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Abdominal aortic aneurysm (AAA) is a complex disease which is incompletely accounted for. Basement membrane (BM) Collagen IV (COL4A1/A2) is abundant in the artery wall, and several lines of evidence indicate a protective role of baseline COL4A1/A2 in AAA development. Using Col4a1/a2 hemizygous knockout mice (Col4a1/a2+/−, 129Svj background) we show that partial Col4a1/a2 deficiency augmented AAA formation. Although unchallenged aortas were morphometrically and biomechanically unaffected by genotype, explorative proteomic analyses of aortas revealed a clear reduction in BM components and contractile vascular smooth muscle cell (VSMC) proteins, suggesting a central effect of the BM in maintaining VSMCs in the contractile phenotype. These findings were translated to human arteries by showing that COL4A1/A2 correlated to BM proteins and VSMC markers in non-lesioned internal mammary arteries obtained from coronary artery bypass procedures. Moreover, in human AAA tissue, MYH11 (VSMC marker) was depleted in areas of reduced COL4 as assessed by immunohistochemistry. Finally, circulating COL4A1 degradation fragments correlated with AAA progression in the largest Danish AAA cohort, suggesting COL4A1/A2 proteolysis to be an important feature of AAA formation. In sum, we identify COL4A1/A2 as a critical regulator of VSMC phenotype and a protective factor in AAA formation.

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.

Introduction Infective endocarditis (IE) has high mortality, partly due to delayed diagnosis. No biomarker can identify IE in patients with fever and clinical picture of infection. To find putative biomarkers we analyzed serum levels of two proteins found in cardiac valves, fibulin-1 (n = 696) and osteoprotegerin (n = 689) among patients on clinical suspicion of IE. Proteomic analyses were performed in 24 patients with bacteremia, 12 patients with definite IE and 12 patients with excluded IE. Methods Fibulin-1 and osteoprotegerin were studied by enzyme linked immunosorbent assay (ELISA). Proteomic analyses were conducted by 2-dimensional polyacrylamid gel electrophoresis (2D-PAGE) and label-free quantitative liquid chromatography - tandem mass spectrometry (LFQ LC-MS/MS). Controls for 2D 2D-PAGE and LFQ LC-MS/MS had bacteremia and excluded IE. Results Osteoprotegerin levels were significantly increased in IE patients compared with non-IE patients. Fibulin-1 showed no difference. 2D-PAGE showed significant differences of 6 proteoforms: haptoglobin, haptoglobin-related protein, α-2-macroglobulin, apolipoprotein A-I and ficolin-3. LFQ LC-MS/MS analysis revealed significant level changes of 7 proteins: apolipoprotein L1, complement C1q subcomponent B and C, leukocyte immunoglobulin-like receptor subfamily A member 3, neuropilin-2, multimerin-1 and adiponectin. Conclusions The concentration changes in a set of proteoforms/proteins suggest that stress and inflammation responses are perturbed in patients with IE compared to patients with bacteremia without IE.

Aims The aims of this study were to investigate the correlation and sex differences between total valve calcium, valve calcium concentration, and aortic valve calcification (AVC) in explanted valves from patients with severe aortic valve stenosis undergoing aortic valve replacement (AVR). Methods and results Sixty-nine patients with severe aortic stenosis (AS) scheduled for elective AVR underwent echocardiography and cardiac computed tomography (CT) prior to surgery (AVCin vivo) and CT of the explanted aortic valve (AVCex vivo). Explanted valves were prepared in acid solution, sonicated, and analysed with Arsenazo III dye to estimate total valve calcium and valve calcium concentration. Median AVCex vivo was 2082 (1421–2973) AU; mean valve calcium concentration was 1.43 ± 0.42 µmol Ca2+/mg tissue; median total valve calcium 156 (111–255) mg Ca2+, and valve calcium density 52 (35–81) mg/cm2. AVC displayed a strong correlation with total valve calcium (R2 = 0.98, P Conclusion AVC score provides a strong estimate for total valve calcium but to a lesser degree calcium concentration in the valve tissue of patients with severe AS. Females presented lower valvular calcium concentrations than males irrespective of AS severity, adding evidence and providing support to the important point that sex differences in valvular calcium concentration in AS does not reflect valvular size.

Purpose Cardiovascular mortality remains high among patients with diabetes compared with the general population. The primary aim was to evaluate the interest in and demand for advanced cardiovascular screening in patients with diabetes; the secondary aim was to explore its efficiency in detecting unprotected subclinical cardiovascular disease (CVD). Patients and methods In a cross-sectional design, randomly selected 40-60-year-old men and women with diabetes were invited to the screening trial. Screening encompassed (1) a comprehensive medical interview; (2) non-contrast computed tomography scanning to quantify coronary artery and aortic valve calcification, to measure left atrial size, to assess heart rhythm and to detect aortic and iliac dilatations; (3) ankle and brachial blood pressure measurements; and (4) blood and urine samples for measurements of HbA1c, lipid profile, renal function, NT-pro B-type natriuretic peptide (pro-BNP) and albuminuria. Primary outcome was participation rate; secondary outcome was rate of unprotected subclinical CVD. Results Of 465 invited patients, 191 (41.1%) attended screening. The participation rate was 40% (95% CI:33-47) for males and 42% (95% CI:36-48) for females. Twenty-four patients were excluded due to previous CVD. The remaining patients' mean age was 52 years; 58% were males. Subclinical CVD was found in 64%, with a male preponderance (males 75% (95% CI:66-83; females 49% (95% CI:37-60)). Presence of severe coronary artery calcification (score ≥ 400) showed a male preponderance (males 19% (95% CI:12-27); females 7% (95% CI:3-16)). Aortic valve calcification, enlarged left atrial volume, atrial fibrillation, aortic dilatations, peripheral artery disease or increased pro-BNP were uncommon, and without any sex differences. Unprotected subclinical CVD was very common, and medical treatment was intensified in 60% (95% CI:53-68) of patients. Conclusion We propose a feasible cardiovascular screening examination from which middle-aged patients with diabetes may benefit. However, the participation rate may be too low to warrant screening.

Background Basement membrane (BM) accumulation is a hallmark of micro-vessel disease in diabetes mellitus (DM). We previously reported marked upregulation of BM components in internal thoracic arteries (ITAs) from type 2 DM (T2DM) patients by mass spectrometry. Here, we first sought to determine if BM accumulation is a common feature of different arteries in T2DM, and second, to identify other effects of T2DM on the arterial proteome. Methods Human arterial samples collected during heart and vascular surgery from well-characterized patients and stored in the Odense Artery Biobank were analysed by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). We included ascending thoracic aortas (ATA) (n = 10 (type 2 DM, T2DM) and n = 10 (non-DM)); laser capture micro-dissected plaque- and media compartments from carotid plaques (n = 10 (T2DM) and n = 9 (non-DM)); and media- and adventitia compartments from ITAs (n = 9 (T2DM) and n = 7 (non-DM)). Results We first extended our previous finding of BM accumulation in arteries from T2DM patients, as 7 of 12 pre-defined BM proteins were significantly upregulated in bulk ATAs consisting of > 90% media. Although less pronounced, BM components tended to be upregulated in the media of ITAs from T2DM patients, but not in the neighbouring adventitia. Overall, we did not detect effects on BM proteins in carotid plaques or in the plaque-associated media. Instead, complement factors, an RNA-binding protein and fibrinogens appeared to be regulated in these tissues from T2DM patients. Conclusion Our results suggest that accumulation of BM proteins is a general phenomenon in the medial layer of non-atherosclerotic arteries in patients with T2DM. Moreover, we identify additional T2DM-associated effects on the arterial proteome, which requires validation in future studies.

