Genome-wide association meta-analysis identifies risk loci for abdominal aortic aneurysm and highlights PCSK9 as a therapeutic target.

Abdominal aortic aneurysm (AAA) is a common disease with substantial heritability. In this study, we performed a genome-wide association meta-analysis from 14 discovery cohorts and uncovered 141 independent associations, including 97 previously unreported loci. A polygenic risk score derived from meta-analysis explained AAA risk beyond clinical risk factors. Genes at AAA risk loci indicate involvement of lipid metabolism, vascular development and remodeling, extracellular matrix dysregulation and inflammation as key mechanisms in AAA pathogenesis. These genes also indicate overlap between the development of AAA and other monogenic aortopathies, particularly via transforming growth factor ß signaling. Motivated by the strong evidence for the role of lipid metabolism in AAA, we used Mendelian randomization to establish the central role of nonhigh-density lipoprotein cholesterol in AAA and identified the opportunity for repurposing of proprotein convertase, subtilisin/kexin-type 9 (PCSK9) inhibitors. This was supported by a study demonstrating that PCSK9 loss of function prevented the development of AAA in a preclinical mouse model.

Cationic proteins from eosinophils bind bone morphogenetic protein receptors promoting vascular calcification and atherogenesis.

AIMS: Blood eosinophil count and eosinophil cationic protein (ECP) concentration are risk factors of cardiovascular diseases. This study tested whether and how eosinophils and ECP contribute to vascular calcification and atherogenesis. METHODS AND RESULTS: Immunostaining revealed eosinophil accumulation in human and mouse atherosclerotic lesions. Eosinophil deficiency in ΔdblGATA mice slowed atherogenesis with increased lesion smooth muscle cell (SMC) content and reduced calcification. This protection in ΔdblGATA mice was muted when mice received donor eosinophils from wild-type (WT), Il4-/-, and Il13-/- mice or mouse eosinophil-associated-ribonuclease-1 (mEar1), a murine homologue of ECP. Eosinophils or mEar1 but not interleukin (IL) 4 or IL13 increased the calcification of SMC from WT mice but not those from Runt-related transcription factor-2 (Runx2) knockout mice. Immunoblot analyses showed that eosinophils and mEar1 activated Smad-1/5/8 but did not affect Smad-2/3 activation or expression of bone morphogenetic protein receptors (BMPR-1A/1B/2) or transforming growth factor (TGF)-ß receptors (TGFBR1/2) in SMC from WT and Runx2 knockout mice. Immunoprecipitation showed that mEar1 formed immune complexes with BMPR-1A/1B but not TGFBR1/2. Immunofluorescence double-staining, ligand binding, and Scatchard plot analysis demonstrated that mEar1 bound to BMPR-1A and BMPR-1B with similar affinity. Likewise, human ECP and eosinophil-derived neurotoxin (EDN) also bound to BMPR-1A/1B on human vascular SMC and promoted SMC osteogenic differentiation. In a cohort of 5864 men from the Danish Cardiovascular Screening trial and its subpopulation of 394 participants, blood eosinophil counts and ECP levels correlated with the calcification scores of different arterial segments from coronary arteries to iliac arteries. CONCLUSION: Eosinophils release cationic proteins that can promote SMC calcification and atherogenesis using the BMPR-1A/1B-Smad-1/5/8-Runx2 signalling pathway.

Artificial intelligence assisted compositional analyses of human abdominal aortic aneurysms ex vivo.

