Effect of erythrophagocytosis-induced ferroptosis during angiogenesis in atherosclerotic plaques.

Intraplaque (IP) angiogenesis is a key feature of advanced atherosclerotic plaques. Because IP vessels are fragile and leaky, erythrocytes are released and phagocytosed by macrophages (erythrophagocytosis), which leads to high intracellular iron content, lipid peroxidation and cell death. In vitro experiments showed that erythrophagocytosis by macrophages induced non-canonical ferroptosis, an emerging type of regulated necrosis that may contribute to plaque destabilization. Erythrophagocytosis-induced ferroptosis was accompanied by increased expression of heme-oxygenase 1 and ferritin, and could be blocked by co-treatment with third generation ferroptosis inhibitor UAMC-3203. Both heme-oxygenase 1 and ferritin were also expressed in erythrocyte-rich regions of carotid plaques from ApoE-/- Fbn1C1039G+/- mice, a model of advanced atherosclerosis with IP angiogenesis. The effect of UAMC-3203 (12.35 mg/kg/day) on atherosclerosis was evaluated in ApoE-/- Fbn1C1039G+/- mice fed a western-type diet (WD) for 12 weeks (n = 13 mice/group) or 20 weeks (n = 16-21 mice/group) to distinguish between plaques without and with established IP angiogenesis, respectively. A significant decrease in carotid plaque thickness was observed after 20 weeks WD (87 ± 19 µm vs. 166 ± 20 µm, p = 0.006), particularly in plaques with confirmed IP angiogenesis or hemorrhage (108 ± 35 µm vs. 322 ± 40 µm, p = 0.004). This effect was accompanied by decreased IP heme-oxygenase 1 and ferritin expression. UAMC-3203 did not affect carotid plaques after 12 weeks WD or plaques in the aorta, which typically do not develop IP angiogenesis. Altogether, erythrophagocytosis-induced ferroptosis during IP angiogenesis leads to larger atherosclerotic plaques, an effect that can be prevented by ferroptosis inhibitor UAMC-3203.

Wide-field time-of-flight measurements of highly scattering media.

We present a setup that makes use of a time-resolved single-photon camera to determine the scattering parameters of media. The measurement is realized in a non-contact way, both for the illumination laser and the detection. By fitting the time-of-flight acquired distributions at different spatial positions with the diffusion equation, we retrieve the reduced scattering coefficients of a highly diffusive isotropic reference media for wavelengths in the range from 540 to 840 nm.

Acetylsalicylic Acid Reduces Passive Aortic Wall Stiffness and Cardiovascular Remodelling in a Mouse Model of Advanced Atherosclerosis.

Acetylsalicylic acid (ASA) is widely used in secondary prevention of cardiovascular (CV) disease, mainly because of its antithrombotic effects. Here, we investigated whether ASA can prevent the progression of vessel wall remodelling, atherosclerosis, and CV complications in apolipoprotein E deficient (ApoE-/-) mice, a model of stable atherosclerosis, and in ApoE-/- mice with a mutation in the fibrillin-1 gene (Fbn1C1039G+/-), which is a model of elastic fibre fragmentation, accompanied by exacerbated unstable atherosclerosis. Female ApoE-/- and ApoE-/-Fbn1C1039G+/- mice were fed a Western diet (WD). At 10 weeks of WD, the mice were randomly divided into four groups, receiving either ASA 5 mg/kg/day in the drinking water (ApoE-/- (n = 14), ApoE-/-Fbn1C1039G+/- (n = 19)) or plain drinking water (ApoE-/- (n = 15), ApoE-/-Fbn1C1039G+/- (n = 21)) for 15 weeks. ApoE-/-Fbn1C1039G+/- mice showed an increased neutrophil-lymphocyte ratio (NLR) compared to ApoE-/- mice, and this effect was normalised by ASA. In the proximal ascending aorta wall, ASA-treated ApoE-/-Fbn1C1039G+/- mice showed less p-SMAD2/3 positive nuclei, a lower collagen percentage and an increased elastin/collagen ratio, consistent with the values measured in ApoE-/- mice. ASA did not affect plaque progression, incidence of myocardial infarction and survival of ApoE-/-Fbn1C1039G+/- mice, but systolic blood pressure, cardiac fibrosis and hypertrophy were reduced. In conclusion, ASA normalises the NLR, passive wall stiffness and cardiac remodelling in ApoE-/-Fbn1C1039G+/- mice to levels observed in ApoE-/- mice, indicating additional therapeutic benefits of ASA beyond its classical use.

