June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Acetylcholine induces proliferation of keratocytes through activation of muscarinic receptors.
Author Affiliations & Notes
  • Marta Sloniecka
    Integrative Medical Biology, Umea University, Umea, Sweden
  • Ludvig J. Backman
    Integrative Medical Biology, Umea University, Umea, Sweden
  • Patrik Danielson
    Integrative Medical Biology, Umea University, Umea, Sweden
  • Footnotes
    Commercial Relationships Marta Sloniecka, None; Ludvig Backman, None; Patrik Danielson, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 723. doi:
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      Marta Sloniecka, Ludvig J. Backman, Patrik Danielson; Acetylcholine induces proliferation of keratocytes through activation of muscarinic receptors.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):723.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: The corneal wound healing response is a complex process involving cytokine-mediated interactions between epithelial cells, keratocytes of the stroma, corneal nerves, tear film, and cells of the immune system. The outcome of the response plays a critical role in the preservation of corneal transparency after injury. The wound healing cascade includes epithelial surface closure, keratocyte apoptosis, proliferation and migration, formation of myofibroblasts, and stromal remodeling.<br /> Acetylcholine (ACh) is regarded as a classical neurotransmitter. However, cells outside of the nervous system have been shown to contain and release ACh. It has been reported that ACh stimulates fibroblast and epithelial cells to proliferate, has an anti-inflammatory effect in macrophages, and upregulates collagen gene expression in fibroblasts. ACh, its muscarinic receptors (mAChRs) and choline acetyltransferase (ChAT; the enzyme responsible for synthesizing ACh) have been shown to be present in corneal epithelium. However, their role in the corneal stroma and corneal injury has not been extensively studied.<br /> We hypothesize that ACh, upon injury, induces corneal stroma cell proliferation, thus promoting the process of wound healing.

Methods: Primary human corneal stroma cells were derived from healthy corneas obtained from the local cornea bank. Immunocytochemistry was performed to delineate intracellular presence of ChAT and to characterize mAChRs. Crystal violet and MTS assay were used to asses ACh induced cell proliferation. Expression of the proliferation markers PCNA and Ki67 was analyzed by western blot. To determine what type of ACh receptors are involved in ACh induced proliferation, atropine and mecamylamine were used to block muscarininc or nicotinic ACh receptors, respectively.

Results: Stromal cells expressed ChAT as well as mAChRs of subtypes M1, M3, M4, and M5. Stimulation of stromal cells with ACh led to increased cell viability and metabolic activity. Expression of PCNA and Ki67 was upregulated in ACh treated cells. Furthermore, mAChRs were the receptor group primarily involved in ACh induced proliferation.

Conclusions: Corneal stroma cells express ChAT and mAChRs. ACh induces stroma cell proliferation through mainly mAChRs, which suggest that ACh may play an important role in corneal wound healing i.e. wound closure and generation on myofibroblasts.

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