Quantification of histological information from excised human abdominal aortic aneurysm (AAA) specimens may provide essential information on the degree of infiltration of inflammatory cells in different regions of the AAA. Such information will support mechanistic insight in AAA pathology and can be linked to clinical measures for further development of AAA treatment regimens. We hypothesize that artificial intelligence can support high throughput analyses of histological sections of excised human AAA. We present an analysis framework based on supervised machine learning. We used TensorFlow and QuPath to determine the overall architecture of the AAA: thrombus, arterial wall, and adventitial loose connective tissue. Within the wall and adventitial zones, the content of collagen, elastin, and specific inflammatory cells was quantified. A deep neural network (DNN) was trained on manually annotated, Weigert stained, tissue sections (14 patients) and validated on images from two other patients. Finally, we applied the method on 95 new patient samples. The DNN was able to segment the sections according to the overall wall architecture with Jaccard coefficients after 65 epocs of 92% for the training and 88% for the validation data set, respectively. Precision and recall both reached 92%. The zone areas were highly variable between patients, as were the outputs on total cell count and elastin/collagen fiber content. The number of specific cells or stained area per zone was deterministically determined. However, combining the masks based on the Weigert stainings, with images of immunostained serial sections requires addition of landmark recognition to the analysis path. The combination of digital pathology, the DNN we developed, and landmark registration will provide a strong tool for future analyses of the histology of excised human AAA. In combination with biomechanical testing and microstructurally motivated mathematical models of AAA remodeling, the method has the potential to be a strong tool to provide mechanistic insight in the disease. In combination with each patients' demographic and clinical profile, the method can be an interesting tool to in supportof a better treatment regime for the patients.

Five-Year Outcomes of the Danish Cardiovascular Screening (DANCAVAS) Trial.

BACKGROUND: Limited data suggest a benefit of population-based screening for cardiovascular disease with respect to the risk of death. METHODS: We performed a population-based, parallel-group, randomized, controlled trial involving men 65 to 74 years of age living in 15 Danish municipalities. The participants were randomly assigned in a 1:2 ratio to undergo screening (the invited group) or not to undergo screening (the control group) for subclinical cardiovascular disease. Randomization was based on computer-generated random numbers and stratified according to municipality. Only the control group was unaware of the trial-group assignments. Screening included noncontrast electrocardiography-gated computed tomography to determine the coronary-artery calcium score and to detect aneurysms and atrial fibrillation, ankle-brachial blood-pressure measurements to detect peripheral artery disease and hypertension, and a blood sample to detect diabetes mellitus and hypercholesterolemia. The primary outcome was death from any cause. RESULTS: A total of 46,611 participants underwent randomization. After exclusion of 85 men who had died or emigrated before being invited to undergo screening, there were 16,736 men in the invited group and 29,790 men in the control group; 10,471 of the men in the invited group underwent screening (62.6%). In intention-to-treat analyses, after a median follow-up of 5.6 years, 2106 men (12.6%) in the invited group and 3915 men (13.1%) in the control group had died (hazard ratio, 0.95; 95% confidence interval [CI], 0.90 to 1.00; P = 0.06). The hazard ratio for stroke in the invited group, as compared with the control group, was 0.93 (95% CI, 0.86 to 0.99); for myocardial infarction, 0.91 (95% CI, 0.81 to 1.03); for aortic dissection, 0.95 (95% CI, 0.61 to 1.49); and for aortic rupture, 0.81 (95% CI, 0.49 to 1.35). There were no significant between-group differences in safety outcomes. CONCLUSIONS: After more than 5 years, the invitation to undergo comprehensive cardiovascular screening did not significantly reduce the incidence of death from any cause among men 65 to 74 years of age. (Funded by the Southern Region of Denmark and others; DANCAVAS ISRCTN Registry number, ISRCTN12157806.).

Cost effectiveness of population screening vs. no screening for cardiovascular disease: the Danish Cardiovascular Screening trial (DANCAVAS).

