Thymic stromal lymphopoietin is a key cytokine for the immunomodulation of atherogenesis with Freund's adjuvant.

Adaptive immune responses regulate the development of atherosclerosis, with a detrimental effect of type 1 but a protective role of type 2 immune responses. Immunization of Apolipoprotein E-deficient (ApoE-/- ) mice with Freund's adjuvant inhibits the development of atherosclerosis. However, the underlying mechanisms are not fully understood. Thymic stromal lymphopoietin (TSLP) is an IL7-like cytokine with essential impact on type 2 immune responses (Th2). Thymic stromal lymphopoietin is strongly expressed in epithelial cells of the skin, but also in various immune cells following appropriate stimulation. In this study, we investigated whether TSLP may be crucial for the anti-atherogenic effect of Freund's adjuvant. Subcutaneous injection of complete Freund's adjuvant (CFA) rapidly led to the expression of TSLP and IL1ß at the site of injection. In male mice, CFA-induced TSLP occurred in immigrated monocytes-and not epithelial cells-and was dependent on NLRP3 inflammasome activation and IL1ß-signalling. In females, CFA-induced TSLP was independent of IL1ß and upon ovariectomy. CFA/OVA led to a more pronounced imbalance of the T cell response in TSLPR-/- mice, with increased INFγ/IL4 ratio compared with wild-type controls. To test whether TSLP contributes to the anti-atherogenic effects of Freund's adjuvant, we treated ApoE-/- and ApoE-/- /TSLPR-/- mice with either CFA/IFA or PBS. ApoE-/- mice showed less atherogenesis upon CFA/IFA compared with PBS injections. ApoE-/- /TSLPR-/- mice had no attenuation of atherogenesis upon CFA/IFA treatment. Freund's adjuvant executes significant immune-modulating effects via TSLP induction. TSLP-TSLPR signalling is critical for CFA/IFA-mediated attenuation of atherosclerosis.

Partial Inhibition of Glycolysis Reduces Atherogenesis Independent of Intraplaque Neovascularization in Mice.

OBJECTIVE: Intraplaque neovascularization is an important feature of unstable human atherosclerotic plaques. However, its impact on plaque formation and stability is poorly studied. Because proliferating endothelial cells generate up to 85% of their ATP from glycolysis, we investigated whether pharmacological inhibition of glycolytic flux by the small-molecule 3PO (3-[3-pyridinyl]-1-[4-pyridinyl]-2-propen-1-one) could have beneficial effects on plaque formation and composition. Approach and Results: ApoE-/- (apolipoprotein E deficient) mice treated with 3PO (50 µg/g, ip; 4×/wk, 4 weeks) showed a metabolic switch toward ketone body formation. Treatment of ApoE-/-Fbn1C1039G+/- mice with 3PO (50 µg/g, ip) either after 4 (preventive, twice/wk, 10 weeks) or 16 weeks of Western diet (curative, 4×/wk, 4 weeks) inhibited intraplaque neovascularization by 50% and 38%, respectively. Plaque formation was significantly reduced in all 3PO-treated animals. This effect was independent of intraplaque neovascularization. In vitro experiments showed that 3PO favors an anti-inflammatory M2 macrophage subtype and suppresses an M1 proinflammatory phenotype. Moreover, 3PO induced autophagy, which in turn impaired NF-κB (nuclear factor-kappa B) signaling and inhibited TNF-α (tumor necrosis factor-alpha)-mediated VCAM-1 (vascular cell adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1) upregulation. Consistently, a preventive 3PO regimen reduced endothelial VCAM-1 expression in vivo. Furthermore, 3PO improved cardiac function in ApoE-/-Fbn1C1039G+/- mice after 10 weeks of treatment. CONCLUSIONS: Partial inhibition of glycolysis restrained intraplaque angiogenesis without affecting plaque composition. However, less plaques were formed, which was accompanied by downregulation of endothelial adhesion molecules-an event that depends on autophagy induction. Inhibition of coronary plaque formation by 3PO resulted in an overall improved cardiac function.

Pharmacological strategies to inhibit intra-plaque angiogenesis in atherosclerosis.