Jes S Lindholt, 1–3 Axel C Diederichsen, 2, 4 Lars M Rasmussen, 2, 5 Lars Frost, 6, 7 Flemming H Steffensen, 8 Jess Lambrechtsen, 8 Grazina Urbonaviciene, 6, 7 Martin Busk, 9 Kenneth Egstrup, 8 Katrine L Kristensen, 1–3 Carsten Behr Andersen, 3 Rikke Søgaard 10 1Department of Cardiothoracic and Vascular Surgery, Odense University Hospital, Odense, Denmark; 2Elitary Centre for Individualized Medicine in Arterial Diseases (CIMA), Odense, Denmark; 3Department of Vascular Surgery, Viborg Hospital, Viborg, Denmark; 4Department of Cardiology, Odense University Hospital, Odense, Denmark; 5Department of Clinical Biochemistry and Pharmacology, Odense University Hospital, Odense, Denmark; 6Department of Cardiology, Diagnostic Centre, Regional Hospital Silkeborg, Silkeborg, Denmark; 7Department of Clinical Medicine, Aarhus University, Aarhus, Denmark; 8Department of Cardiology, Vejle Hospital, Vejle, Denmark; 9Department of Cardiology, University Hospital Odense Svendborg, Svendborg, Denmark; 10Department of Public Health and Department of Clinical Medicine, Aarhus University, Aarhus, DenmarkCorrespondence: Jes S LindholtDepartment of Cardiothoracic and Vascular Surgery, Odense University Hospital, J.B. Winsløv Vej 4, Odense 5000, DenmarkTel +45 2464 1214Email Jes.sanddal.lindholt@rsyd.dkAim: 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.Keywords: abdominal aortic aneurysms, screening, prevalence, survival, progression, smoking

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.

Background The challenge of managing age-related diseases is increasing; routine checks by the general practitioner do not reduce cardiovascular mortality. The aim here was to reduce cardiovascular mortality by advanced population-based cardiovascular screening. The present article reports the organization of the study, the acceptability of the screening offer, and the relevance of multifaceted screening for prevention and management of cardiovascular disease. Methods Danish men aged 65–74 years were invited randomly (1 : 2) to a cardiovascular screening examination using low-dose non-contrast CT, ankle and brachial BP measurements, and blood tests. Results In all, 16 768 of 47 322 men aged 65–74 years were invited and 10 471 attended (uptake 62·4 per cent). Of these, 3481 (33·2 per cent) had a coronary artery calcium score above 400 units. Thoracic aortic aneurysm was diagnosed in the ascending aorta (diameter 45 mm or greater) in 468 men (4·5 per cent), in the arch (at least 40 mm) in 48 (0·5 per cent) and in the descending aorta (35 mm or more) in 233 (2·2 per cent). Abdominal aortic aneurysm (at least 30 mm) and iliac aneurysm (20 mm or greater) were diagnosed in 533 (5·1 per cent) and 239 (2·3 per cent) men respectively. Peripheral artery disease was diagnosed in 1147 men (11·0 per cent), potentially uncontrolled hypertension (at least 160/100 mmHg) in 835 (8·0 per cent), previously unknown atrial fibrillation confirmed by ECG in 50 (0·5 per cent), previously unknown diabetes mellitus in 180 (1·7 per cent) and isolated severe hyperlipidaemia in 48 men (0·5 per cent). In all, 4387 men (41·9 per cent), excluding those with potentially uncontrolled hypertension, were referred for additional cardiovascular prevention. Of these, 3712 (35·5 per cent of all screened men, but 84·6 per cent of those referred) consented and were started on medication. Conclusion Multifaceted cardiovascular screening is feasible and may optimize cardiovascular disease prevention in men aged 65–74 years. Uptake is lower than in aortic aneurysm screening.

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.

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, 10 9 /L, P P 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 + Ly6C hi monocytes, and increased CD11b + Ly6C lo 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.

Objective Pregnancy-associated plasma protein-A (PAPP-A) has been suggested as a proatherogenic enzyme by its ability to locally increase insulin-like growth factor (IGF) activity through proteolytic cleavage of IGF binding protein-4 (IGFBP-4). Recently, stanniocalcin-2 (STC2) was discovered as an inhibitor of PAPP-A. This study aimed to investigate IGFBP-4, PAPP-A, and STC2 as local regulators of IGF bioactivity in the cardiac microenvironment by comparing levels in the pericardial fluid with those in the circulation of patients with cardiovascular disease. Methods Plasma and pericardial fluid were obtained from 39 patients undergoing elective cardiothoracic surgery, hereof 15 patients with type 2 diabetes. Concentrations of IGF-I, intact and fragmented IGFBP-4, PAPP-A, and STC2 were determined by immunoassays and IGF bioactivity by a cell-based assay. Results In pericardial fluid, the concentrations of total IGF-I, intact IGFBP-4, and STC2 were 72 ± 10%, 91 ± 5%, and 40 ± 24% lower than in plasma, while PAPP-A was 15 times more concentrated. The levels of the 2 IGFBP-4 fragments generated by PAPP-A and reflecting PAPP-A activity were elevated by more than 25%. IGF bioactivity was 62 ± 81% higher in the pericardial fluid than plasma. Moreover, pericardial fluid levels of both IGFBP-4 fragments correlated with the concentration of PAPP-A and with the bioactivity of IGF. All protein levels were similar in pericardial fluid from nondiabetic and diabetic subjects. Conclusions PAPP-A increases IGF bioactivity by cleavage of IGFBP-4 in the pericardial cavity of cardiovascular disease patients. This study provides evidence for a distinct local activity of the IGF system, which may promote cardiac dysfunction and coronary atherosclerosis.

