Abstract
Purpose:
Little is understood about the mechanisms that drive scar-formation in diseases characterized by disorganized collagen. Corneal injury, regardless of cause, may ignite a dysregulated wound healing response that commonly leads to corneal scar-formation and loss of visual function. It is demonstrated that collagen-rich tissues similar to the stroma of the cornea, such as tendons, produce and secret neuropeptides, and that this production is increased in diseased states of disorganized collagen. Neurokinin A (NKA) is a neuropeptide that is shown to regulate diverse physiologic processes, including leukocyte-mediated migration. We hypothesize (1) that keratocytes of the corneal stroma express NKA and its preferred receptor, neurokinin receptor 2 (NK-2R), (2) that NKA is up-regulated following wound formation, and (3) that NKA is involved in cell migration during corneal wound healing.
Methods:
Healthy human corneal stroma cells were collected from the cornea bank in Umeå, Sweden. The expression of NKA and NK-2R in the stroma cells in vitro were analyzed both at the level of mRNA and protein using quantitative real time PCR (qPCR) and immunocytochemistry analyses, respectively. The expression changes of NKA following wound formation in vitro was analyzed with enzyme immunoassay. To test cell migration following NKA stimulation (10-6 M, 10-8 M), the CytoSelect™ cell migration and wound healing assay was used. To confirm that any NKA effect is specifically mediated via its receptor, an NK-2R antagonist (GR-159897 10-8M) was used.
Results:
The qPCR and immunocytochemistry analyses confirmed the expression of NKA and NK-2R in stromal cells of the human cornea in vitro. Furthermore, a significant up-regulation of NKA was observed in response to wound formation, whereas NK-2R expression was significantly decreased. Changes seen in cell migration in response to NKA treatment were not significant.
Conclusions:
Keratocytes of the human cornea may increase their NKA-production upon injury. It is also possible that such up-regulated NKA-production influences cell migration during corneal wound healing. The findings of this study warrant further studies in vitro and in vivo to confirm this.