Abstract
Purpose::
To identify the role of FOXO1 in high glucose induced retinal microvascular endothelial cell death.
Methods::
Rat retinal microvascular endothelial cell were isolated and examined at passages 3-8. For apoptosis and RNAi assays, cells were cultured in DMEM media supplemented with 0.25% FBS and different concentrations of high glucose and transfected with or without rat FOXO1 specific siRNA with the target sequence "CCA GCT ATA AAT GCA CAT TTA" or scrambled siRNA (3). Apoptosis was measured by ELISA detection of cytoplasmic histone associated DNA. FOXO1 activation was quantified by ActiveMotif assay. Total RNA was isolated and subjected to focused microarray analysis (SuperArray) followed by data mining with GEArray Expression Analysis Suite. Selected results were validated by real time qPCR. Significance was determined by analysis of variance with Scheffe's post-hoc for comparisons between multiple groups at the P<0.05 level.
Results::
High glucose induced a dose dependent increase in retinal microvascular endothelial cell apoptosis and FOXO1 activation. Transfection with FOXO1 siRNA decreased FOXO1 mRNA levels by 90% and reduced high glucose induced apoptosis by more than 60% (P<0.05). mRNA profiling revealed that high glucose induced proapoptotic genes such as caspase-3, caspase-6, caspase-8 and proinflammatory genes such as ICAM-2, IL-6 and TNF-alpha by more than two fold. FOXO1 RNAi substantially reduced mRNA levels of these pro-apoptotic and pro-inflammatory genes(p<0.05).
Conclusions::
High glucose induced FOXO1 activation, pro-apoptotic gene expression and apoptosis in retinal microvascular cells. Both apoptotic gene expression and apoptosis were significantly reduced when FOXO1 was silenced indicating that FOXO1 plays an important role in microvascular cell death by high glucose. These results give insight into potential mechanisms for the development of acellular capillaries in early diabetic retinopathy.
Keywords: diabetes • transcription factors • apoptosis/cell death