Diacylglycerols and lysophosphatidic acid, enriched on Lipoprotein(a), contribute to monocyte inflammation
Background: OxPLs play a key role in the atherogenic potential of Lp(a), however Lp(a) is a complex particle that warrants research into additional pro-inflammatory mediators. We hypothesized that additional Lp(a)-associated lipids contribute to the atherogenicity of Lp(a). Methods: Untargeted lipidomics was performed on plasma and isolated lipoprotein fractions. The atherogenicity of the observed Lp(a)-associated lipids was tested ex vivo in primary human monocytes by RNAseq, ELISA, Western Blot and transendothelial migratory assays. Using immunofluorescence staining and single-cell RNA sequencing, the phenotype of macrophages was investigated in human atherosclerotic lesions. Results: Compared to healthy individuals with low/normal Lp(a) levels [median 7 mg/dL (18 nmol/L); N=13], individuals with elevated Lp(a) levels [median 87 mg/dL (218 nmol/L); N=12]  demonstrated an increase in lipid species, particularly diacylglycerols (DGs) and lysophosphatidic acid (LPA). DG and the LPA precursor lysophosphatidylcholine were enriched in the Lp(a) fraction. Ex vivo stimulation with DG(40:6) demonstrated a significant upregulation in pro-inflammatory pathways related to leukocyte migration, chemotaxis, NF-?B signalling and cytokine production. Functional assessment showed a dose-dependent increase in the secretion of IL-6, IL-8 and IL-1ß after DG(40:6) and DG(38:4) stimulation, which was in part mediated via the NLRP3-inflammasome. Conversely, LPA-stimulated monocytes did not exhibit an inflammatory phenotype. Furthermore, activation of monocytes by DGs and LPA increased their transendothelial migratory capacity. Human atherosclerotic plaques from patients with high Lp(a) levels demonstrated co-localisation of Lp(a) with M1 macrophages, and an enrichment of CD68+IL18+TLR4+TREM2+ resident macrophages and CD68+CASP1+IL1B+SELL+ inflammatory macrophages compared to patients with low Lp(a). Finally, potent Lp(a) lowering treatment (pelacarsen), resulted in a reduction in specific circulating DG lipid subspecies in cardiovascular disease patients with elevated Lp(a) levels [median 82?mg/dL (205?nmol/L)]. Conclusions: Lp(a)-associated DGs and LPA have a potential role in Lp(a)-induced monocyte inflammation by increasing cytokine secretion and monocyte transendothelial migration. This DG-induced inflammation is in part NLRP3-inflammasome dependent. Overall design: In order to investigate the potential impact of DG and LPA plasma levels on promoting an increased pro-inflammatory monocyte phenotype in individuals with elevated Lp(a) levels, we conducted a functional proof-of concept study using commercially available DGs and LPA. The lipid species DG(38:4) (18:0-20:4; 1-stearoyl-2-arachidonoyl-sn-glycerol), DG(40:6) (18:0-22:6; 1-stearoyl-2-docosahexaenoyl-sn-glycerol) and LPA were selected, as these lipid species are significantly increased in individuals with high levels of Lp(a) (p=0.0007; p=0.0067; p=0.0044, respectively) and were commercially available (Figure 4A and Supplemental Figure 5). Transcriptomic analysis was performed on DG-and LPA-stimulated primary human monocytes.