1Universitätsklinikum Bonn Medizinische Klinik und Poliklinik II Bonn, Deutschland; 2University Hospital Bonn Institute of Innate Immunity Bonn, Deutschland
Background:
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. In a previous study we found a local gradient in 2-AG concentration between the peripheral and coronary circulation.
Methods:
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 multiplexing assays. Transcriptomic changes were quantified through 3' mRNA sequencing and analyzed with a focus on hallmark pathway analysis and Gene Set Enrichment Analysis (GSEA). 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.
Results:
The analysis of transcriptional changes revealed that the top three upregulated hallmark pathways after 4 hours of stimulation were TNFα signaling via the NF-κB pathway, the INF-γ response, and the inflammatory response. The GSEA analysis after 24 hours provided a robust signature of a pro-inflammatory response, leading to the release of cytokines from the monocytes.
Significantly higher concentrations of TNFα, INFγ, and IL-1β were observed in the supernatants of the stimulated monocytes. Multiplexing assays of human plasma samples revealed significantly elevated levels of cardiac remodeling markers, including IP-10, Galectin-3, and Osteopontin, in the coronary circulation compared to samples from the arterial sheath in patients with CAD.
Intriquingly, Pearson’s correlation revealed a significant correlation between the remodelling proteins Galectin-3, IP-10, Osteopontin and plasma 2-AG levels.
Conclusion:
In this study, we identified that stimulation with 2-AG in monocytes activates pro-inflammatory signaling pathways, particularly NF-κB and TNFα, leading to the release of cytokines like IL-1β and TNFα. These cytokines are known to drive cardiac remodeling and are associated with adverse outcomes.
This study demonstrated that cardiac remodeling markers are more concentrated in coronary vessels than in the periphery. The similarity between these remodeling markers and 2-AG encouraged us to correlate them. In patients with acute or chronic coronary syndrome, concentrations of remodeling biomarkers positively correlated with the levels of 2-AG, underscoring the importance of 2-AG in cardiovascular disease.