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James D. Zieske, Xiaoqing Q. Guo, Dimitrios Karamichos, Audrey E. Hutcheon, Tomas Blanco; Localization Of Thrombospondin-1 In A 3D Stromal Model. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3426.
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Thrombospondin-1 (TSP-1) is a large glycoprotein frequently found in extracellular matrices (ECM). It has no known structural role, but has a wide variety of actions, including the activation of latent TGF-β and the inhibition of angiogenesis. We have previously demonstrated that TSP-1 is deposited in the provisional matrix immediately subjacent to the epithelium following wounding in both rat and mouse corneas. In the current study, we examined the expression and localization of TSP-1 by human corneal fibroblasts in a 3D model.
Human corneal fibroblasts (HCF) were cultured with fetal bovine serum (FBS) and stabilized ascorbate (sAsc) and were grown under three different conditions for 4 and 8 weeks: a) Controls: MEM+10%FBS+sAsc; b) MEM+10%FBS+sAsc+TGF-β1 (0.01-0.5ng/ml); and c) Control plus human corneal epithelial cells, which were added for the last week of culture. TSP-1 was localized using indirect-immunofluorescence microscopy (IF).
TSP-1 was present at low levels in the controls and localized throughout the construct in fibrillar arrays that appeared to alternate in directions. Addition of TGF-β1 to the cultures appeared to generate an increase in the amount of TSP-1 and was concentration dependent. Localization of TSP-1 was at highest levels in the top third of the culture and a luxurious pattern of fibrils was seen that resembled the pattern of collagen fibrils. When epithelial cells were added to the culture, the localization of TSP-1 resembled that seen following corneal wounding. TSP-1 levels were higher than in control and appeared to concentrate subjacent to the epithelium.
TSP-1 is secreted and organized into the ECM generated by HCF in a 3D model. TSP-1 is stimulated in a concentration dependent manner by TGF-β1 that also appears to alter the localization. Addition of epithelial cells results in a pattern of TSP-1 that is similar to the pattern seen following the addition of TGF-β1, suggesting that epithelial cells may be secreting TGF-β1. The localization pattern also suggests that TSP-1 associates with collagen fibrils.
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