Dynasore protects the ocular surface against damaging oxidative stress

Abstract

“Vital” dyes such as fluorescein and rose bengal are used clinically to evaluate ocular surface health; however, staining mechanisms remain poorly understood. Recent evidence suggests that sublethal cell damage stimulates fluorescein dye uptake. Since damage can also stimulate reparative plasma membrane remodeling, we hypothesized that dye uptake occurs via endocytic vesicles. Using an oxidative stress model, we show that damage to relatively undifferentiated monolayer cultures of human corneal epithelial cells stimulates uptake of fluorescein and rose bengal dyes and also stimulates endocytosis. Importantly, dye uptake was blocked by co-treatment with three different endocytosis inhibitors. Damage to stratified and differentiated corneal epithelial cell cultures, which are a better model of the ocular surface, also stimulated dye uptake; however, endocytosis was not stimulated in this case, and two of the inhibitors did not block dye uptake. The exception was the inhibitor Dynasore and its more potent analogue Dyngo-4a, small molecules that target dynamin family GTPases, but also have off-target effects on the plasma membrane. Significantly, while Dynasore blocked stress-stimulated dye uptake at the ocular surface of ex vivo mouse eyes when treatment was performed at the same time as eyes were stressed, it had no effect when used after stress was applied and the ocular surface was already damaged. Thus, Dynasore could not be working by inhibiting endocytosis. Employing cytotoxicity and western blotting assays, we demonstrate an alternative mechanism, showing that Dynasore is remarkably protective of cells and their surface glycocalyx, preventing damage due to oxidative stress, and thus precluding dye entry. These unexpected and novel findings provide greater insight into mechanisms of vital dye uptake and emphasize the importance of using a differentiated cell culture model for such studies. They also suggest that Dynasore and analogues might be used therapeutically to protect the ocular surface and to treat ocular surface disease.

Dynasore Blocks Ferroptosis through Combined Modulation of Iron Uptake and Inhibition of Mitochondrial Respiration

“…Interestingly, dynasore has been proposed as a candidate small molecule inhibitor to treat mitochondrial disorders characterized by aberrant accumulation of mitochondrial ROS [ 34 , 42 ]. Furthermore, in human corneal epithelial cells, dynasore prevented oxidative stress-induced cell damage through a non-described independent mechanism of endocytosis inhibition [ 43 ], and it is tempting to speculate as to whether the radical-trapping activity and mitochondrial respiratory inhibition discovered in our study may underlie such tissue protection. Although aberrant accumulation of ROS can cause DNA damage, protein denaturation and lipid peroxidation, certain amounts of iron and ROS are crucial for normal cell function [ 1 , 40 , 41 ].…”

Recombinant Human Clusterin Seals Damage to the Ocular Surface Barrier in a Mouse Model of Ophthalmic Preservative-Induced Epitheliopathy

“…Several recent reports using cultured cells have provided insight into the mechanism of dye uptake by cells [ 72 , 73 , 74 ]. It was noticed that, while all cells in monolayer culture stain with fluorescein [ 72 , 73 ] or rose bengal [ 74 ], a small percentage of the total cells concentrate the dye, becoming very brightly stained. “Hyperstaining” was inhibited by reducing the temperature or by disruption of the plasma membranes [ 73 ].…”

“…“Hyperstaining” was inhibited by reducing the temperature or by disruption of the plasma membranes [ 73 ]. Application of a damaging stress [ 72 , 74 ], or treatment with a multi-purpose contact lens cleaning solution (MPS) [ 73 ], greatly increased the number of hyperstained cells. Hyperstained cells showed characteristics of early apoptosis, whether in monolayer culture, or in the apical epithelial layer of ex vivo rabbit eyes [ 72 ].…”

Chemical Inhibitors of Dynamin Exert Differential Effects in VEGF Signaling

“…As they offer the advantage of a rapid effect, these drugs avoid the manifestation of compensatory trafficking pathways [ 56 ]. However, despite their wide use and their undoubtful inhibitory effect on dynamin, the fact that they are small molecules that could bind to multiple targets raises concerns about possible off-target effects [ 18 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ]. The most compelling evidence in favor of this concern comes from experiments in dynamin triple knockout cells, where, although lack of all three dynamins did not affect fluid-phase endocytosis and membrane ruffling, dynasore and dyngo robustly inhibited these two processes both in wild-type and in the triple knockout cells [ 22 ].…”

“…In addition to dynasore, other dynamin inhibitors, i.e., dyngo 4a and dynole 34-2 [ 19 ], have been widely used to block dynamin-dependent endocytosis, especially when cells are resistant to transfection methods. However, since small molecule inhibitors may bind to multiple targets, it cannot be excluded that these inhibitors exert off-target effects [ 20 , 21 ], similarly to dynasore [ 18 , 22 , 23 , 24 , 25 , 26 , 27 , 28 ]. Here, by comparing the effect of the above drugs in VEGF-induced internalization of VEGFR2 and downstream signaling to ERK1/2 and Akt, we suggest that these inhibitors, besides exerting their specific effect in dynamin-dependent endocytosis, cause inhibitory effects on signaling that are independent of endocytosis; i.e., they are due to off-target effects.…”