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Background and Purpose CD40 and CD40 ligand (CD40L) are costimulatory molecules of the tumor necrosis factor receptor superfamily and well known for their involvement in inflammatory diseases: atherosclerotic mouse models with disrupted CD40 signalling develop lesions of reduced size with a more stable plaque profile. This study investigated the potential of plasma and intraplaque levels of CD40 and CD40L as markers for cardiovascular disease (CVD) in humans and their association with plaque stability.Methods Soluble CD40 and CD40L (sCD40L) were measured in plasma in 1,437 subjects from The SUrrogate markers for Micro- and Macro-vascular hard endpoints for Innovative diabetes Tools (SUMMIT) cohort. Intra-plaque levels of sCD40 and sCD40L were measured in atherosclerotic plaque homogenates from 199 subjects of the Carotid Plaque Imaging Project (CPIP) cohort. Results Both plasma sCD40 and sCD40L levels were elevated in individuals with prevalent stroke, while sCD40 levels also were higher in individuals with a prior acute myocardial infarction. Plasma levels of sCD40 correlated with carotid intima-media thickness and total carotid plaque area and were associated with risk of cardiovascular events over a 3-year follow-up period. Intra-plaque levels of sCD40 and sCD40L were associated with plaque components characteristic for plaque vulnerability and extracellular matrix remodelling.Conclusions Higher plasma sCD40 and sCD40L levels are associated with prevalent CVD. Plasma sCD40 levels also correlate with the severity of carotid atherosclerosis and predict future cardiovascular events, while intra-plaque levels correlate with a vulnerable plaque phenotype. Our findings thus demonstrate that elevated levels of sCD40 and sCD40L are markers of CVD.

Objective: To identify causal pathophysiological mechanisms for atherosclerosis and incident cardiovascular events using protein measurements. Approach and Results: Carotid artery atherosclerosis was assessed by ultrasound, and 86 cardiovascular-related proteins were measured using the Olink CVD-I panel in 7 Swedish prospective studies (11 754 individuals). The proteins were analyzed in relation to intima-media thickness in the common carotid artery (IMT-CCA), plaque occurrence, and incident cardiovascular events (composite end point of myocardial infarction or ischemic stroke) using a discovery/replication approach in different studies. After adjustments for traditional cardiovascular risk factors, 11 proteins remained significantly associated with IMT-CCA in the replication stage, whereas 9 proteins were replicated for plaque occurrence and 17 proteins for incident cardiovascular events. NT-proBNP (N-terminal pro-B-type natriuretic peptide) and MMP (matrix metalloproteinase)-12 were associated with both IMT-CCA and incident events, but the overlap was considerably larger between plaque occurrence and incident events, including MMP-12, TIM-1 (T-cell immunoglobulin and mucin domain 1), GDF (growth/differentiation factor)-15, IL (interleukin)-6, U-PAR (urokinase plasminogen activator surface receptor), LOX-1 (lectin-like oxidized LDL [low-density lipoprotein] receptor 1), and TRAIL-R2 (TNF [tumor necrosis factor]-related apoptosis-inducing ligand receptor 2). Only MMP-12 was associated with IMT-CCA, plaque, and incident events with a positive and concordant direction of effect. However, a 2-sample Mendelian randomization analysis suggested that increased MMP-12 may be protective against ischemic stroke ( P =5.5×10 −7 ), which is in the opposite direction of the observational analyses. Conclusions: The present meta-analysis discovered several proteins related to carotid atherosclerosis that partly differed in their association with IMT-CCA, plaque, and incident atherosclerotic disease. Mendelian randomization analysis for the top finding, MMP-12, suggests that the increased levels of MMP-12 could be a consequence of atherosclerotic burden rather than the opposite chain of events.

Epicardial adipose tissue has a paracrine effect, enhancing coronary artery atherosclerotic plaque development. This study evaluated epicardial fat volume (EFV), adipokines, coronary atherosclerosis, and adverse cardiovascular events in a cohort of asymptomatic patients with type 2 diabetes mellitus (T2DM). Epicardial fat volume was calculated using data from computed tomography coronary angiograms. Adipokines and inflammatory cytokines were also assayed and correlated with EFV. Epicardial fat volume was also assessed as a predictor of coronary artery calcium (CAC) score, number of coronary artery plaques, and significant plaque (>50% luminal stenosis). Data from the EFV analysis were available for 221 (85.7%) participants. Median EFV was 97.4 cm3, mean body mass index was 28.1 kg/m2, and mean duration of T2DM was 13 years. Statistically significant, but weak, correlations were observed between several adipokines, inflammatory cytokines, and EFV. Epicardial fat volume was a significant univariate ( P = .01), but not multivariate, predictor of the number of coronary plaques, but not of CAC score or significant plaque. After a mean follow-up of 22.8 months, 12 adverse cardiovascular events were reported, exclusively in participants with EFV >97.4 cm3. Epicardial fat volume has limited utility as a marker of coronary artery plaque in patients with T2DM and is weakly correlated with adipokine expression.

As cardio metabolic disease manifestations tend to cluster in families there is a need to better understand the underlying mechanisms in order to further develop preventive strategies. In fact, genetic markers used in genetic risk scores, important as they are, will not be able alone to explain these family clusters. Therefore, the search goes on for the so called missing heritability to better explain these associations. Shared lifestyle and social conditions in families, but also early life influences may be of importance. Gene-environmental interactions should be explored. In recent years interest has grown for the role of diet-microbiota associations, as microbiota patterns may be shared by family members. In the Malmö Offspring Study that started in 2013, we have so far been able to examine about 4700 subjects (18–71 years) representing children and grandchildren of index subjects from the first generation, examined in the Malmö Diet Cancer Study during 1991 to 1996. This will provide rich data and opportunities to analyse family traits of chronic disease across three generations. We will provide extensive genotyping and phenotyping including cardiovascular and respiratory function, as well as markers of glucose metabolism. In addition, also cognitive function will be assessed. A 4-day online dietary recall will be conducted and gut as well as oral microbiota analysed. The ambition is to provide one of the first large-scale European family studies with individual data across three generations, which could deepen our knowledge about the role of family traits for chronic disease and its underlying mechanisms. Electronic supplementary material The online version of this article (10.1007/s10654-020-00695-4) contains supplementary material, which is available to authorized users.

Aims and objectives To explore newly graduated registered nurses' experiences and how they manage complex patient situations. Background Newly graduated registered nurses' working in acute care hospital settings are challenged by managing complex patient situations in rapidly changing clinical contexts involving increased patient acuity, comorbidities and staffing shortages. Design Qualitative study design. Methods Data were collected using focus groups interviews of a total of 16 newly graduated registered nurses with clinical work experience of 6 months of direct patient care in an acute care hospital setting. Analyses were conducted using qualitative content analysis. COREQ reporting guidelines were used. Results The analysis resulted in the overarching theme “Not being sufficiently prepared and supported to meet responsibilities and demands.” The theme included three categories: “Responsibility is not in proportion to competence,” “Lack of medical competence and experience complicates patient safety” and “Strives for control to manage and organise nursing care.” Conclusion The results show that newly graduated registered nurses' are not sufficiently supported for the level of responsibility and the demands placed on them when providing nursing in complex patient situations in acute care hospital settings. If they are given sole responsibility for multiple complex patient situations, patient safety may be compromised. Relevance to practice Special attention need to be paid to NGRNs support to medical competence in the areas of assessing, planning, prioritizing, leading, and distributing nursing care in daily clinical settings for at least their first year of professional work.