AIMS: A recent trial has shown that screening of men for cardiovascular disease (CVD) may reduce all-cause mortality. This study assesses the cost effectiveness of such screening vs. no screening from the perspective of European healthcare systems. METHODS AND RESULTS: Randomized controlled trial-based cost-effectiveness evaluation with a mean 5.7 years of follow-up. Screening was based on low-dose computed tomography to detect coronary artery calcification and aortic/iliac aneurysms, limb blood pressure measurement to detect peripheral artery disease and hypertension, telemetric assessment of the heart rhythm to detect atrial fibrillation, and measurements of the cholesterol and HgbA1c levels. Censoring-adjusted incremental costs, life years (LY), and quality-adjusted LY (QALY) were estimated and used for cost-effectiveness analysis. The incremental cost of screening for the entire health care sector was €207 [95% confidence interval (CI) -24; 438, P = 0.078] per invitee for which gains of 0.019 LY (95% CI -0.007; 0.045, P = 0.145) and 0.023 QALY (95% CI -0.001; 0.046, P = 0.051) were achieved. The corresponding incremental cost-effectiveness ratios were of €10 812 per LY and €9075 per QALY, which would be cost effective at probabilities of 0.73 and 0.83 for a willingness to pay of €20 000. Assessment of population heterogeneity showed that cost effectiveness could be more attractive for younger men without CVD at baseline. CONCLUSIONS: Comprehensive screening for CVD is overall cost effective at conventional thresholds for willingness to pay and also competitive to the cost effectiveness of common cancer screening programmes. The screening target group, however, needs to be settled.

Population-Based Risk Factors for Ascending, Arch, Descending, and Abdominal Aortic Dilations for 60-74-Year-Old Individuals.

BACKGROUND: Aortic dilations (ectasias and aneurysms) may occur on any segment of the aorta. Pathogenesis varies between locations, suggesting that etiology and risk factors may differ. Despite this discrepancy, guidelines recommend screening of the whole aorta if 1 segmental dilation is discovered. OBJECTIVES: The purpose of this study was to determine the most dominant predictors for dilations at the ascending, arch, descending, and abdominal part of the aorta, and to establish comprehensive risk factor profiles for each aortic segment. METHODS: Individuals aged 60-74 years were randomly selected to participate in DANCAVAS I+II (Danish Cardiovascular Multicenter Screening Trials). Participants underwent cardiovascular risk assessments, including blood samples, blood pressure readings, medical records, and noncontrast computed tomography scans. Adjusted odds ratios for potential risk factors of dilations were estimated by multivariate logistic analyses. RESULTS: The study population consisted of 14,989 participants (14,235 men, 754 women) with an average age of 68 ± 4 years. The highest adjusted odd ratios for having any aortic dilation were observed when coexisting aortic dilations were present. Other noteworthy predictors included coexisting iliac dilations, hypertension, increasing body surface area, male sex, familial disposition, and atrial fibrillation, which were present in various combinations for the different aortic parts. Smoking and acute myocardial infarction were inversely associated with ascending and abdominal dilations. Diabetes was a shared protective factor. CONCLUSIONS: Risk factors differ for aortic dilations between locations. The most dominant predictor for having a dilation at any aortic segment is the presence of an aortic dilation elsewhere. This supports current guidelines when recommending a full screening of the aorta if a focal aortic dilation is discovered.

Crosslinking of human plasma C-reactive protein to human serum albumin via disulfide bond oxidation.

Inter- and intra-molecular crosslinks can generate protein dysfunction, and are associated with protein aggregate accumulation in aged and diseased tissues. Crosslinks formed between multiple amino acid side chains can be reversible or irreversible. Disulfides formed either enzymatically, or as a result of oxidant-mediated reactions, are a major class of reversible crosslinks. Whilst these are commonly generated via oxidation of Cys thiol groups, they are also formed by 'oxidant-mediated thiol-disulfide reactions' via initial disulfide oxidation to a thiosulfinate or zwitterionic peroxide, and subsequent reaction with another thiol including those on other proteins. This generates new intermolecular protein-protein crosslinks. Here we demonstrate that photooxidation, or reaction with the biological oxidants HOCl and ONOOH, of the single disulfide present in the major human plasma inflammatory protein, C-reactive protein (CRP) can give rise to reversible disulfide bond formation with human serum albumin (HSA). This occurs in an oxidant dose-, or illumination-time-, dependent manner. These CRP-HSA crosslinks are formed both in isolated protein systems, and in fresh human plasma samples containing high, but not low, levels of CRP. The inter-protein crosslinks which involve Cys36 of CRP and Cys34 of HSA, have been detected by both immunoblotting and mass spectrometry (MS). The yield of protein-protein crosslinks depends on the nature and extent of oxidant exposure, and can be reversed by dithiothreitol and tris(2-carboxyethyl)phosphine hydrochloride. These data indicate that oxidation of disulfide bonds in proteins can be a source of novel inter-protein crosslinks, which may help rationalize the accumulation of crosslinked proteins in aged and diseased tissues.

