Endothelial cells (EC) play a key role in atherosclerosis. Although EC are in constant contact with low density lipoproteins (LDL), how EC process LDL and whether this influences atherogenesis, is unclear. Here we show that EC take up and metabolize LDL, and when overburdened with intracellular cholesterol, generate cholesterol crystals (CC). The CC are deposited on the basolateral side, and compromise endothelial function. When hyperlipidemic mice are given a high fat diet, CC appear in aortic sinus within 1 week. Treatment with cAMP-enhancing agents, forskolin/rolipram (F/R), mitigates effects of CC on endothelial function by not only improving barrier function, but also inhibiting CC formation both in vitro and in vivo. A proof of principle study using F/R incorporated into liposomes, designed to target inflamed endothelium, shows reduced atherosclerosis and CC formation in ApoE ^−/− mice. Our findings highlight an important mechanism by which EC contribute to atherogenesis under hyperlipidemic conditions.

Polarization of macrophages is regulated through complex signaling networks. Correlating miRNA and mRNA expression over time after macrophage polarization has not yet been investigated. We used paired RNA-Seq and miRNA-Seq experiments to measure the mRNA and miRNA expression in bone marrow-derived macrophages over a time-series of 8 hours. Bioinformatics analysis identified 31 differentially expressed miRNAs between M1 and M2 polarized macrophages. The top 4 M1 miRNAs (miR-155-3p, miR-155-5p, miR-147-3p and miR-9-5p) and top 4 M2 miRNAs (miR-27a-5p, let-7c-1-3p, miR-23a-5p and miR-23b-5p) were validated by qPCR. Interestingly, M1 specific miRNAs could be categorized to early- and late-response groups, in which three new miRNAs miR-1931, miR-3473e and miR-5128 were validated as early-response miRNAs. M1 polarization led to the enrichment of genes involved in immune responses and signal transduction, whereas M2 polarization enriched genes involved in cell cycle and metabolic processes. C2H2 zinc-finger family members are key targets of DE miRNAs. The integrative analysis between miRNAs and mRNAs demonstrates the regulations of miRNAs on nearly four thousand differentially expressed genes and most of the biological pathways enriched in macrophage polarization. In summary, this study elucidates the expression profiles of miRNAs and their potential targetomes during macrophage polarization.

Monocytes/macrophages respond to external stimuli with rapid changes in the expression of numerous inflammation-related genes to undergo polarisation towards the M1 (pro-inflammatory) or M2 (antiinflammatory) phenotype. We have previously shown that, independently of Toll-like receptor activation, extracellular RNA (eRNA) could exert prothrombotic and pro-inflammatory properties in the cardiovascular system to provoke cytokine mobilisation. Here, mouse bone marrow-derived-macrophages (BMDM) differentiated with mouse macrophage-colony-stimulating factor (M-CSF) were found to be skewed towards the M1 phenotype when exposed to eRNA. This resulted in up-regulated expression of inflammatory markers such as Tnf-α and Il-6, together with Il-12 and iNOS, whereas anti-inflammatory genes such as chitinase-like proteins (Ym1/2) and macrophage mannose receptor-2 (Cd206) were significantly down-regulated. Human peripheral blood monocytes were treated with eRNA and analysed by micro-array analysis of the whole human genome, revealing an up-regulation of 79 genes by at least four-fold; 27 of which are related to signal transduction and 15 genes associated with inflammatory response. In accordance with the proposed actions of eRNA as a pro-inflammatory “alarm signal”, these data shed light on the role of eRNA in the context of chronic inflammatory diseases such as atherosclerosis.

Atherosclerosis, the progressive buildup of plaque within arterial blood vessels, can lead to fatal downstream events, such as heart attack or stroke. A key event contributing to the development of atherosclerosis is the infiltration of monocytes and its associated inflammation, as well as the formation of lipid-laden macrophage foam cells within the vessel wall. IL-37 is recognized as an important anti-inflammatory cytokine expressed especially by immune cells. This study was undertaken to elucidate the role of macrophage-expressed IL-37 in reducing the production and effects of proinflammatory cytokines, preventing foam cell formation, and reducing the development of atherosclerosis. Expression of human IL-37 was achieved with a macrophage-specific overexpression system, using the CD68 promoter in mouse primary bone marrow–derived macrophages via retroviral transduction. Macrophage IL-37 expression in vitro resulted in decreased mRNA (e.g., IL-1B, IL-6, and IL-12) and secreted protein production (e.g., IL-6, M-CSF, and ICAM-1) of key inflammatory mediators. IL-37 expression also inhibited macrophage proliferation, apoptosis, and transmigration, as well as reduced lipid uptake, compared with controls in vitro. The in vivo effects of macrophage-expressed IL-37 were investigated through bone marrow transplantation of transduced hematopoietic stem cells into irradiated atherosclerosis-prone Ldlr^−/− mice. After 10 wk on a high-fat/high-cholesterol diet, mice with IL-37–expressing macrophages showed reduced disease pathogenesis, which was demonstrated by significantly less arterial plaque development and systemic inflammation compared with control mice. The athero-protective effect of macrophage-expressed IL-37 has implications for development of future therapies to treat atherosclerosis, as well as other chronic inflammatory diseases.

Chitinase 1 (CHIT1) is secreted by activated macrophages. Chitinase activity is raised in atherosclerotic patient sera and is present in atherosclerotic plaque. However, the role of CHIT1 in atherosclerosis is unknown. Preliminary studies of atherosclerosis in cynomolgous monkeys revealed CHIT1 to be closely correlated with areas of macrophage infiltration. Thus, we investigated the effects of a chitinase inhibitor, allosamidin, on macrophage function in vitro and on atherosclerotic development in vivo. In RAW264.7 cells, allosamidin elevated monocyte chemoattractant protein 1 and tumor necrosis factor alpha expression, and increased activator protein 1 and nuclear factor-κB transcriptional activity. Although inducible nitric oxide synthase, IL-6, and IL-1β expression were increased, Arg1 expression was decreased by chitinase inhibition, suggesting that suppression of CHIT1 activity polarizes macrophages into a M1 phenotype. Allosamidin decreased scavenger receptor AI, CD36, ABCA1, and ABCG1 expression which led to suppression of cholesterol uptake and apolipoprotein AI-mediated cholesterol efflux in macrophages. These effects were confirmed with CHIT1 siRNA transfection and CHIT1 plasmid transfection experiments in primary macrophages. Apolipoprotein E-deficient hyperlipidemic mice treated for 6 weeks with constant administration of allosamidin and fed an atherogenic diet showed aggravated atherosclerotic lesion formation. These data suggest that CHIT1 exerts protective effects against atherosclerosis by suppressing inflammatory responses and polarizing macrophages toward an M2 phenotype, and promoting lipid uptake and cholesterol efflux in macrophages.