers; whereas, cFN expression was present almost throughout the T3 construct. At week 8, none of the constructs expressed cFN.
All three isoforms stimulate matrix deposition in this 3D model; however, differences in the timing, as well as, the localization of specific proteins associated with scarring, such as collagen III and cFN were observed. The development of a human corneal scar model will allow for the examination of the regulation of corneal scar matrix formation, secretion, and organization.
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