The Fourth Maastricht Consensus Conference on Thrombosis included the following themes. Theme 1: The "coagulome" as a critical driver of cardiovascular disease. Blood coagulation proteins also play... Show moreThe Fourth Maastricht Consensus Conference on Thrombosis included the following themes. Theme 1: The "coagulome" as a critical driver of cardiovascular disease. Blood coagulation proteins also play divergent roles in biology and pathophysiology, related to specific organs, including brain, heart, bone marrow, and kidney. Four investigators shared their views on these organ- specific topics. Theme 2: Novel mechanisms of thrombosis. Mechanisms linking factor XII to fibrin, including their structural and physical properties, contribute to thrombosis, which is also affected by variation in microbiome status. Virus infection-associated coagulopathies perturb the hemostatic balance resulting in thrombosis and/ or bleeding. Theme 3: How to limit bleeding risks: insights from translational studies. This theme included state-of- the- art methodology for exploring the contribution of genetic determinants of a bleeding diathesis; determination of polymorphisms in genes that control the rate of metabolism by the liver of P2Y12 inhibitors, to improve safety of antithrombotic therapy. Novel reversal agents for direct oral anticoagulants are discussed. Theme 4: Hemostasis in extracorporeal systems: the value and limitations of ex vivo models. Perfusion flow chamber and nanotechnology developments are developed for studying bleeding and thrombosis tendencies. Vascularized organoids are utilized for disease modeling and drug development studies. Strategies for tackling extracorporeal membrane oxygenation-associated coagulopathy are discussed. Theme 5: Clinical dilemmas in thrombosis and antithrombotic management. Plenary presentations addressed controversial areas, i. e., thrombophilia testing, thrombosis risk assessment in hemophilia, novel antiplatelet strategies, and clinically tested factor XI(a) inhibitors, both possibly with reduced bleeding risk. Finally, COVID- 19-associated coagulopathy is revisited. Show less
Background:Identification of risk factors and causes of stroke is key to optimize treatment and prevent recurrence. Up to one-third of young patients with stroke have a cryptogenic stroke according... Show moreBackground:Identification of risk factors and causes of stroke is key to optimize treatment and prevent recurrence. Up to one-third of young patients with stroke have a cryptogenic stroke according to current classification systems (Trial of ORG 10172 in Acute Stroke Treatment [TOAST] and atherosclerosis, small vessel disease, cardiac pathology, other causes, dissection [ASCOD]). The aim was to identify risk factors and leads for (new) causes of cryptogenic ischemic stroke in young adults, using the pediatric classification system from the IPSS study (International Pediatric Stroke Study).Methods:This is a multicenter prospective cohort study conducted in 17 hospitals in the Netherlands, consisting of 1322 patients aged 18 to 49 years with first-ever, imaging confirmed, ischemic stroke between 2013 and 2021. The main outcome was distribution of risk factors according to IPSS classification in patients with cryptogenic and noncryptogenic stroke according to the TOAST and ASCOD classification.Results:The median age was 44.2 years, and 697 (52.7%) were men. Of these 1322 patients, 333 (25.2%) had a cryptogenic stroke according to the TOAST classification. Additional classification using the ASCOD criteria reduced the number patients with cryptogenic stroke from 333 to 260 (19.7%). When risk factors according to the IPSS were taken into account, the number of patients with no potential cause or risk factor for stroke reduced to 10 (0.8%).Conclusions:Among young adults aged 18 to 49 years with a cryptogenic ischemic stroke according to the TOAST classification, risk factors for stroke are highly prevalent. Using a pediatric classification system provides new leads for the possible causes in cryptogenic stroke, and could potentially lead to more tailored treatment for young individuals with stroke. Show less
Background: Type A aortic dissections (TAAD) are devastating aortic complications. Patients with Marfan syndrome, a bicuspid aortic valve or a thoracic aortic aneurysm have an increased risk to... Show moreBackground: Type A aortic dissections (TAAD) are devastating aortic complications. Patients with Marfan syndrome, a bicuspid aortic valve or a thoracic aortic aneurysm have an increased risk to develop a TAAD. These predisposing conditions are characterized by a histologically thin intimal layer and hardly any atherosclerosis. Little is known about the susceptibility for atherosclerosis in patients with a type A aortic dissection. Objective: We aim to systematically describe atherosclerotic lesions in TAAD patients. Materials and methods: A total of 51 patients with a TAAD (mean age 62.5 +/- 10.8 years, 49% females) and 17 control patients (mean age 63 +/- 5.5 years, 53% females) were included in this study. Cardiovascular risk factors were assessed clinically. All sections were stained with Movat pentachrome and hematoxylin eosin. Plaque morphology was classified according to the modified AHA classification scheme proposed by Virmani et