Abstract 195: Transcriptional Regulation Of Transcriptional Regulation Of Triggering Receptor Expressed On Myeloid Cells-1 By Low Shear Stress In A Three-dimensional Co-culture Of Coronary Artery Smooth Muscle Cells, Endothelial Cells, And Monocytes

Abstract

Objectives: To identify transcriptional factors involved in the expression of Triggering Receptor Expressed on Myeloid Cells-1 (TREM-1) in response to low shear stress in an in-vitro model of coronary artery atherosclerosis. Methods: Human coronary artery smooth muscle cells were mixed with neutralized type I collagen solution and plated on glass coverslips. The human coronary artery endothelial cells (HCAECs) were then plated on top of the collagen gels followed by plating monocytes (THP-1 cells) on top of the HCAECs. After 48 h, the co-culture was treated with or without low shear stress (LSS, 5±3 dyne/cm 2 ) using the Flexcell Streamer for 30 min. Protein and mRNA of TREM-1 expression was assessed by immunofluorescence staining and real time RT-PCR. The expression of transcriptional factors involved in the expression of TREM-1 was assessed by ChIP assay. Results: LSS significantly stimulated TREM-1 expression as evidenced by IF staining and mRNA expression (fold increase vs control: 43.2 ± 9.7, Fig 1A and 1B ). ChIP assay demonstrated that, under control condition (no flow), NF-κB (p65), PU.1 and activation transcription factor 2 (ATF2) bound to TREM-1 promoter region 2, but not to region 1 ( Fig 2A ). In response to LSS for 30 min, however, binding of the NF-κB (p65), PU.1 and ATF2 to TREM-1 promoter region 2 was significantly enhanced; furthermore, LSS treatment induced binding of NF-κB (p65), PU.1 and ATF2 to TREM-1 promoter region 1 ( Fig 2B) . Conclusions: Transcriptional factors NF-κB (p65), PU.1 and ATF2 are critically involved in the TREM-1 expression in response to LSS stimulation in an in-vitro model of human coronary artery atherosclerosis.