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Aim: The SCORE2 algorithm is recommended to estimate risk of cardiovascular disease (CVD). Coronary artery calcification (CAC) score is expensive but improves the risk prediction. This study aims to determine and compare the additive value of CAC-score and 19 biomarkers in risk prediction. Methods: Traditional cardiovascular (CV) risk factors, CAC-score, and a wide range of biomarkers (including lipids, calcium-phosphate metabolism, troponin, inflammation, kidney function and ankle brachial index (ABI)) were collected from 1211 randomly selected middle-aged men and women in this multicenter prospective cohort in 2009–2010. 10-year follow-up data on CV-events were obtained via the Danish Health Registries. CV-event was defined as stroke, myocardial infarction, hospitalization for heart failure, coronary artery revascularization or death from CVD. The association between SCORE2, CAC-score, biomarkers, and CV-events was assessed using cox proportional hazard rates (HR) and compared using AUC-calculation of ROC-curves. Finally, net reclassification improvement (NRI) was calculated. Results: 92 participants had CV-events. Adjusted for risk factors, CAC-score was significantly associated with events (adjusted HR 1.9 (95%CI:1.1; 3.3), 3.6 (95%CI:1.9; 6.8), and 5. (95%CI:2.6; 10.3) for CAC-score 1–99, CAC-score 100–399 and CAC-score ≥400, respectively. HR for the highest quartile of CRP was 2.3 (95%CI:1.2; 4.5), while none of the remaining biomarkers improved HR. Adjusted for SCORE2, the CAC-score improved AUC (AUCCAC: 0.72, AUCSCORE2: 0.67, p

Background: Pregnancy-associated plasma protein-A (PAPP-A) regulates bioavailability of insulin-like growth factor 1 (IGF1) in various tissues by proteolytic cleavage of a subset of IGF-binding proteins (IGFBPs). Pre-clinical studies have established a role of PAPP-A in atherosclerosis and proposed that targeting the proteolytic activity of PAPP-A has therapeutic value.This study aimed to investigate whether human atherosclerotic plaques contain proteolytically active PAPP-A, a prerequisite for further considering PAPP-A as a therapeutic target in patients. Methods: We obtained carotid (n = 9) and femoral (n = 11) atherosclerotic plaques from patients undergoing vascular surgery and incubated freshly harvested plaque tissue in culture media for 24 h. Subsequently, conditioned media were assayed for PAPP-A, STC2, IGFBP4, and IGF1 using immunoassays. Enzymatic activity of PAPP-A was assessed by its ability to process recombinant IGFBP4-IGF1 complexes - a specific substrate of PAPP-A - by Western blotting. Results: PAPP-A and STC2 were detectable in conditioned media from both carotid and femoral plaques, with higher STC2 concentrations in eluates from carotid plaque incubations (p = 0.02). IGFBP4 and IGF1 were undetectable. Conditioned media from all 20 plaques exhibited PAPP-A proteolytic activity. However, no correlation between PAPP-A concentration and its proteolytic activity was observed, whereas the PAPP-A: STC2 molar ratio correlated with PAPP-A activity (R2 = 0.25, p = 0.03). Conclusion: This study provides evidence for the presence of enzymatically active PAPP-A in atherosclerotic plaques and underscores the need for further investigating potential beneficial effects associated with targeting PAPP-A in atherosclerotic cardiovascular disease.

Abdominal aortic aneurysm (AAA) is a life-threatening condition characterized by the weakening and dilation of the abdominal aorta. Few diagnostic biomarkers have been proposed for this condition. We performed mass spectrometry-based proteomics analysis of affinity-enriched plasma from 45 patients with AAA and 45 matched controls to identify changes to the plasma proteome and potential diagnostic biomarkers. Gene ontology analysis revealed a significant upregulation of the proteins involved in inflammation, coagulation, and extracellular matrix in AAA patients, while proteins related to angiogenesis were among those downregulated. Using recursive feature elimination, we identified a subset of 10 significantly regulated proteins that were highly predictive of AAA. A random forest classifier trained on these proteins achieved an area under the curve (AUC) of 0.93 [95% CI: 0.91–0.95] using cross-validation. Further validation in a larger cohort is necessary to confirm these results.