Retinal Vascular Fractal Dimensions and Their Association with Macrovascular Cardiac Disease.

INTRODUCTION: As the only part of the human vasculature, the retina is available for direct, noninvasive inspection. Retinal vascular fractal dimension (DF) is a method to measure the structure of the retinal vascular tree, with higher noninteger values between 1 and 2 representing a more complex and dense retinal vasculature. Retinal vascular structure has been associated with a variety of systemic diseases, and this study examined the association of DF and macrovascular cardiac disease in a case-control design. METHODS: Retinal fundus photos were captured with Topcon TRC-50X in 38 persons that had coronary artery bypass grafting (CABG, cases) and 37 cardiovascular healthy controls. The semiautomatic software VAMPIRE was used to measure retinal DF. RESULTS: Patients with CABG had lower DF of the retinal main venular vessels compared to the control group (1.15 vs. 1.18, p = 0.01). In a multivariable regression model adjusted for gender and age, eyes in the fourth quartile with higher DF were less likely to have CABG compared to patients in the first (OR, 7.20; 95% confidence interval: 1.63-31.86; p = 0.009) and second (OR, 8.25; 95% confidence interval: 1.70-40.01; p = 0.009) quartiles. CONCLUSIONS: This study demonstrates that lower complexity of main venular vessels associates with higher risk of having CABG. The research supports the hypothesis that the retinal vascular structure can be used to assess nonocular macrovascular disease.

Oxidation of protein disulfide bonds by singlet oxygen gives rise to glutathionylated proteins.

Disulfide bonds play a key function in determining the structure of proteins, and are the most strongly conserved compositional feature across proteomes. They are particularly common in extracellular environments, such as the extracellular matrix and plasma, and in proteins that have structural (e.g. matrix) or binding functions (e.g. receptors). Recent data indicate that disulfides vary markedly with regard to their rate of reaction with two-electron oxidants (e.g. HOCl, ONOOH), with some species being rapidly and readily oxidized. These reactions yielding thiosulfinates that can react further with a thiol to give thiolated products (e.g. glutathionylated proteins with glutathione, GSH). Here we show that these 'oxidant-mediated thiol-disulfide exchange reactions' also occur during photo-oxidation reactions involving singlet oxygen (1O2). Reaction of protein disulfides with 1O2 (generated by multiple sensitizers in the presence of visible light and O2), yields reactive intermediates, probably zwitterionic peroxyl adducts or thiosulfinates. Subsequent exposure to GSH, at concentrations down to 2 µM, yields thiolated adducts which have been characterized by both immunoblotting and mass spectrometry. The yield of GSH adducts is enhanced in D2O buffers, and requires the presence of the disulfide bond. This glutathionylation can be diminished by non-enzymatic (e.g. tris-(2-carboxyethyl)phosphine) and enzymatic (glutaredoxin) reducing systems. Photo-oxidation of human plasma and subsequent incubation with GSH yields similar glutathionylated products with these formed primarily on serum albumin and immunoglobulin chains, demonstrating potential in vivo relevance. These reactions provide a novel pathway to the formation of glutathionylated proteins, which are widely recognized as key signaling molecules, via photo-oxidation reactions.

Eosinophils Protect Mice From Angiotensin-II Perfusion-Induced Abdominal Aortic Aneurysm.

