Abstract
Purpose :
Pericyte dysfunction in the earliest stages of diabetic retinopathy (DR) results in modification of the physical interaction of endothelial cells and pericytes and the infer of pericytes in regulation the endothelial permeability barrier. In this study, using RNA-sequencing we define the transcriptomic profile of pericytes in diabetic animals and provide insight into the role of pericytes in the regulation of vascular cell interaction and function in DR.
Methods :
C57bl/6j mice were made diabetic by streptozotocin injection. Retinal tissue from non-diabetic and diabetic mouse eyes 10 in each group were digested with collagenase D to obtain single cell suspension. Purification of Pericytes was done through fluorescent activated cell sorting (FACS) using pericyte specific fluorescent antibodies, PDGFRb-APC and NG2-PE. For RNA sequencing and qPCR analysis, a cDNA library was generated using template switching oligo and the resulting libraries were sequenced using paired-end Illumina sequencing. Molecular functional pathways were analyzed using differentially expressed genes (DEGs) through gene set enrichment analysis (GSEA).
Results :
Differential expression analysis revealed 246 genes were significantly down and 124 genes were up-regulated in diabetic pericytes. We observed the expression of pericyte enrichment markers such as Ndfip1 and N-Cadherin (Cdh2) genes through the RNA sequencing and validated Pdgfrb gene expression through qPCR. In pericytes from diabetic retinas, we observed a significant increase in Insulin like growth factor 2 (Igf2), an important angiogenic factor. We further observed increased expression of genes important for angiogenesis using GSEA. We also observed a significant reduction of Cdh2 gene expression in diabetic pericytes. GSEA analysis of downregulated gene indicated a compromise in Glycolysis and integrated energy metabolism associated with cell state, membrane trafficking and altered vesicular transport mechanism in diabetic pericytes.
Conclusions :
Our results indicate significant alterations in gene expression pattern of pericytes isolated from the diabetic retina. Further validation of our approach to identify cell specific molecular change through whole transcriptomic approach in diabetic retina will provide novel insight into the pathogenesis of DR and novel therapeutic targets.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.