Objectives This study will compare the incidence of major adverse cardiovascular events (MACEs) with androgen deprivation therapy (ADT) among men with advanced prostate cancer who are being treated with a gonadotropin-releasing hormone (GnRH) antagonist versus a GnRH agonist. Background Treatment of advanced prostate cancer with ADT might increase the risk of subsequent cardiovascular events among men with known atherosclerotic cardiovascular disease (ASCVD), but a recent meta-analysis suggested that this risk might be lower with ADT using a GnRH antagonist versus a GnRH agonist. Methods PRONOUNCE is a multicenter, prospective, randomized, open, blinded endpoint trial that will enroll approximately 900 patients with advanced prostate cancer and pre-existing ASCVD who will be treated with ADT. Participants will be randomized to receive the GnRH antagonist degarelix or the GnRH agonist leuprolide as ADT for 12 months. The primary endpoint is time from randomization to first confirmed, adjudicated occurrence of a MACE, which is defined as a composite of all-cause death, nonfatal myocardial infarction, or nonfatal stroke through 12 months of ADT treatment. Baseline cardiovascular biomarkers (high-sensitivity C-reactive protein, high-sensitivity troponin T, and N-terminal pro-brain natriuretic peptide), as well as serial inflammatory and immune biomarkers, will be evaluated in exploratory analyses. Results As of October 1, 2019, a total of 364 patients have been enrolled. The mean age is 74 years, 90% are white, 80% have hypertension or dyslipidemia, 30% diabetes mellitus, 40% have had a previous myocardial infarction, and 65% have had previous revascularization. Regarding prostate cancer features at randomization, 48% of the patients had localized disease, 23% had locally advanced disease, and 18% had metastatic disease. Conclusions PRONOUNCE is the first prospective cardiovascular outcomes trial in advanced prostate cancer that will delineate whether the risk of subsequent cardiovascular events associated with ADT is lower with a GnRH antagonist versus a GnRH agonist for men with pre-existing ASCVD. (A Trial Comparing Cardiovascular Safety of Degarelix Versus Leuprolide in Patients With Advanced Prostate Cancer and Cardiovascular Disease [PRONOUNCE]; NCT02663908), Central Illustration

An amendment to this paper has been published and can be accessed via the original article.

ObjectivesAtherothrombosis in the carotid arteries is a main cause of ischemic stroke and may depend on plaque propensity to complicate with rupture or erosion, in turn related to vulnerability features amenable to in vivo imaging. This would provide an opportunity for risk stratification and—potentially—local treatment of more vulnerable plaques. We here review current information on this topic.MethodsWe systematically reviewed the literature for concepts derived from pathophysiologic, histopathologic, and clinical studies on imaging techniques attempting at identifying vulnerable carotid lesions.ResultsUltrasound, MRI, CT, and nuclear medicine–based techniques, alone or with multimodality approaches, all have a link to pathophysiology and describe different—potentially complementary—aspects of lesions prone to complications. There is also, however, a true paucity of head-to-head comparisons of such techniques for practical implementation of a thorough and cost-effective diagnostic strategy based on evaluation of outcomes. Especially in asymptomatic patients, major international societies leave wide margins of indecision in the advice to techniques guiding interventions to prevent atherothrombotic stroke.ConclusionsTo improve practical management of such patients—in addition to the patient's vulnerability for systemic reasons—a more precise identification of the vulnerable plaque is needed. A better definition of the diagnostic yield of each imaging approach in comparison with the others should be pursued for a cost-effective translation of the single techniques. Practical translation to guide future clinical practice should be based on improved knowledge of the specific pathophysiologic correlates and on a comparative modality approach, linked to subsequent stroke outcomes.

Objective: CD4 + CD28 null T cells have been shown to be associated with recurrent coronary events and suggested as potential biomarker and therapeutic target. It is unknown whether CD4 + CD28 null T cells associate with first-time cardiovascular events. We examined CD4 + CD28 null T cells in a prospective population-based cohort and in patients with advanced atherosclerosis. Approach and Results: CD4 + CD28 null T cells were quantified in 272 individuals experiencing a first-time coronary event during up to 17 years of follow-up and 272 age- and sex-matched controls in a case-control study, nested within the population-based Malmö Diet and Cancer study. The highest tertile of CD4 + CD28 null T cells was associated with a lower incidence of first-time coronary events compared with the lowest tertile (odds ratio, 0.48 [95% CI, 0.29–0.79], P =0.004) when adjusting for Framingham risk factors. This association remained significant for events recorded after >9 years of follow-up, when most coronary events occurred, but not during the first 9 years of follow-up, despite similar odds ratio. Additionally, we analyzed CD4 + CD28 null T cells in 201 patients with advanced atherosclerosis undergoing carotid endarterectomy. The adjusted hazard ratio for cardiovascular events in patients with advanced atherosclerosis was 2.11 (95% CI, 1.10–4.05, P =0.024), comparing the highest with the lowest CD4 + CD28 null T-cell tertile. Conclusions: Our findings reveal complex associations between CD4 + CD28 null T cells and cardiovascular disease. Although we confirm the reported positive associations with an adverse prognosis in patients with already established disease, the opposite associations with first-time coronary events in the population-based cohort may limit the clinical use of CD4 + CD28 null T cells.

Genetic loci associated with CHD show different relationships with intima-media thickness in the common carotid artery (IMT-CCA) and in the bulb (IMT-bulb). We evaluated if IMT-CCA and IMT-bulb differ also with respect to circulating protein profiles and risk of incident atherosclerotic disease.In three Swedish cohorts (MDC, IMPROVE, PIVUS, total n 7000), IMT-CCA and IMT-bulb were assessed by ultrasound at baseline, and 86 cardiovascular-related proteins were analyzed. In the PIVUS study only, IMT-CCA and IMT-bulb were investigated in relation to incident atherosclerotic disease over 10 years of follow-up.In a meta-analysis of the analysis performed separately in the cohorts, three proteins, matrix metalloproteinase-12 (MMP-12), hepatocyte growth factor (HGF) and N-terminal pro-B-type natriuretic peptide (NT-proBNP), were associated with IMT-CCA when adjusted for traditional cardiovascular risk factors. Five proteins were associated with IMT-bulb (MMP-12, growth/differentiation factor 15 (GDF-15), osteoprotegerin, growth hormone and renin). Following adjustment for cardiovascular risk factors, IMT-bulb was significantly more closely related to incident stroke or myocardial infarction (total number of cases, 111) than IMT-CCA in the PIVUS study (HR 1.51 for 1 SD, 95%CI 1.21-1.87, p 0.001 vs HR 1.17, 95%CI 0.93-1.47, p = 0.16). MMP-12 levels were related to this combined end-point (HR 1.30, 95%CI 1.08-1.56, p = 0.0061).Elevated levels of MMP-12 were associated with both IMT-CCA and IMT-bulb, but other proteins were significantly related to IMT in only one of these locations. The finding that IMT-bulb was more closely related to incident atherosclerotic disease than IMT-CCA emphasizes a difference between these measurements of IMT.

Visual Abstract, Highlights • There is strong experimental evidence that IL-5 has a protective role in atherosclerosis but the clinical importance of this remains poorly studied. • In a prospective study involving 696 subjects with a follow-up of close to 17 years we show that baseline plasma levels of IL-5 do not predict risk for coronary events and stroke. • However, subjects with high levels of IL-5 were less likely to have a carotid plaque at the baseline investigation. • Experimental studies using a shear stress-modifying cast to the carotid artery of Apoe−/− mice deficient for IL-5 showed that lack of IL-5 was associated with increased plaque formation at sites of oscillatory blood flow. • The findings are in line with previous experimental observations of an atheroprotective role of IL-5 but do not support the use of IL-5 measurement in cardiovascular risk prediction., Summary Experimental studies have suggested an atheroprotective role of interleukin (IL)-5 through the stimulation of natural immunoglobulin M antibody expression. In the present study we show that there are no associations between baseline levels of IL-5 and risk for development of coronary events or stroke during a 15.7 ± 6.3 years follow-up of 696 subjects randomly sampled from the Malmö Diet and Cancer study. However, presence of a plaque at the carotid bifurcation was associated with lower IL-5 and IL-5 deficiency resulted in increased plaque development at sites of oscillatory blood flow in Apoe−/− mice suggesting a protective role for IL-5 in plaque development.

Objective— RAGE (receptor for advanced glycation end products) and EMMPRIN (extracellular matrix metalloproteinase inducer) are immune receptors for proinflammatory mediators. These receptors can also be found in a soluble form in the circulation. Soluble RAGE (sRAGE) has shown atheroprotective properties in animal studies, possibly by acting as a decoy receptor for its ligands. Whether sEMMPRIN (soluble EMMPRIN) has similar roles is unknown. We hypothesized that sRAGE and sEMMPRIN might be associated with vascular disease progression, incident coronary events, and mortality. Approach and Results— We measured baseline sRAGE and sEMMPRIN in 4612 cardiovascular disease-free individuals from the population-based Malmö Diet and Cancer cohort. Measurements of intima-media thickness in the common carotid artery were performed at inclusion and after a median of 16.5 years. sRAGE was negatively correlated with carotid intima-media thickness progression, independently of traditional cardiovascular risk factors, kidney function, and hsCRP (high sensitive C-reactive protein). Additionally, sRAGE was associated with decreased risk for major adverse coronary events (hazard ratio=0.90 [0.82–0.97]; P =0.009) and mortality (hazard ratio=0.93 [0.88–0.99]; P =0.011) during a follow-up period of 21 years. The relationship with mortality was independent of all considered potential confounders. We found no correlations between EMMPRIN, intima-media thickness progression, or prognosis. Conclusions— Individuals with high levels of circulating sRAGE have a slower rate of carotid artery disease progression and a better prognosis. Although its predictive value was too weak to promote sRAGE as a useful clinical biomarker in the population, the findings support further research into the potential anti-inflammatory and atheroprotective properties of this soluble receptor.