RATIONALE: Blood eosinophil count and ECP (eosinophil cationic protein) associate with human cardiovascular diseases. Yet, whether eosinophils play a role in cardiovascular disease remains untested. The current study detected eosinophil accumulation in human and murine abdominal aortic aneurysm (AAA) lesions, suggesting eosinophil participation in this aortic disease. OBJECTIVE: To test whether and how eosinophils affect AAA growth. METHODS AND RESULTS: Population-based randomized clinically controlled screening trials revealed higher blood eosinophil count in 579 male patients with AAA than in 5063 non-AAA control (0.236±0.182 versus 0.211±0.154, 109/L, P<0.001). Univariate (odds ratio, 1.381, P<0.001) and multivariate (odds ratio, 1.237, P=0.031) logistic regression analyses indicated that increased blood eosinophil count in patients with AAA served as an independent risk factor of human AAA. Immunostaining and immunoblot analyses detected eosinophil accumulation and eosinophil cationic protein expression in human and murine AAA lesions. Results showed that eosinophil deficiency exacerbated AAA growth with increased lesion inflammatory cell contents, matrix-degrading protease activity, angiogenesis, cell proliferation and apoptosis, and smooth muscle cell loss using angiotensin-II perfusion-induced AAA in Apoe-/- and eosinophil-deficient Apoe-/-ΔdblGATA mice. Eosinophil deficiency increased lesion chemokine expression, muted lesion expression of IL (interleukin) 4 and eosinophil-associated-ribonuclease-1 (mEar1 [mouse EOS-associated-ribonuclease-1], human ECP homolog), and slanted M1 macrophage polarization. In cultured macrophages and monocytes, eosinophil-derived IL4 and mEar1 polarized M2 macrophages, suppressed CD11b+Ly6Chi monocytes, and increased CD11b+Ly6Clo monocytes. mEar1 treatment or adoptive transfer of eosinophil from wild-type and Il13-/- mice, but not eosinophil from Il4-/- mice, blocked AAA growth in Apoe-/-ΔdblGATA mice. Immunofluorescent staining and immunoblot analyses demonstrated a role for eosinophil IL4 and mEar1 in blocking NF-κB (nuclear factor-κB) activation in macrophages, smooth muscle cells, and endothelial cells. CONCLUSIONS: Eosinophils play a protective role in AAA by releasing IL4 and cationic proteins such as mEar1 to regulate macrophage and monocyte polarization and to block NF-κB activation in aortic inflammatory and vascular cells.

Eosinophils improve cardiac function after myocardial infarction.

Clinical studies reveal changes in blood eosinophil counts and eosinophil cationic proteins that may serve as risk factors for human coronary heart diseases. Here we report an increase of blood or heart eosinophil counts in humans and mice after myocardial infarction (MI), mostly in the infarct region. Genetic or inducible depletion of eosinophils exacerbates cardiac dysfunction, cell death, and fibrosis post-MI, with concurrent acute increase of heart and chronic increase of splenic neutrophils and monocytes. Mechanistic studies reveal roles of eosinophil IL4 and cationic protein mEar1 in blocking H2O2- and hypoxia-induced mouse and human cardiomyocyte death, TGF-ß-induced cardiac fibroblast Smad2/3 activation, and TNF-α-induced neutrophil adhesion on the heart endothelial cell monolayer. In vitro-cultured eosinophils from WT mice or recombinant mEar1 protein, but not eosinophils from IL4-deficient mice, effectively correct exacerbated cardiac dysfunctions in eosinophil-deficient ∆dblGATA mice. This study establishes a cardioprotective role of eosinophils in post-MI hearts.

Oxidant-induced glutathionylation at protein disulfide bonds.

Disulfide bonds are a key determinant of protein structure and function, and highly conserved across proteomes. They are particularly abundant in extracellular proteins, including those with critical structural, ligand binding or receptor function. We demonstrate that oxidation of protein disulfides induces polymerization, and results in oxygen incorporation into the former disulfide via thiosulfinate generation. These intermediates, which have half-lives of several hours in vitro, undergo secondary reactions that cleave the disulfide bond, by irreversible hydrolysis to sulfinic and sulfonic acids, or reaction with thiols in a process that yields thiolated proteins (e.g. glutathionylated species in the case of reaction with glutathione). The adducts have been characterized by mass spectrometry (as ions corresponding to the addition of 306 and 712 Da for addition of one and two glutathione molecules, respectively) and immunoblotting. These modifications can be induced by multiple biologically-important oxidants, including HOCl, ONOOH, and H2O2, and on multiple proteins, demonstrating that this is a common disulfide modification pathway. Addition of glutathione to give glutathionylated proteins, can be reversed by reducing systems (e.g. tris(2-carboxyethyl)phosphine), but this does not repair the original disulfide bond. Exposure of human plasma to these modifying agents increases protein glutathionylation, demonstrating potential in vivo relevance. Overall these data provide evidence for a novel and facile route to glutathionylated proteins involving initial oxidation of a disulfide to a thiosulfinate followed by rapid reaction with GSH ('oxidant-mediated thiol-disulfide exchange'). These data elucidate a novel pathway for protein glutathionylation that may have significant implications for redox biology and cell signaling.

