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Laura Soriano-Romaní, Laura Garcia-Posadas, Antonio López-García, Luminita I Paraoan, Yolanda Diebold; In vitro effect of thrombospondin-1 in ocular surface epithelial cell lines. Invest. Ophthalmol. Vis. Sci. 2014;55(13):498.
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© ARVO (1962-2015); The Authors (2016-present)
Thrombospondin-1 (TSP-1) is reported to be involved in ocular surface inflammation. Thus, our aim was to obtain an in vitro protein expression profile of TSP-1 and its receptor CD36 in corneal and conjunctival epithelial cell lines at basal conditions. Also, to analyze the effect of exogenously added TSP-1 on the expression of inflammatory and apoptotic markers.
Basal expression of TSP-1 and CD36 in Human Corneal Epithelial (HCE) and IOBA-Normal Human Conjunctiva (IOBA-NHC) cell lines was determined by enzime-linked immunosorbent assay, immunofluorescence and Western blotting, using human corneal keratocyte primary cells (hCK) as positive controls. Secreted TSP-1 by hCK was quantified and used for further experiments. HCE and IOBA-NHC cells were exposed either to human recombinant TSP-1 (1μg/mL) or secreted TSP-1 (1μg/mL) present in hCK-conditioned medium (hCK-CM), for 24 h. Unexposed cells were used as controls. After TSP-1 exposure, cells were maintained in serum-free medium for 24 h. After TSP-1 treatment and/or the serum-free period cell lysates were obtained and cell culture supernatants collected. Changes in protein expression of CD36, interleukin-6 (IL-6) and activated caspase-3 were analyzed. Statistical significance of p≤0.05 or tendency (p≤0.2) were considered.
At basal conditions, HCE cells secreted a significant amount of TSP-1, similar to that of hCK, while IOBA-NHC cells did not express the protein at detectable levels. Intracellular TSP-1 expression was consistent with quantified secreted levels. CD36 was immunodetected in both cell lines. In HCE cells, but not in IOBA-NHC cells, CD36 expression was significantly increased after TSP-1 exposure compared to that of controls. However, after serum-free period, CD36 levels were higher in recombinant TSP-1-exposed IOBA-NHC cells. IL-6 secreted levels measured after serum-free medium exposure tended to increase in hCK-CM-exposed HCE cells. Interestingly, IL-6 levels were significantly higher in hCK-CM-exposed IOBA-NHC cells. Activated caspase-3 was not immunodetected in any of cell lines at any time analyzed.
In vitro TSP-1 treatment modifies the expression of CD36 in corneal and conjunctival epithelial cells in a different time-dependent manner. Experiments to determine the potential influence of TSP-1 in pro-inflammatory and apoptotic conditions are ongoing using an in vitro model of ocular surface inflammation and apoptosis.
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