Title: LysoPI/GPR55 pathway promotes endothelial activation, vascular inflammation and atherosclerosis Atherosclerosis and its complications are the leading cause of morbidity and mortality in the US. Novel antiinflammatory therapies are urgently needed to inhibit atherosclerosis. The goal of this project is to determine the roles and mechanisms underlying lysophosphatidylinositol (lysoPI) promoted endothelial cell (EC) activation and atherosclerosis. We published numerous papers on EC activation, atherosclerosis and lysophospholipids (lysolipids). Our strong preliminary data and publications show that: 1) we reported a new concept that most lysolipids are new conditional classical damage- associated molecular pattern (DAMPs) that do not bind to classical DAMP receptors but bind to their own receptors to stimulate inflammation; 2) our metabolomics data showed that lysoPI is significantly increased in the aortas and plasma of ApoE-/- mice fed with high fat diet (HF) for three weeks; 3) lysoPI specifically bind to its receptor, G protein coupled receptor 55 (GPR55), and activates primary human aortic endothelial cells (HAECs) by upregulating EC adhesion molecule ICAM-1; 4) Mechanistically, lysoPI induces generation of mitochondrial reactive oxygen species (mtROS) in HAECs and induces proinflammatory proteinarginine methyltransferase 1(PRMT1) activity; 5) our RNA-Seq data showed that significantly different from that induced by lysoPI's relative lysoPC, lysoPI induces sustained EC activation by upregulating DAMP receptors inflammasomes/caspase-1 and proinflammatory secretomes; 6) we established ApoE-/-/GPR55-/- DKO mice; and DKO mice have significantly decreased atherosclerosis in comparison to that in ApoE KO mice fed with HF for 12 weeks; and finally, 7) we generated EC-specific GPR55 KO mice and PRMT1 KO mice. Based on our strong preliminary data and publications, the central hypothesis to be tested is that early hyperlipidemia-induced lysoPI stimulates and sustains aortic EC activation via a GPR55-PRMT1 pathway, proinflammatory monocyte (MC) recruitment, thereby contributing to atherosclerosis. We will test this hypothesis using three aims. Aim 1 will determine expression and function of lysoPI/GPR55-PRMT1 pathway in HAECs activated by hyperlipidemic stimuli and in aortas of ApoE-/- mice (relevant studies). Aim 2 will examine the mechanisms by which lysoPI/GPR55 induces sustained aortic EC activation via inducing PRMT1 upregulation-mtROS generation, which further prolongs EC activation (mechanistic studies). Aim 3 will determine whether inhibition of lysoPI/GPR55 and PRMT1 in EC (EC-specific KO) and other vascular cells (global KO) would decrease atherogenesis in ApoE-/- mice (therapeutic studies).