The diverse leukocyte infiltrate in atherosclerotic mouse aortas was recently analyzed in 9 single cell RNA-Seq (scRNA-Seq) and 2 mass cytometry (CyTOF) studies. In a comprehensive meta-analysis, we demonstrate four macrophage subsets: resident, inflammatory, IFNIC and Trem2 foamy macrophages. We also find that monocytes, neutrophils, dendritic cells, natural killer cells, innate lymphoid cells-2 (ILC2) and CD8 T cells form prominent and separate populations. The CD4 T cells show a large population of Th17-like cells, which also contain γδ T cells. A small number of Tregs and Th1 cells is also identified. The present meta-analysis overcomes limitations of individual studies that, because of their experimental approach, overor under-represent certain cell populations. CyTOF identifies an even larger number of clusters, suggesting that surface markers provide more discriminatory information than transcriptomes. The present analysis provides evidence to further resolve some long-standing controversies in the field. First, Trem2 + foamy macrophages are not pro-inflammatory, but interferon-inducible cell (IFNIC) and inflammatory macrophages are. Second, about half of all foam cells are smooth muscle cell-derived, retaining smooth muscle cell transcripts rather than transdifferentiating to macrophages. Third, Pf4, which had been considered specific for platelets and megakaryocytes, is also prominently expressed in resident vascular macrophages. Finally, the discovery of a prominent ILC2 cluster links the scRNA-Seq work to recent flow cytometry data suggesting a strong atheroprotective role of ILC2 cells. This resolves apparent discrepancies regarding the role of Th2 cells in atherosclerosis based on studies that pre-dated the discovery of ILC2 cells.
Background
Elevated lipoprotein(a) [Lp(a)] is a prevalent, independent cardiovascular risk factor but the underlying mechanisms responsible for its pathogenicity are poorly defined. Since Lp(a) is the prominent carrier of pro-inflammatory oxidized phospholipids (OxPL), part of its atherothrombosis might be mediated through this pathway.
Methods
In vivo imaging techniques MR imaging, 18F-FDG-PET/CT and SPECT/CT were used to measure subsequently atherosclerotic burden, arterial wall inflammation and monocyte trafficking to the arterial wall. Ex vivo analysis of monocytes was performed using FACS analysis, inflammatory stimulation assays and transendothelial migration assays. In vitro studies to the pathophysiology of Lp(a) on monocytes were performed using an in vitro model for trained immunity.
Results
We show that subjects with elevated Lp(a) (108 [50–195] mg/dL; n=30) have increased arterial inflammation and enhanced PBMCs trafficking to the arterial wall, compared with subjects with normal Lp(a) (7 [2–28] mg/dL; n=30). In addition, monocytes isolated from subjects with elevated Lp(a) remain in a long-lasting primed state, as evidenced by an increased capacity to transmigrate and produce pro-inflammatory cytokines upon stimulation (n=15). In vitro studies show that Lp(a) contains OxPL and augments the pro-inflammatory response in monocytes derived from healthy controls (n=6). This effect was markedly attenuated by inactivating OxPL on Lp(a) or removing OxPL on apo(a).
Conclusions
These findings demonstrate that Lp(a) induces monocyte trafficking to the arterial wall and mediates pro-inflammatory responses through its OxPL content. These findings provide a novel mechanism by which Lp(a) mediates cardiovascular disease.
Clinical Trial Registration
URL: http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=5006 Unique Identifier: NTR5006 (VIPER study)
The main process that triggers pathological mineralization of the aortic valve remains elusive. 3 Recently, 3 successive studies with a Mendelian randomization design have reported a significant association between the LPA gene variant (rs10455872), which genetically determines the lipoprotein(a) [Lp(a)] plasma level, and CAVD. [4][5][6] These studies thus suggested a causal relationship between Lp(a) and CAVD risk. Lp(a) is a low-density lipoprotein (LDL)-like particle in which an apolipoprotein(a) is linked by a disulfide bridge to apolipoprotein B. Lp(a) is a major carrier of oxidized phospholipids (OxPLs) and has been associated Background-Mendelian randomization studies have highlighted that lipoprotein(a) [Lp(a)] was associated with calcific aortic valve disease (CAVD). Lp(a) transports oxidized phospholipids with a high content in lysophosphatidylcholine. Autotaxin (ATX) transforms lysophosphatidylcholine into lysophosphatidic acid. We hypothesized that ATXlysophosphatidic acid could promote inflammation/mineralization of the aortic valve. Methods and Results-We have documented the expression of ATX in control and mineralized aortic valves. By using different approaches, we have also investigated the role of ATX-lysophosphatidic acid in the mineralization of isolated valve interstitial cells and in a mouse model of CAVD. Enzyme-specific ATX activity was elevated by 60% in mineralized aortic valves in comparison with control valves. Immunohistochemistry studies showed a high level of ATX in mineralized aortic valves, which colocalized with oxidized phospholipids and apolipoprotein(a). We detected a high level of ATX activity in the Lp(a) fraction in circulation. Interaction between ATX and Lp(a) was confirmed by in situ proximity ligation assay. Moreover, we documented that valve interstitial cells also expressed ATX in CAVD. We showed that ATX-lysophosphatidic acid promotes the mineralization of the aortic valve through a nuclear factor κB/interleukin 6/bone morphogenetic protein pathway. In LDLR -/-/ApoB 100/100 /IGFII mice, ATX is overexpressed and lysophosphatidic acid promotes a strong deposition of hydroxyapatite of calcium in aortic valve leaflets and accelerates the development of CAVD.
Conclusions-ATX
Background: Plasma lipoprotein(a) (Lp(a)) levels can be reduced through proprotein convertase subtilisin/kexin type 9 (PCSK9) through an unknown mechanism. Results: Lp(a) catabolism in hepatoma cells and primary fibroblasts is inhibited by PCSK9 via the low density lipoprotein receptor (LDLR). Conclusion: LDLR mediates the effects of PCSK9 on Lp(a) internalization. Significance: Our results provide a mechanistic explanation for the effects of PCSK9 inhibitors on plasma Lp(a) levels.
TAFI (thrombin-activatable fibrinolysis inhibitor) is a carboxypeptidase zymogen originally identified in plasma. The TAFI pathway helps to regulate the balance between the coagulation and fibrinolytic cascades. Activated TAFI (TAFIa) can also inactivate certain pro-inflammatory mediators, suggesting that the TAFI pathway may also regulate communication between coagulation and inflammation. Expression in the liver is considered to be the source of plasma TAFI. TAFI has also been identified in platelets and CPB2 (the gene encoding TAFI) mRNA has been detected in megakaryocytic cell lines as well as in endothelial cells. We have undertaken a quantitative analysis of CPB2 mRNA and TAFI protein in extrahepatic cell types relevant to vascular disease. Using RT-PCR and quantitative RT-PCR, we detected CPB2 mRNA in the human megakaryoblastic cell lines MEG-01 and Dami, the human monocytoid cell line THP-1 as well as THP-1 cells differentiated into a macrophage-like phenotype, and in primary human umbilical vein and coronary artery endothelial cells. CPB2 mRNA abundance in MEG-01, Dami, and THP-1 cells was modulated by the state of differentiation of these cells. Using a recently developed TAFIa assay, we detected TAFI protein in the lysates of the human hepatocellular carcinoma cell line HepG2 as well as in MEG-01 and Dami cells and in the conditioned medium of HepG2 cells, differentiated Dami cells, and THP-1 macrophages. We have obtained clear evidence for extrahepatic expression of TAFI, which has clear implications for the physiological and pathophysiological functions of the TAFI pathway.
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