Background: The relative cardiovascular safety of gonadotropin-releasing hormone (GnRH) antagonists compared with GnRH agonists in men with prostate cancer and known atherosclerotic cardiovascular disease remains controversial. Methods: In this international, multicenter, prospective, randomized, open-label trial, men with prostate cancer and concomitant atherosclerotic cardiovascular disease were randomly assigned 1:1 to receive the GnRH antagonist degarelix or the GnRH agonist leuprolide for 12 months. The primary outcome was the time to first adjudicated major adverse cardiovascular event (composite of death, myocardial infarction, or stroke) through 12 months. Results: Because of slower-than-projected enrollment and fewer-than-projected primary outcome events, enrollment was stopped before the 900 planned participants were accrued. From May 3, 2016, to April 16, 2020, a total of 545 patients from 113 sites across 12 countries were randomly selected. Baseline characteristics were balanced between study groups. The median age was 73 years, 49.8% had localized prostate cancer; 26.3% had locally advanced disease, and 20.4% had metastatic disease. A major adverse cardiovascular event occurred in 15 (5.5%) patients assigned to degarelix and 11 (4.1%) patients assigned to leuprolide (hazard ratio, 1.28 [95% CI, 0.59–2.79]; P =0.53). Conclusions: PRONOUNCE (A Trial Comparing Cardiovascular Safety of Degarelix Versus Leuprolide in Patients With Advanced Prostate Cancer and Cardiovascular Disease) is the first, international, randomized clinical trial to prospectively compare the cardiovascular safety of a GnRH antagonist and a GnRH agonist in patients with prostate cancer. The study was terminated prematurely because of the smaller than planned number of participants and events, and no difference in major adverse cardiovascular events at 1 year between patients assigned to degarelix or leuprolide was observed. The relative cardiovascular safety of GnRH antagonists and agonists remains unresolved. Registration: URL: https://www.clinicaltrials.gov ; Unique identifier: NCT02663908.

Background The balance between stabilizing and destabilizing atherosclerotic plaque components is used in experimental studies and in imaging studies to identify rupture prone plaques. However, we lack the evidence that this balance predicts future cardiovascular events. Here we explore whether a calculated histological ratio, referred to as vulnerability index (VI), can predict patients at higher risk to suffer from future cardiovascular events. Methods and Results Carotid plaques and clinical information from 194 patients were studied. Tissue sections were used for histological analysis to calculate the VI (CD68 [cluster of differentiation 68], alpha‐actin, Oil red O, Movat pentachrome, and glycophorin A). Postoperative cardiovascular events were identified through the Swedish National Inpatient Health Register (2005–2013). During the follow‐up (60 months) 45 postoperative cardiovascular events were registered. Patients with a plaque VI in the fourth quartile compared with the first to third quartiles had significantly higher risk to suffer from a future cardiovascular event ( P =0.0002). The VI was an independent predictor and none of the 5 histological variables analyzed separately predicted events. In the 13 patients who underwent bilateral carotid endarterectomy, the VI of the right plaque correlated with the VI of the left plaque and vice versa ( r =0.7, P =0.01). Conclusions Our findings demonstrate that subjects with a high plaque VI have an increased risk of future cardiovascular events, independently of symptoms and other known cardiovascular risk factors . This strongly supports that techniques which image such plaques can facilitate risk stratification for subjects in need of more intense treatment.

The parameters derived from reservoir-excess pressure analysis have prognostic utility in several populations. However, evidence in type 2 diabetes (T2DM) remains scarce. We determined if these parameters were associated with T2DM and whether they would predict cardiovascular events in individuals with T2DM. We studied 306 people with T2DM with cardiovascular disease (CVD; DMCVD, 70.4±7.8 years), 348 people with T2DM but without CVD (diabetes mellitus, 67.7±8.4 years), and 178 people without T2DM or CVD (control group [CTRL], 67.2±8.9 years). Reservoir-excess pressure analysis–derived parameters, including reservoir pressure integral, peak reservoir pressure, excess pressure integral, systolic rate constant, and diastolic rate constant, were obtained by radial artery tonometry. Reservoir pressure integral was lower in DMCVD and diabetes mellitus than CTRL. Peak reservoir pressure was lower, and excess pressure integral was greater in DMCVD than diabetes mellitus and CTRL. Systolic rate constant was lower in a stepwise manner among groups (DMCVD< diabetes mellitus P

Matrix metalloproteinases (MMP) and tumor necrosis factor receptor superfamily members have been recently proposed as surrogate markers of atherosclerosis in the general population. The aim of this study was to examine their predictive role in type 2 diabetes (T2D). Plasma levels of MMP-12, MMP-7, CD40 and its ligand (CD40-L) were measured by Proximity Extension Assay in a well-characterized cohort of 1,144 patients with carotid atherosclerosis from the SUMMIT study (age= 69 [64 - 75], males 66.7%, T2D 66.5%). Carotid plaque area was measured by plaque contours using the ultrasound trace function. The associations were adjusted for age, sex, waist, hypertension, history of cardiovascular diseases (CVD), smoke load, plasma triglycerides, LDL-cholesterol (LDL-c) and HDL-cholesterol (HDL-c), and serum creatinine levels. All measured biomarkers were associated with carotid plaque area, independently of potential confounders, with CD40 showing the strongest effect and MMP12 showing a significant interaction with T2D. In subgroup analyses, the effect of MMP-12 and MMP-7 remained significant only in T2D, while the effect of CD40 and CD40-L was similar in both subgroups. This study demonstrates the ability of MMP-12, MMP-7, CD40, and CD40-L in predicting carotid atherosclerotic burden in a population at high cardiovascular risk and identifies MMP-12 as a specific biomarker for T2D. Disclosure S. Leonetti: None. D. Trico: None. I. Goncalves: None. A. Shore: None. F. Khan: None. J. Nilsson: None. A. Natali: None. Funding Innovative Medicines Initiative (IMI-2008/115006)

Background The relationship between growth differentiation factor 15 (GDF-15) and the development of chronic kidney disease (CKD) is still unclear. We sought to examine whether plasma GDF-15 was related to incident CKD and kidney function decline using a large prospective cohort study. Methods 4318 participants of the Malmö Diet and Cancer Study-Cardiovascular Cohort were examined in 1991-1994. Incidence of CKD was followed prospectively by linkage with national patient registers. Estimated glomerular filtration rate (eGFR) was available for all participants at baseline, and was re-measured in a subgroup of 2744 subjects after 16.6 ± 1.49 years. Incidence of CKD was examined in relation to GDF-15 using Cox regression analysis. Logistic regression was used to examine the association of GDF-15 with eGFR change and eGFR-based CKD. Models were carefully corrected for potential confounders including baseline eGFR, N-terminal pro-B-type natriuretic peptide, and competing risk from death. Results 165 patients developed CKD after 19.2 ± 4.04 years of follow-up. The adjusted hazard ratio (95% confidence interval, CI) for CKD in 4th versus 1st quartile of GDF-15 was 2.37 (1.33, 4.24) (p for trend 2 (95% CI, − 1.49 ~ − 0.45; p 2. Conclusions GDF-15 predicted incidence of CKD and eGFR decline in the general population, independent of a wide range of potential risk factors and competing risk of death.

