Dynasore protects the ocular surface against damaging oxidative stress.
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 stimula...
| Main Authors: | , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2018-01-01
|
| Series: | PLoS ONE |
| Online Access: | http://europepmc.org/articles/PMC6179211?pdf=render |
| _version_ | 1819065529550241792 |
|---|---|
| author | Andrew Webster Shravan K Chintala Jasmine Kim Michelle Ngan Tatsuo Itakura Noorjahan Panjwani Pablo Argüeso Joseph T Barr Shinwu Jeong M Elizabeth Fini |
| author_facet | Andrew Webster Shravan K Chintala Jasmine Kim Michelle Ngan Tatsuo Itakura Noorjahan Panjwani Pablo Argüeso Joseph T Barr Shinwu Jeong M Elizabeth Fini |
| author_sort | Andrew Webster |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-12-21T15:47:55Z |
| format | Article |
| id | doaj.art-bd50d6f17f2341efb6b31a56250f4033 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-21T15:47:55Z |
| publishDate | 2018-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-bd50d6f17f2341efb6b31a56250f40332022-12-21T18:58:19ZengPublic Library of Science (PLoS)PLoS ONE1932-62032018-01-011310e020428810.1371/journal.pone.0204288Dynasore protects the ocular surface against damaging oxidative stress.Andrew WebsterShravan K ChintalaJasmine KimMichelle NganTatsuo ItakuraNoorjahan PanjwaniPablo ArgüesoJoseph T BarrShinwu JeongM Elizabeth FiniWater 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.http://europepmc.org/articles/PMC6179211?pdf=render |
| spellingShingle | Andrew Webster Shravan K Chintala Jasmine Kim Michelle Ngan Tatsuo Itakura Noorjahan Panjwani Pablo Argüeso Joseph T Barr Shinwu Jeong M Elizabeth Fini Dynasore protects the ocular surface against damaging oxidative stress. PLoS ONE |
| title | Dynasore protects the ocular surface against damaging oxidative stress. |
| title_full | Dynasore protects the ocular surface against damaging oxidative stress. |
| title_fullStr | Dynasore protects the ocular surface against damaging oxidative stress. |
| title_full_unstemmed | Dynasore protects the ocular surface against damaging oxidative stress. |
| title_short | Dynasore protects the ocular surface against damaging oxidative stress. |
| title_sort | dynasore protects the ocular surface against damaging oxidative stress |
| url | http://europepmc.org/articles/PMC6179211?pdf=render |
| work_keys_str_mv | AT andrewwebster dynasoreprotectstheocularsurfaceagainstdamagingoxidativestress AT shravankchintala dynasoreprotectstheocularsurfaceagainstdamagingoxidativestress AT jasminekim dynasoreprotectstheocularsurfaceagainstdamagingoxidativestress AT michellengan dynasoreprotectstheocularsurfaceagainstdamagingoxidativestress AT tatsuoitakura dynasoreprotectstheocularsurfaceagainstdamagingoxidativestress AT noorjahanpanjwani dynasoreprotectstheocularsurfaceagainstdamagingoxidativestress AT pabloargueso dynasoreprotectstheocularsurfaceagainstdamagingoxidativestress AT josephtbarr dynasoreprotectstheocularsurfaceagainstdamagingoxidativestress AT shinwujeong dynasoreprotectstheocularsurfaceagainstdamagingoxidativestress AT melizabethfini dynasoreprotectstheocularsurfaceagainstdamagingoxidativestress |