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Circadian disturbance (CD) is the consequence of a mismatch between endogenous circadian rhythms, behaviour, and/or environmental cycles, and frequently occurs during shift work. Shift work has been associated with elevated risk for atherosclerotic cardiovascular disease (asCVD) in humans, but evidence for the effectiveness of prevention strategies is lacking.\nHere, we applied time-restricted feeding (TRF) as a strategy to counteract atherosclerosis development during CD in female APOE∗3-Leiden.CETP mice, a well-established model for humanized lipoprotein metabolism. Control groups were subjected to a fixed 12:12 h light-dark cycle, while CD groups were subjected to 6-h phase advancement every 3 days. Groups had either ad libitum (AL) access to food or were subjected to TRF with restricted food access to the dark phase.\nTRF did not prevent the increase in the relative abundance of circulating inflammatory monocytes and elevation of (postprandial) plasma triglycerides during CD. Nonetheless, TRF reduced atherosclerotic lesion size and prevented an elevation in macrophage content of atherosclerotic lesions during CD, while it increased the relative abundance of anti-inflammatory monocytes, prevented activation of T cells, and lowered plasma total cholesterol levels and markers of hepatic cholesterol synthesis. These effects were independent of total food intake.\nWe propose that time restricted eating could be a promising strategy for the primary prevention of asCVD risk in shift workers, which warrants future study in humans.\nThis work was funded by the Novo Nordisk Foundation, the Netherlands Ministry of Social Affairs and Employment, Amsterdam Cardiovascular Sciences, and the Dutch Heart Foundation.

Cardiovascular research 118(17), 3346-3359 (2023). doi:10.1093/cvr/cvac032, Published by Oxford University Press, Oxford

Mast cells have been associated with the progression and destabilization of advanced atherosclerotic plaques. Reducing intraplaque mast cell accumulation upon atherosclerosis progression could be a potent therapeutic strategy to limit plaque destabilization. Leukotriene B4(LTB4) has been reported to induce mast cell chemotaxis in vitro. Here, we examined whether antagonism of the LTB4-receptor BLT1 could inhibit mast cell accumulation in advanced atherosclerosis. Expression of genes involved in LTB4biosynthesis was determined by single-cell RNA sequencing of human atherosclerotic plaques. Subsequently, Western-type diet fed LDLr−/−mice with pre-existing atherosclerosis were treated with the BLT1-antagonist CP105,696 or vehicle control three times per week by oral gavage. In the spleen, a significant reduction in CD11b+myeloid cells was observed, including Ly6Cloand Ly6Chimonocytes as well as dendritic cells. However, atherosclerotic plaque size, collagen and macrophage content in the aortic root remained unaltered upon treatment. Finally, BLT1 antagonism did not affect mast cell numbers in the aortic root. Here, we show that human intraplaque leukocytes may be a source of locally produced LTB4. However, BLT1-antagonism during atherosclerosis progression does not affect either local mast cell accumulation or plaque size, suggesting that other mechanisms participate in mast cell accumulation during atherosclerosis progression.

Increasing evidence has shown that immune checkpoint molecules of the T-cell immunoglobulin and mucin domain (Tim) family are associated with diverse physiologic and pathologic processes. Previous studies of the role of Tim-1 in atherosclerosis using anti-Tim-1 antibodies have yielded contradictory results. We thus aimed to investigate atherosclerosis development in Tim-1 deficient mice.Mice with a specific loss of the Tim-1 mucin-domain (Tim-1Δmucin) and C57BL/6 (WT) mice received a single injection of a recombinant adeno-associated virus encoding murine Pcsk9 (rAAV2/8-D377Y-mPcsk9) and were fed a Western type diet for 13 weeks to introduce atherosclerosis.Tim-1Δmucin mice developed significantly larger lesions in the aortic root compared to WT mice, with significantly more macrophages and a trend towards a larger necrotic core. Furthermore, Tim-1Δmucin mice showed a significant loss of IL-10+ B cells and regulatory B cell subsets and increased pro-atherogenic splenic follicular B cells compared to WT mice. Moreover, Tim-1Δmucin mice displayed a dramatic reduction in Th2-associated immune response compared to controls but we did not observe any changes in humoral immunity.In summary, Tim-1Δmucin mice displayed a profound impairment in IL-10+ B cells and an imbalance in the Th1/Th2 ratio, which associated with exacerbated atherosclerosis.

Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Dutch Heart Foundation Introduction and aim: Mimics of mast cell-derived heparin proteoglycans (HEP-PG) can be tailored to molecules carrying both antiplatelet and anticoagulant properties. These dual antiplatelet and anticoagulant (APAC) constructs can also shield adhesion molecules such as P-selectin and VCAM-1 expressed by endothelial cells upon atherosclerosis development. We hypothesize that via this way, APAC prevents macrophage accumulation and lesion development. In this study, we therefore determined the efficacy of APAC in inhibiting atherosclerosis. Methods Male western-type diet fed apoE-/- mice were equipped with perivascular carotid artery collars to induce atherosclerosis. In this collar model, mRNA expression of adhesion molecules such as ICAM-1, VCAM-1, P-Selectin but also of Platelet Factor 4 (PF4) are significantly upregulated upon lesion development (all P Results APAC treatment did not affect body weight or plasma total cholesterol levels of the mice during the experiment. Interestingly, carotid artery plaque size was reduced by over 50% upon APAC treatment (APAC: 50±10*10E3 versus controls: 102±13*10E3 square µm; P Conclusion We here show that APAC effectively inhibits atherosclerotic lesion development when administered during lesion initiation and may have potential as therapeutic agent to prevent atherosclerosis.