al. Results: In the TAAD group thirty-seven percent were overweight (BMI > 25). Diabetes and peripheral arterial disease were not present in any of the patients. Fifty-nine percent of the patients had a history of hypertension. The intima in TAAD patients was significantly thinner as compared to the control group (mean thickness 143 +/- 126.5 mu m versus 193 +/- 132 mu m, p < 0.023). Seven TAAD patients had a normal intima without any form of adaptive or pathological thickening. Twenty-three TAAD patients demonstrated adaptive intimal thickening. Fourteen had an intimal xanthoma, also known as fatty streaks. A minority of 7 TAAD patients had progressive atherosclerotic lesions, 4 of which demonstrated pathological intimal thickening, 3 patients showed early fibroatheroma, late fibroatheroma and thin cap fibroatheroma. In the control group the majority of the patients exhibited progressive atherosclerotic lesions: three pathologic intimal thickening, two early fibroatheroma, six late fibroatheroma, one healed rupture and two fibrotic calcified plaque. Discussion: This study shows that TAAD patients hardly exhibit any form of progressive atherosclerosis. The majority of TAAD patients showcase non-progressive intimal lesions, whereas the control group mostly demonstrated progressive intimal atherosclerotic lesions. Findings are independent of age, sex, or the presence of (a history of) hypertension. Show less
Vos, D.Y.; Wijers, M.; Smit, M.; Huijkman, N.; Kloosterhuis, N.J.; Wolters, J.C.; ... ; Sluis, B. van de 2023
Background:The copper metabolism MURR1 domains/coiled-coil domain containing 22/coiled-coil domain containing 93 (CCC) complex is required for the transport of low-density lipoprotein receptor ... Show moreBackground:The copper metabolism MURR1 domains/coiled-coil domain containing 22/coiled-coil domain containing 93 (CCC) complex is required for the transport of low-density lipoprotein receptor (LDLR) and LRP1 (LDLR-related protein 1) from endosomes to the cell surface of hepatocytes. Impaired functioning of hepatocytic CCC causes hypercholesterolemia in mice, dogs, and humans. Retriever, a protein complex consisting of subunits VPS26C, VPS35L, and VPS29, is associated with CCC, but its role in endosomal lipoprotein receptor transport is unclear. We here investigated the contribution of retriever to hepatocytic lipoprotein receptor recycling and plasma lipids regulation. Methods:Using somatic CRISPR/Cas9 gene editing, we generated liver-specific VPS35L or VPS26C-deficient mice. We determined total and surface levels of LDLR and LRP1 and plasma lipids. In addition, we studied the protein levels and composition of CCC and retriever. Results: Hepatocyte VPS35L deficiency reduced VPS26C levels but had minimal impact on CCC composition. VPS35L deletion decreased hepatocytic surface expression of LDLR and LRP1, accompanied by a 21% increase in plasma cholesterol levels. Hepatic VPS26C ablation affected neither levels of VPS35L and CCC subunits, nor plasma lipid concentrations. However, VPS26C deficiency increased hepatic LDLR protein levels by 2-fold, probably compensating for reduced LRP1 functioning, as we showed in VPS26C-deficient hepatoma cells. Upon PCSK9 (proprotein convertase subtilisin/kexin type 9)-mediated LDLR elimination, VPS26C ablation delayed postprandial triglyceride clearance and increased plasma triglyceride levels by 26%. Conclusions: Our study suggests that VPS35L is shared between retriever and CCC to facilitate LDLR and LRP1 transport from endosomes to the cell surface. Conversely, retriever subunit VPS26C selectively transports LRP1, but not LDLR, and thereby may control hepatic uptake of postprandial triglyceride-rich lipoprotein remnants. Show less
Schonke, M.; Ying, Z.X.; Kovynev, A.; Panhuis, W.I.H.; Binnendijk, A.; Poel, S. van der; ... ; Rensen, P.C.N. 2023
The metabolic and inflammatory processes that are implicated in the development of cardiovascular diseases are under control of the biological clock. While skeletal muscle function exhibits... Show moreThe metabolic and inflammatory processes that are implicated in the development of cardiovascular diseases are under control of the biological clock. While skeletal muscle function exhibits circadian rhythms, it is unclear to what extent the beneficial health effects of exercise are restricted to unique time windows. We aimed to study whether the timing of exercise training differentially modulates the development of atherosclerosis and elucidate underlying mechanisms. We endurance-trained atherosclerosis-prone female APOE*3-Leiden.CETP mice fed a Western-type diet, a well-established human-like model for cardiometabolic diseases, for 1 h five times a week for 4 weeks either in their early or in their late active phase on a treadmill. We monitored metabolic parameters, the development of atherosclerotic lesions in the aortic root and assessed the composition of the gut microbiota. Late, but not early, exercise training reduced fat mass by 19% and the size of early-stage atherosclerotic lesions by as much as 29% compared to sedentary animals. No correlation between cholesterol exposure and lesion size was evident, as no differences in plasma lipid levels were observed, but circulating levels of the pro-inflammatory markers ICAM-1 and VCAM-1 were reduced with late exercise. Strikingly, we observed a time-of-day-dependent effect of exercise training on the composition of the gut microbiota as only late training increased the abundance of gut bacteria producing short-chain fatty acids with proposed anti-inflammatory properties. Together, these findings indicate that timing is a critical factor to the beneficial anti-atherosclerotic effects of exercise with a great potential to further optimize training recommendations for patients. Show less