Novel methods to characterize the plasma proteome has made it possible to examine a wide range of proteins in large longitudinal cohort studies, but the complexity of the human proteome makes it difficult to identify robust protein-disease associations. Nevertheless, identification of individuals at high risk of early mortality is a central issue in clinical decision making and novel biomarkers may be useful to improve risk stratification. With adjustment for established risk factors, we examined the associations between 138 plasma proteins measured using two proximity extension assays and long-term risk of all-cause mortality in 3,918 participants of the population-based Malmö Diet and Cancer Study. To examine the reproducibility of protein-mortality associations we used a two-step random-split approach to simulate a discovery and replication cohort and conducted analyses using four different methods: Cox regression, stepwise Cox regression, Lasso-Cox regression, and random survival forest (RSF). In the total study population, we identified eight proteins that associated with all-cause mortality after adjustment for established risk factors and with Bonferroni correction for multiple testing. In the two-step analyses, the number of proteins selected for model inclusion in both random samples ranged from 6 to 21 depending on the method used. However, only three proteins were consistently included in both samples across all four methods (growth/differentiation factor-15 (GDF-15), N-terminal pro-B-type natriuretic peptide, and epididymal secretory protein E4). Using the total study population, the C-statistic for a model including established risk factors was 0.7222 and increased to 0.7284 with inclusion of the most predictive protein (GDF-15; P

Background and Purpose:VIM (vimentin) is a cytoskeletal intermediate filament protein, which has been linked to atherosclerosis and thrombosis; both are important causes of stroke. We examined the relationship between circulating VIM and incidence of stroke, and if carotid plaque could modify the association in a prospective population-based cohort.Methods:This prospective study was based on the Malmö Diet and Cancer Cohort. A total of 4688 participants (39.7% men; mean age, 57.6 years) were examined and blood samples were collected between 1991 and 1994. Incidence of stroke was followed up to 2018. Cox’ proportional hazards regression was used to assess the relationship between VIM and stroke.Results:During a mean follow-up of 22.0 years, a total of 528 subjects were diagnosed with stroke, among which 434 were ischemic stroke. Participants in the highest quartile (vs 1stquartile) had 1.34× higher risk of total stroke (95% CI, 1.03–1.74) and 1.47× higher of ischemic stroke (95% CI, 1.10–1.98) after adjustment for potential confounders. A significant interaction was found between carotid plaque and VIM with respect to incidence of both total stroke and ischemic stroke (P=0.041 and 0.011, respectively). After stratifying by carotid plaque, high VIM had stronger association with stroke in participants with carotid plaque, especially for the risk of ischemic stroke (adjusted hazard ratio,1.66 [95% CI, 1.23–2.25] for quartile 4 versus quartile 1 to 3).Conclusions:VIM is positively associated with the incidence of stroke, especially in individuals with carotid plaque. Further studies are needed to confirm the observed associations.

Background: Acute cardiovascular events arise when the arterial wall fails to repair the damage caused by cardiovascular risk factors. Hepatocyte growth factor (HGF) stimulates growth of vascular cells but has been associated with increased risk of cardiovascular disease (CVD) in epidemiological studies. Here we tested the hypothesis that this association reflects activation of a protective vascular repair response. Methods: Small interfering RNA (siRNA) and recombinant human HGF were used to assess the role of HGF in regulation of human umbilical cord endothelial and coronary artery smooth muscle cell proliferation, migration and viability. Proximity extension assay was used to determine HGF and other biomarkers in 4742 subjects participating in the Malmo Diet and Cancer study and in extracts of 200 human carotid plaques. Findings: HGF promoted migration, proliferation and survival of human endothelial and smooth muscle cells. Human atherosclerotic plaques with a high content of HGF had a more stable phenotype. Cultured vascular cells expressed HGF in response to stress. Plasma HGF correlated with circulating levels of cellular stress biomarkers such as soluble TRAIL receptor-2 and caspase-8. Decreased ability to respond with elevated HGF levels to cellular stress, as assessed by the ratios of HGF to soluble TRAIL receptor-2 and caspase-8, associated with increased risk of cardiovascular events during a follow-up period of 19.4±5.0 years. Interpretations: These findings provide support for a protective role of HGF in CVD and show that subjects with a low ability to express HGF in response to stress have an increased risk of cardiovascular events. Funding Information: The work was supported by the Swedish Society for Medical Research, Emil and Wera Cornell foundation, Hjelt foundation, the Swedish Research Council, the Swedish Foundation for International Cooperation in Research and Higher Education, Crafoord foundation, the Swedish Medical Society, Diabetes foundation, Swedish Heart and Lung Foundation, Diabetes Research and Wellness foundation, Albert Pahlssons foundation, SUS foundations and funds, Lund University Infrastructure grant ”Malmo population-based cohorts” (STYR 2019/2046) and Lund University Diabetes Center (Swedish Research Council - Strategic Research Area Exodiab Dnr 2009-1039 and the Swedish Foundation for Strategic Research Dnr IRC15-006). Declaration of Interests: None. Ethics Approval Statement: All studies were approved by the respective Regional Ethical Review Boards and conducted in accordance with the Helsinki Declaration. All subjects gave written consent.

Importance: Human genetics and studies in experimental models support a key role of monocyte-chemoattractant protein-1 (MCP-1) in atherosclerosis. Yet, the associations of circulating MCP-1 levels with risk of coronary heart disease and cardiovascular death in the general population remain largely unexplored. Objective: To explore whether circulating levels of MCP-1 are associated with risk of incident coronary heart disease, myocardial infarction, and cardiovascular mortality in the general population. Data Sources and Selection: Population-based cohort studies, identified through a systematic review, that have examined associations of circulating MCP-1 levels with cardiovascular end points. Data Extraction and Synthesis: Using a prespecified harmonized analysis plan, study-specific summary data were obtained from Cox regression models after excluding individuals with overt cardiovascular disease at baseline. Derived hazard ratios (HRs) were synthesized using random-effects meta-analyses. Main Outcomes and Measures: Incident coronary heart disease (myocardial infarction, coronary revascularization, and unstable angina), nonfatal myocardial infarction, and cardiovascular death (from cardiac or cerebrovascular causes). Results: The meta-analysis included 7 cohort studies involving 21401 individuals (mean [SD] age, 53.7 [10.2] years; 10012 men [46.8%]). Mean (SD) follow-up was 15.3 (4.5) years (326392 person-years at risk). In models adjusting for age, sex, and race/ethnicity, higher MCP-1 levels at baseline were associated with increased risk of coronary heart disease (HR per 1-SD increment in MCP-1 levels: 1.06 [95% CI, 1.01-1.11]; P =.01), nonfatal myocardial infarction (HR, 1.07 [95% CI, 1.01-1.13]; P =.02), and cardiovascular death (HR, 1.12 [95% CI, 1.05-1.20]; P

The hepatokine follistatin is elevated in patients with type 2 diabetes (T2D) and promotes hyperglycemia in mice. Here we explore the relationship of plasma follistatin levels with incident T2D and mechanisms involved. Adjusted hazard ratio (HR) per standard deviation (SD) increase in follistatin levels for T2D is 1.24 (CI: 1.04–1.47, p, Follistatin promotes in type 2 diabetes (T2D) pathogenesis in model animals and is elevated in patients with T2D. Here the authors report that plasma follistatin associates with increased risk of incident T2D in two longitudinal cohorts, and show that follistatin regulates insulin-induced suppression lipolysis in cultured human adipocytes.

Placental growth factor (PlGF) is a mitogen for endothelial cells, but it can also act as a proinflammatory cytokine. Because it promotes early stages of plaque formation in experimental models of atherosclerosis and was implicated in epidemiological associations with risk of cardiovascular disease (CVD), PlGF has been attributed a pro-atherogenic role. Here, we investigated whether PlGF has a protective role in CVD and whether elevated PlGF reflects activation of repair processes in response to vascular stress. In a population cohort of 4742 individuals with 20 years of follow-up, high baseline plasma PlGF was associated with increased risk of cardiovascular death, myocardial infarction, and stroke, but these associations were lost or weakened when adjusting for cardiovascular risk factors known to cause vascular stress. Exposure of cultured endothelial cells to high glucose, oxidized low-density lipoprotein (LDL) or an inducer of apoptosis enhanced the release of PlGF. Smooth muscle cells and endothelial cells treated with PlGF small interference RNA demonstrated that autocrine PlGF stimulation plays an important role in vascular repair responses. High expression of PlGF in human carotid plaques removed at surgery was associated with a more stable plaque phenotype and a lower risk of future cardiovascular events. When adjusting associations of PlGF with cardiovascular risk in the population cohort for plasma soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor-2, a biomarker of cellular stress, a high PlGF/TRAIL receptor-2 ratio was associated with a lower risk. Our findings provide evidence for a protective role of PlGF in CVD.