Background The recently updated pre-test probability (PTP) model for diagnosing chronic coronary syndrome suggested by the European Society of Cardiology (ESC) was designed to predict the presence of obstructive coronary artery disease (CAD). In addition to this model, identification of non-obstructive CAD and utilization of preventive interventions may also lower rates of death and non-fatal myocardial infarction. Opposite to the ESC PTP, the minimal risk tool (MRT) is a new model developed to identify individuals without CAD but symptoms suggestive of CAD. We explored a combined use of the 2 models to predict the absence or presence of obstructive CAD. Methods This was a sub-study of the Danish study of Non-Invasive testing in Coronary Artery Disease (Dan-NICAD) which included patients with low-intermediate PTP of CAD. Minimal risk was defined as having a coronary calcium score of 0, no evidence of coronary atherosclerosis at coronary computed tomography angiography, and no cardiovascular (CV) events defined as myocardial infarction, death or revascularization in the mean observation period of 3.1 [2.7–3.4] years. Obstructive CAD was defined as a fractional flow reserve 90% lumen reduction). The risk factors included in the MRT were age, sex, smoking history, diabetes mellitus, dyslipidaemia, family history of premature CAD, hypertension, symptoms related to stress, and high-density lipoprotein concentration. Based on a point-system ranging from 0–5, the MRT and the ESC PTP were combined (dual-PTP) (figure 1). A dual-PTP ≤1 indicated very low risk. Using both minimal risk and obstructive CAD as references, the dual PTP was compared to the MRT and the ESC PTP through tests of model discrimination. Results Of the 1544 eligible patients, 710 (46%) had normal coronary arteries and no CV events. Obstructive CAD was diagnosed in 152 (10%). Equivalent to a dual-PTP 50% or ESC PTP 5–15% and MRT >75% were classified as very low risk. Of these patients, 84% were at true minimal risk (red area figure 1). Furthermore, only 6 patients would have been diagnosed with obstructive CAD at ICA, and 0 events would be missed. The dual-PTP was non-inferior to the MRT and the ESC PTP in identifying patients having minimal risk and obstructive CAD, respectively (minimal risk: c-statistics 0.74 (0.72–0.77) vs. 0.76 (0.73–0.78); obstructive CAD: c-statistics 0.66 (0.62–0.70) vs. (0.67 (0.63–0.72)). The dual-PTP was superior to the ESC PTP in discriminating patients at minimal risk (c-statistics 0.74 (0.72–0.77) vs. 0.69 (0.67–0.71). Conclusions Combining the ESC PTP and the MRT, the dual-PTP seems to enable accurate prediction of both patients with minimal risk and patients with obstructive CAD. Based on the dual-PTP, patients can safely be deferred from or referred for diagnostic testing Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): Aarhus University, Health Research Fund of Central Denmark Region

Background and aims: Hyperlipidemia is a suggested risk factor for abdominal aortic aneurysm (AAA). However, whether hyperlipidemia is causally involved in AAA progression remains elusive. Here, we tested the hypothesis that hyperlipidemia aggravates AAA formation in the widely used porcine pancreatic elastase (PPE) model of AAA in mice with varying levels of plasma lipids. Methods: Prior to PPE-surgery, 8-week-old male C57BL/6J mice (n = 32) received 1·1011 viral genomes of rAAV8-D377Y-mPcsk9 or control rAAV8 via the tail vein. Mice were fed either western type diet or regular chow. At baseline and during the 28 days following PPE-surgery, mice underwent weekly ultrasonic assessment of AAA progression. Experiments were repeated using Apolipoprotein E knockout (ApoE−/−) mice (n = 7) and wildtype C57BL/6J mice (n = 5). Results: At sacrifice, maximal intergroup plasma cholesterol and non-HDL/HDL ratio differences were >5-fold and >20-fold, respectively. AAA diameters expanded to 150% of baseline, but no intergroup differences were detected. This was verified in an independent experiment comparing 8-week-old male ApoE−/− mice with wildtype mice. Histological evaluation of experimental AAA lesions revealed accumulated lipid in neointimal and medial layers, and analysis of human AAA lesions (n = 5) obtained from open repair showed medial lipid deposition. Conclusions: In summary, we find that lipid deposition in the aortic wall is a feature of PPE-induced AAA in mice as well as human AAA lesions. Despite, our data do not support the hypothesis that hyperlipidemia contributes to AAA progression.

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.

AIM: Acute myocardial infarction (AMI) remains a major cause of mortality and morbidity, and cardiogenic shock (CS) a major cause of hospital mortality after AMI. Especially for ST elevation myocardial infarction (STEMI) patients, fast intervention is essential.Few proteins have proven clinically applicable for AMI. Most proposed biomarkers are based on a priori hypothesis-driven studies of single proteins, not enabling identification of novel candidates. For clinical use, the ability to predict AMI is important; however, studies of proteins in prediction models are surprisingly scarce.Consequently, we applied proteome data for identifying proteins associated with definitive STEMI, CS, and all-cause mortality after admission, and examined the ability of the proteins to predict these outcomes. METHODS AND RESULTS: Proteome-wide data of 497 patients with suspected STEMI were investigated; 381 patients were diagnosed with STEMI, 35 with CS, and 51 died during the first year. Data analysis was conducted by logistic and Cox regression modeling for association analysis, and by multivariable LASSO regression models for prediction modeling.Association studies identified 4 and 29 proteins associated with definitive STEMI or mortality, respectively. Prediction models for CS and mortality (holding two and five proteins, respectively) improved the prediction ability as compared with protein-free prediction models; AUC of 0.92 and 0.89, respectively. CONCLUSION: The association analyses propose individual proteins as putative protein biomarkers for definitive STEMI and survival after suspected STEMI, while the prediction models put forward sets of proteins with putative predicting ability of CS and survival. These proteins may be verified as biomarkers of potential clinical relevance.

Aims Vitamin K antagonists (VKAs) are suspected of causing aortic valve calcification (AVC). The objective of this study was to clarify whether patients undergoing VKA treatment have increased AVC scores compared to patients treated with new oral anticoagulants (NOACs) and patients who never have been treated with VKA/NOAC. Methods and results We included participants from the population-based DANCAVAS trial (n = 15 048). Information on confounders was collected, and the AVC scores were measured on non-contrast computed tomography scans. The participants’ medication data, including VKA and NOAC data, were collected from the Danish National Health Service Prescription Database. The final population consisted of 14 604 participants (67.4 years, 95% men) of whom 873 had been treated with VKA and 602 with NOAC. The association between AVC score and duration of anticoagulant use was investigated in an adjusted zero-inflated negative binomial regression model. For every year treated with VKA, the AVC score increased, on average, by 6% [ratio of expected counts (RECs) = 1.06; 95% confidence interval (CI) 1.02–1.10] compared to non-use. The results were consistent in sensitivity analyses excluding patients with known cardiovascular disease and statin users (REC = 1.07; 95% CI 1.02–1.11 and REC = 1.10; 95% CI 1.03–1.17, respectively). NOAC treatment was not significantly associated with AVC score in any of the corresponding models (REC = 1.03, 1.02, and 0.96). Conclusion Compared to no treatment with anticoagulants, VKA use was associated with increased AVC score, while a similar association could not be established for NOAC.

