2-arachidonoylglycerol (2-AG) is an endogenous mediator of inflammatory processes and a ligand of the endocannabinoid system. There is evidence for its key role in cardiovascular diseases, especially atherogenesis and cardiac remodelling, but the molecular mechanisms are still poorly understood.

 

We used primary CD14+ human monocytes, isolated from blood of healthy donors, as a model to mimic 2-AG-induced monocyte activation in cardiovascular diseases. Upon stimulation with 2-AG for 4 to 24 hours, cytokine release was measured using multiplex assays. Changes in the transcriptome were quantified by using 3’ mRNA sequencing and analysed with our in-house bioinformatics platform. Using this approach, we identified key pathways and their respective downstream targets, which were subsequently validated in human plasma samples. Blood samples were drawn from patients with either stable coronary artery disease (CAD) or non-ST-segment elevation myocardial infarction (NSTEMI). The samples were drawn from the arterial sheath and from the culprit coronary artery to account for local concentration gradients, which have been reported for 2-AG. We correlated the identified biomarkers to the levels of circulating 2-AG.

 

Stimulation of human monocytes with 2-AG enhanced the release of pro-inflammatory cytokines, especially IL-1β, CXCL-10 and TNFα. Analysis of transcriptional changes (GO-term and pathway analysis) revealed the upregulation of pro-inflammatory programs, like the NF-kB signalling pathway and the TNF signalling pathway. Furthermore, transcription of CXCL-10 was significantly upregulated.

In human plasma samples, multiplex assays revealed a significantly increased concentration of CXCL-10 in the coronary circulation compared to samples from the sheath in patients with CAD. Additionally, we confirmed that plasma levels of Galectin-3 and Osteopontin, both linked to IL-1β-, TNFα-, and NF-kB signalling and involved in adverse cardiac remodelling, were significantly enhanced. Pearson’s correlation revealed a significant correlation between the levels of circulating 2-AG and CXCL-10, Galectin-3, Osteopontin and Pentraxin-3.

 

The present study investigates the mechanism by which 2-AG promotes inflammation and leads to cardiac remodelling. Due to elevated concentrations of 2-AG, inflammation is promoted via the canonical pathway, leading to increased NF-kB signalling and to an enhanced release of IL-1β and TNFα. This causes increased transcription and release of the pro-inflammatory cytokine CXCL-10. Other biomarkers for cardiac remodelling, related to NF-kB/TNFα or IL-1β signalling, were also increased, suggesting a pivotal role for 2-AG upstream in this process. In patients suffering from acute or chronic coronary syndrome, concentrations of remodelling biomarkers correlated positively with the concentrations of 2-AG, again highlighting the importance of 2-AG in cardiovascular disease.