Impact of oxidative stress on the cytoskeleton and barrier integrity of the corneal endothelium

Abstract

Purpose : Oxidative stress (OS) is implicated in the pathophysiology of Fuchs Endothelial Corneal Dystrophy (FECD). It is known to induce microtubule disassembly in other cell types, leading to remodelling of the apical junctional complex via actomyosin contraction and eventually to barrier failure (Srinivas; EER, 2012). This study aims to investigate the impact of experimental oxidative stress on the barrier properties of the corneal endothelium (CE).

Methods : All experiments were carried out with primary cultured cells of porcine/bovine CE. For oxidative stress, cells were exposed to H₂O₂ (100 μM, 1 h) after holding the cells in a culture medium containing 2% serum for 24 hrs. Immunocytochemical techniques were employed to assess the disassembly of the microtubules and the organization of ZO-1. The loss of barrier function was assessed by TER, which was measured by electric cell-substrate impedance sensing with bovine CE grown on gold electrodes that were coated with an ECM cocktail. In these experiments, the culture medium was supplemented with 50 µM Riboflavin (Rf) and exposed to UV-A (365 nm; 30 min, 0.5 mW/cm2) to induce the Type-1 photochemical reactions and thereby cause an in situ release of H₂O₂.

Results : Exposure to H₂O₂ for 1 h led to microtubule disassembly (immunocytochemistry with a-tubulin), which could be inhibited by pre-treatment with 10 µM of SB-203580 (a p38 MAPK inhibitor; 1 h). In independent experiments, exposure to H₂O₂ led to a loss of the contiguous appearance of ZO-1 (Fig. 1B), which marks the breakdown of tight junctions. Accordingly, a sustained loss of TER (over 10-12 h) was induced when cells on gold electrodes, bathed in Rf-supplemented medium, were exposed to UV-A. Inclusion of catalase (7000 U/mL), which prevents the accumulation of H₂O₂, abolished the loss in TER induced by UV-A.

Conclusions : We have demonstrated that acute oxidative stress induces microtubule disassembly, leading to a destruction of apical junctional complex and barrier integrity in cultured CE. The effects of p38 MAPK confirms a role for the stress kinase downstream of oxidative stress. Thus, the response to oxidative stress is reminiscent of the TNF-a-induced breakdown of barrier failure reported in CE (Srinivas; EER, 2012).

This is a 2021 ARVO Annual Meeting abstract.

 

Figure 1: Immunostaining of ZO-1 in cultured porcine corneal endothelial cells. (A) Control; (B) H₂O₂ treatment (100µM, 1 h)