Schonke, M.; Ying, Z.X.; Kovynev, A.; Panhuis, W.I.H.; Binnendijk, A.; Poel, S. van der; ... ; Rensen, P.C.N. 2023
The metabolic and inflammatory processes that are implicated in the development of cardiovascular diseases are under control of the biological clock. While skeletal muscle function exhibits... Show moreThe metabolic and inflammatory processes that are implicated in the development of cardiovascular diseases are under control of the biological clock. While skeletal muscle function exhibits circadian rhythms, it is unclear to what extent the beneficial health effects of exercise are restricted to unique time windows. We aimed to study whether the timing of exercise training differentially modulates the development of atherosclerosis and elucidate underlying mechanisms. We endurance-trained atherosclerosis-prone female APOE*3-Leiden.CETP mice fed a Western-type diet, a well-established human-like model for cardiometabolic diseases, for 1h five times a week for 4weeks either in their early or in their late active phase on a treadmill. We monitored metabolic parameters, the development of atherosclerotic lesions in the aortic root and assessed the composition of the gut microbiota. Late, but not early, exercise training reduced fat mass by 19% and the size of early-stage atherosclerotic lesions by as much as 29% compared to sedentary animals. No correlation between cholesterol exposure and lesion size was evident, as no differences in plasma lipid levels were observed, but circulating levels of the pro-inflammatory markers ICAM-1 and VCAM-1 were reduced with late exercise. Strikingly, we observed a time-of-day-dependent effect of exercise training on the composition of the gut microbiota as only late training increased the abundance of gut bacteria producing short-chain fatty acids with proposed anti-inflammatory properties. Together, these findings indicate that timing is a critical factor to the beneficial anti-atherosclerotic effects of exercise with a great potential to further optimize training recommendations for patients. Show less
Schönke, M.; Ying, Z.X.; Kovynev, A.; Panhuis, W.I.H.; Binnendijk, A.; Poel, S. van der; ... ; Rensen, P.C.N. 2023
The metabolic and inflammatory processes that are implicated in the development of cardiovascular diseases are under control of the biological clock. While skeletal muscle function exhibits... Show moreThe metabolic and inflammatory processes that are implicated in the development of cardiovascular diseases are under control of the biological clock. While skeletal muscle function exhibits circadian rhythms, it is unclear to what extent the beneficial health effects of exercise are restricted to unique time windows. We aimed to study whether the timing of exercise training differentially modulates the development of atherosclerosis and elucidate underlying mechanisms. We endurance-trained atherosclerosis-prone female APOE*3-Leiden.CETP mice fed a Western-type diet, a well-established human-like model for cardiometabolic diseases, for 1 h five times a week for 4 weeks either in their early or in their late active phase on a treadmill. We monitored metabolic parameters, the development of atherosclerotic lesions in the aortic root and assessed the composition of the gut microbiota. Late, but not early, exercise training reduced fat mass by 19% and the size of early-stage atherosclerotic lesions by as much as 29% compared to sedentary animals. No correlation between cholesterol exposure and lesion size was evident, as no differences in plasma lipid levels were observed, but circulating levels of the pro-inflammatory markers ICAM-1 and VCAM-1 were reduced with late exercise. Strikingly, we observed a time-of-day-dependent effect of exercise training on the composition of the gut microbiota as only late training increased the abundance of gut bacteria producing short-chain fatty acids with proposed anti-inflammatory properties. Together, these findings indicate that timing is a critical factor to the beneficial anti-atherosclerotic effects of exercise with a great potential to further optimize training recommendations for patients. Show less
Klei, V.M.G.T.H. van der; Poortvliet, R.K.E.; Bogaerts, J.M.K.; Blom, J.W.; Mooijaart, S.P.; Teh, R.; ... ; Gussekloo, J. 2022
