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Transthyretin Exerts Pro-Apoptotic Effects in Human Retinal Microvascular Endothelial Cells Through a GRP78-Dependent
Jun Shao1, Ying Yin1, Xiaowen Yin1
1Department of Ophthalmology, Wuxi People's Hospital affiliated to Nanjing Medical University, Wuxi, China.
Background/Aims:
Diabetic retinopathy (DR) is one of the main causes of blindness in the world. Our previous study showed that transthyretin (TTR) regulates key genes in the Tie2 pathway and inhibits the development of neovascularization in DR, but the mechanism is still unclear. Here, we investigated how TTR affects the progression of neovascularization in DR.
Methods:
Natural and simulated DR media (hyperglycemia and hypoxia) were used to culture human retinal microvascular endothelial cells (hRECs). Flow cytometry was employed to investigate the effect of TTR on apoptosis of hRECs. Fluorescent labeling and immunofluorescence staining were used to determine the TTR distribution in hRECs. The membrane proteins of hRECs were extracted and applied to a sepharose-TTR column, and the captured proteins were identified by Mass Spectrometric analysis. Gene knock-down and western blotting assays were used to study the key signal pathway of the most abundant identified protein.
Results:
TTR induced apoptosis of hRECs in an environment that simulated hypoxia. Immunofluorescent staining showed that TTR could enter the nuclei of hRECs. A total of 30 unique TTR-captured proteins were identified by Mass Spectrometry, and glucose-regulated protein 78 (GRP78) was one of the most abundant. Western blotting and gene knock-down indicated that TTR might upregulate GRP78 and facilitate apoptosis through the eIF2α/CHOP pathway.
Conclusions:
In the DR environment (hyperglycemia and hypoxia), TTR was shown to repress neovascularization by promoting apoptosis of hRECs through a GRP78-dependent pathway.
Insights
Transthyretin (TTR) promotes human retinal microvascular endothelial cell (hREC) apoptosis in diabetic retinopathy (DR) environments. This process, mediated by glucose-regulated protein 78 (GRP78), inhibits neovascularization, offering a potential therapeutic target for DR.
Area of Science:
- Ophthalmology
- Endocrinology
- Molecular Biology
Background:
- Diabetic retinopathy (DR) is a leading cause of blindness globally.
- Transthyretin (TTR) previously showed potential in inhibiting DR neovascularization.
- The precise mechanism of TTR's action in DR remained unclear.
Purpose of the Study:
- To investigate the mechanism by which TTR influences neovascularization in diabetic retinopathy.
- To determine TTR's effect on human retinal microvascular endothelial cells (hRECs) under DR-simulating conditions.
Main Methods:
- Cultured hRECs in natural and simulated DR media (hyperglycemia, hypoxia).
- Utilized flow cytometry for apoptosis analysis and immunofluorescence for TTR localization.
- Identified TTR-interacting proteins via Mass Spectrometry and validated pathways using gene knockdown and Western blotting.
Main Results:
- TTR induced hREC apoptosis under hypoxic conditions.
- TTR was found to enter the nuclei of hRECs.
- Mass Spectrometry identified 30 TTR-binding proteins, including abundant glucose-regulated protein 78 (GRP78).
- TTR upregulates GRP78, facilitating apoptosis via the eIF2α/CHOP pathway.
Conclusions:
- TTR represses neovascularization in DR by promoting hREC apoptosis.
- This effect is mediated through a GRP78-dependent pathway.
- TTR's role in DR progression involves GRP78 and the eIF2α/CHOP signaling cascade.
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