Introduction: The knowledge of the etiology and associated mortality of undifferentiated shock in the emergency department (ED) is limited. We aimed to describe the etiology-based proportions and incidence rates (IR) of shock, as well as the associated mortality in the ED. Methods: Population-based cohort study at a University Hospital ED in Denmark from January 1, 2000, to December 31, 2011. Patients aged ≥18 years living in the ED-catchment area (N = 225,000) with a first-time ED presentation with shock (n = 1,553) defined as hypotension (systolic blood pressure ≤100 mm Hg) and ≥1 organ failures were included. Discharge diagnoses defined the etiology and were grouped as follows: distributive septic shock (SS), distributive non-septic shock (NS), cardiogenic shock (CS), hypovolemic shock (HS), obstructive shock (OS), and other conditions (OC). Outcomes were etiology-based characteristics, annual IR per 100,000 person-years at risk (95% confidence intervals [CIs]), mortality at 0 to 7-, and 0 to 90 days (95% CIs) and hazard rates (HR) at 0 to 7, 8 to 90 days (95% CIs). Poisson and Cox regression models were used for analyses. Results: Among 1,553 shock patients: 423 (27.2%) had SS, 363 (23.4%) NS, 217 (14.0%) CS, 479 (30.8%) HS, 14 (0.9%) OS, and 57 (3.7%) OC. The corresponding IRs were 16.2/100,000 (95% CI: 14.8–17.9), 13.9/100,000 (95% CI: 12.6–15.4), 8.3/100,000 (95% CI: 7.3–9.5), 18.4/100,000 (95% CI: 16.8–20.1), 0.5/100,000 (95% CI: 0.3–0.9), and 2.2/100,000 (95% CI: 1.7–2.8). SS IR increased from 8.4 to 28.5/100,000 during the period 2000 to 2011. Accordingly, the 7-, and 90-day mortalities of SS, NS, CS, and HS were 30.3% (95% CI: 25.9–34.7) and 56.2% (95% CI: 50.7–61.5), 12.7% (95% CI: 9.2–16.1) and 22.6% (95% CI: 18.1–27.7), 34.6% (95% CI: 28.2–40.9) and 52.3% (95% CI: 44.6–59.8), 19.2% (95% CI: 15.7–22.7), and 36.8% (95% CI: 33.3–43.3). SS (HR = 1.46 [95% CI: 1.03–2.07]), and CS (HR = 2.15 [95% CI: 1.47–3.13]) were independent predictors of death within 0 to 7 days, whereas SS was a predictor within 8 to 90 days (HR = 1.66 [95% CI: 1.14–2.42]). Conclusion: HS and SS are frequent etiological characteristics followed by NS and CS, whereas OS is a rare condition. We confirm the increasing trend of SS, as previously reported. Seven-day mortality ranged from 12.7% to 34.6%, while 90-day mortality ranged from 22.6% to 56.2%. The underlying etiology was an independent predictor of mortality.

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-β-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., Blood eosinophil (EOS) counts may serve as risk factors for human coronary heart diseases. Here the authors show that increased circulating and myocardial EOS after myocardial infarction play a cardioprotective role by reducing cardiomyocyte death, cardiac fibroblast activation and fibrosis, and endothelium activation-mediated inflammatory cell accumulation.

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.

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 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.

Background Type 2 diabetes mellitus (T2DM) is an independent risk factor of cardiovascular disease (CVD), however, the underlying mechanisms are largely unknown. Using non-atherosclerotic internal thoracic arteries (ITAs) obtained from coronary artery bypass grafting, we previously identified a distinct elevation in the level of proteins comprising the arterial basement membrane in T2DM patients not treated with metformin. Altered transcription of genes encoding these proteins has not been observed, indicating alternative mechanisms of dysregulation. Methods In this study we screened for differential expression of arterial microRNAs (miRNAs) in T2DM patients to test the hypothesis that the arterial protein signature of diabetic patients is associated with dysregulation at the miRNA level, and further to lay the foundation for novel hypotheses addressing the increased CVD risk of T2DM patients. MiRNA isolated from fresh frozen ITAs [from 18 T2DM- (10 of which were subject to metformin treatment) and 30 non-diabetes mellitus (non-DM) patients] were analyzed by microarray, and miRNAs isolated from formalin-fixated paraffin-embedded (FFPE) ITAs were analyzed by quantitative PCR (qPCR) in an independent study group [26 T2DM- (15 of which were subject to metformin treatment) and 26 non-DM patients] to determine expression levels of miRNAs in a pre-defined panel of 12 miRNAs. Results Unexpectedly, no miRNAs were found to be affected by T2DM status in either of the two study groups. Conclusions Our data suggest that alternatives to microRNA dysregulation underlie T2DM-associated protein changes in non-atherosclerotic arteries. Electronic supplementary material The online version of this article (10.1186/s12933-018-0715-y) contains supplementary material, which is available to authorized users.

The prevalence of diabetes mellitus is increased in patients with porphyria cutanea tarda. Different tests are available for diagnosing and screening for type II diabetes mellitus, however choosing the most suitable test is challenging. The pitfalls in the different tests along with the interfering comorbidities and treatments concerning patients with porphyria cutanea tarda complicate diagnosing these patients with diabetes mellitus. HbA1c, fasting glucose, or oral glucose tolerance are the current available tests, with HbA1c as first choice. Measuring HbA1c requires no fasting, however HbA1c can be false low if the patient is treated with phlebotomy or has liver cirrhosis or chronic hepatitis. Instead fasting glucose and oral glucose tolerance tests can be used if the patient is not acutely ill. If either of the tests give a result in the diagnostic range, the test should be repeated if the patient has no clinical symptoms of diabetes. Diagnosing diabetes mellitus is important for the purpose of early intervention, and this review provides the knowledge needed to diagnose this special patient group properly.

BACKGROUND: Primary to validate a commercial semi-automated computed tomography angiography (CTA) -software for vulnerable plaque detection compared to histology of carotid endarterectomy (CEA) specimens and secondary validating calcifications scores by in vivo CTA with ex vivo non-contrast enhanced computed tomography (NCCT).METHODS: From January 2014 to October 2016 53 patients were included retrospectively, using a cross-sectional design. All patients underwent both CTA and CEA. Sixteen patients had their CEA specimen NCCT scanned. The semi-automated CTA software analyzed carotid stenosis using different HU values defining plaque components. The predictive values of CTA based detection of vulnerable plaques were calculated. Quantification of calcifications on CTA using region of interest (ROI)-function and mathematical equations was done manually, and validated by NCCT of the CEA specimen.RESULTS: The semi-automated CTA software had a sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 89.1% (95% CI, 73.6% - 96.4%), 31.3% (95% CI, 12.1% - 58.5%), 75% (95% CI, 59.3% - 86.2%) and 55.6% (95% CI, 22.6% - 84.6%). Strong correlation between in vivo CTA and ex vivo NCCT in quantification of calcification was observed, but CTA systematically underestimated calcificationsscore (CALS) with increasing calcification.CONCLUSION: The CTA-software cannot be used in risk assessment of patients, due to poor specificity and NPV. The correlation between in vivo CTA and ex vivo NCCT was strong, proposing it to be used in both scientifically and clinical settings, but studies with larger sample sizes are needed.