Objectives: Previous findings suggest a vascular foundation underlying apathy, but transdiagnostic and prospective evidence on vascular apathy is scarce. This study examines the association between... Show moreObjectives: Previous findings suggest a vascular foundation underlying apathy, but transdiagnostic and prospective evidence on vascular apathy is scarce. This study examines the association between vascular disease and the presence and development of apathy symptoms in the very old.Methods: Four cohorts of the Towards Understanding Longitudinal International older People Studies (TULIPS)-consortium were included in a two-staged, individual participant data meta-analysis using generalized linear mixed models, Vascular disease was defined as a history of any clinical atherosclerotic pathology (angina pectoris, myocardial infarction, intermittent claudication, transient ischemic attack, stroke or related surgeries) and was related to apathy symptoms as repeatedly measured by the Geriatric Depression Scale (GDS-3A >= 2) over a maximum of 5 years.Results: Of all 1868 participants (median age 85 years old), 53.9% had vascular disease and 44.3% experienced apathy symptoms. Participants with vascular disease had a 76% higher risk of apathy symptoms at baseline (odds ratio (OR) 1.76, 95% confidence interval (CI) 1.32-2.35), irrespective of depressive symptoms and only partially explained by stroke. Conversely, there was no association of vascular disease with the occurrence of apathy symptoms longitudinally, both in those with apathy at baseline (OR 1.00, 95% CI 0.84-1.20) and without (OR 0.96, 95% CI 0.841.09).Conclusions: Vascular disease in the very old is associated with apathy symptoms cross-sectionally, but not proven longitudinally, independent of depressive symptoms. These findings query a vascular cause underlying apathy symptoms. However, the consistency of our cross-sectional findings in direction and magnitude across the TULIPS-consortium do emphasize international relevance of the interplay of vascular factors and apathy in advanced age, which meaning needs further unravelling. Show less
Phosphorylcholine (PC) is one of the main oxLDL epitopes playing a central role in atherosclerosis, due to its atherogenic and proinflammatory effects. PC can be cleared by natural IgM antibodies... Show morePhosphorylcholine (PC) is one of the main oxLDL epitopes playing a central role in atherosclerosis, due to its atherogenic and proinflammatory effects. PC can be cleared by natural IgM antibodies and low levels of these antibodies have been associated with human vein graft (VG) failure. Although PC antibodies are recognized for their anti-inflammatory properties, their effect on intraplaque angiogenesis (IPA) and intraplaque hemorrhage (IPH)-interdependent processes contributing to plaque rupture-are unknown. We hypothesized that new IgG phosphorylcholine antibodies (PC-mAb) could decrease vulnerable lesions in murine VGs.Therefore, hypercholesterolemic male ApoE3*Leiden mice received a (donor) caval vein interposition in the carotid artery and weekly IP injections of (5 mg/kg) PCmAb (n = 11) or vehicle (n = 12) until sacrifice at day 28. We found that PCmAb significantly decreased vein graft media (13%), intima lesion (25%), and increased lumen with 32% compared to controls. PCmAb increased collagen content (18%) and decreased macrophages presence (31%). PCmAb resulted in 23% decreased CD163+ macrophages content in vein grafts whereas CD163 expression was decreased in Hb:Hp macrophages. PCmAb significantly lowered neovessel density (34%), EC proliferation and migration with/out oxLDL stimulation. Moreover, PCmAb enhanced intraplaque angiogenic vessels maturation by increasing neovessel pericyte coverage in vivo (31%). Together, this resulted in a 62% decrease in IPH. PCmAb effectively inhibits murine atherosclerotic lesion formation in vein grafts by reducing IPA and IPH via decreased neovessel density and macrophages influx and increased neovessel maturation. PC-mAb therefore holds promise as a new therapeutic approach to prevent vein graft disease. Show less
Introduction: Low ankle-brachial index (ABI) <= 0. 9 is a marker for generalized atherosclerosis and a risk factor for cognitive decline in the general population. Objective: To evaluate the... Show moreIntroduction: Low ankle-brachial index (ABI) <= 0. 9 is a marker for generalized atherosclerosis and a risk factor for cognitive decline in the general population. Objective: To evaluate the impact of ABI <= 0.9 on cognitive function up to 3 years after first-ever ischemic stroke. Methods: Data was used from the "PROspective Cohort with Incident Stroke-Berlin" (PROSCIS-B; NCT01363856). ABI was measured at baseline and categorized into normal (1.4-0.9) vs. low (<= 0.9). Cognitive function was assessed with the Montreal Cognitive Assessment (MoCA) and the Mini-Mental-State-Examination (MMSE) at baseline and with the Telephone Interview for Cognitive Status-modified (TICS-m) at 1-3 years of follow-up. We performed confounder adjusted generalized linear models (GLM) to calculate relative risks (RR) for cognitive impairment at baseline (MMSE <= 26; MoCA <= 25) and linear mixed models (LMM) to estimate the impact of low ABI on TICS-m over time. Results: We included 325 patients [mean age: 66 (SD = 13); 38% female, median NIHSS = 2 (IQR = 1-4), ABI <= 0.9: 59 (18%)). Patients with tow ABI were at increased risk of cognitive impairment at baseline (adjusted RR for MoCA <= 25 = 1.98; 95%-CI:1.24 to 3.16). TICS-m scores were consistently lower over time in patients with low ABI (adjusted beta = -1.96; 95%-CI:-3.55 to -0.37). Independent of ABI, cognitive function did not decline over time (adjusted 95%-CI:-0.06 to 0.64). Conclusion: In patients with mild to moderate first-ever ischemic stroke, low ABI is associated with reduced cognitive function over a 3-year follow-up. Show less
