Atherosclerosis is a progressive disease resulting in the formation of an arterial plaque. Despite lipid lowering, recurrent cardiovascular events remain a risk. While atherosclerosis is primarily... Show moreAtherosclerosis is a progressive disease resulting in the formation of an arterial plaque. Despite lipid lowering, recurrent cardiovascular events remain a risk. While atherosclerosis is primarily lipid-driven, the immune system plays a critical role in the pathophysiology. Additional treatment could be achieved via immunomodulation. We aimed to identify potential biomarkers for monitoring of immunomodulatory drugs in future clinical trials and investigated pharmacological modulation of atherogenic pathways. We identified smokers and elderly healthy people as suitable groups for future clinical trials. We investigated the impact of sample aging on LPS responses, and optimized methodology for evaluation of LPS-driven neutrophil responses, in vitro and in vivo. As potential anti-atherogenic strategy, we evaluated the effect of pneumococcal vaccination on circulating oxLDL-IgM levels in man. The immunomodulatory impact of hydroxychloroquine, a drug with potential anti-atherogenic effects, was evaluated in healthy volunteers. A novel OX40L inhibitor was tested in healthy volunteers, since the OX40-OX40L axis may play a role in atherogenesis. OX40L inhibition was safe and effectively reduced T cell activity. Lastly, we showed that PD-1 agonism reduced atherosclerosis in Ldlr-/- mice. This thesis adds to the future development of effective and specific immunomodulatory treatments for atherosclerosis. Show less
Current guidelines barely support marine omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in cardiology, mainly because results of large trials were equivocal. Most... Show moreCurrent guidelines barely support marine omega-3 fatty acids eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in cardiology, mainly because results of large trials were equivocal. Most large trials have tested EPA alone or EPA + DHA combined as a drug, thereby disregarding the relevance of their blood levels. These levels are frequently assessed with the Omega-3 Index (percentage of EPA + DHA in erythrocytes), which is determined using a specific standardised analytical procedure. EPA and DHA are present in every human being at unpredictable levels (even in the absence of intake), and their bioavailability is complex. Both facts need to be incorporated into trial design and should direct clinical use of EPA and DHA. An Omega-3 Index in the target range of 8-11% is associated with lower total mortality, fewer major adverse cardiac and other cardiovascular events. Moreover, functions of organs such as the brain benefit from an Omega-3 Index in the target range, while untoward effects, such as bleeding or atrial fibrillation, are minimised. In pertinent intervention trials, several organ functions were improved, with improvements correlating with the Omega-3 Index. Thus, the Omega-3 Index is relevant in trial design and clinical medicine, which calls for a widely available standardised analytical procedure and a discussion on possible reimbursement of this test. Show less
Currently immunomodulatory compounds are under investigation for use in patients with cardiovascular disease, caused by atherosclerosis. These trials, using recurrent cardiovascular events as... Show moreCurrently immunomodulatory compounds are under investigation for use in patients with cardiovascular disease, caused by atherosclerosis. These trials, using recurrent cardiovascular events as endpoint, require enrollment of large patient groups. We investigated the effect of key risk factors for atherosclerosis development, ageing and smoking, on the immune system, with the objective to identify biomarkers differentiating between human populations, and potentially serving as endpoints for future phase 1B trials with immunomodulatory compounds. Blood was collected from young healthy volunteers (aged 18-25 years, n=30), young smokers (18-25 years, n=20), elderly healthy volunteers (>60 years, n=20), heavy smokers (>45 years, 15 packyears, n=11) and patients with stable coronary artery disease (CAD) (>60 years, n=27). Circulating immune cell subsets were characterized by flow cytometry, and collected plasma was evaluated by proteomics (Olink). Clear ageing effects were observed, mostly illustrated by a lower level in CD8+ and naïve CD4+ and CD8+ T cells, with an increase in CD4+ and CD8+ effector memory T cells in elderly healthy volunteers compared to young healthy volunteers. Heavy smokers showed a more inflammatory cellular phenotype, especially a shift in Th1/Th2 ratio: higher Th1 and lower Th2 percentages compared to young healthy volunteers. A significant decrease in circulating atheroprotective oxLDL-specific IgM was found in patients with CAD compared to young healthy volunteers. Elevated pro-inflammatory and chemotactic proteins TREM1 and CCL11 were observed in elderly volunteers compared to young volunteers. In addition, heavy smokers had an increase in pro-inflammatory cytokine IL-6 and lysosomal protein LAMP3. These data show that ageing and smoking are associated with an inflammatory immunophenotype, and that heavy smokers or aged individuals may serve as potential populations for future clinical trials investigating immunomodulatory drugs targeted for cardiovascular disease. Show less
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... Show moreIncreasing 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. Show less
Ingen, E. van; Foks, A.C.: Woudenberg, T.; Bent, M.L. van der; Jong, A. de; Hohensinner, P.J.; Wojta, J.; ... ; Nossent, A.Y. 2021
