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An In Vitro Model of Diabetic Retinal Vascular Endothelial Dysfunction and Neuroretinal Degeneration

BACKGROUND: Diabetic retinopathy (DR) is a leading cause of blindness in working-age populations. Proper in vitro DR models are crucial for exploring pathophysiology and identifying novel therapeutic targets. This study establishes a rational in vitro diabetic retinal neuronal-endothelial dysfunctio...

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Autores principales: Wang, Qiyun, Zhang, Xinyuan, Wang, Kaiyue, Zhu, Ling, Qiu, Bingjie, Chen, Xiaosi, Lin, Xiao, Nie, Yao
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Hindawi 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8598328/
https://www.ncbi.nlm.nih.gov/pubmed/34805414
http://dx.doi.org/10.1155/2021/9765119
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author Wang, Qiyun
Zhang, Xinyuan
Wang, Kaiyue
Zhu, Ling
Qiu, Bingjie
Chen, Xiaosi
Lin, Xiao
Nie, Yao
author_facet Wang, Qiyun
Zhang, Xinyuan
Wang, Kaiyue
Zhu, Ling
Qiu, Bingjie
Chen, Xiaosi
Lin, Xiao
Nie, Yao
author_sort Wang, Qiyun
collection PubMed
description BACKGROUND: Diabetic retinopathy (DR) is a leading cause of blindness in working-age populations. Proper in vitro DR models are crucial for exploring pathophysiology and identifying novel therapeutic targets. This study establishes a rational in vitro diabetic retinal neuronal-endothelial dysfunction model and a comprehensive downstream validation system. METHODS: Human retinal vascular endothelial cells (HRMECs) and retinal ganglion cells (RGCs) were treated with different glucose concentrations with mannitol as matched osmotic controls. Cell proliferation and viability were evaluated by the Cell Counting Kit-8. Cell migration was measured using a transwell migration assay. Cell sprouting was assessed by a tube formation assay. The VEGF expression was assessed by ELISA. RGCs were labeled by neurons and RGC markers TUJ1 and BRN3A for quantitative and morphological analysis. Apoptosis was detected using PI/Hoechst staining and TUNEL assay and quantified by ImageJ. RESULTS: Cell proliferation and migration in HRMECs were significantly higher in the 25 mM glucose-treated group (p < 0.001) but lower in the 50 mM and 100 mM groups (p < 0.001). The permeability and the apoptotic index in HRMECs were statistically higher in the 25 mM, 50 mM, and 100 mM groups (p < 0.05). The tube formation assay found that all the parameters were significantly higher in the 25 mM and 50 mM groups (p < 0.001) concomitant with the elevated VEGFA expression in HRMECs (p = 0.016). Cell viability was significantly lower in the 50 mM, 100 mM, and 150 mM groups in RGCs (p(50mM) = 0.013, p(100mM) = 0.019, and p(150mM) = 0.002). Apoptosis was significantly elevated, but the proportion of RGCs with neurite extension was significantly lower in the 50 mM, 100 mM, and 150 mM groups (p(50mM) < 0.001, p(100mM) < 0.001, and p(150mM) < 0.001). CONCLUSIONS: We have optimized glucose concentrations to model diabetic retinal endothelial (25-50 mM) or neuronal (50-100 mM) dysfunction in vitro, which have a wide range of downstream applications.
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spelling pubmed-85983282021-11-18 An In Vitro Model of Diabetic Retinal Vascular Endothelial Dysfunction and Neuroretinal Degeneration Wang, Qiyun Zhang, Xinyuan Wang, Kaiyue Zhu, Ling Qiu, Bingjie Chen, Xiaosi Lin, Xiao Nie, Yao J Diabetes Res Research Article BACKGROUND: Diabetic retinopathy (DR) is a leading cause of blindness in working-age populations. Proper in vitro DR models are crucial for exploring pathophysiology and identifying novel therapeutic targets. This study establishes a rational in vitro diabetic retinal neuronal-endothelial dysfunction model and a comprehensive downstream validation system. METHODS: Human retinal vascular endothelial cells (HRMECs) and retinal ganglion cells (RGCs) were treated with different glucose concentrations with mannitol as matched osmotic controls. Cell proliferation and viability were evaluated by the Cell Counting Kit-8. Cell migration was measured using a transwell migration assay. Cell sprouting was assessed by a tube formation assay. The VEGF expression was assessed by ELISA. RGCs were labeled by neurons and RGC markers TUJ1 and BRN3A for quantitative and morphological analysis. Apoptosis was detected using PI/Hoechst staining and TUNEL assay and quantified by ImageJ. RESULTS: Cell proliferation and migration in HRMECs were significantly higher in the 25 mM glucose-treated group (p < 0.001) but lower in the 50 mM and 100 mM groups (p < 0.001). The permeability and the apoptotic index in HRMECs were statistically higher in the 25 mM, 50 mM, and 100 mM groups (p < 0.05). The tube formation assay found that all the parameters were significantly higher in the 25 mM and 50 mM groups (p < 0.001) concomitant with the elevated VEGFA expression in HRMECs (p = 0.016). Cell viability was significantly lower in the 50 mM, 100 mM, and 150 mM groups in RGCs (p(50mM) = 0.013, p(100mM) = 0.019, and p(150mM) = 0.002). Apoptosis was significantly elevated, but the proportion of RGCs with neurite extension was significantly lower in the 50 mM, 100 mM, and 150 mM groups (p(50mM) < 0.001, p(100mM) < 0.001, and p(150mM) < 0.001). CONCLUSIONS: We have optimized glucose concentrations to model diabetic retinal endothelial (25-50 mM) or neuronal (50-100 mM) dysfunction in vitro, which have a wide range of downstream applications. Hindawi 2021-11-10 /pmc/articles/PMC8598328/ /pubmed/34805414 http://dx.doi.org/10.1155/2021/9765119 Text en Copyright © 2021 Qiyun Wang et al. https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Wang, Qiyun
Zhang, Xinyuan
Wang, Kaiyue
Zhu, Ling
Qiu, Bingjie
Chen, Xiaosi
Lin, Xiao
Nie, Yao
An In Vitro Model of Diabetic Retinal Vascular Endothelial Dysfunction and Neuroretinal Degeneration
title An In Vitro Model of Diabetic Retinal Vascular Endothelial Dysfunction and Neuroretinal Degeneration
title_full An In Vitro Model of Diabetic Retinal Vascular Endothelial Dysfunction and Neuroretinal Degeneration
title_fullStr An In Vitro Model of Diabetic Retinal Vascular Endothelial Dysfunction and Neuroretinal Degeneration
title_full_unstemmed An In Vitro Model of Diabetic Retinal Vascular Endothelial Dysfunction and Neuroretinal Degeneration
title_short An In Vitro Model of Diabetic Retinal Vascular Endothelial Dysfunction and Neuroretinal Degeneration
title_sort in vitro model of diabetic retinal vascular endothelial dysfunction and neuroretinal degeneration
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8598328/
https://www.ncbi.nlm.nih.gov/pubmed/34805414
http://dx.doi.org/10.1155/2021/9765119
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