BackgroundThe Danish cardiovascular screening (DANCAVAS) trial, a nationwide trial designed to investigate the impact of cardiovascular screening in men, did not decrease all-cause mortality, an outcome decided by the investigators. However, the target group may have varied preferences. In this study, we aimed to evaluate whether men aged 65 to 74 years requested a CT-based cardiovascular screening examination and to assess its impact on outcomes determined by their preferences.Methods and findingsThis is a post hoc study of the randomised DANCAVAS trial. All men 65 to 74 years of age residing in specific areas of Denmark were randomised (1:2) to invitation-to-screening (16,736 men, of which 10,471 underwent screening) or usual-care (29,790 men). The examination included among others a non-contrast CT scan (to assess the coronary artery calcium score and aortic aneurysms). Positive findings prompted preventive treatment with atorvastatin, aspirin, and surveillance/surgical evaluation. The usual-care group remained unaware of the trial and the assignments. The user-defined outcome was based on patient preferences and determined through a survey sent in January 2023 to a random sample of 9,095 men from the target group, with a 68.0% response rate (6,182 respondents). Safety outcomes included severe bleeding and mortality within 30 days after cardiovascular surgery. Analyses were performed on an intention-to-screen basis. Prevention of stroke and myocardial infarction was the primary motivation for participating in the screening examination. After a median follow-up of 6.4 years, 1,800 of 16,736 men (10.8%) in the invited-to-screening group and 3,420 of 29,790 (11.5%) in the usual-care group experienced an event (hazard ratio (HR), 0.93 (95% confidence interval (CI), 0.88 to 0.98; p = 0.010); number needed to invite at 6 years, 148 (95% CI, 80 to 986)). A total of 324 men (1.9%) in the invited-to-screening group and 491 (1.7%) in the usual-care group had an intracranial bleeding (HR, 1.17; 95% CI, 1.02 to 1.35; p = 0.029). Additionally, 994 (5.9%) in the invited-to-screening group and 1,722 (5.8%) in the usual-care group experienced severe gastrointestinal bleeding (HR, 1.02; 95% CI, 0.95 to 1.11; p = 0.583). No differences were found in mortality after cardiovascular surgery. The primary limitation of the study is that exclusive enrolment of men aged 65 to 74 renders the findings non-generalisable to women or men of other age groups.ConclusionIn this comprehensive population-based cardiovascular screening and intervention program, we observed a reduction in the user-defined outcome, stroke and myocardial infarction, but entail a small increased risk of intracranial bleeding.Trial registrationISRCTN Registry number, ISRCTN12157806 https://www.isrctn.com/ISRCTN12157806.

Background A paradoxical protective effect of diabetes on the development and progression of abdominal aortic aneurysms (AAA) has been known for years. This study aimed to investigate whether the protective role of diabetes on AAAs has evolved over the years. Methods A cross-sectional study, a systematic review and meta-analysis. This study was based on two large, population-based, randomised screening trials of men aged 65–74; VIVA (2008–2011) and DANCAVAS (2014–2018), including measurement of the abdominal aorta by ultrasound or CT, respectively. Analyses were performed using multiple logistic regressions to estimate the odds ratios (ORs) for AAAs in men with diabetes compared to those not having diabetes. Moreover, a systematic review and meta-analysis of population-based screening studies of AAAs to visualise a potential change of the association between diabetes and AAAs. Studies reporting only on women or Asian populations were excluded. Results In VIVA, the prevalence of AAA was 3.3%, crude OR for AAA in men with diabetes 1.04 (95% confidence interval, CI, 0.80-1.34), and adjusted OR 0.64 (CI 0.48-0.84). In DANCAVAS, the prevalence of AAA was 4.2%, crude OR 1.44 (CI 1.11-1.87), and adjusted OR 0.78 (CI 0.59-1.04). Twenty-three studies were identified for the meta-analysis (N = 224 766). The overall crude OR was 0.90 (CI 0.77-1.05) before 2000 and 1.16 (CI 1.03-1.30) after 1999. The overall adjusted OR was 0.63 (CI 0.59-0.69) before 2000 and 0.69 (CI 0.57-0.84) after 1999. Conclusion Both the crude and adjusted OR showed a statistically non-significant trend towards an increased risk of AAA by the presence of diabetes. If this represents an actual trend, it could be due to a change in the diabetes population. Trial registration DANCAVAS: Current Controlled Trials: ISRCTN12157806. VIVA: ClinicalTrials.gov NCT00662480.

