The Role of Transforming Growth Factor-Beta in Retinal Ganglion Cells with Hyperglycemia and Oxidative Stress

Chen, Hsin‐Yi; Ho, Yi‐Jung; Chou, Hsiu‐Chuan; Liao, En‐Chi; Tsai, Yi‐Ting; Wei, Yu‐Shan; Lin, Li‐Hsun; Lin, Ming-Tzer; Wang, Yi‐Shiuan; Ko, Mei–Lan; Chan, Hong‐Lin

doi:10.3390/ijms21186482

“…In this study, we found that the cell variability of RGCs is significantly lower in the 50 mM to 150 mM and the linear declined trend was evidence from 5.5 mM to 150 mM, which is consistent with previous studies [ 41 ]. RGCs were further labeled with neurons and RGC-specific markers TUJ1 and BRN3A, respectively, followed by the ImageJ quantitative analysis [ 42 ].…”

Section: Discussionsupporting

confidence: 93%

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

Wang

¹

,

Zhang

²

,

Wang

³

et al. 2021

Journal of Diabetes Research

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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 50 mM = 0.013 , p 100 mM = 0.019 , and p 150 mM = 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 50 mM < 0.001 , p 100 m M < 0.001 , and p 150 mM < 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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“…ALDH3A1 can oxidize various aldehydes to their corresponding acids and is involved in the detoxification of alcohol-derived acetaldehyde and metabolism of corticosteroids, biogenic amines, neurotransmitters, and lipid peroxidation. The expression of ALDH3A1 is suggested to play an important role in the stress response and the anti-oxidation pathway for the protection of RGCs, transforming growth factor-beta in the treatment of diabetic retinopathy [47,48] and corneal cells against ultra-violet damage [49]. Our local network clusters analysis also indicated that there is an interaction between Aldh3a1, Oplah, and Mgst3, which are also believed to be related to the glutathione metabolic process.…”

Section: Discussionmentioning

confidence: 57%

Differential Retinal Protein Expression in Primary and Secondary Retinal Ganglion Cell Degeneration Identified by Integrated SWATH and Target-Based Proteomics

Kwong

¹

,

Sze

²

,

Yu

³

et al. 2021

IJMS

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To investigate the retinal proteins associated with primary and secondary retinal ganglion cell (RGC) degeneration and explore their molecular pathways, SWATH label-free and target-based mass spectrometry was employed to identify the proteomes in various retinal locations in response to localized optic nerve injury. Unilateral partial optic nerve transection (pONT) was performed on adult Wistar rats and their retinas were harvested 2 weeks later. To confirm the separation of primary and secondary RGC degeneration, immunohistochemistry of RNA binding protein with multiple splicing (RBPMS) and glial fibrillary acidic protein (GFAP) was performed on retinal whole-mounts. Retinal proteomes in the temporal and nasal quadrants were evaluated with high resolution hybrid quadrupole time-of-flight mass spectrometry (QTOF-MS), and SWATH-based acquisition, and their expression was compared to the corresponding retinal quadrant in contralateral control eyes and further validated by multiple reaction monitoring mass spectrometry (MRM-MS). A total of 3641 proteins (FDR < 1%) were identified using QTOF-MS. The raw data are available via ProteomeXchange with the identifier PXD026783. Bioinformatics data analysis showed that there were 37 upregulated and 25 downregulated proteins in the temporal quadrant, whereas 20 and five proteins were upregulated and downregulated, respectively, in the nasal quadrant, respectively (n = 4, p < 0.05; fold change ≥ 1.4-fold or ≤0.7). Six proteins were regulated in both the temporal and the nasal quadrants, including CLU, GFAP, GNG5, IRF2BPL, L1CAM, and CPLX1. Linear regression analysis indicated a strong association between the data obtained by means of SWATH-MS and MRM-MS (temporal, R2 = 0.97; nasal, R2 = 0.96). Gene ontology analysis revealed statistically significant changes in the biological processes and cellular components of primary RGC degeneration. The majority of the significant changes in structural, signaling, and cell death proteins were associated with the loss of RGCs in the area of primary RGC degeneration. The combined use of SWATH-MS and MRM-MS methods detects and quantifies regional changes of retinal protein expressions after localized injury. Future investigation with this integrated approach will significantly increase the understanding of diverse processes of progressive RGC degeneration from a proteomic prospective.

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“…However, sulfur antioxidants prevent necroptosis of RGC-5 cell lines by eliminating reactive oxygen species [ 37 ]. These drugs protect cells by blocking the signaling pathway that causes necroptosis from the beginning [ 38 ]. However, few experiments have been conducted on either of the drugs in vivo [ 39 ].…”

Section: Discussionmentioning

confidence: 99%

Investigation on the expression regulation of RIPK1/RIPK3 in the retinal ganglion cells (RGCs) cultured in high glucose

Gao

¹

,

Huang

²

,

Zhang

³

et al. 2021

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Diabetic retinopathy (DR) represents the most typical complication of type 2 diabetes mellitus and one of the most primary oculopathy causing blindness. However, the mechanism of DR remains unknown. RIPK1/RIPK3, as homologous serine/threonine kinases, are key elements in mediating necroptosis and may have functions in DR development. To clarify the relationship between DR and RIPK1/RIPK3, this study established a model of apoptosis using high-glucose induced RGCs, which were treated with 7.5, 19.5, and 35 mM D-glucose for 12, 24, and 48 h, respectively. Subsequently, the expression of RIPK1/RIPK3 was determined and the protective effect of necrostatin-1 on RGCs injury induced by high glucose was explored. The results demonstrated that the expression of RIPK1 and RIPK3 in the cells was increased markedly following 12 h treatment with 19.5 mM D-glucose. Additionally, following an addition of 100 μM necrostatin-1 in 19.5 mM D-glucose medium for RGCs treatment 12 h, the protein expression of RIPK1 and RIPK3 was decreased markedly, and the number of Nissl bodies in cells was increased substantially. The findings of the present study indicated that high glucose could induce the expression of RIPK1/ RIPK3, and necrostatin-1 could effectively protect RGCs from D-glucose-induced cell necrosis.

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The Role of Transforming Growth Factor-Beta in Retinal Ganglion Cells with Hyperglycemia and Oxidative Stress

Cited by 28 publications

References 43 publications

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

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

Differential Retinal Protein Expression in Primary and Secondary Retinal Ganglion Cell Degeneration Identified by Integrated SWATH and Target-Based Proteomics

Investigation on the expression regulation of RIPK1/RIPK3 in the retinal ganglion cells (RGCs) cultured in high glucose

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