Seidel, F.; Kleemann, R.; Duyvenvoorde, W. van; Trigt, N. van; Keijzer, N.; Kooij, S. van der; ... ; Morrison, M.C. 2022
Background: Chronic inflammation is an important driver in the progression of non-alcoholic steatohepatitis (NASH) and atherosclerosis. The complement system, one of the first lines of defense in... Show moreBackground: Chronic inflammation is an important driver in the progression of non-alcoholic steatohepatitis (NASH) and atherosclerosis. The complement system, one of the first lines of defense in innate immunity, has been implicated in both diseases. However, the potential therapeutic value of complement inhibition in the ongoing disease remains unclear. Methods: After 20 weeks of high-fat diet (HFD) feeding, obese Ldlr-/-.Leiden mice were treated twice a week with an established anti-C5 antibody (BB5.1) or vehicle control. A separate group of mice was kept on a chow diet as a healthy reference. After 12 weeks of treatment, NASH was analyzed histopathologically, and genome-wide hepatic gene expression was analyzed by next-generation sequencing and pathway analysis. Atherosclerotic lesion area and severity were quantified histopathologically in the aortic roots. Results: Anti-C5 treatment considerably reduced complement system activity in plasma and MAC deposition in the liver but did not affect NASH. Anti-C5 did, however, reduce the development of atherosclerosis, limiting the total lesion size and severity independently of an effect on plasma cholesterol but with reductions in oxidized LDL (oxLDL) and macrophage migration inhibitory factor (MIF). Conclusion: We show, for the first time, that treatment with an anti-C5 antibody in advanced stages of NASH is not sufficient to reduce the disease, while therapeutic intervention against established atherosclerosis is beneficial to limit further progression. Show less
Background: In patients with large vessel occlusion stroke of the anterior circulation, underlying cause is a determinant of outcome. Whether this is the case for posterior circulation large vessel... Show moreBackground: In patients with large vessel occlusion stroke of the anterior circulation, underlying cause is a determinant of outcome. Whether this is the case for posterior circulation large vessel occlusion stroke has yet to be determined. We aimed to report on cause in patients with posterior circulation stroke treated with endovascular thrombectomy and to analyze the association with functional outcome. Methods: We used data of patients with posterior circulation stroke included in the MR CLEAN (Multicenter Randomized Controlled Trial of Endovascular Treatment for Acute Ischemic Stroke in the Netherlands) registry, a prospective multicenter observational study, between 2014 and 2018. Stroke cause was categorized into large artery atherosclerosis (LAA), cardioembolism, arterial dissection, embolic stroke of undetermined source (ESUS), other determined cause, or undetermined cause. For primary analysis on the association between cause and outcome, we used multivariable ordinal logistic regression analysis to estimate the adjusted common odds ratio for a shift towards a better functional outcome on the modified Rankin Scale at 90 days with LAA as a reference group. Secondary outcomes included favorable functional outcome (modified Rankin Scale score 0-3), National Institutes of Health Stroke Scale score at 24 to 48 hours, reperfusion on digital subtraction angiography, and stroke progression. Results: Of 264 patients with posterior circulation stroke, 84 (32%) had LAA, 48 (18%) cardioembolism, 31 (12%) dissection, and 14 (5%) ESUS. Patients with a dissection were younger (48 [interquartile range, 43-60] years) and had a lower National Institutes of Health Stroke Scale at baseline (12 [interquartile range, 6-31]) than patients with other cause. Functional outcome was better for patients with cardioembolism and ESUS compared to LAA (modified Rankin Scale adjusted common odds ratio, 2.4 [95% CI, 1.1-5.2], respectively adjusted common odds ratio, 3.1 [95% CI, 1.0-9.3]). Patients with a dissection had a lower chance of successful reperfusion compared with LAA (adjusted odds ratio, 0.20 [95% CI, 0.06-0.70]). Conclusions: Unlike the anterior circulation, most frequent cause in our posterior large vessel occlusion stroke cohort is LAA followed by cardioembolism, dissection, and ESUS. Patients with cardioembolism and ESUS have a better prognosis for functional outcome after endovascular thrombectomy than patients with LAA. Show less