Abstract Alcohol‐associated liver fibrosis accumulates over decades, driven by hepatic inflammation and cell death. We investigated the diagnostic accuracy of keratin‐18 degradation, measured using serum M30 and M65 levels, and the ActiTest for hepatic inflammatory activity in patients with compensated alcohol‐associated liver disease (ALD). Furthermore, we evaluated the prognostic accuracy of markers for liver‐related events and all‐cause mortality. All findings were compared with routine liver function tests: Aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma‐glutamyltransferase. Our prospective, biopsy‐controlled, single‐center study included 265 patients with ongoing or prior excessive alcohol intake, representing the full spectrum of compensated ALD. We defined hepatic inflammatory activity as a combined score of lobular inflammation and ballooning. For severe hepatic inflammatory activity (n = 40), we found excellent diagnostic accuracy for M30 (area under the receiver operating characteristics curve [AUROC] = 0.90), M65 (AUROC = 0.86), and AST (AUROC = 0.86). Elevated M30 (M30 > 240 U/L) had the highest positive predictive value (PPV) and specificity, significantly higher than M65, ActiTest and ALT, but not AST (M30: sensitivity = 83%, specificity = 82%, positive predictive value = 45%, negative predictive value = 95%). Patients were followed up for 1445 patient‐years. All markers, except for ALT, significantly predicted liver‐related events and all‐cause mortality. After adjusting for advanced fibrosis, drinking behavior and body mass index, M30 and M65 remained significant predictors of liver‐related events, whereas M30 and AST were significant predictors of all‐cause mortality. Conclusion: M30 and AST accurately detect severe hepatic inflammatory activity in patients with compensated ALD. M30 was the only significant predictor of both liver‐related events and all‐cause mortality after adjusting for advanced fibrosis, body mass index, and drinking behavior at inclusion.

Introduction Coronary artery calcification (CAC) and especially progression in CAC is a strong predictor of acute myocardial infarction and cardiovascular mortality. Supplementation with vitamin K2 and D3 has been suggested to have a protective role in the progression of CAC. In this study, we will examine the effect of vitamins K2 and D3 in men and women with severe CAC. We hypothesise that supplementation with vitamins K2 and D3 will slow down the calcification process.Method and analysis In this multicentre and double-blinded placebo-controlled study, 400 men and women with CAC score≥400 are randomised (1:1) to treatment with vitamin K2 (720 µg/day) and vitamin D3 (25 µg/day) or placebo treatment (no active treatment) for 2 years. Among exclusion criteria are treatment with vitamin K antagonist, coagulation disorders and prior coronary artery disease. To evaluate progression in coronary plaque, a cardiac CT-scan is performed at baseline and repeated after 12 and 24 months of follow-up. Primary outcome is progression in CAC score from baseline to follow-up at 2 years. Among secondary outcomes are coronary plaque composition and cardiac events. Intention-to-treat principle is used for all analyses.Ethics and dissemination There are so far no reported adverse effects associated with the use of vitamin K2. The protocol was approved by the Regional Scientific Ethical Committee for Southern Denmark and the Data Protection Agency. It will be conducted in accordance with the Declaration of Helsinki. Positive as well as negative findings will be reported.Trial registration number NCT05500443.

Abstract 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.

BackgroundTumor necrosis factor (TNF) is pathologically elevated in human abdominal aortic aneurysms (AAA). Non-selective TNF inhibition-based therapeutics are approved for human use but have been linked to several side effects. Compounds that target the proinflammatory soluble form of TNF (solTNF) but preserve the immunomodulatory capabilities of the transmembrane form of TNF (tmTNF) may prevent these side effects. We hypothesize that inhibition of solTNF signaling prevents AAA expansion.MethodsThe effect of the selective solTNF inhibitor, XPro1595, and the non-selective TNF inhibitor, Etanercept (ETN) was examined in porcine pancreatic elastase (PPE) induced AAA mice, and findings with XPro1595 was confirmed in angiotensin II (ANGII) induced AAA in hyperlipidemic apolipoprotein E (Apoe) –/– mice.ResultsXPro1595 treatment significantly reduced AAA expansion in both models, and a similar trend (p = 0.06) was observed in PPE-induced AAA in ETN-treated mice. In the PPE aneurysm wall, XPro1595 improved elastin integrity scores. In aneurysms, mean TNFR1 levels reduced non-significantly (p = 0.07) by 50% after TNF inhibition, but the histological location in murine AAAs was unaffected and similar to that in human AAAs. Semi-quantification of infiltrating leucocytes, macrophages, T-cells, and neutrophils in the aneurysm wall were unaffected by TNF inhibition. XPro1595 increased systemic TNF levels, while ETN increased systemic IL-10 levels. In ANGII-induced AAA mice, XPro1595 increased systemic TNF and IL-5 levels. In early AAA development, proteomic analyses revealed that XPro1595 significantly upregulated ontology terms including “platelet aggregation” and “coagulation” related to the fibrinogen complex, from which several proteins were among the top regulated proteins. Downregulated ontology terms were associated with metabolic processes.ConclusionIn conclusion, selective inhibition of solTNF signaling reduced aneurysm expansion in mice, supporting its potential as an attractive treatment option for AAA patients.

