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Peter Boman, Sandrine Le Roux, Patrik Danielson, ; Substance P potentiates Anti-Fas induced apoptosis in human corneal fibroblasts. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5227.
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Substance P (SP), a neuropeptide originally associated with the nervous system, has also been found in non-neuronal cells of collagenous tissues like tendons, where its proliferative effect has been studied. In the cornea, mRNA for SP and its preferred receptor, the Neurokinin 1 Receptor (NK-1 R), has been found in both epithelial and stromal cells (keratocytes), but SP’s role in the corneal stroma has not been extensively investigated. In the process of corneal wound healing, one of the first events occurring is the apoptosis of the keratocytes around the wound area. It is known that SP has apoptois-modulating effects in other cell types, where it protects against apoptosis induced by the Fas ligand. This effect of SP in Anti-Fas induced apoptosis involves phosphorylation of the protein Akt. In this study we hypothesize that SP impacts corneal fibroblast turn-over, which is important in the wound healing process. The aims were to study 1) if Anti-Fas treatment is a good apoptosis model for human corneal fibroblasts, 2) the role of SP in Anti-Fas induced apoptosis, and 3) by which mechanisms SP mediates its possible response.
Primary fibroblast cultures were derived from healthy corneal stroma, obtained from donated transplantation graft leftovers. Immunocytochemistry was performed to assess the presence of SP, NK-1 R, and the Fas receptor (FasR). Apoptosis markers, Akt and NK-1 R were investigated by western blot. Gene expression was performed using qPCR.
Gene expression of SP and NK-1 R in human corneal fibroblasts was verified in the primary cultures. The presence of SP was also confirmed at the protein level using immunocytochemistry. Western blot and immunocytochemistry showed that the full length isoform of NK-1 R is the dominant isoform. Anti-Fas treatment induced apoptosis in the cells, and SP augmented this response, as seen by an increase in activated apoptotic enzymes. Treating cells with SP also lead to a decreased amount of phosphorylated/activated Akt. The effects of SP were reduced with a specific NK-1 R antagonist.
SP is produced by human corneal fibroblasts. Anti-Fas treatment induces apoptosis in these cells and SP augments this response through a NK-1 R specific pathway, by reducing phosphorylation of Akt. This suggests that SP may play a role in the apoptosis occurring in corneal wound healing.
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