Background Adolescents with chronic disease are often exposed to inflammatory, metabolic, and hemodynamic risk factors for early atherosclerosis. Since postmortem studies have shown that... Show moreBackground Adolescents with chronic disease are often exposed to inflammatory, metabolic, and hemodynamic risk factors for early atherosclerosis. Since postmortem studies have shown that atherogenesis starts in the aorta, the CDACD (Cardiovascular Disease in Adolescents with Chronic Disease) study investigated preclinical aortic atherosclerosis in these adolescents. Methods and Results The cross-sectional CDACD study enrolled 114 adolescents 12 to 18 years old with chronic disorders including juvenile idiopathic arthritis, cystic fibrosis, obesity, corrected coarctation of the aorta, and healthy controls with a corrected atrial septal defect. Cardiovascular magnetic resonance was used to assess aortic pulse wave velocity and aortic wall thickness, as established aortic measures of preclinical atherosclerosis. Cardiovascular magnetic resonance showed a higher aortic pulse wave velocity, which reflects aortic stiffness, and higher aortic wall thickness in all adolescent chronic disease groups, compared with controls (P<0.05). Age (beta=0.253), heart rate (beta=0.236), systolic blood pressure (beta=-0.264), and diastolic blood pressure (beta=0.365) were identified as significant predictors for aortic pulse wave velocity, using multivariable linear regression analysis. Aortic wall thickness was predicted by body mass index (beta=0.248) and fasting glucose (beta=0.242), next to aortic lumen area (beta=0.340). Carotid intima-media thickness was assessed using ultrasonography, and was only higher in adolescents with coarctation of the aorta, compared with controls (P<0.001). Conclusions Adolescents with chronic disease showed enhanced aortic stiffness and wall thickness compared with controls. The enhanced aortic pulse wave velocity and aortic wall thickness in adolescents with chronic disease could indicate accelerated atherogenesis. Our findings underscore the importance of the aorta for assessment of early atherosclerosis, and the need for tailored cardiovascular follow-up of children with chronic disease. Show less
Rykaczewska, U.; Zhao, Q.Y.; Saliba-Gustafsson, P.; Lengquist, M.; Kronqvist, M.; Bergman, O.; ... ; Matic, L. 2022
Background: Understanding the processes behind carotid plaque instability is necessary to develop methods for identification of patients and lesions with stroke risk. Here, we investigated... Show moreBackground: Understanding the processes behind carotid plaque instability is necessary to develop methods for identification of patients and lesions with stroke risk. Here, we investigated molecular signatures in human plaques stratified by echogenicity as assessed by duplex ultrasound. Methods: Lesion echogenicity was correlated to microarray gene expression profiles from carotid endarterectomies (n=96). The findings were extended into studies of human and mouse atherosclerotic lesions in situ, followed by functional investigations in vitro in human carotid smooth muscle cells (SMCs). Results: Pathway analyses highlighted muscle differentiation, iron homeostasis, calcification, matrix organization, cell survival balance, and BCLAF1 (BCL2 [B-cell lymphoma 2]-associated transcription factor 1) as the most significant signatures. BCLAF1 was downregulated in echolucent plaques, positively correlated to proliferation and negatively to apoptosis. By immunohistochemistry, BCLAF1 was found in normal medial SMCs. It was repressed early during atherogenesis but reappeared in CD68+ cells in advanced plaques and interacted with BCL2 by proximity ligation assay. In cultured SMCs, BCLAF1 was induced by differentiation factors and mitogens and suppressed by macrophage-conditioned medium. BCLAF1 silencing led to downregulation of BCL2 and SMC markers, reduced proliferation, and increased apoptosis. Transdifferentiation of SMCs by oxLDL (oxidized low-denisty lipoprotein) was accompanied by upregulation of BCLAF1, CD36, and CD68, while oxLDL exposure with BCLAF1 silencing preserved MYH (myosin heavy chain) 11 expression and prevented transdifferentiation. BCLAF1 was associated with expression of cell differentiation, contractility, viability, and inflammatory genes, as well as the scavenger receptors CD36 and CD68. BCLAF1 expression in CD68+/BCL2+ cells of SMC origin was verified in plaques from MYH11 lineage-tracing atherosclerotic mice. Moreover, BCLAF1 downregulation associated with vulnerability parameters and cardiovascular risk in patients with carotid atherosclerosis. Conclusions: Plaque echogenicity correlated with enrichment of distinct molecular pathways and identified BCLAF1, previously not described in atherosclerosis, as the most significant gene. Functionally, BCLAF1 seems necessary for survival and transdifferentiation of SMCs into a macrophage-like phenotype. The role of BCLAF1 in plaque vulnerability should be further evaluated. Show less
Aims: Atherosclerosis develops progressively and worsens over time, yet event risk patterns vary in the left circumflex (LCx), right coronary artery (RCA) and left anterior descending (LAD). The... Show moreAims: Atherosclerosis develops progressively and worsens over time, yet event risk patterns vary in the left circumflex (LCx), right coronary artery (RCA) and left anterior descending (LAD). The aim of this analysis was to examine varying progressive disease alterations between the three major coronary arteries. Methods and results: Patients were included from a prospective, international registry of consecutive patients who underwent serial CCTA at a median interval of 3.3 years. Annual progression of quantitative total and compositional plaque volume were compared between the three coronary arteries (LCx, LAD, and RCA). Other analyses compared stenosis >= 50% and new high-risk plaque (HRP; >= 2 of the following: spotty calcification, positive remodelling, napkin-ring sign, and low-attenuation plaque) on follow-up. Generalized estimating equations and marginal Cox regression models