Diabetes Is Not Associated with the Risk of Rupture Among Patients with Abdominal Aortic Aneurysms - Results From a Large Danish Register Based Matched Case Control Study From 1996 to 2016.

OBJECTIVE: Numerous studies have shown a paradoxical protective effect of diabetes on the development and progression of abdominal aortic aneurysm (AAA). The aim of this study was to investigate whether the protective role of diabetes on AAA extends to rupture, given the presence of an AAA. METHODS: This was a register based case control study. Patients with ruptured AAA (RAAA) were matched 1:1 with patients undergoing elective surgery for AAA by sex, age, and year of diagnosis. Multiple conditional logistic regression was performed to estimate the odds ratio (OR) associating a diagnosis of diabetes with RAAA. No protocol was registered. RESULTS: From 1996 to 2016, there were 6293 potential people with RAAA. A total of 898 people with a RAAA were excluded since no matching controls existed. This left 5 395 cases in the study. The cases had a median age of 75, and 85.4% were men. Diabetes was defined by hospital diagnosis or the redemption of antidiabetic prescriptions within one year. Comparing cases with controls and the presence of diabetes, a significant crude OR of 0.82 (95% confidence interval [CI] 0.71-0.95) was found. When adjusting for confounders OR increased to 0.97 (CI 0.83-1.14). Stratifying by age and year of diagnosis did not change the results markedly. OR associating RAAA with diabetes was significantly elevated in women (adjusted OR 1.82 [CI 1.17-2.81]). Of the 5395 cases, the overall 30 days mortality was 58% (n = 3145). Using Cox regression, a crude hazard ratio (HR) of 1.06 (CI 0.93-1.22) was found for the 30 day mortality and having diabetes compared with not having diabetes. Adjusting for index year, male sex, and age had little effect on this estimate (HR 1.11 [CI 0.97-1.28]). CONCLUSION: Diabetes was not found to protect against RAAA, given the presence of an AAA. Furthermore, diabetes did not increase the risk of dying within 30 days of RAAA.

Survival, Prevalence, Progression and Repair of Abdominal Aortic Aneurysms: Results from Three Randomised Controlled Screening Trials Over Three Decades.

AIM: The prevalence and mortality of abdominal aortic aneurysms (AAA) has been reported to decline. The aim of this study is to compare survival, prevalence, and repair rate of AAA in Denmark in the 1990s, the 2000s and the 2010s - and to examine any change in factors known to influence the prevalence. METHODS: Baseline status and up to 5-year outcomes of 34,079 general population men aged 65-74 were obtained from three RCTs; the Viborg study (1994-1998, n=4,860), the Viborg Vascular (VIVA) trial (2008-2011, n=18,748), and the Danish Cardiovascular (DANCAVAS) trial (2015-2018, n=10,471). After the millennium (VIVA and DANCAVAS) men with AAA were further offered low dose aspirin and statins. Follow-up data were not available for the DANCAVAS trial yet. RESULTS: Across the three decades, the AAA prevalence was 3.8% (Reference), 3.3% (p<0.001) and 4.2% (p=0.882), the proportion of smokers were 62%, 42% and 34% (p<0.001) amongst men with AAA, but AAA risk associations with smoking increased during the decades suggesting increased tobacco consumption of smokers. In addition, the proportions of attenders with ischemic heart disease or stroke increased significantly. The aneurysmal progression rate in the 1990s was 2.90 vs 2.98 mm/year in the 2000s (p=0.91). The need for preventive AAA repair increased insignificantly in the 2000s (Age adj. HR= 1.29, 95% C.I.: 0.95; 1.71, p=0.10), and mortality of men with screen-detected AAA was lower in the 2000s compared to the 1990s (Age-adj. HR= 0.28, 95% C.I.: 0.22; 0.36, p<0.001). CONCLUSION: The Danish prevalence of AAA today compares to the nineties. Unchanged aneurysmal progression rates combined with improved survival of men at risk of AAA leave them in longer time to develop an AAA, be diagnosed and to need later aneurysmal repair or experience rupture. CLINICAL TRIAL REGISTRATIONS: Viborg study: No possibility of registration in the nineties. VIVA: NCT00662480, URL: https://clinicaltrials.gov/show/NCT00662480, DANCAVAS: ISRCTN12157806, URL: http://www.isrctn.com/ISRCTN12157806.

