Abstract
Purpose :
Histatins are low molecular weight salivary and lacrimal peptides with wound healing, anti-inflammatory, anti-apoptotic, and antimicrobial properties. In a rabbit model of anterior keratectomy, topically administered histatin-1 accelerated epithelial wound repair, inhibited and restored loss of stromal keratocytes and corneal endothelial cells, and stimulated regeneration of corneal nerves (Burke, ARVO 2022). Histatin-1 also markedly decreased endothelial cell loss under stressed temperature conditions in donor human cornea buttons and reduced associated biomarkers of apoptosis (Sawant, ARVO 2023). We examined whether histatin-1 could stimulate growth of human corneal endothelial cells as a potential therapy for corneal endothelial dystrophies.
Methods :
Human primary corneal endothelial cells were grown for 24 hrs in 10% serum in coated 6-well tissue plates to 90% confluency. A single scratch was made in each well, then low serum media (1%) was added with histatin-1 concentrations ranging from 1x10-9M to 1x10-5M or PBS as a control. Cells were incubated for up to 48 hours. Cell proliferation was determined at 24 and 48 hours using the Click-iT EdU Assay (Thermo Fisher). Imaging software was used to quantify the percent of scratch area regenerated or EDU-stained cell counts.
Results :
Histatin-1 increased the rate of regeneration (migration or proliferation) over PBS control at all 3 incubation times. Maximal efficacy was at 1x10-8 M for all 3 timepoints, and were 120%, 93% and 77% over PBS control for 6 hrs, 24 hrs and 48 hrs, respectively. This increase in regeneration in the scratch area was associated with concentration-related increases in proliferation based on EDU-stained cell counts. The percentage change over PBS control for 1x10-9M to 1x10-5M histatin-1 ranged from 7% to 48% at 24 hrs and from 12% to 130% at 48 hrs.
Conclusions :
This study showed for the 1st time that histatin-1 rapidly regenerates human primary corneal endothelial cells in a wound repair setting, and that cell proliferation is a key component of the regenerative effect. These results add to a growing literature that supports a potential therapeutic utility of histatins in the eye.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.