were used to compare progression, with covariate adjustment by the baseline atherosclerotic cardiovascular disease risk score, statin use, and plaque burden. Quantitative plaque measurements were calculated in 1344 patients (age 60 +/- 9 years, 57% men). Plaque progression occurred less often in the LCx (41.0%) as compared to the RCA (52.7%) and LAD (77.4%, P < 0.001). Odds for annual plaque burden increase >= population mean were 1.98- and 1.43-fold as high in the LAD (P < 0.001) and RCA (P < 0.001) as compared to the LCx. Similarly, the LAD was associated with a 2.45 higher risk of progression to obstructive CAD (P < 0.001), as compared to the LCx; with no differences between the RCA and LCx (P = 0.13). New HRP lesions formed least often in the LCx (3.4%), followed by the RCA (8.1%) and most often in the LAD (10.1%; P < 0.001). Conclusions: Our findings reveal novel insights into varied patterns of atherosclerotic plaque progression within the LCx as compared to the other epicardial coronary arteries. These varied patterns reflect differing stages in the disease process or differing pathogenic milieu across the coronary arteries. Show less
Loef, M.; Geest, R.J. van der; Lamb, H.J.; Mutsert, R. de; Cessie, S. le; Rosendaal, F.R.; Kloppenburg, M. 2021
Objective. We investigated the role of blood pressure, vessel wall stiffness [pulse wave velocity (PWV)] and subclinical atherosclerosis markers [carotid intima-media thickness (cIMT), popliteal... Show moreObjective. We investigated the role of blood pressure, vessel wall stiffness [pulse wave velocity (PWV)] and subclinical atherosclerosis markers [carotid intima-media thickness (cIMT), popliteal vessel wall thickness (pVWT)] as mediators of the association of obesity with OA.Methods. We used cross-sectional data from a subset of the population-based NEO study (n = 6334). We classified clinical hand and knee OA by the ACR criteria, and structural knee OA, effusion and bone marrow lesions on MRI (n = 1285). cIMT was assessed with ultrasonography. pVWT was estimated on knee MRI (n = 1285), and PWV by abdominal velocity-encoded MRIs (n = 2580), in subpopulations. Associations between BMI and OA were assessed with logistic regression analyses, adjusted for age, sex and education. Blood pressure, cIMT, pVWT and PWV were added to the model to estimate mediation.Results. The population consisted of 55% women, with a mean (S.D.) age of 56(6) years. Clinical hand OA was present in 8%, clinical knee OA in 10%, and structural knee OA in 12% of participants. BMI was positively associated with all OA outcomes. cIMT partially mediated the association of BMI with clinical hand OA [10.6 (6.2; 30.5)%], structural knee OA [3.1 (1.9; 7.3)%] and effusion [10.8 (6.0; 37.6)%]. Diastolic blood pressure [2.1 (1.6; 3.0)%] minimally mediated the association between BMI and clinical knee OA. PWV and pVWT did not mediate the association between BMI and OA.Conclusions. cIMT and diastolic blood pressure minimally mediated the association of BMI with OA. This suggests that such mediation is trivial in the middle-aged population. Show less
Calcification is a prominent feature of late-stage atherosclerosis, but the mechanisms driving this process are unclear. Using a biobank of carotid endarterectomies, we recently showed that... Show moreCalcification is a prominent feature of late-stage atherosclerosis, but the mechanisms driving this process are unclear. Using a biobank of carotid endarterectomies, we recently showed that Proteoglycan 4 (PRG4) is a key molecular signature of calcified plaques, expressed in smooth muscle cell (SMC) rich regions. Here, we aimed to unravel the PRG4 role in vascular remodeling and intimal calcification. PRG4 expression in human carotid endarterectomies correlated with calcification assessed by preoperative computed tomographies. PRG4 localized to SMCs in early intimal thickening, while in advanced lesions it was found in the extracellular matrix, surrounding macro-calcifications. In experimental models, Prg4 was upregulated in SMCs from partially ligated ApoE(-/-) mice and rat carotid intimal hyperplasia, correlating with osteogenic markers and TGFb1. Furthermore, PRG4 was enriched in cells positive for chondrogenic marker SOX9 and around plaque calcifications in ApoE(-/-) mice on warfarin. In vitro, PRG4 was induced in SMCs by IFNg, TGFb1 and calcifying medium, while SMC markers were repressed under calcifying conditions. Silencing experiments showed that PRG4 expression was driven by transcription factors SMAD3 and SOX9. Functionally, the addition of recombinant human PRG4 increased ectopic SMC calcification, while arresting cell migration and proliferation. Mechanistically, it suppressed endogenous PRG4, SMAD3 and SOX9, and restored SMC markers' expression. PRG4 modulates SMC function and osteogenic phenotype during intimal remodeling and macro-calcification in response to TGFb1 signaling, SMAD3 and SOX9 activation. The effects of PRG4 on SMC phenotype and calcification suggest its role in atherosclerotic plaque stability, warranting further investigations. Show less
Vreeken, D.; Bruikman, C.S.; Stam, W.; Cox, S.M.L.; Nagy, Z.; Zhang, H.Y.; ... ; Gils, J.M. van 2021
