Transthyretin Exerts Pro-Apoptotic Effects in Human Retinal Microvascular Endothelial Cells Through a GRP78-Dependent Pathway in Diabetic Retinopathy

Abstract

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 glucoseregulated 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.

Transthyretin Upregulates Long Non-Coding RNA MEG3 by Affecting PABPC1 in Diabetic Retinopathy

“…Our previous work has exhibited that TTR inhibits retinal vascular proliferation [14][15][16][17]. In this current work, lncRNA-MEG3 levels were increased by adding TTR ( Figure 3A).…”

“…Regarding some clinical investigations of ocular diseases, myopia is suggested to prevent patients from suffering DR [11,12]; therefore, in our previous work, we studied the relationship between the higher vitreous TTR level of high myopia patients [13] and the DR protection phenomenon. It is interesting that the serum and vitreous TTR levels in DR patients should be associated with DR progression [15], and in vitro, TTR has been proved to prevent the progression of DR [14], and repress angiogenesis by the Tie signaling pathway in hyperglycemia [16] or promote the apoptosis of hRECs by the GRP78-dependent pathway [17]. Additionally, TTR has also been proved to suppress the proliferation of hRECs by a TTR/STAT4/miR-223-3p/FBXW7 signaling pathway and could further affect the content of Notch1 [32].…”

“…In our previous work, although TTR could inhibit the proliferation and induce the apoptosis of hRECs in vitro [14][15][16][17]32], the effect of TTR on DR progression in vivo was unclear. Therefore, in this work, the DR mice model was induced with STZ, and after intravitreal injection of adeno-associated virus (AAV) vector containing TTR cDNA, the pathological progression of DR was partially reversed with overexpressed TTR (Figure 1).…”

“…Our previous work has demonstrated that higher vitreous TTR content of high myopia patient [13] might help to prevent the progression of DR [14]. The serum and vitreous TTR levels in DR patients were associated with DR progression [15]; TTR suppressed angiogenesis by affecting the angiopoietin-Tie signaling pathway in hyperglycemia [16], and enhanced the apoptosis of hRECs through a hypoxia-associated 78-kDa glucose-regulated protein (GRP78)-dependent pathway [17]. Still, the regulatory mechanisms involving TTR in DR are not entirely clear.Long non-coding RNAs (lncRNAs) regulate targeted mRNA expression via the microRNA (miRNA) response element known as competing endogenous RNA (ceRNA) [18,19].…”

Identification of O-GlcNAcylation Modification in Diabetic Retinopathy and Crosstalk with Phosphorylation of STAT3 in Retina Vascular Endothelium Cells

“…In addition to hyperglycemia, hypoxia caused by capillary abnormalities is another vital factor to DR [30]. We cultured primary BRVECs in high glucose combined with hypoxic …”