Serum Corticosterone and Insulin Resistance as Early Biomarkers in the hAPP23 Overexpressing Mouse Model of Alzheimer's Disease.

Increasing epidemiological evidence highlights the association between systemic insulin resistance and Alzheimer's disease (AD). As insulin resistance can be caused by high-stress hormone levels and since hypercortisolism appears to be an important risk factor of AD, we aimed to investigate the systemic insulin functionality and circulating stress hormone levels in a mutant humanized amyloid precursor protein (APP) overexpressing (hAPP23+/-) AD mouse model. Memory and spatial learning of male hAPP23+/- and C57BL/6 (wild type, WT) mice were assessed by a Morris Water Maze (MWM) test at the age of 4 and 12 months. The systemic metabolism was examined by intraperitoneal glucose and insulin tolerance tests (GTT, ITT). Insulin and corticosterone levels were determined in serum. In the hippocampus, parietal and occipital cortex of hAPP23+/- brains, amyloid-beta (Aß) deposits were present at 12 months of age. MWM demonstrated a cognitive decline in hAPP23+/- mice at 12 but not at 4 months, evidenced by increasing total path lengths and deteriorating probe trials compared to WT mice. hAPP23+/- animals presented increased serum corticosterone levels compared to WT mice at both 4 and 12 months. hAPP23+/- mice exhibited peripheral insulin resistance compared to WT mice at 4 months, which stabilized at 12 months of age. Serum insulin levels were similar between genotypes at 4 months of age but were significantly higher in hAPP23+/- mice at 12 months of age. Peripheral glucose homeostasis remained unchanged. These results indicate that peripheral insulin resistance combined with elevated circulating stress hormone levels could be potential biomarkers of the pre-symptomatic phase of AD.

Linking CD11b (+) Dendritic Cells and Natural Killer T Cells to Plaque Inflammation in Atherosclerosis.

Atherosclerosis remains the leading cause of death and disability in our Western society. To investigate whether the dynamics of leukocyte (sub)populations could be predictive for plaque inflammation during atherosclerosis, we analyzed innate and adaptive immune cell distributions in blood, plaques, and lymphoid tissue reservoirs in apolipoprotein E-deficient (ApoE(-/-)) mice and in blood and plaques from patients undergoing endarterectomy. Firstly, there was predominance of the CD11b(+) conventional dendritic cell (cDC) subset in the plaque. Secondly, a strong inverse correlation was observed between CD11b(+) cDC or natural killer T (NKT) cells in blood and markers of inflammation in the plaque (including CD3, T-bet, CCR5, and CCR7). This indicates that circulating CD11b(+) cDC and NKT cells show great potential to reflect the inflammatory status in the atherosclerotic plaque. Our results suggest that distinct changes in inflammatory cell dynamics may carry biomarker potential reflecting atherosclerotic lesion progression. This not only is crucial for a better understanding of the immunopathogenesis but also bares therapeutic potential, since immune cell-based therapies are emerging as a promising novel strategy in the battle against atherosclerosis and its associated comorbidities. The cDC-NKT cell interaction in atherosclerosis serves as a good candidate for future investigations.

Elastin fragmentation in atherosclerotic mice leads to intraplaque neovascularization, plaque rupture, myocardial infarction, stroke, and sudden death.