Objective: Besides hyperlipidemia, inflammation is an important determinant in the initiation and the progression of atherosclerosis. As Neuroimmune Guidance Cues (NGCs) are emerging as regulators... Show moreObjective: Besides hyperlipidemia, inflammation is an important determinant in the initiation and the progression of atherosclerosis. As Neuroimmune Guidance Cues (NGCs) are emerging as regulators of atherosclerosis, we set out to investigate the expression and function of inflammation-regulated NGCs.Methods and results: NGC expression in human monocytes and endothelial cells was assessed using a publicly available RNA dataset. Next, the mRNA levels of expressed NGCs were analyzed in primary human monocytes and endothelial cells after stimulation with IL1 beta or TNF alpha. Upon stimulation a total of 14 and 19 NGCs in monocytes and endothelial cells, respectively, were differentially expressed. Since plexin A4 (PLXNA4) was strongly downregulated in endothelial cells under inflammatory conditions, the role of PLXNA4 in endothelial function was investigated. Knockdown of PLXNA4 in endothelial cells markedly impaired the integrity of the monolayer leading to more elongated cells with an inflammatory phenotype. In addition, these cells showed an increase in actin stress fibers and decreased cell-cell junctions. Functional assays revealed decreased barrier function and capillary network formation of the endothelial cells, while vascular leakage and trans-endothelial migration of monocytes was increased.Conclusion: The current study demonstrates that pro-inflammatory conditions result in differential expression of NGCs in endothelial cells and monocytes, both culprit cell types in atherosclerosis. Specifically, endothelial PLXNA4 is reduced upon inflammation, while PLXNA4 maintains endothelial barrier function thereby preventing vascular leakage of fluids as well as cells. Taken together, PLXNA4 may well have a causal role in atherogenesis that deserves further investigation. Show less
Objective:Brown fat activation attenuates atherosclerosis development by accelerating triglyceride-rich lipoprotein turnover and/or stimulation of reverse cholesterol transport via the SRB1 ... Show moreObjective:Brown fat activation attenuates atherosclerosis development by accelerating triglyceride-rich lipoprotein turnover and/or stimulation of reverse cholesterol transport via the SRB1 (scavenger receptor class B type 1). The aim of this study was to investigate the specific role of hepatic SRB1 in the atheroprotective properties of brown fat activation.Approach and Results:APOE*3-Leiden.CETP mice, a well-established model of human-like lipoprotein metabolism and atherosclerosis, were treated with vehicle or adenoassociated virus serotype 8-short hairpin RNA, which decreased hepatic SRB1 protein levels by 40% to 55%. After 2 weeks, mice without or with hepatic SRB1 knockdown were treated with vehicle or the beta 3-adrenergic receptor agonist CL316 243 to activate brown fat for 4 weeks to determine HDL (high-density lipoprotein) catabolism and for 9 weeks to evaluate atherosclerosis. Surprisingly, hepatic SRB1 knockdown additively improved the beneficial effects of beta 3-adrenergic receptor agonism on atherosclerosis development. In fact, hepatic SRB1 knockdown per se not only increased HDL-cholesterol levels but also reduced plasma triglyceride and non-HDL-cholesterol levels, thus explaining the reduction in atherosclerosis development. Mechanistic studies indicated that this is due to increased lipolytic processing and hepatic uptake of VLDL (very low density lipoprotein) by facilitating VLDL-surface transfer to HDL.Conclusions:Hepatic SRB1 knockdown in a mouse model with an intact ApoE (apolipoprotein E)-LDLR (low density lipoprotein receptor) clearance pathway, relevant to human lipoprotein metabolism, reduced atherosclerosis and improved the beneficial effect of brown fat activation on atherosclerosis development, explained by pleiotropic effects of hepatic SRB1 knockdown on lipolytic processing and hepatic uptake of VLDL. Brown fat activation could thus be an effective strategy to treat cardiovascular disease also in subjects with impaired SRB1 function. Show less
The vascular endothelium is a highly specialized barrier that controls passage of fluids and migration of cells from the lumen into the vessel wall. Endothelial cells assist leukocytes to... Show moreThe vascular endothelium is a highly specialized barrier that controls passage of fluids and migration of cells from the lumen into the vessel wall. Endothelial cells assist leukocytes to extravasate and despite the variety in the specific mechanisms utilized by different leukocytes to cross different vascular beds, there is a general principle of capture, rolling, slow rolling, arrest, crawling, and ultimately diapedesis via a paracellular or transcellular route. In atherosclerosis, the barrier function of the endothelium is impaired leading to uncontrolled leukocyte extravasation and vascular leakage. This is also observed in the neovessels that grow into the atherosclerotic plaque leading to intraplaque hemorrhage and plaque destabilization. This review focuses on the vascular endothelial barrier function and the interaction between endothelial cells and leukocytes during transmigration. We will discuss the role of endothelial dysfunction, transendothelial migration of leukocytes and plaque angiogenesis in atherosclerosis. Show less