Atherosclerosis is a complex multifactorial disease that affects large and medium-sized arteries. Rupture of atherosclerotic plaques and subsequent acute cardiovascular complications remain a leading cause of death and morbidity in the Western world. There is a considerable difference in safety profile between a stable and a vulnerable, rupture-prone lesion. The need for plaque-stabilizing therapies is high, and for a long time the lack of a suitable animal model mimicking advanced human atherosclerotic plaques made it very difficult to make progress in this area. Evidence from human plaques indicates that intra-plaque (IP) angiogenesis promotes atherosclerosis and plaque destabilization. Although neovascularization has been widely investigated in cancer, studies on the pharmacological inhibition of this phenomenon in atherosclerosis are scarce, mainly due to the lack of an appropriate animal model. By using ApoE-/- Fbn1C1039G+/- mice, a novel model of vulnerable plaques, we were able to investigate the effect of pharmacological inhibition of various mechanisms of IP angiogenesis on plaque destabilization and atherogenesis. In the present review, we discuss the following potential pharmacological strategies to inhibit IP angiogenesis: (1) inhibition of vascular endothelial growth factor signalling, (2) inhibition of glycolytic flux, and (3) inhibition of fatty acid oxidation. On the long run, IP neovascularization might be applicable as a therapeutic target to induce plaque stabilization on top of lipid-lowering treatment.

Everolimus depletes plaque macrophages, abolishes intraplaque neovascularization and improves survival in mice with advanced atherosclerosis.

BACKGROUND AND AIMS: Inhibition of the mechanistic target of rapamycin (mTOR) is a promising approach to halt atherogenesis in different animal models. This study evaluated whether the mTOR inhibitor everolimus can stabilize pre-existing plaques, prevent cardiovascular complications and improve survival in a mouse model of advanced atherosclerosis. METHODS: ApoE-/-Fbn1C1039G+/- mice (n = 24) were fed a Western diet (WD) for 12 weeks. Subsequently, mice were treated with everolimus (1.5 mg/kg daily) or vehicle for another 12 weeks while the WD continued. RESULTS: Despite hypercholesterolemia, everolimus treatment was associated with a reduction in circulating Ly6Chigh monocytes (15 vs. 28% of total leukocytes, p = 0.046), a depletion of plaque macrophages (2.1 vs. 4.1%, p = 0.040) and an abolishment of intraplaque neovascularization, which are all indicative of a more stable plaque phenotype. Moreover, everolimus reduced hypoxic brain damage and improved cardiac function, which led to increased survival (100 vs. 67% of animals, p = 0.038). CONCLUSIONS: Everolimus enhances features of plaque stability and counters cardiovascular complications in ApoE-/-Fbn1C1039G+/- mice, even when administered at a later stage of the disease.

Axitinib attenuates intraplaque angiogenesis, haemorrhages and plaque destabilization in mice.

AIM: An increased density of intraplaque (IP) microvessels in ruptured versus nonruptured human plaques suggests that IP neovascularization has a major causative effect on plaque development and instability. Possibly, vascular endothelial growth factor (VEGF) or other angiogenic factors mediate IP microvessel growth and plaque destabilization. Because apolipoprotein deficient mice with a heterozygous mutation (C1039G+/-) in the fibrillin-1 gene (ApoE-/-Fbn1C1039G+/-) manifest substantial IP neovascularization, they represent a unique tool to further investigate angiogenesis and its role in atherosclerosis. Here, we examined whether administration of axitinib (inhibitor of VEGF receptor-1,-2 and -3) inhibits IP neovascularization and stabilizes atherosclerotic plaques. METHODS: ApoE-/-Fbn1C1039G+/- mice were fed a western diet (WD) for 20weeks. After 14weeks WD, mice received axitinib (35µg/g) or solvent i.p. 4×/week for 6weeks. Cardiac function was monitored to evaluate the effect of axitinib on atherosclerosis-driven complications such as myocardial infarction. RESULTS: Axitinib significantly reduced IP neovascularization, with subsequent less prevalence of IP haemorrhages. The smooth muscle cell content doubled, whereas the amount of macrophages decreased. Overall cardiac function was improved in axitinib-treated animals. Moreover, the number of animals with myocardial infarction was decreased by 40%. Coronary plaque formation was observed in almost all control animals whereas treated animals showed a 30% reduction in the occurrence of coronary plaques. CONCLUSIONS: Inhibition of VEGF receptor signalling by axitinib attenuates intraplaque angiogenesis and plaque destabilization in mice.

Cytoprotective effects of transgenic neuroglobin overexpression in an acute and chronic mouse model of ischemic heart disease.