Background: The knowledge of the frequency and associated mortality of shock in the emergency department (ED) is limited. The aim of this study was to describe the incidence, all-cause mortality and factors associated with death among patients suffering shock in the ED. Methods: Population-based cohort study at an University Hospital ED in Denmark from January 1, 2000, to December 31, 2011. All patients aged =18 years living in the hospital catchment area with a first time ED presentation with shock (n = 1553) defined as hypotension (systolic blood pressure (SBP) ≤100 mmHg)) and =1 organ failures. Outcomes were annual incidence per 100,000 person-years at risk (pyar), all-cause mortality at 0-7, and 8-90 days and risk factors associated with death. Results: We identified 1553 of 438,191 (0.4%) ED patients with shock at arrival. Incidence of shock increased from 53.6-74.8 cases per 100,000 pyar. The 7-day, and 90-day mortality was 23.3% (95% CI: 21.2-25.4) and 41.1% (95% CI: 38.6-43.5), respectively. Independent predictors of 7-day mortality were: age (adjusted HR 1.03 (95% CI: 1.03-1.04), and number of organ failures (≥3 organ failures; adjusted HR 3.30 95% CI: 2.33-4.66). Age, comorbidity level and number of organ failure were associated with 90-day mortality. Conclusion: Shock is a frequent and critical finding in the ED, carrying a 7- and, 90- day mortality of 23.3% and 41.1%, respectively. Age and number of organ failures are independent prognostic factors for death within 7 days, whereas age, comorbidity and organ failures are of significance within 8-90 days.

BACKGROUND: Diabetes mellitus type 2 (T2DM) is a significant risk factor for the development of cardiovascular diseases (CVDs). In a previous microarray study of internal mammary arteries from patients with and without T2DM, we observed several elastin-related genes with altered mRNA-expression in diabetic patients, namely matrix metalloproteinase 2 (MMP-2), lysyl oxidase (LOX) and elastin itself. In this study we investigate whether the serum concentrations of elastin-related proteins correlate to signs of CVD in patients with T2DM.METHODS: Blood samples from 302 type 2 diabetic patients were analysed for MMP-2, LOX, and the elastin degradation products ELM and ELM2. The results were investigated for correlations to signs of CVD in different vascular territories, as determined by myocardial perfusion scintigraphy, carotid artery thickness and ankle-brachial blood pressure index.RESULTS: T2DM patients with peripheral arterial disease (low ankle-brachial index) (PAD) display higher levels of MMP-2 and ELM compared to patients without PAD. However, none of the proteins or degradation products correlated with myocardial ischemia or a combined measure of CVD-signs, including myocardial ischemia, increased carotid thickness and decreased ankle-brachial blood pressure.CONCLUSIONS: Our results suggest that the diabetic environment affects the circulating amounts of MMP-2 and ELM in patients with PAD. However, the same connection could not be seen in diabetic patients with CVD broadly identified in three vascular territories. LOX and ELM-2 did not correlate to any type of CVD. Overall, serum levels of elastin-related molecules are only remotely related to CVD in type 2 diabetes.

The impact of disease-related changes in the extracellular matrix (ECM) on the mechanical properties of human resistance arteries largely remains to be established. Resistance arteries from both pig and human parietal pericardium (PRA) display a different ECM microarchitecture compared with frequently used rodent mesenteric arteries. We hypothesized that the biaxial mechanics of PRA mirror pressure-induced changes in the ECM microarchitecture. This was tested using isolated pig PRA as a model system, integrating vital imaging, pressure myography, and mathematical modeling. Collagenase and elastase digestions were applied to evaluate the load-bearing roles of collagen and elastin, respectively. The incremental elastic modulus linearly related to the straightness of adventitial collagen fibers circumferentially and longitudinally (both R2 ≥ 0.99), whereas there was a nonlinear relationship to the internal elastic lamina elastin fiber branching angles. Mathematical modeling suggested a collagen recruitment strain (means ± SE) of 1.1 ± 0.2 circumferentially and 0.20 ± 0.01 longitudinally, corresponding to a pressure of ~40 mmHg, a finding supported by the vital imaging. The integrated method was tested on human PRA to confirm its validity. These showed limited circumferential distensibility and elongation and a collagen recruitment strain of 0.8 ± 0.1 circumferentially and 0.06 ± 0.02 longitudinally, reached at a distending pressure below 20 mmHg. This was confirmed by vital imaging showing negligible microarchitectural changes of elastin and collagen upon pressurization. In conclusion, we show here, for the first time in resistance arteries, a quantitative relationship between pressure-induced changes in the extracellular matrix and the arterial wall mechanics. The strength of the integrated methods invites for future detailed studies of microvascular pathologies. NEW & NOTEWORTHY This is the first study to quantitatively relate pressure-induced microstructural changes in resistance arteries to the mechanics of their wall. Principal findings using a pig model system were confirmed in human arteries. The combined methods provide a strong tool for future hypothesis-driven studies of microvascular pathologies.

Introduction Chronic kidney disease (CKD) is associated with high cardiovascular morbidity and mortality. Recent evidence suggests that increases in both serum and intracellular magnesium (Mg) can slow or even prevent the development of vascular calcification seen in CKD. Serum calcification propensity (T50) is a novel functional test, which is associated with all-cause mortality in CKD and measures the ability of serum to delay the formation of crystalline nanoparticles. Theoretically, increasing serum Mg should improve T50 and thereby reduce the propensity towards ectopic calcification. Methods We conducted a randomized placebo-controlled double-blinded clinical trial to investigate the safety of 2 different doses of oral Mg supplementation in subjects with CKD stages 3 and 4 as well as their effects on intracellular Mg and T50. Thirty-six subjects with CKD stages 3 and 4 were randomized to one of 3 groups (placebo, elemental Mg 15 mmol/d or elemental Mg 30 mmol/d) given as slow-release Mg hydroxide and followed for 8 weeks. Results Thirty-four subjects completed the trial. Intracellular Mg remained stable throughout the trial despite significant increases in both serum and urine Mg. T50 increased significantly by 40 min from 256 ± 60 (mean ± SD) to 296 ± 64 minutes (95% confidence interval, 11–70, P < 0.05) in the Mg 30 mmol/d group after 8 weeks. No serious adverse events related to the study medication were reported during the study. Discussion Oral Mg supplementation was safe and well tolerated in CKD stages 3 and 4 and improved T50, but did not increase intracellular Mg. Further studies are needed to investigate the long-term effects of Mg supplementation in CKD stage 3 and 4 and whether improvement in calcification propensity is related to clinical endpoints.