AIMS: There is a need for animal models of plaque rupture. We previously reported that elastin fragmentation, due to a mutation (C1039G(+/-)) in the fibrillin-1 (Fbn1) gene, promotes atherogenesis and a highly unstable plaque phenotype in apolipoprotein E deficient (ApoE(-/-)) mice on a Western-type diet (WD). Here, we investigated whether plaque rupture occurred in ApoE(-/-)Fbn1(C1039G+/-) mice and was associated with myocardial infarction, stroke, and sudden death. METHODS AND RESULTS: Female ApoE(-/-)Fbn1(C1039G+/-) and ApoE(-/-) mice were fed a WD for up to 35 weeks. Compared to ApoE(-/-) mice, plaques of ApoE(-/-)Fbn1(C1039G+/-) mice showed a threefold increase in necrotic core size, augmented T-cell infiltration, a decreased collagen I content (70 ± 10%), extensive neovascularization, intraplaque haemorrhage, and a significant increase in matrix metalloproteinase-2, -9, -12, and -13 expression or activity. Plaque rupture was observed in 70% of ascending aortas and in 50% of brachiocephalic arteries of ApoE(-/-)Fbn1(C1039G+/-) mice. In ApoE(-/-) mice, plaque rupture was not seen in ascending aortas and only in 10% of brachiocephalic arteries. Seventy percent of ApoE(-/-)Fbn1(C1039G+/-) mice died suddenly, whereas all ApoE(-/-) mice survived. ApoE(-/-)Fbn1(C1039G+/-) mice showed coronary plaques and myocardial infarction (75% of mice). Furthermore, they displayed head tilt, disorientation, and motor disturbances (66% of cases), disturbed cerebral blood flow (73% of cases; MR angiograms) and brain hypoxia (64% of cases), indicative of stroke. CONCLUSIONS: Elastin fragmentation plays a key role in plaque destabilization and rupture. ApoE(-/-)Fbn1(C1039G+/-) mice represent a unique model of acute plaque rupture with human-like complications.

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.

Advancements in risk stratification and management strategies in primary cardiovascular prevention.

Atherosclerotic cardiovascular disease (ASCVD) remains a leading cause of morbidity and mortality worldwide, highlighting the urgent need for advancements in risk assessment and management strategies. Although significant progress has been made recently, identifying and managing apparently healthy individuals at a higher risk of developing atherosclerosis and those with subclinical atherosclerosis still poses significant challenges. Traditional risk assessment tools have limitations in accurately predicting future events and fail to encompass the complexity of the atherosclerosis trajectory. In this review, we describe novel approaches in biomarkers, genetics, advanced imaging techniques, and artificial intelligence that have emerged to address this gap. Moreover, polygenic risk scores and imaging modalities such as coronary artery calcium scoring, and coronary computed tomography angiography offer promising avenues for enhancing primary cardiovascular risk stratification and personalised intervention strategies. On the other hand, interventions aiming against atherosclerosis development or promoting plaque regression have gained attention in primary ASCVD prevention. Therefore, the potential role of drugs like statins, ezetimibe, proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors, omega-3 fatty acids, antihypertensive agents, as well as glucose-lowering and anti-inflammatory drugs are also discussed. Since findings regarding the efficacy of these interventions vary, further research is still required to elucidate their mechanisms of action, optimize treatment regimens, and determine their long-term effects on ASCVD outcomes. In conclusion, advancements in strategies addressing atherosclerosis prevention and plaque regression present promising avenues for enhancing primary ASCVD prevention through personalised approaches tailored to individual risk profiles. Nevertheless, ongoing research efforts are imperative to refine these strategies further and maximise their effectiveness in safeguarding cardiovascular health.

Empagliflozin decreases ageing-associated arterial stiffening and vascular fibrosis under normoglycemic conditions.

Arterial stiffness is a hallmark of vascular ageing and results in increased blood flow pulsatility to the periphery, damaging end-organs such as the heart, kidneys and brain. Treating or "reversing" arterial stiffness has therefore become a central target in the field of vascular ageing. SGLT2 inhibitors, initially developed in the context of type 2 diabetes mellitus, have become a cornerstone of heart failure treatment. Additionally, effects on the vasculature have been reported. Here, we demonstrate that treatment with the SGLT2 inhibitor empagliflozin (7 weeks, 15 mg/kg/day) decreased ageing-induced arterial stiffness of the aorta in old mice with normal blood glucose levels. However, no universal mechanism was identified. While empagliflozin reduced the ageing-associated increase in collagen type I in the medial layer of the abdominal infrarenal aorta and decreased medial TGF-ß deposition, this was not observed in the thoracic descending aorta. Moreover, empagliflozin was not able to prevent elastin fragmentation. In conclusion, empagliflozin decreased arterial stiffness in aged mice, indicating that SGLT2 inhibition could be a valuable strategy in mitigating vascular ageing. Further research is warranted to unravel the underlying, possibly region-specific, mechanisms.