Neuroglobin (NGB) is an oxygen-binding protein that is mainly expressed in nervous tissues where it is considered to be neuroprotective during ischemic brain injury. Interestingly, transgenic mice overexpressing NGB reveal cytoprotective effects on tissues lacking endogenous NGB, which might indicate a therapeutic role for NGB in a broad range of ischemic conditions. In the present study, we investigated the effect of NGB overexpression on survival as well as on the size and occurrence of myocardial infarctions (MI) in a mouse model of acute MI (AMI) and a model of advanced atherosclerosis (ApoE -/- Fbn1 C1039G+/- mice), in which coronary plaques and MI develop in mice being fed a Western-type diet. Overexpression of NGB significantly enhanced post-AMI survival and reduced MI size by 14% 1 week after AMI. Gene expression analysis of the infarction border showed reduction of tissue hypoxia and attenuation of hypoxia-induced inflammatory pathways, which might be responsible for these beneficial effects. In contrast, NGB overexpression did not affect survival or occurrence of MI in the atherosclerotic mice although the incidence of coronary plaques was significantly reduced. In conclusion, NGB proved to act cytoprotectively during MI in the acute setting while this effect was less pronounced in the atherosclerosis model.

Intraplaque neovascularization as a novel therapeutic target in advanced atherosclerosis.

INTRODUCTION: Atherosclerosis is a lipid-driven inflammatory process with a tremendously high mortality due to acute cardiac events. There is an emerging need for new therapies to stabilize atherosclerotic lesions. Growing evidence suggests that intraplaque (IP) neovascularisation and IP hemorrhages are important contributors to plaque instability. AREAS COVERED: Neovascularization is a complex process that involves different growth factors and inflammatory mediators of which their individual significance in atherosclerosis remains poorly understood. This review discusses different aspects of IP neovascularization in atherosclerosis including the potential treatment opportunities to stabilize advanced plaques. Furthermore, we highlight the development of accurate and feasible in vivo imaging modalities for IP neovascularization to prevent acute events. EXPERT OPINION: Although lack of a valuable animal model of IP neovascularization impeded the investigation of a causal and straightforward link between neovascularization and atherosclerosis, recent evidence shows that vein grafts in ApoE*3 Leiden mice as well as plaques in ApoE(-/-) Fbn1(C1039G+/-) mice are useful models for intraplaque neovessel research. Even though interference with vascular endothelial growth factor (VEGF) signalling has been widely investigated, new therapeutic opportunities have emerged. Cell metabolism, in particular glycolysis and fatty acid oxidation, appears to perform a crucial role in the development of IP neovessels and thereby serves as a promising target.

Development of atherosclerotic plaques in a mouse model of pseudoxanthoma elasticum.

OBJECTIVE: Pseudoxanthoma elasticum (PXE) is an autosomal recessive disorder, characterized by extensive mineralization of connective tissues and fragmentation of elastin fibres. PXE patients may sporadically suffer from severe cardiovascular complications caused by accelerated atherosclerosis. Consistent with this finding, recent evidence suggests that elastin fragmentation in arteries of atherosclerotic mice leads to unstable plaques and human-like complications such as myocardial infarction, stroke and sudden death. Because Abcc6-/- mice manifest the human features of PXE including the fragmentation of elastin fibres, Abcc6-/- mice were crossbred with ApoE-/- mice to investigate the level of plaque formation and potential complications. METHODS AND RESULTS: ApoE-/- and ApoE-/- Abcc6-/- mice were fed a Western-type diet (WD) for 25 weeks to induce plaque formation.WD-fed animals showed neither signs of neurological dysfunction nor sudden death. Cardiac function of ApoE-/- Abcc6-/- mice, as assessed by echocardiography, was not different from ApoE-/- control mice. Histochemical analysis did not reveal elastin fragmentation or pronounced mineral deposition in the vessel wall. Plaques from the proximal ascending aorta and brachiocephalic artery of ApoE-/- Abcc6-/- mice were similar in size and composition as compared to ApoE-/- mice. Moreover, en face oil red O stainings of the aortic arch and descending thoracic aorta did not reveal enhanced plaque formation in ApoE-/- Abcc6-/- mice as compared to ApoE-/-controls. CONCLUSION: ApoE-/-Abcc6-/- mice do not represent an adequate model of accelerated atherosclerosis and therefore are not useful to study atherosclerosis-related complications as observed in PXE patients.