Background Vitamin K antagonists (VKA) are the most frequently prescribed oral anticoagulants worldwide although new oral anticoagulants (NOAC) have become an important alternative. VKA inhibits Vitamin K1 necessary to produce coagulation factors but also Vitamin K2, which is essential in the activation of matrix-Gla protein, thought to be a strong local inhibitor of arterial calcifications. Purpose The aim was to investigate, whether VKA treatment is associated with coronary artery calcification (CAC) in a population with no prior cardiovascular disease (CVD). Methods We collected data on cardiovascular risk factors and CAC scores from cardiac CT scans performed as part of clinical examinations (n=9,672) or research studies (n=14,166) in the period 2007–2017. Data on use of VKA and NOAC was obtained from the Danish National Health Service Prescription Database. The association between VKA treatment duration and categorized CAC score was investigated by ordered logistic regression while adjusting for covariates. The independent variables included in the model were: age, gender, smoking, body mass index (BMI), diabetes mellitus, hypertension, hypercholesterolemia and/or statin treatment, family history of CVD, estimated glomerular filtration rate, VKA treatment duration and NOAC treatment duration. The categorisation of CAC was: 0, 1–99, 100–399 and ≥400 AU, corresponding to no, mild, moderate and severe atherosclerotic plaque burden, respectively. Results The final study population consisted of 17,254 participants (median 67 years old, 75% males) with no prior CVD, of which 1,748 (10%) and 1,144 (7%) had been treated with VKA or NOAC, respectively. A longer duration of VKA treatment was associated with higher CAC categories (Figure). For each cumulative year of VKA treatment, the odds of being in a higher CAC category, i.e. having more severe atherosclerosis, increased (odds ratio (OR)=1.032, 95% CI 1.009–1.057). All traditional cardiovascular risk factors were also associated with CAC. In contrast, NOAC treatment duration was not associated with CAC category (OR=1.004, 95% CI 0.937–1.075). In a sensitivity analysis of patients without statin treatment (n=12,143), the association between VKA treatment and CAC category remained unchanged. There was no significant interaction between VKA treatment duration and age on CAC category. Conclusion Adjusted for cardiovascular risk factors, VKA treatment – in contrast to NOAC - is associated with more severe CAC. Additional studies are required to clarify the clinical importance of this association in terms of hard cardiovascular endpoints. Acknowledgement/Funding Novo Nordisk Foundation and Independent Research Fund Denmark

The objective of this study was to determine if plasma CCN2 is associated with abdominal aorta aneurysm (AAA), and future need for AAA repair, and further to assess the potential clinical value of CCN2 in predicting disease outcome. CCN2 was quantified in plasma samples obtained from a cohort of 679 men aged 65–74 at initial ultrasound screening for AAA in the Viborg Vascular (VIVA) screening trial. Plasma CCN2 was correlated with need for future surgical repair in the whole study population (HR = 1.457 (1.081–1.962), p =.013) and in the AAA group alone (HR = 1.431 (1.064–1.926), p =.018), yet the predictive value (CCN2 > 0 and

AimsIn aortic stenosis (AS), there is poor association between symptoms and conventional markers of AS severity or left ventricular (LV) systolic function. This may reflect that symptoms arise from LV diastolic dysfunction or that aortic valve area (AVA) and transvalvular gradient do not reflect afterload. We aimed to study the impact of afterload (end-systolic wall stress [ESWS]) on the presence of symptoms in AS and to test whether symptoms are related to increased ESWS or LV remodelling.Methods and resultsIn a prospective study, ESWS was estimated by measuring LV wall thickness from MRI and estimated LV end systolic pressure from echocardiographic mean gradient and systolic blood pressure in 78 patients with severe AS scheduled for aortic valve replacement and 91 patients with asymptomatic severe AS. Symptomatic patients had lower indexed AVA (0.40±0.11 vs 0.45±0.09 cm2/m2, p=0.009). They had undergone more extensive remodelling (MRI LV mass index [LVMi]: 85±24 vs 69±17 g/m2, p2, p=0.003). Multivariate logistic regression identified echocardiographic relative wall thickness, tricuspid gradient, mitral deceleration time, early diastolic strain rate, MRI LVMi, MRI LV end-diastolic volume index and ESWS as independently associated with being symptomatic.ConclusionESWS can be estimated from multimodality imaging combining MRI and echocardiography. It is correlated with LV remodelling and neurohormonal activation and is independently associated with symptomatic status in AS.

Aim: Heparin administration affects the concentrations of many plasma proteins through their displacement from the endothelial glycocalyx. A differentiated protein response in diabetes will therefore, at least partly, reflect glycocalyx changes. This study aims at identifying biomarkers of endothelial dysfunction in diabetes by statistical exploration of plasma proteome data for interactions between diabetes status and heparin treatment. Methods: Diabetes-by-heparin interactions in relation to protein levels were inspected by regression modelling in plasma proteome data from 497 patients admitted for acute angiography. Analyses were conducted separately for all 273 proteins and as set-based analyses of 44 heparin-relevant proteins identified by gene ontology analysis and 42 heparin-influenced proteins previously reported. Results: Seventy-five patients had diabetes and 361 received heparin before hospitalization. The proteome-wide analysis displayed no proteins with diabetes-heparin interaction to pass correction for multiple testing. The overall set-based analyses revealed significant association for both protein sets (p-values−4), while constraining on opposite directions of effect in diabetics and none-diabetics was insignificant (p-values = 0.11 and 0.17). Conclusions: Our plasma proteome-wide interaction approach supports that diabetes influences heparin effects on protein levels, however the direction of effects and individual proteins could not be definitively pinpointed, likely reflecting a complex protein-basis for glycocalyx dysfunction in diabetes.

AIMS/HYPOTHESIS: Accelerated migration and proliferation of vascular smooth muscle cells (VSMCs) enhances arterial restenosis after angioplasty in insulin resistance and diabetes. Elevation of Src homology 2-containing protein tyrosine phosphatase 1 (SHP-1) induces apoptosis in the microvasculature. However, the role of SHP-1 in intimal hyperplasia and restenosis has not been clarified in insulin resistance and diabetes.METHODS: We used a femoral artery wire injury mouse model, rodent models with insulin resistance and diabetes, and patients with type 2 diabetes. Further, we modulated SHP-1 expression using a transgenic mouse that overexpresses SHP-1 in VSMCs (Shp-1-Tg). SHP-1 agonists were also employed to study the molecular mechanisms underlying the regulation of SHP-1 by oxidised lipids.RESULTS: Mice fed a high-fat diet (HFD) exhibited increased femoral artery intimal hyperplasia and decreased arterial SHP-1 expression compared with mice fed a regular diet. Arterial SHP-1 expression was also decreased in Zucker fatty rats, Zucker diabetic fatty rats and in patients with type 2 diabetes. In primary cultured VSMCs, oxidised LDL suppressed SHP-1 expression by activating Mek-1 (also known as Map2k1) and increased DNA methylation of the Shp-1 promoter. VSMCs from Shp-1-Tg mice exhibited impaired platelet-derived growth factor (PDGF)-stimulated tyrosine phosphorylation with a concomitant decrease in PDGF-stimulated VSMC proliferation and migration. Similarly, HFD-fed Shp-1-Tg mice and mice treated with the SHP-1 inducer, Icariside II, were protected from the development of intimal hyperplasia following wire injury.CONCLUSIONS/INTERPRETATION: Suppression of SHP-1 by oxidised lipids may contribute to the excessive VSMC proliferation, inflammatory cytokine production and intimal hyperplasia observed in arteries from diabetes and insulin resistance. Augmenting SHP-1 levels is a potential therapeutic strategy to maintain stent patency in patients with insulin resistance and diabetes.