Background Type 2 diabetes (T2D) patients are at a greater risk of cardiovascular events due to aggravated atherosclerosis. Oxidized LDL (oxLDL) has been shown to be increased in T2D plaques and suggested to contribute to plaque ruptures. Despite intensified statin treatment during the last decade the higher risk for events remains. Here, we explored if intensified statin treatment was associated with reduced oxLDL in T2D plaques and if oxLDL predicts cardiovascular events, to elucidate whether further plaque oxLDL reduction would be a promising therapeutic target. Methods Carotid plaque OxLDL levels and plasma lipoproteins were assessed in 200 patients. Plaque oxLDL was located by immunohistochemistry. Plaque cytokines, cells and scavenger receptor gene expression were quantified by Luminex, immunohistochemistry and RNA sequencing, respectively. Clinical information and events during follow-up were obtained from national registers. Results Plaque oxLDL levels correlated with markers of inflammatory activity, endothelial activation and plasma LDL cholesterol (r = 0.22-0.32 and p ≤ 0.01 for all). T2D individuals exhibited lower plaque levels of oxLDL, sLOX-1(a marker of endothelial activation) and plasma LDL cholesterol (p = 0.001, p = 0.006 and p = 0.009). No increased gene expression of scavenger receptors was identified in T2D plaques. The lower oxLDL content in T2D plaques was associated with a greater statin usage (p = 0.026). Supporting this, a linear regression model showed that statin treatment was the factor with the strongest association to plaque oxLDL and plasma LDL cholesterol (p Conclusions This study points out the importance of statin treatment in affecting plaque biology in T2D. It also implies that other biological components, beyond oxLDL, need to be identified and targeted to further reduce the risk of events among T2D patients receiving statin treatment.

BACKGROUND AND AIMS After aortic coarctation (CoA) repair, patients still suffer from cardiovascular complications. The aim of this study was to measure cardiovascular markers, intima-media thickness (IMT) and plaques in controls and patients with CoA. METHODS Sixty-four patients with CoA (66% male, mean age 48 ± 15 years) and controls (54% men, mean age 47 ± 16 years) underwent ultrasound of their arteries. A multiplex platform to analyze circulating blood levels biomarkers reflecting inflammation, tissue remodeling and repair was used. RESULTS In men following CoA repair, a significantly increased carotid bulb IMT was observed in comparison to the control group (1.05 [0.72-1.24] vs. 0.67 [0.59-0.95] mm; p = 0.003). Median common carotid artery (CCA) IMT was increased in men compared to controls (0.82 [0.61-0.97] mm vs. 0.58 [0.53-0.76] mm, p

Macrophages are a functionally heterogeneous group of immune cells abundant in atherosclerotic plaques. Macrophages expressing CD163 are associated with intraplaque hemorrhage and have previously been considered atheroprotective. However, in a recent study CD163-deficient atherosclerotic ApoE−/− mice exhibited smaller and less complex plaques, suggesting a proatherogenic role of CD163. Previous smaller studies on CD163+ macrophages and plaque stability in humans have yielded diverging results. Here we assessed the association of CD163+ cells to plaque vulnerability in a large cohort of human carotid plaques. CD163 protein expression was analyzed by immunohistochemistry in 200 human carotid plaques removed by endarterectomy from 103 patients with and 93 patients without cerebrovascular symptoms. Furthermore, CD163 mRNA expression was analyzed in 66 of the plaques. Both protein and mRNA expression of CD163 was higher in plaques from symptomatic patients and in plaques with high vulnerability index. CD163+ macrophages were primarily found in shoulder regions and in the center of the plaques. The present data show that CD163 is associated with increased plaque vulnerability in human carotid plaques, supporting the notion that CD163+ macrophages could contribute to clinical events.

Background Previous studies have proposed growth differentiation factor-15 (GDF-15) as a predictor of adverse cardiovascular outcomes and mortality. The present study aimed to determine if such associations remain after accounting for death as a competing risk, and if GDF-15 provides superior prediction performance than other biomarkers. Methods Plasma GDF-15 levels and cardiovascular risk factors were measured in individuals without cardiovascular diseases (n = 4,143, aged 57.4 ± 5.96 years, 38.6 % men) from Malmo Diet and Cancer-Cardiovascular Cohort and were followed up for more than 20 years. Incidence of coronary events, ischemic stroke, cardiovascular mortality, and all-cause mortality was studied in relation to GDF-15 using Cox proportional hazards regression, with adjustment for potential confounders. Confounding from death as competing risk was carefully checked using the Fine and Gray subdistribution hazard model. Predictive capabilities were further evaluated using C-statistics, continuous net reclassification improvement, and integrated discrimination improvement. Results During follow-up, 424 coronary events, 327 ischemic stroke, 368 cardiovascular deaths, and 1,308 all-cause deaths occurred. After controlling for death from other causes as competing events, only all-cause mortality remained significantly related to GDF-15. The addition of GDF-15 significantly improved prediction for all-cause mortality in addition to the traditional risk factors, high-sensitive C-reactive protein and N-terminal prohormone of brain natriuretic peptide. Only N-terminal prohormone of brain natriuretic peptide improved prediction for CVD mortality. Conclusions GDF-15 is a robust biomarker for all-cause mortality but less reliable for coronary event, ischemic stroke or cardiovascular mortality. Competing risk from death is an important consideration when interpreting the results.

Aims: Hypertension is the leading cause for the development of heart failure (HF). Here, we aimed to identify cardiomyocyte stretch-induced circulating biomarkers for predicting hypertension-associated HF. Methods and results: Circulating levels of 149 proteins were measured by proximity extension assay at baseline examination in 4742 individuals from the Malmo Diet and Cancer study. Protein levels were compared with stretch-activated gene expression changes in cultured neonatal rat ventricular myocytes (NRVMs) in response to 1–48 h of mechanical stretch. We also studied the association between protein levels and hypertension and HF incidence using respectively binary logistic and Cox regressions. Levels of 35 proteins were differentially expressed after Bonferroni correction in incident HF vs. control (P < 3.4E−4). Growth differentiation factor-15 (GDF-15), interleukin-6 (IL-6), IL-1 receptor type 1, and urokinase plasminogen activator surface receptor had corresponding mRNA levels up-regulated by stretch in NRVMs at all time points (P < 0.05). These four proteins were individually associated with increased risk of HF after age and sex adjustment [hazard ratio (HR) per standard deviation: 1.19 ≤ HR ≤ 1.49, P ≤ 4.90E−3]. GDF-15 and IL-6 were associated with HF independently of each other (1.22 ≤ HR ≤ 1.33, P ≤ 0.001). In subjects with hypertension, these associations remained significant after further adjustment for N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels (1.23 ≤ HR ≤ 1.45, P ≤ 0.001). A higher fasting value of a GDF-15, IL-6 score aggregate was associated with increased risk of hypertensive HF after adjustment for all traditional risk factors for HF and NT-proBNP (HR = 1.31, P = 2.19E−4). Conclusions: Cardiomyocyte mRNA levels of GDF-15 and IL-6 are consistently up-regulated by stretch, and their circulating protein levels predict HF in hypertensive subjects independently of NT-proBNP during long-term follow-up. Our results encourage further studies on lower blood pressure goals in hypertensive subjects with high GDF-15 and IL-6, and interventions targeted at stretch-induced cardiomyocyte expressed biomarkers. (Less)

Background The kidney injury molecule-1 (KIM-1) has previously been associated with kidney function in rodents and humans. Yet its role as a predictive marker for future decline in kidney function has remained less clear. Methods At baseline (1991–1994), fasting plasma KIM-1 (p-KIM-1) was measured in 4739 participants of the population-based Malmö Diet and Cancer Study. Creatinine and cystatin C were used to calculate estimated glomerular filtration rate (eGFR) according to Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) Collaboration 2012 creatinine–cystatin C equation at baseline and follow-up examination (2007–2012). Incident CKD was defined as an eGFR Results During a mean follow-up time of 16.6 years, high p-KIM-1 levels were associated with a greater decline in eGFR (quartile 1 −1.36 versus quartile 4 −1.54 mL/min/1.73 m2; P Conclusion Our results show that p-KIM-1 predicts the future decline of eGFR and risk of CKD in healthy middle-aged participants. Whether p-KIM-1 can be used to prioritize preventive action that needs to be further investigated.