Platelet aggregation is not altered among men with diabetes mellitus.

AIMS: Platelets are pivotal in arterial thrombosis, and platelet hyperresponsiveness may contribute to the increased incidence of cardiovascular events in diabetes mellitus. Consequently, we hypothesized that increased in vitro platelet aggregation responses exist in men with diabetes mellitus. METHODS: The Danish Cardiovascular Screening Trial (DANCAVAS) is a community-based cardiovascular screening trial including men aged 65-74 years. Platelet aggregation was tested using 96-well light transmission aggregometry with thrombin receptor-activating peptide (TRAP), adenosine diphosphate, collagen type 1, arachidonic acid and protease-activated receptor-4 in three concentrations. Further, cardiovascular risk factors and coronary artery calcification (CAC), estimated by CT scans and ankle-brachial index, were obtained. RESULTS: Included were 720 men aged 65-74 years, 110 with diabetes mellitus. Overall, there was no difference in platelet aggregation among men with versus without diabetes mellitus when adjusting for or excluding platelet inhibitor treatment and men with established cardiovascular disease (CVD). This was true for all agonists, e.g., 10 µM TRAP-induced platelet aggregation of median 69% (IQR 53-75) versus 70% (IQR 60-76) in men with versus without diabetes mellitus. Platelet aggregation did not correlate with HbA1c or CAC. Men with diabetes mellitus displayed higher CAC, median 257 Agatston units (IQR 74-1141) versus median 111 Agatston units (IQR 6-420) in the remaining individuals, p < 0.0001. CONCLUSIONS: Among outpatients with diabetes mellitus, but no CVD and no platelet inhibitor treatment, neither are platelets hyperresponsive in diabetes mellitus, nor is platelet aggregation associated with glycemic status or with the degree of coronary atherosclerosis. TRIAL REGISTRATION: ISRCTN12157806.

Periarterial fat from two human vascular beds is not a source of aldosterone to promote vasoconstriction.

Mouse adipocytes have been reported to release aldosterone and reduce endothelium-dependent relaxation. It is unknown whether perivascular adipose tissue (PVAT) releases aldosterone in humans. The present experiments were designed to test the hypothesis that human PVAT releases aldosterone and induces endothelial dysfunction. Vascular reactivity was assessed in human internal mammary and renal segmental arteries obtained at surgery. The arteries were prepared with/without PVAT, and changes in isometric tension were measured in response to the vasoconstrictor thromboxane prostanoid receptor agonist U46619 and the endothelium-dependent vasodilator acetylcholine. The effects of exogenous aldosterone and of mineralocorticoid receptor (MR) antagonist eplerenone were determined. Aldosterone concentrations were measured by ELISA in conditioned media incubated with human adipose tissue with/without angiotensin II stimulation. Presence of aldosterone synthase and MR mRNA was examined in perirenal, abdominal, and mammary PVAT by PCR. U46619 -induced tension and acetylcholine-induced relaxation were unaffected by exogenous and endogenous aldosterone (addition of aldosterone and MR blocker) in mammary and renal segmental arteries, both in the presence and absence of PVAT. Aldosterone release from incubated perivascular fat was not detectable. Aldosterone synthase expression was not consistently observed in human adipose tissues in contrast to that of MR. Thus, exogenous aldosterone does not affect vascular reactivity and endothelial function in ex vivo human arterial segments, and the tested human adipose tissues have no capacity to synthesize/release aldosterone. In perspective, physiologically relevant effects of aldosterone on vascular function in humans are caused by systemic aldosterone originating from the adrenal gland.