BACKGROUND AND AIMS: High serum calcium-phosphate levels are associated with increased risk of cardiovascular disease (CVD) in patients with chronic kidney disease. Recent studies have demonstrated this relationship also in subjects with normal kidney function. Our aim was to examine whether calcium-phosphate metabolism is associated with the presence and extent of coronary artery calcification (CAC) in asymptomatic and apparently healthy individuals.METHODS: Serum samples from 1088 randomly recruited middle-aged men and women without known CVD and diabetes (DM), from the general population, were analysed for total calcium, phosphate, parathyroid hormone (PTH) and 25-hydroxyvitamin D (25(OH)D). CAC was measured by a non-contrast cardiac CT scan and categorised into four groups: 0, 1-99, 100-399, ≥400 Agatston units. The association of calcium-phosphate metabolism with CAC was evaluated by a multiple ordered logistic regression model. All the multiple regression analyses were performed in the entire cohort as well as in men and women separately.RESULTS: In the study population, 96% of the serum calcium values, 93% of the PTH values, 90% of the phosphate values, and only 64% of the 25(OH)D values were placed within the normal range. In men, the odds of being in a higher CAC category, i.e. having more severe CAC, increased by 30% when serum calcium concentration increased by 0.1 mmol/l (95% CI: 1.04-1.61, p = 0.019), independently of traditional cardiovascular risk factors. In women, no significant association between serum calcium and CAC was identified (OR 0.99, 95% CI: 0.81-1.21, p = 0.91). Neither phosphate, PTH nor 25(OH)D was significantly associated with CAC in men, in women or when performed in the entire cohort.CONCLUSIONS: Serum calcium, even with values within normal range and independent of traditional risk factors, was significantly associated with CAC in asymptomatic and apparently healthy middle-aged men, but not in women.

IntroductionAortic stenosis is a common heart valve disease, and due to the growing elderly population, the prevalence is increasing. The disease is progressive with increasing calcification of the valve cusps. A few attempts with medical preventive treatment have failed; thus, presently, the only effective treatment of aortic stenosis is surgery. This study will examine the effect of menaquinone-7 (MK-7) supplementation on progression of aortic valve calcification (AVC). We hypothesise that MK-7 supplementation will slow down the calcification process.Methods and analysisIn this multicenter and double-blinded, placebo-controlled study, 400 men aged 65–74 years with substantial AVC are randomised (1:1) to treatment with MK-7 (720 µg/day) supplemented by the recommended daily dose of vitamin D (25 µg/day) or placebo treatment (no active treatment) for 2 years. Exclusion criteria are treatment with vitamin K antagonist or coagulation disorders. To evaluate AVC score, a non-contrast CT scan is performed at baseline and repeated after 12 and 24 months of follow-up. Primary outcome is difference in AVC score from baseline to follow-up at 2 years. Intention-to-treat principle is used for all analyses.Ethics and disseminationThere are no reported adverse effects associated with the use of MK-7. The protocol is approved by the Regional Scientific Ethical Committee for Southern Denmark (S-20170059) and the Data Protection Agency (17/19010). It is conducted in accordance with the Declaration of Helsinki. Positive as well as negative findings will be reported.Trial registration numberNCT03243890.

Abdominal aortic aneurysm AAA is a chronic dilatation of the abdominal aorta. It is a potentially life threatening disease as it develops asymptomatic and is often first discovered at the time of rupture. One key element in AAA formation is degradation of the elastin fibers in the aortic wall. When elastin is degraded small bioactive elastin derived peptides (EDPs) are released into the circulation. These EDPs affect vascular smooth muscle cells in the aortic wall and attract monocytes to the aortic wall resulting in further degradation of elastin. We hypothesize that the level of circulating elastin derived peptides (EDPs) augments AAA expansion and that inhibition of circulating EDPs will inhibit AAA expansion. We tested the hypothesis in an elastase induced aneurysm murine model by applying synthetic EDPs or scrambles peptides and measure the AAA size. In addition, EDP-neutralizing antibody or control IgG was given to angiotensin II induced AAA in ApoE-/- mice and AAA size was determined 28 days later. Fourteen days after elastase infusion the abdominal aortic diameter was significantly increased in the EDP treated group when compared to the scramble peptid treated control group (1.33±0.07 vs. 1.13±0.06 mm, n=17-19, p

BACKGROUND: Diabetes is related to increased risk of cardiovascular disease, and arterial stiffness and its consequences may be the factor connecting the two. Arterial stiffness is often measured by carotid-femoral pulse wave velocity (cf-PWV), but no plasma biomarker reflecting arterial stiffness is available. Fibulin-1 is an extracellular matrix protein, up-regulated in arterial tissue and in plasma in patients with type 2 diabetes. We aimed to evaluate the association between plasma fibulin-1 and arterial stiffness measured by cf PWV in a group of patients with diabetes, and one without, all undergoing coronary artery bypass grafting.METHODS: Pulse wave velocity (PWV) and pulse wave analysis including augmentation index (Aix75) was measured in 273 patients, who subsequently underwent a coronary by-pass operation. Plasma samples were drawn and information was gathered on diabetes status, HbA1c, lipids, medication, body mass index, co-morbidities and smoking status. Carotid artery intima-media thickness, as well as estimation of carotid artery plaque burden, and distal blood pressure was also obtained.RESULTS: Sixty three patients had diabetes, and this group had significantly higher levels of plasma fibulin-1, PWV and Aix75, compared to the 210 patients who did not have diabetes. In univariate analysis fibulin-1 and pulse wave velocity were not correlated in either group whereas fibulin-1 in patients without diabetes was correlated to Aix75.CONCLUSION: Fibulin-1 and arterial stiffness indices are not directly related in patients with cardiac disease, despite the fact that both measures are increased among patients with diabetes.