Inflammation and elastin degradation are key hallmarks in the pathogenesis of abdominal aortic aneurysms (AAAs). It has been acknowledged that activation of alpha7 nicotinic acetylcholine receptors (α7nAChRs) attenuates inflammation, termed the cholinergic anti-inflammatory pathway (CAP). Thus, we hypothesize that low-dose nicotine impairs the progression of elastase-induced AAAs in rats by exerting anti-inflammatory and anti-oxidative stress properties. Male Sprague–Dawley rats underwent surgical AAA induction with intraluminal elastase infusion. We compared vehicle rats with rats treated with nicotine (1.25 mg/kg/day), and aneurysm progression was monitored by weekly ultrasound images for 28 days. Nicotine treatment significantly promoted AAA progression (p = 0.031). Additionally, gelatin zymography demonstrated that nicotine significantly reduced pro-matrix metalloproteinase (pro-MMP) 2 (p = 0.029) and MMP9 (p = 0.030) activity in aneurysmal tissue. No significant difference was found in the elastin content or the score of elastin degradation between the groups. Neither infiltrating neutrophils nor macrophages, nor aneurysmal messenger RNA (mRNA) levels of pro- or anti-inflammatory cytokines, differed between the vehicle and nicotine groups. Finally, no difference in mRNA levels of markers for anti-oxidative stress or the vascular smooth muscle cells’ contractile phenotype was observed. However, proteomics analyses of non-aneurysmal abdominal aortas revealed that nicotine decreased myristoylated alanine-rich C-kinase substrate and proteins, in ontology terms, inflammatory response and reactive oxygen species, and in contradiction to augmented AAAs. In conclusion, nicotine at a dose of 1.25 mg/kg/day augments AAA expansion in this elastase AAA model. These results do not support the use of low-dose nicotine administration for the prevention of AAA progression.

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.

The pathogenesis of abdominal aortic aneurysm involves vascular inflammation and elastin degradation. Astragalusradix contains cycloastragenol, which is known to be anti-inflammatory and to protect against elastin degradation. We hypothesized that cycloastragenol supplementation inhibits abdominal aortic aneurysm progression. Abdominal aortic aneurysm was induced in male rats by intraluminal elastase infusion in the infrarenal aorta and treated daily with cycloastragenol (125 mg/kg/day). Aortic expansion was followed weekly by ultrasound for 28 days. Changes in aneurysmal wall composition were analyzed by mRNA levels, histology, zymography and explorative proteomic analyses. At day 28, mean aneurysm diameter was 37% lower in the cycloastragenol group (p < 0.0001). In aneurysm cross sections, elastin content was insignificantly higher in the cycloastragenol group (10.5% ± 5.9% vs. 19.9% ± 16.8%, p = 0.20), with more preserved elastin lamellae structures (p = 0.0003) and without microcalcifications. Aneurysmal matrix metalloprotease-2 activity was reduced by the treatment (p = 0.022). Messenger RNA levels of inflammatory- and anti-oxidative markers did not differ between groups. Explorative proteomic analysis showed no difference in protein levels when adjusting for multiple testing. Among proteins displaying nominal regulation were fibulin-5 (p = 0.02), aquaporin-1 (p = 0.02) and prostacyclin synthase (p = 0.007). Cycloastragenol inhibits experimental abdominal aortic aneurysm progression. The suggested underlying mechanisms involve decreased matrix metalloprotease-2 activity and preservation of elastin and reduced calcification, thus, cycloastragenol could be considered for trial in abdominal aortic aneurysm patients.

Abstract 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.

