USC Institute for Genetic Medicine, Keck School of Medicine of USC, University of Southern California, Los Angeles, CA, United States of America.
Program in Biological Sciences, USC Dana and David Dornsife College of Letters, Arts and Sciences, University of Southern California, Los Angeles, CA, United States of America.
PLoS One. 2018 Oct 10;13(10):e0204288. doi: 10.1371/journal.pone.0204288. eCollection 2018.
Water soluble "vital" dyes are commonly used clinically to evaluate health of the ocular surface; however, staining mechanisms remain poorly understood. Recent evidence suggests that sublethal damage stimulates vital dye uptake by individual living cells. Since cell damage can also stimulate reparative plasma membrane remodeling, we hypothesized that dye uptake occurs via endocytic vesicles. In support of this idea, we show here that application of oxidative stress to relatively undifferentiated monolayer cultures of human corneal epithelial cells stimulates both dye uptake and endocytosis, and that dye uptake is blocked by co-treatment with three different endocytosis inhibitors. Stress application 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, and two of the endocytosis inhibitors did not block dye uptake. The exception was Dynasore and its more potent analogue Dyngo-4a, both small molecules developed to target dynamin family GTPases, but also having 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 went on to demonstrate an alternative mechanism. We show that Dynasore is remarkably protective of cells and their surface glycocalyx, preventing damage due to stress, and thus precluding dye entry. These unexpected and novel findings provide greater insight into the mechanisms of vital dye uptake and point the direction for future study. Significantly, they also suggest that Dynasore and its analogues might be used therapeutically to protect the ocular surface and to treat ocular surface disease.
水溶性“生命”染料常用于临床评估眼表健康状况，但染色机制仍知之甚少。最近的证据表明，亚致死性损伤会刺激活细胞摄取生命染料。由于细胞损伤也会刺激修复质膜重塑，我们假设染料摄取是通过内吞小泡进行的。为了支持这一观点，我们在这里表明，应用氧化应激于相对未分化的单层人角膜上皮细胞培养物中，会刺激染料摄取和内吞作用，并且用三种不同的内吞抑制剂共同处理会阻断染料摄取。将应激应用于分层和分化的角膜上皮细胞培养物（更能模拟眼表）也会刺激染料摄取；然而，内吞作用没有被刺激，并且两种内吞抑制剂都不能阻断染料摄取。例外的是 Dynasore 和它的更有效类似物 Dyngo-4a，都是针对动力蛋白家族 GTPases 开发的小分子，但其对质膜也有非靶向作用。重要的是，虽然 Dynasore 在对活体小鼠眼表面同时进行应激处理时，可以阻断应激刺激的染料摄取，但在应激后应用时，即在眼表面已经受损时，它没有效果。因此，Dynasore 不能通过抑制内吞作用发挥作用。通过细胞毒性和 Western blot 测定，我们进一步证明了另一种机制。我们表明，Dynasore 对细胞及其表面糖萼具有显著的保护作用，可防止应激引起的损伤，从而防止染料进入。这些出乎意料和新颖的发现为深入了解生命染料摄取的机制提供了更多的见解，并为未来的研究指明了方向。重要的是，它们还表明 Dynasore 及其类似物可用于治疗眼表疾病，以保护眼表。
