Abstract
Purpose :
Histatins are a family of endogenous antimicrobial peptides that are known to have wound healing properties in oral and skin epithelia. We recently reported that Histatin-1 (H1) can promote wound healing in human corneal epithelia. The family of histatins contains two genes HTN1 and HTN3 and 13 protein products. H1, H2 and H3 are thought to have wound healing properties attributed to their C terminal. The critical residues associated with wound healing in the C’ of H1 was determined using scratch assays and serial truncation experiments that determined that residues 20-32 (SHREFPFYGDYGS) were the minimal active wound healing domain of H1. Histatin-5 (H5) is a proteolytic product of H3 that retains SHR…..G.Y of the C’ fragment and retains the EKHH domain N’ of SHR. Given the similarities in other functions of these family members we sought to determine whether the C terminal of H5 maintained the wound healing properties of these in corneal epithelia.
Methods :
Human corneal epithelial (HCE) cells were cultured and a scratch mark was made with a sterile P200 pipette tip as described prior. The cells were then washed twice with Phosphate-buffered saline (PBS) to remove cellular debris. Wounded areas were then treated with Hst5, truncation forms and scrambled peptide (SP) controls in MEM media with reduced serum conditions (0.5% FBS) Scratches were photographed microscopically every hour over the course of the experiment. The wound areas were measured using ImageJ software.
Results :
To determine which portions of Hst5 were needed to promote epithelial cell migration, we performed a serial truncation experiment by progressively deleting residues in Hst5 and testing its efficacy. We found that the C terminal residues were necessary and sufficient to promote migration. All peptides were tested at 80µM, based on our results in HCE. Truncated versions of Hst5 which did not include C terminus sequence showed no significant increase in wound closure rates. However, constructs containing an intact C terminal sequence showed significant increases in scratch closure rates. Scrambled peptides of the C terminal sequence did not increase wound closure rates. Thus, the C terminal sequence is necessary and sufficient to promote epithelial migration rates in in vitro scratch assays.
Conclusions :
In this report, we describe a new functional domain of Hst5 which is necessary and sufficient for promotion of human corneal epithelial wound healing in vitro.
This is a 2020 ARVO Annual Meeting abstract.