Rationale: Proinflammatory cytokines have been identified as potential targets for lowering vascular risk. Experimental evidence and Mendelian randomization suggest a role of MCP-1 (monocyte chemoattractant protein-1) in atherosclerosis and stroke. However, data from large-scale observational studies are lacking. Objective: To determine whether circulating levels of MCP-1 are associated with risk of incident stroke in the general population. Methods and Results: We used previously unpublished data on 17 180 stroke-free individuals (mean age, 56.7±8.1 years; 48.8% men) from 6 population-based prospective cohort studies and explored associations between baseline circulating MCP-1 levels and risk of any stroke, ischemic stroke, and hemorrhagic stroke during a mean follow-up interval of 16.3 years (280 522 person-years at risk; 1435 incident stroke events). We applied Cox proportional-hazards models and pooled hazard ratios (HRs) using random-effects meta-analyses. After adjustments for age, sex, race, and vascular risk factors, higher MCP-1 levels were associated with increased risk of any stroke (HR per 1-SD increment in ln-transformed MCP-1, 1.07; 95% CI, 1.01–1.14). Focusing on stroke subtypes, we found a significant association between baseline MCP-1 levels and higher risk of ischemic stroke (HR, 1.11 [1.02–1.21]) but not hemorrhagic stroke (HR, 1.02 [0.82–1.29]). The results followed a dose-response pattern with a higher risk of ischemic stroke among individuals in the upper quartiles of MCP-1 levels as compared with the first quartile (HRs, second quartile: 1.19 [1.00–1.42]; third quartile: 1.35 [1.14–1.59]; fourth quartile: 1.38 [1.07–1.77]). There was no indication for heterogeneity across studies, and in a subsample of 4 studies (12 516 individuals), the risk estimates were stable after additional adjustments for circulating levels of IL (interleukin)-6 and high-sensitivity CRP (C-reactive protein). Conclusions: Higher circulating levels of MCP-1 are associated with increased long-term risk of stroke. Our findings along with genetic and experimental evidence suggest that MCP-1 signaling might represent a therapeutic target to lower stroke risk.Visual Overview: An online visual overview is available for this article.

Type 2 diabetes mellitus is associated with an elevated risk of cardiovascular disease, but the mechanism through which diabetes contributes to cardiovascular disease development remains incompletely understood. In this study, we compared the association of circulating regulatory T cells, naïve T cells, effector memory T cells or central memory T cells with cardiovascular disease in patients with and without type 2 diabetes mellitus. Percentage of circulating T cell subsets was analysed by flow cytometry in type 2 diabetes mellitus subjects with and without prevalent cardiovascular disease as well as in non-diabetic subjects with and without prevalent cardiovascular disease from the Malmö SUMMIT cohort. Subjects with type 2 diabetes mellitus had elevated percentages of effector memory T cells (CD4+CD45RO+CD62L–; 21.8% ± 11.2% vs 17.0% ± 9.2% in non-type 2 diabetes mellitus, p < 0.01) and central memory T cells (CD4+CD45RO+CD62L+; 38.0% ± 10.7% vs 36.0% ± 9.5% in non-type 2 diabetes mellitus, p < 0.01). In contrast, the frequency of naïve T cells was reduced (CD4+CD45RO–CD62L+, 35.0% ± 16.5% vs 42.9% ± 14.4% in non-type 2 diabetes mellitus, p < 0.001). The proportion of effector memory T cells was increased in type 2 diabetes mellitus subjects with cardiovascular disease as compared to those without (26.4% ± 11.5% vs 18.4% ± 10.2%, p < 0.05), while no difference in regulatory T cells was observed between these two patient groups. This study identifies effector memory T cells as a potential cellular biomarker for cardiovascular disease among subjects with type 2 diabetes mellitus, suggesting a state of exacerbated immune activation in type 2 diabetes mellitus patients with cardiovascular disease.

Aims/hypothesis Growth differentiation factor 15 (GDF-15) is an anti-inflammatory cytokine of the transforming growth factor-β superfamily. Circulating levels of GDF-15 are associated with hyperglycaemia among people with obesity or diabetes, but longitudinal evidence on the association between GDF-15 levels and diabetes risk is scarce. Our aim was to explore whether circulating levels of GDF-15 at baseline are positively associated with future diabetes incidence in a middle-aged urban population. Methods Between 1991 and 1994, baseline fasting plasma GDF-15 levels were measured in 4360 individuals without diabetes (mean age 57.4 ± 5.96 years, 38.6% men) who were participants in the Malmö Diet and Cancer–Cardiovascular Cohort. After a follow-up of 19.0 ± 5.16 years (mean ± SD), Cox proportional hazards regression analysis was used for the study of the relationship between baseline GDF-15 and incident diabetes, with adjustment for established confounders. A sensitivity analysis included further adjustment for levels of C-reactive protein (CRP). Results During the follow-up period, 621 individuals developed diabetes. The multivariate-adjusted HR for diabetes incidence was 1.43 (95% CI 1.11, 1.83; p for trend = 0.007) for the fourth compared with the first quartile of GDF-15, and was 1.17 (95% CI 1.07, 1.28; p 55 and ≤60) and >60 years, respectively. With adjustment for levels of CRP, the HR per SD increase of GDF-15 (1.21, 95% CI 1.09, 1.35) was significant (p = 0.015), but the HR for the fourth compared with the first quartile of GDF-15 was not significant (HR 1.30; 95% CI 1.01, 1.67; p for trend = 0.061). Conclusions/interpretation GDF-15 may be useful for identification of people with a risk of incident diabetes, especially if those people are ≤60 years old.

ObjectiveVascular endothelial growth factor D (VEGF-D) has important functions in lymphangiogenesis and angiogenesis. High plasma levels of VEGF-D have been associated with incidence of heart failure. The association of VEGF-D with atrial fibrillation (AF) and stroke is unclear and we hypothesised that VEGF-D could also be associated with incidence of AF and ischaemic stroke.MethodsVEGF-D was measured in fasting blood samples of 4689 subjects (40% men) without a history of AF from the Malmö Diet and Cancer Study, a prospective, population-based study in Sweden. Median age was 58 years (range 46–68). Cox regression analyses, adjusted for multiple risk factors, was used to assess AF and ischaemic stroke risk in relation to VEGF-D levels.ResultsDuring a median follow-up time of 20.6 years, there were 637 cases of incident AF and 322 cases of first ischaemic stroke. After adjustment, VEGF-D was significantly associated with AF (HR 1.13(95% CI 1.04 to 1.23) per 1 SD increase) and ischaemic stroke (HR 1.14(95% CI 1.02 to 1.28) per 1 SD). The association with ischaemic stroke was explained by an increased incidence of AF-related stroke. HRs per 1 SD were 1.34 (95% CI 1.04 to 1.71) for AF-related ischaemic stroke and 1.04 (95% CI 0.90 to 1.19) for ischaemic stroke without AF.ConclusionsIncreased VEGF-D concentrations were associated with AF and ischaemic stroke. The relationship with ischaemic stroke was more pronounced in subjects with a diagnosis of AF.

Measures to reduce the spread of COVID-19 have been associated with reduction in other respiratory infections. Results of a national Swedish cohort study of infant pertussis during April 2020–September 2021 were compared with those during January 2014–March 2020. The number of pertussis cases decreased significantly during the COVID-19 pandemic, from an average of 21 infant cases per quarter of a year before the pandemic to an average of 1 case per quarter during the pandemic. Swedish strategies to mitigate the spread of COVID-19 seem to have had an impact on pertussis incidence in infants.