We have previously shown that treatment with third-generation antisense oligonucleotides against miR-494-3p (3GA-494) reduces atherosclerotic plaque progression and stabilizes lesions, both in... Show moreWe have previously shown that treatment with third-generation antisense oligonucleotides against miR-494-3p (3GA-494) reduces atherosclerotic plaque progression and stabilizes lesions, both in early and established plaques, with reduced macrophage content in established plaques. Within the plaque, different subtypes of macrophages are present. Here, we aimed to investigate whether miR-494-3p directly influences macrophage polarization and activation. Human macrophages were polarized into either proinflammatory M1 or anti-inflammatory M2 macrophages and simultaneously treated with 3GA-494 or a control antisense (3GA-ctrl). We show that 3GA-494 treatment inhibited miR-494-3p in M1 macrophages and dampened M1 polarization, while in M2 macrophages miR-494-3p expression was induced and M2 polarization enhanced. The proinflammatory marker CCR2 was reduced in 3GA-494-treated atherosclerosis-prone mice. Pathway enrichment analysis predicted an overlap between miR-494-3p target genes in macrophage polarization and Wnt signaling. We demonstrate that miR-494-3p regulates expression levels of multiple Wnt signaling components, such as LRP6 and TBL1X. Wnt signaling appears activated upon treatment with 3GA-494, both in cultured M1 macrophages and in plaques of hypercholesterolemic mice. Taken together, 3GA-494 treatment dampened M1 polarization, at least in part via activated Wnt signaling, while M2 polarization was enhanced, which is both favorable in reducing atherosclerotic plaque formation and increasing plaque stability. Show less
The thesis discussed several strategies that may contribute to further CVD risk reduction in the future. We described two novel lipid-lowering strategies, we unraveled (part of) the etiology of the... Show moreThe thesis discussed several strategies that may contribute to further CVD risk reduction in the future. We described two novel lipid-lowering strategies, we unraveled (part of) the etiology of the cardiovascular safety issues of TKIs that are used for the treatment of CML, and we investigated the dose effects of PFOA on lipoprotein metabolism. Looking forward, we developed a novel mouse model that can be used for the study of diabetic macrovascular complications, and we evaluated the potential of OSM as novel target in CVD. Show less
Cardiovascular diseases are the primary cause of death in the world with atherosclerosis as primary underlying cause. Atherosclerosis is characterized by cholesterol accumulation in the vessel wall... Show moreCardiovascular diseases are the primary cause of death in the world with atherosclerosis as primary underlying cause. Atherosclerosis is characterized by cholesterol accumulation in the vessel wall and inflammation of the vessel wall of medium to large size arteries. Both cholesterol accumulation and inflammation are pathogenic in the context of atherosclerosis. Current treatment regimens are tailored to reduce cholesterol levels in the blood. However, even a successful lowering of cholesterol is in many patients not sufficient to prevent a major cardiovascular event due to unresolved inflammation. Therefore, the immune system provides an interesting therapeutic target for the treatment of atherosclerosis. In this thesis we have explored the effect on atherosclerosis of several immunomodulatory strategies in pre-clinical models.As cholesterol is not soluble in water, cholesterol is transported in the bloodstream in particles called lipoproteins. The low-density lipoprotein (LDL) carries the highest concentration of cholesterol and accumulates in the vessel wall where a pathogenic specific immune response against LDL is instigated. In this thesis we have used several strategies to modulate the specific immune response against LDL, inducing LDL-specific regulatory T cells, antibodies, and cytotoxic T cells. Through immunoproteasomal inhibition we assessed the effect of general immune inhibition on atherosclerosis. Show less
The studies described in this thesis show that inflammation and CETP are both important factors in lipid metabolism and atherosclerosis. In the first part of this thesis we showed that high dietary... Show moreThe studies described in this thesis show that inflammation and CETP are both important factors in lipid metabolism and atherosclerosis. In the first part of this thesis we showed that high dietary cholesterol can induce hepatic inflammation via disturbed cholesterol homeostasis and ER stress, revealing new targets for the treatment of metabolic inflammation. Next, we demonstrated that intervention in both systemic and vascular inflammation can reduce atherosclerosis progression and/ or induce regression, highlighting the importance of developing drugs targeting the inflammatory component of atherosclerotic disease. In the second part of this thesis we showed that CETP inhibition per se may be anti-atherogenic, but that combination therapy of the CETP inhibitor torcetrapib with atorvastatin may have obscured its atheroprotective effect. Furthermore, we showed that the VLDL-increasing effect of CETP largely explains its atherogenic effect, at least in APOE*3-Leiden.CETP mice, and that CETP inhibition may negatively affect lesion stability. Our data suggest that CETP inhibition may not be the most optimal strategy to increase HDL-C levels and thereby reduce atherosclerosis. We anticipate that strategies improving HDL functionality, rather than raising the HDL level, are more likely to effectively reduce CVD. Show less