Doxorubicin-induced cardiovascular toxicity: a longitudinal evaluation of functional and molecular markers.

AIMS: Apart from cardiotoxicity, the chemotherapeutic doxorubicin (DOX) induces vascular toxicity, represented by arterial stiffness and endothelial dysfunction. Both parameters are of interest for cardiovascular risk stratification as they are independent predictors of future cardiovascular events in the general population. However, the time course of DOX-induced cardiovascular toxicity remains unclear. Moreover, current biomarkers for cardiovascular toxicity prove insufficient. Here, we longitudinally evaluated functional and molecular markers of DOX-induced cardiovascular toxicity in a murine model. Molecular markers were further validated in patient plasma. METHODS AND RESULTS: DOX (4 mg/kg) or saline (vehicle) was administered intra-peritoneally to young, male mice weekly for 6 weeks. In vivo cardiovascular function and ex vivo arterial stiffness and vascular reactivity were evaluated at baseline, during DOX therapy (Weeks 2 and 4) and after therapy cessation (Weeks 6, 9, and 15). Left ventricular ejection fraction (LVEF) declined from Week 4 in the DOX group. DOX increased arterial stiffness in vivo and ex vivo at Week 2, which reverted thereafter. Importantly, DOX-induced arterial stiffness preceded reduced LVEF. Further, DOX impaired endothelium-dependent vasodilation at Weeks 2 and 6, which recovered at Weeks 9 and 15. Conversely, contraction with phenylephrine was consistently higher in the DOX-treated group. Furthermore, proteomic analysis on aortic tissue identified increased thrombospondin-1 (THBS1) and alpha-1-antichymotrypsin (SERPINA3) at Weeks 2 and 6. Up-regulated THBS1 and SERPINA3 persisted during follow-up. Finally, THBS1 and SERPINA3 were quantified in plasma of patients. Cancer survivors with anthracycline-induced cardiotoxicity (AICT; LVEF < 50%) showed elevated THBS1 and SERPINA3 levels compared with age-matched control patients (LVEF ≥ 60%). CONCLUSIONS: DOX increased arterial stiffness and impaired endothelial function, which both preceded reduced LVEF. Vascular dysfunction restored after DOX therapy cessation, whereas cardiac dysfunction persisted. Further, we identified SERPINA3 and THBS1 as promising biomarkers of DOX-induced cardiovascular toxicity, which were confirmed in AICT patients.

Distinct Features of Vascular Diseases in COVID-19.

The Coronavirus Disease 2019 (COVID-19) pandemic was declared in early 2020 after several unexplained pneumonia cases were first reported in Wuhan, China, and subsequently in other parts of the world. Commonly, the disease comprises several clinical features, including high temperature, dry cough, shortness of breath, and hypoxia, associated with findings of interstitial pneumonia on chest X-ray and computer tomography. Nevertheless, severe forms of acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) are not limited to the respiratory tract but also may be extended to other systems, including the cardiovascular system. The bi-directional relationship between atherosclerosis and COVID-19 is accompanied by poor prognosis. The immune response hyperactivation due to SARS-CoV-2 infection causes an increased secretion of cytokines, endothelial dysfunction, and arterial stiffness, which promotes the development of atherosclerosis. Also, due to the COVID-19 pandemic, access to healthcare amenities was reduced, resulting in increased morbidity and mortality in patients at risk. Furthermore, as lockdown measures were largely adopted worldwide, the sedentary lifestyle and the increased consumption of processed nutrients or unhealthy food increased, and in the consequence, we might observe even 70% of overweight and obese population. Altogether, with the relatively low ratio of vaccinated people in many countries, and important health debt appeared, which is now and will be for next decade a large healthcare challenge. However, the experience gained in the COVID-19 pandemic and the new methods of patients' approaching have helped the medical system to overcome this crisis and will hopefully help in the case of new possible epidemics.