Uremia accelerates atherosclerosis, but little is known about affected pathways in human vasculature. This study aimed to identify differentially expressed arterial transcripts in patients with chronic kidney disease (CKD). Global mRNA expression was estimated by microarray hybridization in iliac arteries ( n = 14) from renal transplant recipients and compared with renal arteries from healthy living kidney donors ( n = 19) in study 1. Study 2 compared nonatherosclerotic internal mammary arteries (IMA) from five patients with elevated plasma creatinine levels and age- and sex-matched controls with normal creatinine levels. Western blotting and immunohistochemistry for selected proteins were performed on a subset of study 1 samples. Fifteen gene transcripts were significantly different between the two groups in study 1 [fold changes (FC) > 1.05 and false discovery rates (FDR) < 0.005]. Most upregulated mRNAs associated with cellular signaling, apoptosis, TNFα/NF-κB signaling, smooth muscle contraction, and 10 other pathways were significantly affected. To focus attention on genes from genuine vascular cells, which dominate in IMA, concordant deregulated genes in studies 1 and 2 were examined and included 23 downregulated and eight upregulated transcripts (settings in study 1: FC > 1.05 and FDR < 0.05; study 2: FC > 1.2 and P < 0.2). Selected deregulated gene products were investigated at the protein level, and whereas HIF3α confirmed mRNA upregulation, vimentin showed upregulation in contrast to the mRNA results. We conclude that arteries from CKD patients display change in relatively few sets of genes. Many were related to differentiated vascular smooth muscle cell phenotype. These identified genes may contribute to understanding the development of arterial injury among patients with CKD.

Objective The pathogenesis of abdominal aortic aneurysms (AAA) involves a disturbed balance of breakdown and buildup of arterial proteins. We envision that individuals with AAA carry generalized arterial protein alterations either because of effects of genetically or environmental AAA risk factors or because of compensatory changes due to signaling molecules released from the affected aneurysmal tissue. Approach Protein extraction and quantitative proteome analysis by LC-MS/MS (liquid chromatography-mass spectrometry) was done on individual samples from the internal mammary artery from 11 individuals with AAA and 33 sex- and age-matched controls without AAA. Samples were selected from a biobank of leftover internal mammary arterial tissue gathered at coronary by-pass operations. Results We identified and quantitated 877 proteins, of which 44 were differentially expressed between the two groups (nominal p-values without correction for multiple testing). Some proteins related to the extracellular matrix displayed altered concentrations in the AAA group, particularly among elastin-related molecules [elastin, microfibrillar-associated protein 4 (MFAP4), lysyl oxidase]. In addition, several histones e.g. (e.g. HIST1H1E, HIST1H2BB) and other vascular cell proteins (e.g. versican, type VI collagen) were altered. Conclusions Our results support the notion that generalized alterations occur in the arterial tree in patients with AAA. Elastin-related proteins and histones seem to be part of such changes, however these preliminary results require replication in an independent set of specimens and validation by functional studies.

In the realm of clinical chemistry, the field of clinical proteomics, that is, the application of proteomic methods for understanding mechanisms and enabling diagnosis, prediction, measurement of activity, and treatment response in disease, is first and foremost a discovery and research tool that feeds assay development downstream. Putative new assay candidates generated by proteomics discovery projects compete with well-established assays with known indications, well-described performance, and of known value in specific clinical settings. Careful attention to the many options available in the design, execution, and interpretation of clinical proteomics studies is thus necessary for translation into clinical practice. We here review and discuss important options associated with clinical proteomics endeavors stretching from the planning phases to the final use in clinical chemistry.

Background— Severe aortic stenosis (AS) most often presents with reduced aortic valve area (2 ), normal stroke volume index (≥35 mL/m 2 ), and either high mean gradient (≥40 mm Hg; normal-flow high-gradient AS) or low mean gradient (normal-flow low-gradient [NFLG] AS). The benefit of aortic valve replacement (AVR) among NFLG patients is controversial. We compared the impact of NFLG condition on preoperative left ventricular (LV) remodeling and myocardial fibrosis and postoperative remodeling and symptomatic benefit. Methods and Results— Eighty-seven consecutive patients with reduced aortic valve area and normal stroke volume index undergoing AVR underwent echocardiography, magnetic resonance imaging, a 6-minute walk test, and measurement of natriuretic peptides before and 1 year after AVR. Myocardial fibrosis was assessed from magnetic resonance imaging. Patients were stratified as NFLG or normal-flow high-gradient. In total, 33 patients (38%) had NFLG. Before AVR, they were characterized by similar symptom burden but less severe AS measured by aortic valve area index (0.50±0.09 versus 0.40±0.08 cm 2 /m 2 ; P 2 ; P =0.01), but the same degree of myocardial fibrosis. After AVR, NFLG had a smaller reduction in LV mass index (−3±10 versus −±18 g/m 2 ; P Conclusions— Patients with NFLG had less severe AS and LV remodeling than patients with normal-flow high-gradient. Furthermore, NFLG patients experienced less reverse remodeling but the same symptomatic benefit. Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique identifier: NCT02316587.

BACKGROUND Glioblastoma (GBM) is the most common and aggressive primary brain tumor having a median overall survival below 15 months. To improve the prognosis, biomarkers are needed for early detection of GBM progression. In the present study we identified differentially expressed microRNAs (miRNAs) in plasma samples from GBM patients and investigated their potential to function as noninvasive biomarkers.

An aortic dilation is a potentially lethal condition because it may lead to rupture or dissection [(1)][1]. Dilations on the ascending aorta (AsAo) develop secondary to cystic medial necrosis, whereas dilations on the descending aorta (DeAo) and abdominal aorta (AbAo) are often associated with

Aim: The water channel aquaporin 1 (AQP1) promotes endothelial cell migration. It was hypothesized that AQP1 promotes neovascularization and growth of atherosclerotic plaques. Methods: AQP1 immunoreactivity and protein abundance was examined in human and murine atherosclerotic lesions and aortic aneurysms. Apolipoprotein E (ApoE) knockout (-/-) and AQP1-/-ApoE-/- mice were developed and fed Western diet (WD) for 8 and 16 weeks to accelerate the atherosclerosis process. In ApoE-/- and AQP1-/-ApoE-/- mice abdominal aortic aneurysms (AAA) were induced by angiotensin II (ANGII) infusion by osmotic minipumps for 4 weeks. Results: In human atherosclerotic lesions and AAA, AQP1 immunoreactive protein was associated with intralesional small vessels. In ApoE-/- mouse aorta, APQ1 mRNA levels were increased with time on WD (n = 7-9, P < 0.003). Both in murine lesions at the aortic root and in the abdominal aortic aneurysmal wall, AQP1 immunoreactivity was associated with microvascular structures. The atherosclerotic lesion burden was enhanced significantly in ANGII-infused AQP1-/-ApoE-/- mice compared with ApoE-/- mice, but neither incidence nor progression of AAA was different. The aortic lesion burden increased with time on WD but was not different between ApoE-/- and AQP1-/-ApoE-/- mice at either 8 or 16 weeks (n = 13-15). Baseline blood pressure and ANGII-induced hypertension were not different between genotypes. Conclusion: AQP1 is expressed in atherosclerotic lesion neovasculature in human and mouse arteries and AQP1 deficiency augments lesion development in ANGII-promoted atherosclerosis in mice. Normal function of AQP1 affords cardiovascular protection.