Abstract
Purpose :
To examine the detailed mechanism of PPAR alpha activation in human umbilical vein endothelial cell (HUVEC) by genome-wide analysis, and to confirm the effect of pemafibrate for retinal inflammation in diabetic rats.
Methods :
The genome wide transcriptome and PPARa distribution in HUVEC treated with pemafibrate were analyzed by DNA microarray and chromatin-immunoprecipitation sequencing (ChIP-seq). To confirm the results of genome-wide analysis, ChIP-qPCR and immunoblot analysis were performed. In vivo experiments, streptozotocin-induced diabetic rats were treated with pemafibrate and THBD siRNA or control siRNA, and were examined retinal inflammation by immunoblot, vascular leukostasis and vascular leakage.
Results :
In microarray analysis, 1062 genes were >1.5-fold upregulated by pemafibrate treatment compared with DMSO treatment (control) 24 hour after treatment. In ChIP-seq analysis, ChIP-seq calling by SICER identified 6017 genomic regions as significant binding sites of PPAR alpha in pemafibrate-treated HUVEC. PPAR alpha binding sites, which were annotated to 4186 proximal genes, 1062 pemafibrate-induced genes with an overlapped of 221 genes. These genes included THBD, which encodes thrombomodulin (TM). Immunoblot analysis showed that TM was upregulated by pemafibrate treatment in HUVEC and human retinal microvascular endothelial cell. Results of ChIP-qPCR showed that PPAR alpha was significantly bound the promoter region of THBD by pemafibrate treatment compared with vehicle treatment (p<0.05). In vivo experiments, the protein levels of retinal inflammatory molecules in retina were significantly decreased in pemafibrate-treated diabetic rats, and were attenuated by intravitreal injection of THBD siRNA (p<0.05, respectively). Treatment of pemafibrate significantly reduced retinal vascular leukostasis and leakage in diabetic rats, compared with the untreated diabetic rats. However, vascular leukostasis and leakage were significantly increased in the THBD siRNA treated rats compared with control siRNA treated rats (p<0.05, respectively).
Conclusions :
TM is directly regulated by PPAR alpha activation in endothelial cell. Treatment of pemafibrate suppressed the inflammatory response in diabetic retina, and these effects were dependent on expression of TM.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.