The GPR55 antagonist CID16020046 protects against ox-LDL-induced inflammation in human aortic endothelial cells (HAECs)

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Abstract

Atherosclerosis is a commonplace cardiovascular disease which affects most people in old age. While its causes are currently poorly understood, continuous study is being performed in order to elucidate both the pathogenesis and treatment of this insidious disease. Atherosclerosis is presently thought to be linked to several factors such as endothelial dysfunction, monocyte adhesion to the intima of the artery, and increased oxidative stress. Oxidized low-density lipoprotein (ox-LDL), colloquially known as the “bad cholesterol”, is known to play a critical role in the previously mentioned atherosclerotic processes. In this study, our goal was to elucidate the role of the lysophospholipid receptor G protein-coupled receptor 55 (GPR55) and its antagonist, the cannabinoid CID16020046, in endothelial dysfunction. While their existence and especially their role in atherosclerosis has only semi-recently been elucidated, a growing body of research has begun to link their interaction to antiatherosclerosis. In our research, we found CID16020046 to have distinct atheroprotective properties such as anti-inflammation, antioxidant, and inhibition of monocyte attachment to endothelial cells. While there was previously a small body of research regarding the potential of cannabinoids to treat or prevent atherosclerosis, studies on the treatment potential of CID16020046 were even fewer. Thus, this study is one of the first to explore the effects of cannabinoids in atherosclerosis. Our findings in the present study provide a strong argument for the use of CID16020046 in the treatment of atherosclerosis as well as a basis for further experimentation using cannabinoids as therapy against atherosclerosis.

Introduction

Oxidized low-density lipoprotein (ox-LDL) is associated with endothelial cell dysfunction and plays an important role in the pathogenesis of atherosclerosis [1,2]. One of the consequences of endothelial dysfunction is an enhanced inflammatory response via increased expression of proinflammatory cytokines, including interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) [3]. Overproduction of ox-LDL leads to an increase in the rate of the attachment of monocytes to the arterial endothelium, which is a major cause of the characteristic plaque buildup seen in atherosclerosis. This is a result of the expression of vascular adhesion molecules such as vascular cellular adhesion molecule-1 (VCAM-1) and endothelial selectin (E-selectin) [4,5]. Additionally, ox-LDL inhibits the activation of the Krüppel-like factor 2 (KLF2) signaling pathway, which has recently been shown to be heavily involved in preventing atherosclerosis and mediating the effects of other test items in this study, including inflammation and monocyte attachment to the endothelium [[6], [7], [8], [9]]. Interestingly, other lipids, including endocannabinoids, ysophospholipids and sphingolipids, have been reported to differentially regulate athero-progression and plaque vulnerability via the expression of the two main cannabinoid receptors type-1 and type-2 (CB1R and CB2R) [10,11]. However, the physiological functions of CB1R and CB2R in atherosclerosis are both potentially valuable and controversial [12,13]. Opposite results and conclusions have been presented in independent studies. Recently, researches have paid more attention to the putative "type 3" cannabinoid receptor GPR55. GPR55 is a G protein-coupled receptor, which was identified as having a connection to cannabinoids for the first time in 2001 [14,15]. It has long been thought to be a potential treatment target for symptoms of pain and inflammation owing to its analgesic properties [16]. The endocannabinoid neurotransmitter l-α-lysophosphatidylinositol is a ligand and agonist of GPR55 which was initially found to have a link to GPR55 in 2007 [17]. The GPR55 antagonist CID16020046 (4-[4,6-dihydro-4-(3-hydroxyphenyl)-3-(4-methylphenyl)-6-oxopyrrolo[3,4-c]pyrazol-5(1H)-yl]-benzoic acid) is known for its anti-inflammatory properties in the context of various diseases and in various areas of the body, such as the intestines and, as part of the focus of this study, the vasculature [18]. Importantly, a recent study demonstrated that GPR55 is expressed in human macrophages and is increased by ox-LDL. Agonism of GPR55 exacerbated the accumulation of lipids and blocked cholesterol efflux induced by ox-LDL, while GPR55 antagonism counteracted these effects, suggesting that CID16020046 may have clinical potential [19]. Here, we investigated the effects of antagonism of GPR55 on physiological profiles of human aortic endothelial cells (HAECs) under stimulation with ox-LDL, including cell viability, oxidative stress, inflammation, adhesion of monocytes to endothelial cells, and activation of the KLF2 transcription factor. Our results indicate a promising role for CID16020046 in the prevention of endothelial insults caused by ox-LDL.

Section snippets

Cell culture and treatment

Human aortic endothelial cells (HAECs) used in all experiments were purchased from Lonza (Basel, Switzerland). The cells were then cultured in endothelial growth media (EGM2). Human monocytic leukemia cell line THP-1 cells from American Type Culture Collection (ATCC) were maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum. Cells used in all experiments were incubated in a 5% CO2 incubator overnight at a temperature of 37 °C. HAECs were treated with

Agonism of GPR55 mediates ox-LDL-induced cell death

We found that ox-LDL was capable of upregulating GPR55 in a dose-dependent manner, with the highest dose augmenting the mRNA expression level by 3.5-fold. A similar pattern was observed in the protein expression level of GPR55, which was upregulated by approximately 3-fold by the higher dose (Fig. 1). Furthermore, we found that ox-LDL-induced overexpression of GPR55 resulted in increased cell death of HAECs. We used the GPR55 agonist 1-α-lysophosphatidylinositol (LPI) to demonstrate the

Discussion

Cannabinoids have long suffered cultural stigma due to their origin in the cannabis plant, which has been treated as a controlled substance in many countries throughout the world. However, an increasing body of research is demonstrating numerous medical benefits of cannabinoids in a vast array of diseases, such as in the case of muscular dystrophy, epilepsy, cardiovascular diseases including atherosclerosis, and others [[20], [21], [22]]. However, the use and assessed medical value of cannabis

CRediT authorship contribution statement

Yaowen Wang: Conceptualization, Investigation, Resources, Methodology, Software, Data curation. Wei Pan: Investigation, Software, Data curation. Yan Wang: Visualization, Supervision, Writing - review & editing. Yuehui Yin: Conceptualization, Supervision, Validation, Writing - review & editing.

Acknowledgement

The National Nature Science Foundation of China (81860643).

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