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Dhara Shah, Kyung-No Son, Sushma Kalmodia, Marwan Ali, Arun Balasubramaniam, Brian Layden, Vinay Kumar Aakalu; Pertussis Toxin Sensitive Wound Healing Effects of Histatin Peptides in Corneal Epithelia. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3878.
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Histatin peptides are a family of endogenous antimicrobial peptides that are known to have wound healing properties in oral and skin epithelia. The wound healing effects of histatin peptides in non-ocular epithelia are thought to be EGF independent and G-protein coupled receptor (GPCR) mediated. The precise pathways and critical receptors for these functions of histatin peptides are unknown. We have demonstrated that histatin peptides can enhance corneal epithelial migration in vitro. We hypothesized that histatin peptide enhancement of wound healing in ocular epithelia could be GPCR dependent. We sought to investigate this possibility through the application of a GPCR pathway inhibitor, Pertussis toxin (PTX), to demonstrate the requirement of Gαi/Gαo signaling in these effects, using scratch assays and GPCR signaling reporters.
A time lapse imaging scratch assay was performed using Human corneal epithelial (HCE) cells. HCE cells were cultured as confluent monolayers and incubated in 1% FBS for 16 h co-treated with PTX and wounded in a line across the well with a 200-μl pipette tip. Experimental groups included: Untreated, PTX only (0.2µg/ml), Histatin-1 (H1), Histatin-5 (H5), H1 + PTX and H5 + PTX. Scratches were photographed microscopically (Zeiss, CA, USA) at 0, 4, 8, 12, 16, 20 and 24 hours. WimScratch software by IBIDI (Germany) was used to analyze the % wound closure. Cyclic AMP (cAMP) levels were measured using cAMP-Glo™ Max Assay kit with or without histatin peptides after induction by benzalkonium chloride (BAK), an agent which is toxic to HCE and may impair wound healing.
H1 increased rates of scratch closure, and this effect was abrogated by PTX treatment. PTX alone did not alter wound healing. Similar results were found with H5 application (Fig 1a-b). Thus, the effects of H1 and H5 on HCE wound healing are sensitive to PTX and may indicate a role of a Gαi/Gαo coupled GPCR in histatin mediated enhancement of corneal epithelial wound healing. We also found that histatin peptides could affect levels of downstream GPCR signaling elements (cAMP) in corneal epithelia (Fig 1c).
Corneal epithelial scratch closure was accelerated by histatin peptides, in a PTX sensitive fashion. Effects of histatins on HCE may be GPCR mediated, involve Gi/o coupled pathways, and require further investigation to determine critical receptors and mediators.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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