Hippo pathway inhibition induces regeneration of retinal pigment epithelium

Abstract

Purpose : The purpose of this study was to investigate whether Hippo pathway inhibition using a Lats kinase inhibitor (LKI) is sufficient to induce regeneration of the retinal pigment epithelium (RPE) in in vitro and in vivo models of RPE injury.

Methods : An in vitro wound healing assay was performed on RPE monolayers to assess RPE wound closure and RPE proliferation with TDI-011536, a novel LKI, or vehicle. Wound closure was quantified as the percentage change in wound area over time. RPE proliferation was quantified as the percentage of 5-ethynyl-2’-deoxyuridine positive (EdU+) cells in 800 µm² regions at varying distances from the site of the wound 96 hours after wound induction. To assess the effect of LKI on RPE in vivo, laser injury was induced in Dutch belted rabbits with laser photocoagulation. Following laser, animals received intravitreal TDI-011536 or vehicle. Fundus photography and optical coherence tomography (OCT) was performed after laser as well as at 1- and 4-week time points. Following imaging, immunohistochemistry (IHC) was performed to characterize the effect of LKI on histology.

Results : In vitro RPE monolayers demonstrated increased wound healingwith TDI-011536 (n=28 regions) compared to control (n=30 regions) at 24 hours (19.5±6.3 v. 12.7±8.5; P=0.001), 48 hours (52.4±9.5 v. 34.7±11.8; P<0.0001), 72 hours (81.9±8.0 v. 56.7±13.4; P<0.0001), and 96 hours (88.8±7.13 v. 71.1±14.2; P<0.0001). RPE monolayers showed increased cell proliferation with TDI-011536 (n=39 regions) compared to control (n=45 regions) in scratch regions (43.1±17.8 v. 32.9±19.0; P=0.01) and proximal regions (13.3±13.1 v. 3.9±4.7; P<0.0001) but not distal regions (3.3±3.2 v. 2.1±3.1; P=0.09) (Figure 1A-D). Four weeks after laser injury in an in vivo model of retinal degeneration, eyes treated with TDI-011536 (n=8 eyes) demonstrated hyperpigmentation of laser spots on fundus photos (n=8 eyes). On OCT, TDI-011536-treated eyes showed thickening of the RPE with increased shadowing underlying the laser spots. On IHC, TDI-011536-treated eyes demonstrated hypertrophy of the RPE layer at sites of laser damage (Figure 2A-B).

Conclusions : TDI-011536 induced RPE wound healing in an in vitro model of RPE injury. In addition, intravitreal LKI demonstrated changes consistent with RPE regeneration on fundus photos, OCT, and IHC in an in vivo model of retinal damage. These findings prompt further study of LKIs to treat degenerative retinal disorders.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.