Abstract
Purpose :
Clinical evidence suggests that diabetic macular edema occurs at least in part due to breakdown of the inner blood retinal barrier (iBRB). Our understanding of the molecular composition of the iBRB and how this is disrupted during diabetes remains incomplete. In this study we have begun to address this knowledge gap by using bulk RNA-Seq to analyze the barrier genes expressed in HRECs under conditions mimicking key aspects of the diabetic milieu.
Methods :
HRECs were cultured under normoglycemic or hyperglycemic conditions for 4w and then treated with VEGF165 (25ng/ml) for 24h. RNA was extracted, libraries prepared, and sequencing performed on an Illumina NextSeq system. Changes in barrier gene expression were determined using Limma-Voom and mapped onto an in-silico model of the iBRB created using Cytoscape and GENEmania. For barrier genes downregulated by hyperglycemia, CiiiDER software was used to identify over-represented transcription factor (TF) binding sites. Under normoglycemic conditions, siRNA knockdown studies were performed for several of the identified TFs to investigate their involvement in regulating HREC barrier gene expression.
Results :
A detailed in silico model of the junctional and structural genes involved in forming the HREC barrier was built and gene expression levels mapped onto this.Treatment with hyperglycemia caused a significant downregulation of many junctional genes including CDH5,6 and 11, CLDN1,and 14, CADM1 and FLRT3. In comparison, VEGF elicited only minor changes, both in the absence and presence of hyperglycemia. Various over-represented TF-binding sites were discovered in barrier genes downregulated in hyperglycemia, including ZBTB12, THAP1, TCFL5 and ZNF317. siRNA studies of those TF suggested that there is no significant change in barrier genes expression.
Conclusions :
Hyperglycemia has a greater downregulation effect on barrier gene expression in HRECs than VEGF. These effects could contribute to iBRB breakdown during diabetes. RNA-seq data allowed us to identify enriched TF binding sites within barrier genes downregulated in hyperglycemia. However, knockdown of these TFs under normoglycemic conditions had no broad-ranging effect on barrier gene expression. These results suggest that hyperglycemia-induced downregulation of barrier gene expression in HRECs is not likely to be mediated primarily by activation of an individual TF.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.