Background An increased rate of cell death by apoptosis has been implicated in both diabetes and atherosclerosis. Apoptosis can be induced through activation of the death receptors TNF receptor 1 (TNFR-1), TRAIL receptor 2 (TRAILR-2) and Fas. Soluble forms of these receptors are found in plasma. The objective of this study was to determine if soluble death receptors are markers of receptor-activated apoptosis and predict risk for development of diabetes and cardiovascular events. Methods Fas ligand was used to induce apoptosis in peripheral blood mononuclear cells and INS-1 pancreatic β-cells and release of TNFR-1, TRAILR-2 and Fas measured by ELISA. Proximity Extension Assay was used to analyze plasma levels of TNFR-1, TRAILR-2 and Fas in baseline samples of 4742 subjects in the Malmö Diet and Cancer Study and related to development of diabetes and cardiovascular events during 19.2 years of follow-up. Results Activation of apoptosis by Fas ligand was associated with release of soluble Fas, TNFR-1 and TRAILR-2 in both cell types. Circulating levels of all three receptors were higher in subjects with diabetes and correlated with markers of impaired glucose metabolism in non-diabetic subjects. Among the latter, those in the highest tertile of soluble Fas, TNFR-1 and TRAILR-2 had increased risk for development of diabetes and cardiovascular events. These associations became weaker when adjusting for cardiovascular risk factors in Cox regression models, but remained significant for TRAILR-2 with incident diabetes, cardiovascular mortality, myocardial infarction and ischemic stroke, and for TNFR-1 with myocardial infarction. Conclusion The present study demonstrates an association between several cardiovascular risk factors and elevated levels of circulating markers of apoptotic cell death. It also shows that subjects with high levels of these biomarkers have increased risk of diabetes and CVD. This implies that soluble death receptors are markers of β-cell and vascular injury and potentially could be used as surrogate markers of therapeutic efficiency in risk factor interventions., Graphic Abstract Associations between metabolic stress, release of soluble death receptors and development of diabetes and cardiovascular disease.Image 1, Highlights • Receptor-activated apoptosis is associated with release of soluble death receptors that act as biomarkers of apoptosis • Several cardiovascular risk factors including markers of impaired glucose metabolism associate with elevated plasma levels of death receptors • Subjects with high plasma levels of death receptors have an increased risk of diabetes and cardiovascular disease Atherosclerosis has been proposed to develop in response to chronic arterial injury caused by cardiovascular risk factors. The present study provides clinical evidence for this hypothesis by demonstrating an association between several cardiovascular risk factors and elevated levels of circulating markers of apoptotic cell death and that subjects with high levels of these biomarkers have increased risk of cardiovascular mortality, MI and stroke. These observations point to the possibility that the plasma level of soluble death receptors can be used as surrogate markers of arterial injury and atherosclerotic disease activity in cardiovascular interventions. Finally, our findings imply that soluble death receptors also may serve as biomarkers of the damage caused by metabolic stress to β-cells and risk for development of type 2 diabetes.

Background and Purpose— ECP (eosinophil cationic protein) is a marker of eosinophil activity and degranulation, which has been linked to atherosclerosis and cardiovascular disease. We examined the relationship between ECP, carotid plaque, and incidence of stroke in a prospective population-based cohort. Methods— The subjects participated in the Malmö Diet and Cancer Study between 1991 and 1994. A total of 4706 subjects with no history of stroke were included (40% men; mean age, 57.5 years). Carotid plaque was determined by B-mode ultrasound of the right carotid artery. Incidence of stroke was followed up during a mean period of 16.5 years in relation to plasma ECP levels. Results— Subjects in the third tertile (versus first tertile) of ECP tended to have higher prevalence of carotid plaque (odds ratio: 1.18; 95% confidence interval: 1.003–1.39; P =0.044 after multivariate adjustments). A total of 258 subjects were diagnosed with ischemic stroke (IS) during follow-up. ECP was associated with increased incidence of IS after risk factor adjustment (hazard ratio, 1.57; 95% confidence interval: 1.13–2.18; for third versus first tertile; P =0.007). High ECP was associated with increased risk of IS in subjects with carotid plaque. The risk factor–adjusted hazard ratio for IS was 1.86 (95% confidence interval: 1.32–2.63) in subjects with carotid plaque and ECP in the top tertile, compared with those without plaque and ECP in the first or second tertiles. Conclusions— High ECP is associated with increased incidence of IS. The association between ECP and IS was also present in the subgroup with carotid plaque.

Background Oxidation of LDL particles entrapped in the extracellular matrix of the arterial wall is a key factor in the development of atherosclerosis. Lipid oxidation products, such as malondialdehyde (MDA), react with surrounding extracellular matrix proteins and cause modifications that are recognized by the immune system. MDA modification of collagen type IV is increased in carotid lesions from symptomatic patients and correlates with autoantibodies against MDA-modified collagen type IV in plasma. Objective The aim of this study was to determine whether autoantibodies against MDA-modified collagen type IV predict risk of development of myocardial infarction (MI). Methods Plasma levels of MDA-modified collagen type IV IgM and IgG antibodies were analysed by enzyme-linked immunosorbent assay in 385 subjects with incident MI during 13 years of follow-up and 410 age- and sex-matched controls in the Malmo Diet and Cancer study. Results MDA-modified collagen type IV IgG levels were higher in cases with incident MI than in controls. Subjects in the highest tertile of MDA-modified collagen type IV IgG had an increased risk of MI (hazard ratio 1.56, 95% confidence interval 1.22–2.00, P for trend 0.0004). This association remained significant after adjusting for factors included in the Framingham risk score and diabetes. High levels of MDA-collagen type IV IgG were associated with increased carotid intima-media thickness and elevated plasma levels of matrix metalloproteinase 10 and 12. Conclusions Immune responses against MDA-modified collagen type IV are associated with more severe carotid disease and increased risk of MI. These immune responses may reflect LDL oxidation in the artery wall, but could also affect the atherosclerotic disease process.

Individually consistent behavioral and physiological responses to stressful situations (often referred to as coping styles) has been reported in many animal species. Differences in hypothalamic-pituitary axis reactivity characterize individuals, and it has been proposed that the glucocorticoid (gr) and mineralocorticoid (mr) receptors are fundamental in regulating coping styles. We sorted individuals into reactive and proactive coping styles by collapsing behavioral outputs from net restraint and confinement stress tests in a principal component analysis. We then analyzed plasma cortisol levels, serotonin neurochemistry and the relative mRNA expression of gr1 and mr in stressed individuals per coping style. Proactive fish were characterized as having a lower serotonergic activity and being more active under stress. In addition, proactive fish had higher hypothalamic gr1 and mr abundance and a higher mr/gr1 ratio, compared to reactive fish. We found no significant differences in cortisol or telencephalic mRNA, gr1 and mr expression, or their ratio. Brain MR and GR have been proven to have an important role in the appraisal, coping and adaptation to stressful stimuli, so that a higher expression of these receptors in proactive fish suggests increased tolerance and performance under stress, compared to reactive individuals. We present evidence of a conserved neuroendocrine mechanism associated with coping styles in a fish species which is ecologically very diverse and considered to be the most cold-adapted fish in freshwater. We propose that this may be a first step into exploiting this model in order to better understand climate-change related effects in sub populations and ecophenotypes.

Objective— To investigate the relationship between 3 markers of apoptosis, that is, FADD (Fas-associated death domain–containing protein), caspase-3, and caspase-8, and incidence of coronary events (CEs) in a population-based cohort study. Approach and Results— In vitro experiments were performed to assess the response of the apoptotic biomarkers after Fas stimulation of peripheral blood mononuclear cells. The experiments showed significantly increased releases of FADD, caspase-3, and caspase-8 after Fas stimulation. The relationship between FADD, caspase-3, and caspase-8, respectively, and incidence of CEs was studied in 4284 subjects from the population-based Malmö Diet and Cancer Study. Cox’ proportional hazards regression was used to examine the association between the apoptotic biomarkers and incidence of CE over a mean follow-up of 19 years. A total of 381 individuals had CE during the follow-up. High FADD at baseline was significantly associated with incident CE. In the highest compared with the lowest quartile of FADD, the risk factor adjusted hazards ratio for CE was 1.82 (95% confidence interval, 1.35–2.46; P for trend P for trend Conclusions— High levels of FADD and caspase-8, but not caspase-3, were associated with increased incidence of CE in subjects from the general population. The in vitro experiments support the view that these biomarkers could reflect activation of the extrinsic apoptotic pathway.

Life style changes and improved medical therapy have decreased cardiovascular mortality in many countries over the last decades. This has been accompanied by changes in disease characteristics including more non-ST segment elevation myocardial infraction and less vulnerable plaques as assessed by histological analysis of surgical specimens. However, many patients with established disease still suffer from recurrent cardiovascular events in spite of treatment with state-of-the-art-therapy including statins. It is likely that this reflects a state of the disease in which statins control the pro-inflammatory effects of lipids allowing other statin-unresponsive disease mechanisms to become increasingly important. If this assumption is correct it means that patients with established disease with time will get insuffient protection by current therapies alone. Against this background it is critical to reach a better understanding of alternative mechanisms for plaque vulnerability. Examples of such mechanisms include altered patterns of blood flow caused by plaque stenosis resulting in down-regulation of the anti-inflammatory and anti-thrombotic signals in the endothelium, impaired vascular repair associated with diabetes and plaque inflammation driven by cholesterol crystals, infectious pathogens as well as autoimmune responses against modified plaque components. Novel biomarkers and other diagnostics are needed to establish the clinical importance of these mechanisms as well as to determine how they are affected by current treatments. Consequently, there will also be a need for development of new treatments targeting these mechanisms and that can act in concert with current therapies.

