Abstract Wed148: The impact of TMAO and Oxidized Linoleic Acid on selected Calcification Pathways in Endothelial Cells

Introduction: A complex interaction between vascular and osteogenic genes controls calcification in both bone and vascular tissues. Trimethylamine-N-oxide (TMAO), Linoleic Acid (LA) and oxidized linoleic acid (Ox-LA) have been linked to endothelial dysfunction and vascular calcification. The purpose of this study is to investigate the role of TMAO, and Ox-LA combination on endothelial cells calcium regulations. The human endothelial cells were treated with TMAO and in combination with Ox-LA to mimic the gut microbiota environment. Methods: Human Endothelial cells were cultured in Lonza’s Endothelial Cell Growth Medium at 37°C in a 5% CO2 humidified incubator. Cells were treated with linoleic acid (LA), oxidized linoleic acid (ox-LA), and trimethylamine-N-oxide (TMAO) at concentrations of 100uM for 12 and 18 hrs. Calcification related genes such as RUNX-2, RANK, BMP-2, Sm22a, a-sma, OPN were determined by real-time quantitative (RT-PCR). cDNA was synthesized using iScript Reverse Transciptase Supermix, and RT-PCR was performed using SYBR Green with specific designed primers. Gene expression was normalized to GAPDH, and relative quantification was calculated using the ΔΔCt method. Results: Our results showed upregulation of calcification genes RUNX-2, Osteopontin, and BMP-2 following treatment with Ox-LA and TMAO compared to LA with TMAO which suggests a role for gut microbiota Ox-LA in vascular calcification. BMP-2, which is necessary for bone formation, was downregulated three times, as did RUNX2, a transcription factor that is crucial for osteoblast differentiation. A significant loss in mineralization potential was indicated by the approximately 3.5-fold decrease in OPN expression. The calcific responses were exacerbated by the additive effect of Ox-LA in a time dependent manner. Most notably is the nearly 6-fold downregulation of SM22a, demonstrating a significant inhibition of this gene's expression. Conclusion: Upregulation of the calcification genes and downregulation of α-SMA and SM22a suggests a change from contractile to osteogenic phenotype of endothelial cells suggesting their potential role in the calcification progression.