Pharmacological modulation of vascular ageing: A review from VascAgeNet.

Vascular ageing, characterized by structural and functional changes in blood vessels of which arterial stiffness and endothelial dysfunction are key components, is associated with increased risk of cardiovascular and other age-related diseases. As the global population continues to age, understanding the underlying mechanisms and developing effective therapeutic interventions to mitigate vascular ageing becomes crucial for improving cardiovascular health outcomes. Therefore, this review provides an overview of the current knowledge on pharmacological modulation of vascular ageing, highlighting key strategies and promising therapeutic targets. Several molecular pathways have been identified as central players in vascular ageing, including oxidative stress and inflammation, the renin-angiotensin-aldosterone system, cellular senescence, macroautophagy, extracellular matrix remodelling, calcification, and gasotransmitter-related signalling. Pharmacological and dietary interventions targeting these pathways have shown potential in ameliorating age-related vascular changes. Nevertheless, the development and application of drugs targeting vascular ageing is complicated by various inherent challenges and limitations, such as certain preclinical methodological considerations, interactions with exercise training and sex/gender-related differences, which should be taken into account. Overall, pharmacological modulation of endothelial dysfunction and arterial stiffness as hallmarks of vascular ageing, holds great promise for improving cardiovascular health in the ageing population. Nonetheless, further research is needed to fully elucidate the underlying mechanisms and optimize the efficacy and safety of these interventions for clinical translation.

Gasdermin D Deficiency Limits the Transition of Atherosclerotic Plaques to an Inflammatory Phenotype in ApoE Knock-Out Mice.

Gasdermin D (GSDMD) is the key executor of pyroptotic cell death. Recent studies suggest that GSDMD-mediated pyroptosis is involved in atherosclerotic plaque destabilization. We report that cleaved GSDMD is expressed in macrophage- and smooth muscle cell-rich areas of human plaques. To determine the effects of GSDMD deficiency on atherogenesis, ApoE-/- Gsdmd-/- (n = 16) and ApoE-/-Gsdmd+/+ (n = 18) mice were fed a western-type diet for 16 weeks. Plaque initiation and formation of stable proximal aortic plaques were not altered. However, plaques in the brachiocephalic artery (representing more advanced lesions compared to aortic plaques) of ApoE-/- Gsdmd-/- mice were significantly smaller (115 ± 18 vs. 186 ± 16 × 103 µm2, p = 0.006) and showed features of increased stability, such as decreased necrotic core area (19 ± 4 vs. 37 ± 7 × 103 µm2, p = 0.03) and increased αSMA/MAC3 ratio (1.6 ± 0.3 vs. 0.7 ± 0.1, p = 0.01), which was also observed in proximal aortic plaques. Interestingly, a significant increase in TUNEL positive cells was observed in brachiocephalic artery plaques from ApoE-/- Gsdmd-/- mice (141 ± 25 vs. 62 ± 8 cells/mm2, p = 0.005), indicating a switch to apoptosis. This switch from pyroptosis to apoptosis was also observed in vitro in Gsdmd-/- macrophages. In conclusion, targeting GSDMD appears to be a promising approach for limiting the transition to an inflammatory, vulnerable plaque phenotype.

Pleiotropic Effects of Atorvastatin Result in a Downregulation of the Carboxypeptidase U System (CPU, TAFIa, CPB2) in a Mouse Model of Advanced Atherosclerosis.