Proteomic Discovery and Validation of the Confounding Effect of Heparin Administration on the Analysis of Candidate Cardiovascular Biomarkers.

BACKGROUND: Several plasma proteins have been suggested as markers for a variety of cardiovascular conditions but fail to qualify in independent patient cohorts. This may relate to interference of medication on plasma protein concentrations. We used proteomics to identify plasma proteins that changed in concentration with heparin administration and therefore potentially may confound their evaluation as biomarkers in situations in which heparin is used. METHODS: We used a proteomic approach based on isobaric tagging and nano-LC-MS/MS analysis to quantify several hundred proteins in a discovery study in which individual plasma samples from 9 patients at intravascular ultrasound follow-up 12 months after an acute myocardial infarction before heparin administration and 2, 15, and 60 min after heparin administration; we validated our findings in 500 individual plasma samples obtained at admission from patients with suspected ST segment elevation myocardial infarction (STEMI), of whom 363 were treated with heparin before admission. RESULTS: In the discovery study, 25 of 653 identified plasma proteins displayed a changed concentration after heparin administration (Bonferroni-corrected P value at P < 7.66 × 10-5). Fourteen of the proteins changed significantly among heparin-treated patients in the validation study (nominal significance level of P < 6.92 × 10-5). Among heparin-affected proteins in both the discovery study and the validation study were midkine, spondin 1, secreted frizzled-like protein 1, lipoprotein lipase, and follistatin, all previously associated with STEMI. CONCLUSIONS: Medications such as heparin administration given before blood sampling may confound biomarker discovery and should be carefully considered in such studies.

External validity of a cardiovascular screening including a coronary artery calcium examination in middle-aged individuals from the general population.

Background Coronary artery calcium is important in cardiovascular risk stratification, but this knowledge is based on studies with a significant selection bias. This study aims to evaluate the external validity of a screening programme including coronary artery calcium examination, and the association between coronary artery calcium and cardiovascular events. Design Multi-centre population based study. Methods Randomly selected middle-aged men and women ( N = 1751) free of cardiovascular disease were invited to the examination during 2009-2010. Participation rate in the examination was 70%. Participants ( n = 1227) and non-participants ( n = 524) were compared regarding: cardiovascular medical treatment, Charlson comorbidity index and socioeconomic status (evaluated by cohabitation, gross income and education). Study endpoints were cardiovascular events and mortality. Results Non-participants had a significant higher comorbidity ( p = 0.003) and a lower socioeconomic status ( p < 0.0001), while cardiovascular medical treatment was alike. Over a median follow-up time of 6.5 years the cardiovascular event and mortality rates were equal (6.7% vs. 6.4%, p = 0.80 and 0.4% vs. 0.5%, p = 0.76, respectively). Adjusted hazard ratio was 0.90 (95% confidence interval (CI) 0.63-1.37). Among participants, the extent of coronary artery calcium was significantly associated with increased risk of cardiovascular events (hazard ratio 1.92, 95% CI 1.03-3.54, hazard ratio 3.66, 95% CI 1.82-7.32, hazard ratio 6.51, 95% CI 3.17-13.36 for coronary artery calcium scores 1-99, 100-399, ≥400 AU, respectively). Conclusions Non-participants had a higher comorbidity index and a lower socioeconomic status, but the cardiovascular event and mortality rates were equal to those of participants. Thus, a screening programme including a coronary artery calcium examination had a high external validity regarding cardiovascular risk, but also a significant social imbalance.