Hippo pathway inhibition induces regeneration of retinal pigment epithelium monolayers in vitro

Abstract

Purpose : The Hippo signaling pathway is an important regulator of numerous processes including cell proliferation and tissue regeneration. This study aimed to determine whether Hippo pathway inhibition via a novel Lats kinase inhibitor (LKI) is sufficient to induce regenerative cell proliferation in retinal pigment epithelium (RPE) monolayers in vitro.

Methods : Primary human fetal RPE cells were grown on 96-well plates until they formed confluent monolayers. To assess the effect of Hippo pathway inhibition on cell proliferation in the absence of injury, monolayers were incubated with 5-ethynyl-2’-deoxyuridine (EdU) for 96 hours with or without 1 μM TDI-011536, a recently developed LKI. To assess cell proliferation in the setting of injury, monolayers were subjected to a linear scratch with a pipette tip and then cultured for 96 hours. To assess the effect of growth media on cell proliferation, RPE were cultured in media with or without fetal bovine serum (FBS). Cell proliferation was quantified as the ratio of EdU+ to DAPI+ nuclei within 800 mm² regions of interest after 96 hours.

Results : In the absence of LKI or scratch, there was minimal cell proliferation with or without FBS (1.1±0.4% and 0.2±0.1%, respectively). Addition of the LKI significantly increased cell proliferation with or without FBS (11.9±2.7% and 10.1±1.3%, respectively; P<0.0001). Scratched wells had increased cell proliferation compared to non-scratched wells regardless of LKI or FBS (all P<0.0001). However, addition of the LKI further potentiated cell proliferation in scratched wells (P<0.0001). Within scratched wells, most cell proliferation occurred at the scratch area with less outside the scratch area (P<0.0001).

Conclusions : Hippo pathway inhibition via a novel LKI induces cell proliferation in confluent RPE monolayers in vitro. The magnitude of this effect is increased in an RPE injury model. These findings should prompt further study of this LKI in vivo to stimulate endogenous RPE repair in degenerative retinal disorders such as atrophic age-related macular degeneration.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.

 

Figure 1. In non-scratched wells, addition of the LKI increased RPE proliferation both with FBS (11.9±2.7% v. 1.1±0.4%, P<0.0001) and without FBS (10.1±1.3% v. 0.2±0.1%, P<0.0001).

 

Figure 2. In scratched wells, addition of the LKI increased RPE proliferation both with FBS (38.5±1.8% v. 14.5±2.5%, P<0.0001) and without FBS (32.5±3.7% v. 4.9±0.8%, P<0.0001).