Statins (hydroxymethyl-glutaryl-CoA-reductase inhibitors) lower procarboxypeptidase U (proCPU, TAFI, proCPB2). However, it is challenging to prove whether this is a lipid or non-lipid-related pleiotropic effect, since statin treatment decreases cholesterol levels in humans. In apolipoprotein E-deficient mice with a heterozygous mutation in the fibrillin-1 gene (ApoE-/-Fbn1C1039G+/-), a model of advanced atherosclerosis, statins do not lower cholesterol. Consequently, studying cholesterol-independent effects of statins can be achieved more straightforwardly in these mice. Female ApoE -/-Fbn1C1039G+/- mice were fed a Western diet (WD). At week 10 of WD, mice were divided into a WD group (receiving WD only) and a WD + atorvastatin group (receiving 10 mg/kg/day atorvastatin +WD) group. After 15 weeks, blood was collected from the retro-orbital plexus, and the mice were sacrificed. Total plasma cholesterol and C-reactive protein (CRP) were measured with commercially available kits. Plasma proCPU levels were determined with an activity-based assay. Total plasma cholesterol levels were not significantly different between both groups, while proCPU levels were significantly lower in the WD + atorvastatin group. Interestingly proCPU levels correlated with CRP and circulating monocytes. In conclusion, our results confirm that atorvastatin downregulates proCPU levels in ApoE-/-Fbn1C1039G+/- mice on a WD, and evidence was provided that this downregulation is a pleiotropic effect of atorvastatin treatment.

Defective Autophagy in Vascular Smooth Muscle Cells Alters Vascular Reactivity of the Mouse Femoral Artery.

Autophagy is an important cellular survival process that enables degradation and recycling of defective organelles and proteins to maintain cellular homeostasis. Hence, defective autophagy plays a role in many age-associated diseases, such as atherosclerosis, arterial stiffening and hypertension. Recently, we showed in mice that autophagy in vascular smooth muscle cells (VSMCs) of large elastic arteries such as the aorta is important for Ca2+ mobilization and vascular reactivity. Whether autophagy plays a role in the smaller muscular arteries, such as the femoral artery, and thereby contributes to for example, blood pressure regulation is currently unknown. Therefore, we determined vascular reactivity of femoral artery segments of mice containing a VSMC specific deletion of the essential autophagy gene Atg7 (Atg7F/F SM22α-Cre+) and compared them to femoral artery segments of corresponding control mice (Atg7+/+ SM22α-Cre+). Our results indicate that similar to the aorta, femoral artery segments showed enhanced contractility. Specifically, femoral artery segments of Atg7F/F SM22α-Cre+ mice showed an increase in phasic phenylephrine (PE) induced contractions, together with an enhanced sensitivity to depolarization induced contractions. In addition, and importantly, VSMC sensitivity to exogenous nitric oxide (NO) was significantly increased in femoral artery segments of Atg7F/F SM22α-Cre+ mice. Notwithstanding the fact that small artery contractility is a significant pathophysiological determinant for the development of hypertension, 7 days of treatment with angiotensin II (AngII), which increased systolic blood pressure in control mice, was ineffective in Atg7F/F SM22α-Cre+ mice. It is likely that this was due to the increased sensitivity of VSMCs to NO in the femoral artery, although changes in the heart upon AngII treatment were also present, which could also be (partially) accountable for the lack of an AngII-induced rise in blood pressure in Atg7F/F SM22α-Cre+ mice. Overall, our study indicates that apart from previously shown effects on large elastic arteries, VSMC autophagy also plays a pivotal role in the regulation of the contractile and relaxing properties of the smaller muscular arteries. This may suggest a role for autophagy in vascular pathologies, such as hypertension and arterial stiffness.

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.

Dietary Polyphenols Targeting Arterial Stiffness: Interplay of Contributing Mechanisms and Gut Microbiome-Related Metabolism.

Increased arterial stiffness is a degenerative vascular process, progressing with age that leads to a reduced capability of arteries to expand and contract in response to pressure changes. This progressive degeneration mainly affects the extracellular matrix of elastic arteries and causes loss of vascular elasticity. Recent studies point to significant interference of dietary polyphenols with mechanisms involved in the pathophysiology and progression of arterial stiffness. This review summarizes data from epidemiological and interventional studies on the effect of polyphenols on vascular stiffness as an illustration of current research and addresses possible etiological factors targeted by polyphenols, including pathways of vascular functionality, oxidative status, inflammation, glycation, and autophagy. Effects can either be inflicted directly by the dietary polyphenols or indirectly by metabolites originated from the host or microbial metabolic processes. The composition of the gut microbiome, therefore, determines the resulting metabolome and, as a consequence, the observed activity. On the other hand, polyphenols also influence the intestinal microbial composition, and therefore the metabolites available for interaction with relevant targets. As such, targeting the gut microbiome is another potential treatment option for arterial stiffness.