The human plasma proteome mirrors the physiological state of the cardiovascular system, a fact that has been used to analyze plasma biomarkers in routine analysis for the diagnosis and monitoring of cardiovascular diseases for decades. These biomarkers address, however, only a very limited subset of cardiovascular diseases, such as acute myocardial infarct or acute deep vein thrombosis, and clinical plasma biomarkers for the diagnosis and stratification cardiovascular diseases that are growing in incidence, such as heart failure and abdominal aortic aneurysm, do not exist and are urgently needed. The discovery of novel biomarkers in plasma has been hindered by the complexity of the human plasma proteome that again transforms into an extreme analytical complexity when it comes to the discovery of novel plasma biomarkers. This complexity is, however, addressed by recent achievements in technologies for analyzing the human plasma proteome, thereby facilitating the possibility for novel biomarker discoveries. The aims of this article is to provide an overview of the recent achievements in technologies for proteomic analysis of the human plasma proteome and their applications in cardiovascular medicine.

Specific plasma proteins serve as valuable markers for various diseases and are in many cases routinely measured in clinical laboratories by fully automated systems. For safe diagnostics and monitoring using these markers, it is important to ensure an analytical quality in line with clinical needs. For this purpose, information on the analytical and the biological variation of the measured plasma protein, also in the context of the discovery and validation of novel, disease protein biomarkers, is important, particularly in relation to for sample size calculations in clinical studies. Nevertheless, information on the biological variation of the majority of medium-to-high abundant plasma proteins is largely absent. In this study, we hypothesized that it is possible to generate data on inter-individual biological variation in combination with analytical variation of several hundred abundant plasma proteins, by applying LC-MS/MS in combination with relative quantification using isobaric tagging (10-plex TMT-labeling) to plasma samples. Using this analytical proteomic approach, we analyzed 42 plasma samples prepared in doublets, and estimated the technical, inter-individual biological, and total variation of 265 of the most abundant proteins present in human plasma thereby creating the prerequisites for power analysis and sample size determination in future clinical proteomics studies. Our results demonstrated that only five samples per group may provide sufficient statistical power for most of the analyzed proteins if relative changes in abundances >1.5-fold are expected. Seventeen of the measured proteins are present in the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Biological Variation Database, and demonstrated remarkably similar biological CV’s to the corresponding CV’s listed in the EFLM database suggesting that the generated proteomic determined variation knowledge is useful for large-scale determination of plasma protein variations.

Abstract 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.

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 and aimsVitamin K antagonists (VKA) remain the most frequently prescribed oral anticoagulants worldwide despite the introduction of non-vitamin K antagonist oral anticoagulants (NOAC). VKA interfere with the regeneration of Vitamin K1 and K2, essential to the activation of coagulation factors and activation of matrix-Gla protein, a strong inhibitor of arterial calcifications. This study aimed to clarify whether VKA treatment was associated with the extent of coronary artery calcification (CAC) in a population with no prior cardiovascular disease (CVD).MethodsWe 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 anticoagulation were obtained from the Danish National Health Service Prescription Database. The association between duration of anticoagulation and categorized CAC score (0, 1-99, 100-399, ≥400) was investigated by ordered logistic regression adjusting for covariates.ResultsThe final study population consisted of 17,254 participants with no prior CVD, of whom 1,748 and 1,144 had been treated with VKA or NOAC, respectively. A longer duration of VKA treatment was associated with higher CAC categories. For each year of VKA treatment, the odds of being in a higher CAC category increased (odds ratio (OR) = 1.032, 95%CI 1.009-1.057). In contrast, NOAC treatment duration was not associated with CAC category (OR = 1.002, 95%CI 0.935-1.074). There was no significant interaction between VKA treatment duration and age on CAC category.ConclusionsAdjusted for cardiovascular risk factors, VKA treatment-contrary to NOAC-was associated to higher CAC category.

