Abstract
Purpose :
We have recently identified activation of the integrated stress response (ISR) in keratoconic corneas and in vitro fibroblast cultures. We sought to investigate the effect of modulating the activation state of normal and keratoconic fibroblasts in an effort to recapitulate the diseased phenotype in healthy cells and further probe the nature of this activation.
Methods :
Donor corneal fibroblasts (DN) were extracted from limbal rings and corneal buttons and expanded in serum containing media. These were then treated with tunicamycin (0.1-1μg/ml) or the cell-permeable eIF-2a phosphatase inhibitor SAL003 (10-0.5μM). Cell survival, was monitored by resazurin reduction. MMP production was monitored with Gelatin zymography and Collagen production was measured via hydroxyproline and sirius red incorporation. qPCR, immunohistochemistry and Western Blotting was used to measure ATF4 activity and ISR status.
Results :
Activation of the ISR (SAL003/Tunicamycin) did not cause profound cell loss over 7 dayas in culture. However, we observed increase in MMP2 production (n=4) in response to tunicamycin (7.4±0.8) and SAL003 (4.7±0.4), when normalized to final cell number. We also observe a dose dependent decrease in collagen production over 7 days by both sirius red and hydroxyproline production with an Ic50 of 5µM (n=5) for SAL003. We observe increases in the production of CRP55 and CTAGE5 in treated cells as we have previously published for keratoconic fibroblasts. We have also correlated this with PERK phosphorylation in ex vivo keratoconic corneas and in vitro keratoconic fibroblasts.
Conclusions :
Our results demonstrate that the activation of the ISR in healthy fibroblasts recapitulates critical aspects of the observed in vitro keratoconic fibroblast phenotype. We have demosntrated that triggering an unfolded protein response, simialr to that observed ex vivo triggers an increase in catabolic enzymes, decrease in collagen production and eventual cell death. We believe that targetting the integrated stress reponse represents a novel therapeutic opportunity for keratoconus.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.