Nitric oxide donor molsidomine favors features of atherosclerotic plaque stability and reduces myocardial infarction in mice.

Nitric oxide (NO) donors are commonly used for the prevention and treatment of ischemic heart disease. Besides their effects on the heart, NO donors may also prevent hypoxic brain damage and exert beneficial effects on atherosclerosis by favoring features of plaque stability. We recently described that apolipoprotein E (ApoE) deficient mice with a mutation in the fibrillin-1 (Fbn1) gene (ApoE-/-Fbn1C1039G+/-) develop accelerated atherosclerosis, plaque rupture, myocardial infarction, cerebral hypoxia and sudden death. In the present study, we evaluated the effects of chronic treatment with the NO donor molsidomine on atherosclerotic plaque stability, cardiac function, neurological symptoms and survival in the ApoE-/-Fbn1C1039G+/- mouse model. Female ApoE-/-Fbn1C1039G+/- mice were fed a Western diet (WD). After 8 weeks of WD, the mice were divided into two groups receiving either molsidomine via the drinking water (1 mg/kg/day; n = 34) or tap water (control; n = 36) until 25 weeks of WD. Survival tended to increase after molsidomine treatment (68% vs. 58% in controls). Importantly, atherosclerotic plaques of molsidomine-treated mice had a thicker fibrous cap (11.1 ±â€¯1.2 vs. 8.1 ±â€¯0.7 µm) and showed an increased occurrence of plaque macrocalcifications (30% vs. 0%), indicative of a more stable phenotype. Molsidomine also improved cardiac function, as fractional shortening was increased (40 ±â€¯2% vs. 27 ±â€¯2%) combined with a decreased end diastolic (3.1 ±â€¯0.2 vs. 3.9 ±â€¯0.2 mm) and end systolic diameter (1.9 ±â€¯0.1 vs. 2.9 ±â€¯0.2 mm). Furthermore, perivascular fibrosis (23 ±â€¯2 vs. 30 ±â€¯2%) and the occurrence of myocardial infarctions (12% vs. 36%) was significantly reduced. Track width, a measure of the animal's hind limb base of support and representative of hypoxic brain damage, was also normalized as a result of molsidomine treatment (2.54 ±â€¯0.04 vs. 2.91 ±â€¯0.09 cm in controls). These findings demonstrate that the NO donor molsidomine improves cardiac function, reduces neurological symptoms and enhances atherosclerotic plaque stability.

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.

Defective Autophagy in Atherosclerosis: To Die or to Senesce?

Autophagy is a subcellular process that plays an important role in the degradation of proteins and damaged organelles such as mitochondria (a process termed "mitophagy") via lysosomes. It is crucial for regulating protein and mitochondrial quality control and maintaining cellular homeostasis, whereas dysregulation of autophagy has been implicated in a wide range of diseases including atherosclerosis. Recent evidence has shown that the autophagic process becomes dysfunctional during the progression of atherosclerosis, regardless of whether there are many autophagy-stimulating factors (e.g., reactive oxygen species, oxidized lipids, and cytokines) present within the atherosclerotic plaque. This review highlights the recent insights into the causes and consequences of defective autophagy in atherosclerosis, with a special focus on the role of autophagy and mitophagy in plaque macrophages, vascular smooth muscle cells (VSMCs), and endothelial cells (ECs). It has been shown that defective autophagy can promote apoptosis in macrophages but that it accelerates premature senescence in VSMCs. In the ECs, defective autophagy promotes both apoptosis and senescence. We will discuss the discrepancy between these three cell types in their response to autophagy deficiency and underline the cell type-dependent role of autophagy, which may have important implications for the efficacy of autophagy-targeted treatments for atherosclerosis.