Aims In 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 results In 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, p

Introduction: Arterial stiffening is associated with structural and biomechanical alterations in the aorta. However, there are still gaps in our understanding as to how the structure and properties of arteries across the vasculature are altered with high PWV.Objective: To determine whether altered ultrastructural and nanomechanical properties are exhibited in the internal mammary artery (IMA) in high PWV patients. Methods: Human IMA biopsies were obtained from patients with known carotid-femoral PWV. Patients were grouped as low PWV (8.5 ± 0.7 ms−1, n = 8) and high PWV (13.4 ± 3.0 ms−1, n = 9). With Peakforce QNM atomic force microscopy (AFM) the nanomechanical (elastic modulus) and morphological properties (collagen fibril diameter and D-Period) of the IMA were measured. Principal component analysis (PCA) was used to determine the relationship of nanomechanical and structural data with proteomics data (small leucine rich proteoglycans, SLRPs) [1] and patient metadata. Results: PCA analysis shows that the nano-scale elastic modulus was one of the key variables which separated low and high PWV groups and was correlated with PWV. Furthermore, nano-scale alterations in adventitial collagen fibrils were evident. D-Period and collagen fibril diameter were found to be negatively correlated. Most SLRPs were closely grouped in the PCA analysis. Conclusions: Although the IMA is not involved in the carotid-femoral pathway, patients with high PWV exhibited distinct alterations in the IMA at the nano-scale relative to those with low PWV. Our approach provides new insight into systemic structure-property changes in the vasculature, and also provides a novel method for characterizing small biopsy samples for arterial stiffening studies.

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.

Background: Arterial stiffening, occurring as part of the natural aging process of the artery, is well- established as a powerful predictor of cardiovascular disease. However, little is known about how localised changes in the extracellular matrix and mechanical properties of arterial tissue contribute to gross stiffening in the vasculature, particularly in the adventitia. The mechanical properties of the adventitia are attributed to the collagen fibrils which exhibit high tensile strength when an axial load is placed on the vessel. Objective: To determine the relationship between the adventitial collagen fibril properties and carotid-femoral pulse wave velocity (PWV). Methods: 16 patients were split into high PWV (13.6±1.1ms−1) and low (8.5±0.3ms−1) PWV groups (t-test, P < 0.001). Internal mammary arteries (IMAs) which were collected during coronary artery bypass grafting (CABG) were used to nano-scale characterisation of the tissue with atomic force microscopy (AFM). AFM was used to determine nanomechanical properties and collagen fibril morphology. Results: Abundant, highly oriented collagen fibrils were observed in the adventitial layer in both groups. The adventitia had high elastic modulus values in the high PWV group (Low PWV = 2298.64±75.38MPa; High PWV = 2734.63±95.52MPa, P < 0.001). The collagen fibril diameters were found to be higher in patients with high PWV (Low PWV = 117.23±22.19nm, High PWV = 119.18±21.96nm, P < 0.001). Conclusion: Nanomechanical properties and collagen fibril morphology in arterial tissue associated with carotid-femoral PWV. Nano-scale changes in the IMA are therefore indicative of systematic changes in arterial stiffness in the vasculature.

Background: Arterial stiffening occurs as part of the natural ageing process. Degradation of the extracellular matrix (ECM) in the medial layer is typically implicated in arterial stiffening. However, little is known about how localised changes in arteries in terms of both structure and mechanical properties contribute to the overall stiffening of arteries. Aim: To determine localised differences in the nano-structure and mechanical properties in the medial layer of internal mammary arteries (IMA) in patients with high and low pulse wave velocity (PWV). Methods: IMAs were collected form coronary bypass operations from 7 patients with high (13.8 3.3 m/s) and 7 patients with low (8.6 0.7 m/s) PWV. The samples were cryo-sectioned to a nominal thickness of 5 μm for atomic force microscopy (AFM) measurement. All the samples were tested hydrated. Histological analysis was used to determine collagen and elastin content. Data are presented as means SEMs. Results: The medial layers of IMAs in the high PWV group were significant stiffer than in the low PWV group (Low 228.4 15.6 kPa, High 735.8 108.8 kPa,) (p < 0.0001). Topographical features as visualised with AFM were similar in both groups but the higher nanomechanical stiffness was found to correlate with histological data. Conclusions: Nanomechanical properties of the medial layer in the IMA associate with PWV data. Changes in composition in the ECM drive the profound localized changes in tissue stiffness.

Proteins are the main molecular components of the arterial wall. Alterations in the amounts of specific proteins in both the extracellular matrix and in vascular cells are believed to be associated with different arterial pathologies, however only sparse data is currently available, particularly in relation to human arteries. Proteome analysis is large scale analysis of the quantity of many proteins in a single analytical run from biological samples. Combining “state of the art” proteome analysis by LC-MS (liquid chromatography-mass spectrometry) with access to samples from a large human artery biobank, we have obtained knowledge about protein changes in arteries from patients with various cardiovascular conditions. Specific alterations in matrix proteins are for example present in relation to increased arterial stiffness and to diabetes, whereas alterations in non-matrix proteins are associated with the growth rate of aortic aneurysms. Such new knowledge about changes of arterial proteins in specific vascular conditions can direct our attention towards pathophysiological understandings and display routes to new potential treatment targets and novel biomarkers for arterial diseases.