(Ultra) Small superparamagnetic iron oxide nanoparticles, (U)SPIO, are widely used as magnetic resonance imaging contrast media and assumed to be safe for clinical applications in cardiovascular disease. As safety tests largely relied on normolipidemic models, not fully representative of the clinical setting, we investigated the impact of (U)SPIOs on disease-relevant endpoints in hyperlipidemic models of atherosclerosis.RAW264.7 foam cells, exposed in vitro to Ferumoxide (dextran-coated SPIO), Ferumoxtran (dextran-coated USPIO), or Ferumoxytol (carboxymethyl dextran-coated USPIO) (all 1 mg Fe/ml) showed increased apoptosis and ROS accumulation for Ferumoxide and Ferumoxtran, whereas Ferumoxytol was tolerated well. Pro-apoptotic (TUNEL+) and pro-oxidant activity of Ferumoxide (0.3 mg Fe/kg) and Ferumoxtran (1 mg Fe/kg) were confirmed in plaque, spleen, and liver of hyperlipidemic ApoE-/- (n = 9/group) and LDLR-/- (n = 9-16/group) mice that had received single IV injections compared to saline-treated controls. Again, Ferumoxytol treatment (1 mg Fe/kg) failed to induce apoptosis or oxidative stress in these tissues. Concomitant antioxidant treatment (EUK-8/EUK-134) largely prevented these effects in vitro (-68%, P Ferumoxide and Ferumoxtran, but not Ferumoxytol, induced apoptosis of lipid-laden macrophages in human and murine atherosclerosis, potentially impacting disease progression in patients with advanced atherosclerosis.

We have previously shown that third-generation antisense (3GA) inhibition of 14q32 microRNA (miRNA)-494 reduced early development of atherosclerosis. However, patients at risk of atherosclerotic complications generally present with advanced and unstable lesions. Here, we administered 3GAs against 14q32 miRNA-494 (3GA-494), miRNA-329 (3GA-329), or a control (3GA-ctrl) to mice with advanced atherosclerosis. Atherosclerotic plaque formation in LDLr−/− mice was induced by a 10-week high-fat diet and simultaneous carotid artery collar placement. Parallel to 3GA-treatment, hyperlipidemia was normalized by a diet switch to regular chow for an additional 5 weeks. We show that, even though plasma cholesterol levels were normalized after diet switch, carotid artery plaque progression continued in 3GA-ctrl mice. However, treatment with 3GA-494 and, in part, 3GA-329 halted plaque progression. Furthermore, in the aortic root, intra-plaque collagen content was increased in 3GA-494 mice, accompanied by a reduction in the intra-plaque macrophage content. Pro-atherogenic cells in the circulation, including inflammatory Ly6Chi monocytes, neutrophils, and blood platelets, were decreased upon miRNA-329 and miRNA-494 inhibition. Taken together, treatment with 3GA-494, and in part with 3GA-329, halts atherosclerotic plaque progression and promotes stabilization of advanced lesions, which is highly relevant for human atherosclerosis.

Investigating the in vivo effects of miR-494 inhibition on progression and stability of advanced atherosclerotic lesions.LDLr-/- mice were fed a Western Type Diet (WTD) for 10 weeks to induce atherosclerosis. Semi-constrictive collars were placed around both carotid arteries 4 weeks after start of WTD and 6 weeks after collar placement, a subset of mice (N=10) was sacrificed to analyze baseline plaque size and composition. For the remaining mice, WTD was replaced by normal chow and 3rd Generation Antisense (3GA) against miR-494 (3GA-494; N=10) or negative control (3GA-ctrl; N=10) were administered (i.v., 1 mg/mouse) immediately after and at 2 and 4 weeks after diet switch. Mice were sacrificed one week after final injection.3GA-ctrl mice showed increased carotid artery plaque size compared to baseline, indicating continued atherogenesis, even after lowering plasma cholesterol levels by diet-replacement (before: 863±115 mg/dL vs. after: 214±13 mg/dL). 3GA-494 mice however, showed a significant decrease in carotid artery plaque size compared to control; in fact, 3GA-494 mice had similar plaque sizes to baseline mice (baseline: 30±8*103 µm2, 3GA-ctrl: 56±16*103 µm2 vs. 3GA-494: 23±9*103 µm2, PThese results show that treatment with 3GA-494 halts plaque progression in the carotid artery and increases plaque stability in aortic root plaques. Furthermore, 3GA-494 treatment reduces the platelet count, as well as plasma cholesterol levels, which is an additional improvement of cardiovascular risk factors.