Macrophage P2Y6 receptor deletion attenuates atherosclerosis by limiting foam cell formation through phospholipase Cß/store-operated calcium entry/calreticulin/scavenger receptor A pathways.

BACKGROUND AND AIMS: Macrophage-derived foam cells play a causal role during the pathogenesis of atherosclerosis. P2Y6 receptor (P2Y6R) highly expressed has been considered as a disease-causing factor in atherogenesis, but the detailed mechanism remains unknown. This study aims to explore P2Y6R in regulation of macrophage foaming, atherogenesis, and its downstream pathways. Furthermore, the present study sought to find a potent P2Y6R antagonist and investigate the feasibility of P2Y6R-targeting therapy for atherosclerosis. METHODS: The P2Y6R expression was examined in human atherosclerotic plaques and mouse artery. Atherosclerosis animal models were established in whole-body P2Y6R or macrophage-specific P2Y6R knockout mice to evaluate the role of P2Y6R. RNA sequencing, DNA pull-down experiments, and proteomic approaches were performed to investigate the downstream mechanisms. High-throughput Glide docking pipeline from repurposing drug library was performed to find potent P2Y6R antagonists. RESULTS: The P2Y6R deficiency alleviated atherogenesis characterized by decreasing plaque formation and lipid deposition of the aorta. Mechanically, deletion of macrophage P2Y6R significantly inhibited uptake of oxidized low-density lipoprotein through decreasing scavenger receptor A expression mediated by phospholipase Cß/store-operated calcium entry pathways. More importantly, P2Y6R deficiency reduced the binding of scavenger receptor A to CALR, accompanied by dissociation of calreticulin and STIM1. Interestingly, thiamine pyrophosphate was found as a potent P2Y6R antagonist with excellent P2Y6R antagonistic activity and binding affinity, of which the pharmacodynamic effect and mechanism on atherosclerosis were verified. CONCLUSIONS: Macrophage P2Y6R regulates phospholipase Cß/store-operated calcium entry/calreticulin signalling pathway to increase scavenger receptor A protein level, thereby improving foam cell formation and atherosclerosis, indicating that the P2Y6R may be a potential therapeutic target for intervention of atherosclerotic diseases using P2Y6R antagonists including thiamine pyrophosphate.

P2Y6 Receptor Activation Aggravates NLRP3-dependent Microglial Pyroptosis via Downregulation of the PI3K/AKT Pathway in a Mouse Model of Intracerebral Hemorrhage.

Pro-inflammatory signals generated after intracerebral hemorrhage (ICH) trigger a form of regulated cell death known as pyroptosis in microglia. White matter injury (WMI) refers to the condition where the white matter area of the brain suffers from mechanical, ischemic, metabolic, or inflammatory damage. Although the p2Y purinoceptor 6 (P2Y6R) plays a significant role in the control of inflammatory reactions in central nervous system diseases, its roles in the development of microglial pyroptosis and WMI following ICH remain unclear. In this study, we sought to clarify the role of P2Y6R in microglial pyroptosis and WMI by using an experimental mouse model of ICH. Type IV collagenase was injected into male C57BL/6 mice to induce ICH. Mice were then treated with MRS2578 and LY294002 to inhibit P2Y6R and phosphatidylinositol 3-kinase (PI3K), respectively. Bio-conductivity analysis was performed to examine PI3K/AKT pathway involvement in microglial pyroptosis. Quantitative Real-Time PCR, immunofluorescence staining, and western blot were conducted to examine microglial pyroptosis and WMI following ICH. A modified Garcia test, corner turning test, and forelimb placement test were used to assess neurobehavior. Hematoxylin-eosin staining (HE) was performed to detect cells damage around hematoma. Increases in the expression of P2Y6R, NLRP3, ASC, Caspase-1, and GSDMD were observed after ICH. P2Y6R was only expressed on microglia. MRS2578, a specific inhibitor of P2Y6R, attenuated short-term neurobehavioral deficits, brain edema and hematoma volume while improving both microglial pyroptosis and WMI. These changes were accompanied by decreases in pyroptosis-related proteins and pro-inflammatory cytokines both in vivo and vitro. Bioinformatic analysis revealed an association between the PI3K/AKT pathway and P2Y6R-mediated microglial pyroptosis. The effects of MRS2578 were partially reversed by treatment with LY294002, a specific PI3K inhibitor. P2Y6R inhibition alleviates microglial pyroptosis and WMI and ameliorates neurological deficits through the PI3K/AKT pathway after ICH. Consequently, targeting P2Y6R might be a promising approach for ICH treatment.

Inflammatory neuronal loss in the substantia nigra induced by systemic lipopolysaccharide is prevented by knockout of the P2Y6 receptor in mice.