Aim: Arterial stiffening occurs as part of the natural ageing process, and is thought to be related to the accumulation of collagen and degradation of elastin. However, little is known about how regional variations in arterial structure and mechanical properties contribute to arterial stiffening. This study compared localised differences in the nano-structure and mechanical properties in the internal mammary arteries (IMA) from patients with high and low PWV. Methods: 6 IMAs were collected from coronary bypass operations and the patients were grouped according to their carotid-femoral PWV; high (14.6 ± 1.4 m/s) and low (8.7 ± 0.5 m/s). The nano-topography and elastic modulus were determined by atomic force microscopy (AFM) using 5 μm cryosections. Data are presented as means ± SEMs. Results: Overall, IMAs in the high PWV group were significantly stiffer than in the low PWV group (High; 2234.7 ± 72.3 MPa, Low; 2015.3 ± 58.4 MPa), (p < 0.0001). Although no significant difference was found in the intimal or medial layers, the adventitia was stiffer in the high PWV group (High; 2597.6 ± 135.7 MPa, Low;2215.6 ± 110.2 MPa), (p < 0.001). Furthermore, the collagen fibrils in the adventitia of the high PWV group were found to have a smaller diameter (High; 118.44 ± 1.1 nm, Low; 123.81 ± 1.3 nm), (p < 0.01)). Conclusion: Overall, the nanomechanical data associates with PWV data. The high PWV group exhibited higher nanomechanical stiffness alongside morphological alterations within the adventitial layer.

Affinity purification, immunoprecipitation, gel electrophoresis and mass spectrometry were used to identify fibulin-1C, C1 esterase inhibitor and glucose regulated protein 75, grp75, as binding partners of the CREC proteins, calumenin and reticulocalbin. Surface plasmon resonance was used to verify the interaction of all three proteins with each of the CREC proteins. Fibulin-1C interacts with calumenin and reticulocalbin with an estimated dissociation constant around 50-60 nM. The interaction, at least for reticulocalbin, was not dependent upon the presence of Ca2+. C1 esterase inhibitor interacted with both proteins with an estimated dissociation constant at 1 μM for reticulocalbin and 150 nM for calumenin. The interaction, at least for calumenin, was dependent upon the presence of Ca2+ with strong interaction at 3.5 mM while no detectable interaction could be found at 0.1 mM. Grp75 binds with an affinity of approximately 3-7 nM with reticulocalbin as well as with calumenin. These interactions suggest functional participation of the CREC proteins in chaperone activity, cell proliferation and transformation, cellular aging, haemostasis and thrombosis as well as modulation of the complement system in fighting bacterial infection.

Microfibrillar-associated protein 4 (MFAP4) is located in the extracellular matrix (ECM). We sought to identify tissues with high levels of MFAP4 mRNA and MFAP4 protein expression. Moreover, we aimed to evaluate the significance of MFAP4 as a marker of cardiovascular disease (CVD) and to correlate MFAP4 with other known ECM markers, such as fibulin-1, osteoprotegerin (OPG), and osteopontin (OPN). Quantitative real-time PCR demonstrated that MFAP4 mRNA was more highly expressed in the heart, lung, and intestine than in other elastic tissues. Immunohistochemical studies demonstrated high levels of MFAP4 protein mainly at sites rich in elastic fibers and within blood vessels in all tissues investigated. The AlphaLISA technique was used to determine serum MFAP4 levels in a clinical cohort of 172 patients consisting of 5 matched groups with varying degrees of CVD: 1: patients with ST elevation myocardial infarction (STEMI), 2: patients with non-STEMI, 3: patients destined for vascular surgery because of various atherosclerotic diseases (stable atherosclerotic disease), 4: apparently healthy individuals with documented coronary artery calcification (CAC-positive), and 5: apparently healthy individuals without signs of coronary artery calcification (CAC-negative). Serum MFAP4 levels were significantly lower in patients with stable atherosclerotic disease than CAC-negative individuals (p