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Y. Yang, H. Yang, Z. Wang, Z. Pan, F. Zhang, J. Yuan, J. Wolosin, P. Reinach; Functional Expression of Trpv1 in Human Corneal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6227.
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© ARVO (1962-2015); The Authors (2016-present)
Transient receptor potential channels in the vanilloid subfamily (TRPV) are expressed in human corneal epithelial cells. Activation of one of their subtypes, TRPV1, induces proinflammatory cytokine release.1 As TRPV1 activation by severe injury in mice contributes to inappropriate healing,2 we determined if part of this response may be accounted for by TRPV1 activation on stromal fibroblasts.
Fresh human cadaver corneas were obtained from New York Upstate Transplant Service. Stromal fibroblasts were isolated and cultured following a described method.3 Immunocytochemistry staining, quantitative RT-PCR and western blot assays probed for TRPV1 expression. Calcium imaging and ELISA assay determined if they exhibit functional TRPV1 expression.
Immunostaining identified TRPV1 plasma membrane and nuclear localization. Quantitative RT-PCR assay revealed that TRPV1 mRNA gene was up to nearly 1% of the level of actin expression. Western blots documented TRPV1 protein expression. TRPV1 is functional since exposure to a selective TRPV1 agonist, capsaicin (10 µM) induced a transient 1.8 -fold of the fura2 f340/f380 nm ratio in dye loaded fibroblasts. Such rises were blocked if the cells were preexposed first to a TRPV1 antagonist, capsazepine (1µM). Capsaicin (CAP) induced dose dependent increases in interleukin-8 (IL-8) release. After 24 h exposure to 10 µM CAP, they maximally rose 2.5 -fold above the control level.
TRPV1 is functionally expressed in cultured human stromal fibroblasts since CAP induced Ca 2+ signaling and increases in IL-8 chemoattractant release. This realization suggests that drug-induced suppression of TRPV1 expression on fibroblasts could improve in a clinical setting corneal wound healing outcome.
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