Inflammation may contribute to multiple brain pathologies. One cause of inflammation is lipopolysaccharide/endotoxin (LPS), the levels of which are elevated in blood and/or brain during bacterial infections, gut dysfunction and neurodegenerative diseases, such as Parkinson's disease. How inflammation causes neuronal loss is unclear, but one potential mechanism is microglial phagocytosis of neurons, which is dependent on the microglial P2Y6 receptor. We investigated here whether the P2Y6 receptor was required for inflammatory neuronal loss. Intraperitoneal injection of LPS on 4 successive days resulted in specific loss of dopaminergic neurons (measured as cells staining with tyrosine hydroxylase or NeuN) in the substantia nigra of wild-type mice, but no neuronal loss in cortex or hippocampus. This supports the hypothesis that neuronal loss in Parkinson's disease may be driven by peripheral LPS. By contrast, there was no LPS-induced neuronal loss in P2Y6 receptor knockout mice. In vitro, LPS-induced microglial phagocytosis of cells was prevented by inhibition of the P2Y6 receptor, and LPS-induced neuronal loss was reduced in mixed glial-neuronal cultures from P2Y6 receptor knockout mice. This supports the hypothesis that microglial phagocytosis contributes to inflammatory neuronal loss, and can be prevented by blocking the P2Y6 receptor, suggesting that P2Y6 receptor antagonists might be used to prevent inflammatory neuronal loss in Parkinson's disease and other brain pathologies involving inflammatory neuronal loss.

Microglia Purinoceptor P2Y6: An Emerging Therapeutic Target in CNS Diseases.

The purinergic receptor P2Y6 is expressed in immune cells, including the microglia that are implicated in neurological disorders. Its ligand, UDP, is a signaling molecule that can serve as an "find-me" signal when released in significant quantities by damaged/dying cells. The binding of UDP by P2Y6R leads to the activation of different biochemical pathways, depending on the disease context and the pathological environment. Generally, P2Y6R stimulates phagocytosis. However, whether or not phagocytosis coincides with cell activation or the secretion of pro-inflammatory cytokines needs further investigation. The current review aims to discuss the various functions of P2Y6R in some CNS disorders. We present evidence that P2Y6R may have a detrimental or beneficial role in the nervous system, in the context of neurological pathologies, such as ischemic stroke, Alzheimer's disease, Parkinson's disease, radiation-induced brain injury, and neuropathic pain.

Purinergic receptor P2Y6 contributes to 1-methyl-4-phenylpyridinium-induced oxidative stress and cell death in neuronal SH-SY5Y cells.

Oxidative stress and neural degeneration have been shown to be involved in the pathogenesis of Parkinson's disease (PD). The P2Y6 purinergic receptor (P2Y6R) has been shown to participate in the activation of microglia and the production of pro-inflammatory factors induced by lipopolysaccharide to cause neuronal loss. However, the function of P2Y6R during oxidative stress in neurons is unclear. In the present study, 1-methyl-4-phenylpyridinium (MPP+ ) treatment increased the level of UDP/P2Y6R on neuronal SH-SY5Y cells. Importantly, pharmacological inhibition of P2Y6R or knockdown of P2Y6R using a siRNA exerted an increased protective effect by preventing MPP+ -induced increases in the levels of reactive oxygen species (ROS), superoxide anion, inducible nitric oxide synthase (iNOS), and malondialdehyde (MDA) and down-regulation of superoxide dismutase 1 (SOD1) expression. UDP, an agonist of P2Y6R, enhanced the effects of MPP+ , which was also inhibited by apyrase or MRS2578. Additionally, P2Y6R knockdown also significantly reversed both the loss of cell viability and the increase in the levels of phosphorylated extracellular signal-regulated protein kinase (p-ERK1/2) and p38 (p-p38) caused by MPP+ stimulation. However, the inhibition of the ERK1/2 and p38 kinase signaling pathways had no effect on P2Y6R expression. Taken together, these results support the hypothesis that P2Y6R expressed on neuronal SH-SY5Y cell is associated with the progression of oxidative stress and cell death induced by MPP+ , suggesting that P2Y6R may play an important role in the pathogenesis of PD.

Uridine adenosine tetraphosphate acts as a proangiogenic factor in vitro through purinergic P2Y receptors.

Uridine adenosine tetraphosphate (Up4A), a dinucleotide, exerts vascular influence via purinergic receptors (PR). We investigated the effects of Up4A on angiogenesis and the putative PR involved. Tubule formation assay was performed in a three-dimensional system, in which human endothelial cells were cocultured with pericytes with various Up4A concentrations for 5 days. Expression of PR subtypes and angiogenic factors was assessed in human endothelial cells with and without P2Y6R antagonist. No difference in initial tubule formation was detected between Up4A stimulation and control conditions at day 2 In contrast, a significant increase in vascular density in response to Up4A was observed at day 5 Up4A at an optimal concentration of 5 µM promoted total tubule length, number of tubules, and number of junctions, all of which were inhibited by the P2Y6R antagonist MRS2578. Higher concentrations of Up4A (10 µM) had no effects on angiogenesis parameters. Up4A increased mRNA level of P2YRs (P2Y2R, P2Y4R, and P2Y6R) but not P2XR (P2X4R and P2X7R) or P1R (A2AR and A2BR), while Up4A upregulated VEGFA and ANGPT1, but not VEGFR2, ANGPT2, Tie1, and Tie2. In addition, Up4A increased VEGFA protein levels. Transcriptional upregulation of P2YRs by Up4A was inhibited by MRS2578. In conclusion, Up4A is functionally capable of promoting tubule formation in an in vitro coculture system, which is likely mediated by pyrimidine-favored P2YRs but not P2XRs or P1Rs, and involves upregulation of angiogenic factors.