Hypoxia and Hyperglycemia-induced proteomic changes in retinal pigment epithelial cells: implications for tight junctions and vascular integrity

Purpose- The retina is characterized by the presence of both an outer and inner blood-retinal barrier, which are essential for establishing immune privilege within the ocular environment. Outer blood-retinal barrier (oBRB) is composed of retinal pigment epithelium (RPE) cells that are interconnected by tight junctions. In Diabetic Macular Edema (DME), integrity of these cellular connections in retinal blood vessels are compromised due to hypoxia and hyperglycemia resulting in the leakage of fluid. We investigated molecular and pathway alterations in the proteome of RPE cells upon exposure to hypoxia, hyperglycaemia, and their combination.

Method- In order to examine the molecular environment of oBRB, we grew RPE cells under normal glucose concentration of 5.5mM and glycaemic conditions 25 mM for 5 days. The cells were further either exposed to normoxia (20% O2) or hypoxia (1% O2) for 24 hours to mimic DME conditions in oBRB. The proteins extracted from cell pellets were subjected to enzymatic digestion, quantification and analysis using shotgun LC-MS/MS. Proteome Discoverer 2.0 used for identification of proteins. The proteins were further analysed with MetaboAnalyst 6.0, Reactome, string etc.

Results- We have got more than 5000 proteins, upon analysis 4081 proteins were with high FDR and 1,658 of them being statistically significant within three groups. Reactome analysis revealed that proteins linked to tight junctions and cell adhesion pathways were disrupted in hypoxic and hyperglycemic cells as compared to normoxic cells. This disruption points to a possible stress-related degradation in the tight connection integrity of RPE cells. Analysing further showed the presence of angiogenin/F-actin pathway and its interactors in normoxic cells, validating its role in preserving vascular integrity. Notably, we found two novel proteins in RPE cells: junction plakoglobin and cofilin. Consistent with earlier research in rat models that linked this protein with neovascularization, cofilin was markedly increased under hypoxia and hyperglycemia. On the other hand, normoxic conditions increased junction plakoglobin, emphasizing its function in preserving cellular integrity under normal physiological situations.

Conclusion- Our results show that tight junction proteins and RPE cell adhesion are altered by hypoxia and hyperglycemia. Subsequent analysis has revealed that angiogenin/F-actin provides protection to the blood vessels in the retina against stress. Cofilin and junction plakoglobin were formerly unknown in RPE cells. DME studies on rats showed increased level of cofilin, similar observation seen in RPE stressed cells validating connection between neovascularization and this protein. Increased levels of junction plakoglobin levels in normoxic conditions, demonstrates its significance for cell integrity.