TTHX1114 Rescues Cells Against DNA damage and Cell Loss in Primary Rabbit Corneal Endothelial Cells

Posterboard#: B0598

Abstract Number: 4146 - B0598

AuthorBlock: David Eveleth¹, Jessica Weant¹, Anna Stuhlfire¹, Lindsey Myers¹, Sarah Pizzuto^1
1Trefoil Therapeutics, California, United States;

DisclosureBlock: David Eveleth, Code E (Employment) Trefoil Therapeutics, Code I (Personal Financial Interest) Trefoil Therapeutics, Jessica Weant, Code E (Employment) Trefoil Therapeutics, Code I (Personal Financial Interest) Trefoil Therapeutics, Anna Stuhlfire, Code E (Employment) Trefoil Therapeutics, Lindsey Myers, Code E (Employment) Trefoil Therapeutics, Sarah Pizzuto, Code C (Consultant/Contractor) Trefoil Therapeutics

Purpose
Cataract surgery is associated with a 10-40% loss of corneal endothelial cells, often resulting in edema that can be persistent. Phacoemulsification results in the formation of hydroxyl radicals that can elicit an oxidative stress response causing corneal endothelial cell death. Herein we interrogate a oxidative stress model using tBHP to investigate if a synthetic analog of FGF1 - TTHX1114 (TTHX), is effective at safeguarding or rescuing primary rabbit corneal endothelial cells (pRCEnCs) from oxidative stress associated DNA damage and cell death

Methods
pRCEnCs were harvested from Descemet’s Membranes. Cells were subjected to oxidative stress using tBHP insult at IC40 and IC10 concentrations for 2 hours. Cells either received 50 ng/mL TTHX or serum free media. The cells were looked at immediately after insult, 24 and 48 hrs post insult. The MTT assay was used to quantify cell metabolism. Cells were fixed, permeabilized, and stained with TUNEL, ZO-1, and Hoechst. Cell density and TUNEL counts were performed with ImageJ. Statistical analysis performed in PRISM

Results
MTT absorbance data show loss of pRCEnC viability and loss of cell metabolic function following tBHP insult. Pre or post TTHX treatment maintains or restores metabolic function at both the IC40 and IC10 tBHP insult concentrations. Cell counts for the TTHX treatment/insult paradigms are similar to the uninsulted growth media condition. Fluorescently stained cell images show TTHX treated cells have clearly defined and regular ZO-1 borders. TUNEL staining was increased in cells that were fixed directly following a 2 hr insult. A reduction in TUNEL staining was observed in all conditions over time with the greatest reduction in TUNEL staining occurring at the 48 hrs post TTHX treatment paradigms.

Conclusions
A pre and / or post treatment with 50 ng/mL TTHX after an oxidative stress insult that results in 10-40% cell loss is sufficient for protecting and rescuing pRCEnCs from DNA damage and cell death