G-PROTEIN COUPLED RECEPTOR 55: A FACILIATOR OF ATHEROSCLEROSIS DEVELOPMENT

Abstract

Cardiovascular diseases (CVDs) have been the leading cause of mortality in the United States since 1975. Owing to the pervasive nature of risk factors such as contemporary high-fat diets and stationary lifestyles, deaths attributable to CVDs surpassed those from cancer in 2015. Atherosclerosis, a chronic inflammatory condition, is characterized by the accumulation of plaque in major arteries and arterial stiffness. Its manifestation varies depending on the affected artery and serves as a pivotal pathogenic factor underlying various CVDs. Therefore, atherosclerosis and its associated complications significantly reduce life expectancy and rank high in global morbidity and mortality. Current treatments for atherosclerosis fail to prevent over 70% of clinical events related to CVDs. This is often due to off-target effects, acute injuries, or complications such as in-stent restenosis following surgery. Hence, the development of more effective therapies is urgently needed. Atherosclerosis initiates when endothelial cells (ECs) respond to danger-associated molecular patterns derived from excessive lipid particles and inflammation by upregulating adhesive molecules, marking the onset of EC activation and dysfunction. However, the precise mechanisms through which EC activation promotes atherosclerosis development have not been fully characterized.Metabolomic reprogramming plays a pivotal role in the pathogenesis of multifactorial diseases like atherosclerosis, offering deep insights into the effects of genetic and protein changes. In our research, we conducted a comprehensive examination of metabolite shifts in various tissues of atherogenic apolipoprotein E-deficient (ApoE-/-) mice, which were subjected to a high-fat diet for three weeks, in comparison with wild-type (WT) mice maintained on a normal chow (NC) diet. We observed a marked increase in lysolipid-associated metabolites, especially lysophosphatidylinositols (lysoPIs), in the ApoE-/- mice. This finding suggested metabolic shifts in the early atherosclerosis development. Transcriptomic analysis via microarray revealed alterations in lysoPI levels, presumably due to changed phospholipase expression and activity, which could be a contributing factor. Further investigations of mouse plasma using mass spectrometry confirmed a consistent increase in lysoPI concentrations over time in ApoE-/- mice fed a high-fat diet, highlighting their critical influence on the progression of atherosclerosis. G-protein-coupled receptor 55 (GPR55) is identified as a specific receptor for LysoPI species and is widely expressed in various human and mouse tissues. GPCRs represent a large family of membrane receptors crucial for transmitting external signals into cellular responses, thereby influencing a wide range of pathophysiological functions. Currently, approximately 60% of pharmaceuticals target GPCRs, underscoring their importance in drug discovery and development. Consequently, our subsequent studies primarily focused on the role of GPR55 in the development of atherosclerosis. Our research detected a significantly heightened protein expression of GPR55 within the aortas of ApoE-/- mice on a high-fat diet over a period of 12 weeks by Western blot analyses. This finding emphasizes the potential importance of GPR55 in the atherosclerosis pathogenesis. Additionally, the deficiency of GPR55 resulted in a marked decrease in atherosclerotic lesions in the aortas of global GPR55/ApoE double knockout (gDKO) mice, alongside reduce monocyte adhesion to the endothelium as observed via intravital microscopy, which indicates GPR55 may also play an important role in EC during atherosclerosis development. This hypothesis was further corroborated by the normalized appearance of endothelial cell morphology in gDKO comparison to that observed in ApoE-/- mice, as determined by Transmission Electron Microscopy. Further in vitro experiments indicated that LysoPI activation of GPR55 might increase adhesion molecule expression, pro-inflammatory cytokine secretion, and mitochondrial reactive oxygen species (mtROS) production. These findings, studied by RT-PCR, western blotting, cytokine array analysis, Fluorescence-Activated Cell Sorting (FACS), and Seahorse Mito Stress tests, provide the potential mechanisms into how GPR55 activation may promote atherosclerosis by driving endothelial cell activation and inflammation. Our project marks a significant advance in the comprehensive exploration of early hyperlipidemia-induced metabolomic reprogramming and the impact of lysoPI-GPR55 signaling—a newly identified danger-associated molecular pattern—on endothelial cells at the onset of atherosclerosis. Utilizing cutting-edge methodologies and informed RNA sequencing analysis, we have identified GPR55 as a potential therapeutic target. This receptor may be instrumental in regulating critical aspects of endothelial cell activation, such as lipid metabolism, monocyte recruitment, and inflammatory responses, which are integral to the development of atherosclerosis.