The notion that atherosclerosis can be prevented or mitigated by vaccination is now moving towards clinical trials. This strategy is based on the existence of autoimmunity to LDL, the cholesterol-carrying particles that accumulate in arteries. In this Comment, we discuss the underlying concepts, research basis and challenges for the development of a vaccine against atherosclerosis.

Background High red cell distribution width (RDW) has been increasingly recognized as a risk factor for cardiovascular diseases (CVDs), but the underlying mechanisms remain unknown. Our aim was to explore the associations between RDW and plasma proteins implicated in the pathogenesis of CVD using a targeted proteomics panel. Methods RDW and 88 plasma proteins were measured in a population-based cohort study (n = 4726), Malmö Diet and Cancer-Cardiovascular Cohort (MDC-CC). A random 2/3 of the cohort was used as discovery sample and remaining 1/3 was used for replication. Multiple linear regression was used to assess the associations between RDW and plasma proteins, with adjustments for age, sex, and other potential confounders. Proteins with Bonferroni-corrected significant associations with RDW in the discovery sub-cohort were validated in the replication cohort. Results Thirteen of 88 plasma proteins had significant associations with RDW in the discovery sample, after multivariate adjustments. Eleven of them were also significant in the replication sample, including SIR2-like protein 2 (SIRT2), stem cell factor (SCF, inversely), melusin (ITGB1BP2), growth differentiation factor-15 (GDF-15), matrix metalloproteinase-7 (MMP-7), hepatocyte growth factor (HGF), chitinase-3-like protein 1 (CHI3L1), interleukin-8 (IL-8), CD40 ligand (CD40-L), urokinase plasminogen activator surface receptor (U-PAR) and matrix metalloproteinase-3 (MMP-3). Conclusions Several proteins from this targeted proteomics panel were associated with RDW in this cohort. These proteins could potentially be linked to the increased cardiovascular risk in individuals with high RDW.

Background and aims: A vulnerable plaque is an atherosclerotic plaque that is rupture-prone with a higher risk to cause cardiovascular symptoms such as myocardial infarction or stroke. Mimecan or osteoglycin is a small leucine-rich proteoglycan, important for collagen fibrillogenesis, that has been implicated in atherosclerotic disease, yet the role of mimecan in human atherosclerotic disease remains unknown. Methods: 196 human atherosclerotic carotid plaques were immunostained for mimecan. Smooth muscle cells, macrophages and intraplaque haemorrhage were also measured with immunohistochemistry. Neutral lipids were stained with Oil Red O and calcium deposits were quantified. Plaque homogenate levels of MCP-1, IL-6 and MIP-1β were measured using a Proximity Extension Assay and MMP-9 levels were measured using Mesoscale. Glycosaminoglycans, collagen and elastin were assessed by colorimetric assays and TGF-β1, β2 and β3 were measured using a multiplex assay. Mimecan gene expression in THP-1 derived macrophages was quantified by qPCR and protein expression in vitro was visualized with immunofluorescence. Cardiovascular events were registered using medical charts and national registers during follow-up. Results: Mimecan correlated positively with plaque area of lipids, macrophages, intraplaque haemorrhage and inversely with smooth muscle cell staining. Mimecan also correlated positively with plaque levels of MMP-9 and MCP-1. Mimecan was upregulated in THP-1 derived macrophages upon stimulation with MCP-1. Patients with high levels of mimecan (above median) had higher risk for cardiovascular death. Conclusions: This study indicates that mimecan is associated with a vulnerable plaque phenotype, possibly regulated by plaque inflammation. In line, plaque levels of mimecan independently predict future cardiovascular death. (Less)

Purpose It is unclear whether plasma homocysteine (Hcy) has a direct noxious impact on the cardiovascular (CV) system or whether its association with cardiovascular events (CVEs) is mediated by established risk factors. To explore the role of Hcy in CV impairment, the study evaluated cross-sectional relationships between plasma Hcy and indices of CV organ damage together with the associations of these indices with the history of CVEs. Methods In 269 patients with a high prevalence of diabetes, dyslipidemia, and hypertension, the carotid intima-media thickness, ankle–brachial index (ABI), reactive hyperemic index, carotid–femoral pulse wave velocity (cfPWV), left ventricular (LV) mass, and cardiac index were measured. Results 132 patients had carotid plaque, 31 ABI Conclusion This study demonstrated an independent association between Hcy and LV mass as well as between LVH and a history of CVEs and suggests that LVH may represent 1 of the pathophysiologic links between Hcy and CV risk.

Circulating proteins are vital in human health and disease and are frequently used as biomarkers for clinical decision-making or as targets for pharmacological intervention. Here, we map and replicate protein quantitative trait loci (pQTL) for 90 cardiovascular proteins in over 30,000 individuals, resulting in 451 pQTLs for 85 proteins. For each protein, we further perform pathway mapping to obtain trans-pQTL gene and regulatory designations. We substantiate these regulatory findings with orthogonal evidence for trans-pQTLs using mouse knockdown experiments (ABCA1 and TRIB1) and clinical trial results (chemokine receptors CCR2 and CCR5), with consistent regulation. Finally, we evaluate known drug targets, and suggest new target candidates or repositioning opportunities using Mendelian randomization. This identifies 11 proteins with causal evidence of involvement in human disease that have not previously been targeted, including EGF, IL-16, PAPPA, SPON1, F3, ADM, CASP-8, CHI3L1, CXCL16, GDF15 and MMP-12. Taken together, these findings demonstrate the utility of large-scale mapping of the genetics of the proteome and provide a resource for future precision studies of circulating proteins in human health.

AIM: To assess self-reported clinical competence and the need for further training among newly graduated registered nurses (NGRNs) working in Swedish acute care hospital settings. BACKGROUND: NGRNs are expected to take full responsibility for patients' nursing care in an increasingly complex clinical context, and professional nurses' clinical competence is critical in providing high-quality and safe nursing care. DESIGN: A cross-sectional design. METHODS: Data were collected using the 50-item ProffNurse SAS II. A total of 85 NGRNs who had recently commenced working with direct patient care at three hospitals in central Sweden participated in the study. The response rate was 69%. The STROBE cross-sectional reporting guidelines were used. RESULTS: The NGRNs assessed their clinical competence as being highest in areas relating to team collaboration and ethics and lowest in areas relating to professional development and direct clinical practice. The need for further training was greatest in areas such as direct clinical practice and patient safety and lowest in areas such as team collaborating and ethics. CONCLUSION: The use of instruments to identify NGRNs' self-assessed clinical competence is of value when designing and evaluating introductory programmes for NGRNs taking on positions in acute care hospital settings. The availability of experienced nurses from whom NGRNs can gain clinical competence and learn from is of importance, both from the perspective of the NGRNs themselves and patient safety. RELEVANCE TO CLINICAL PRACTICE: An understanding of NGRNs' clinical competence and their need for further training may assist in both planning and organising nursing programmes and in making clinical policy decisions when designing introduction programmes in acute care settings.

Aim To explore and describe changes in self‐assessed clinical competence and the need for further training among newly graduated Registered Nurses during their first 15 months of professional work in acute care hospital settings. Design Quantitative longitudinal design. Methods The 50‐item Professional Nurse Self‐Assessment Scale of clinical core competencies II was used. A total of 45 newly graduated Registered Nurses answered the questionnaire at four different occasions. Data were collected after 2, 5, 9 and 15 months of working experience. Result The components “ethical decision‐making,” “cooperation and consultation” and “clinical leadership” were rated highest in clinical competence and lowest in need for further training. The components “professional development” and “critical thinking” were rated lowest in clinical competence and “direct clinical practice” rated highest in need for further training. The clinical competence increased significant between 9–15 months, with the exception of “critical thinking” and need for further training decreased significantly between 9–15 months, with the exception of “critical thinking.”

Background and Purpose— Extracellular matrix proteins are important in atherosclerotic disease by influencing plaque stability and cellular behavior but also by regulating inflammation. COMP (cartilage oligomeric matrix protein) is present in healthy human arteries and expressed by smooth muscle cells. A recent study showed that transplantation of COMP-deficient bone marrow to apoE −/− mice increased atherosclerotic plaque formation, indicating a role for COMP also in bone marrow–derived cells. Despite the evidence of a role for COMP in murine atherosclerosis, knowledge is lacking about the role of COMP in human atherosclerotic disease. Methods— In the present study, we investigated if COMP was associated with a stable or a vulnerable human atherosclerotic plaque phenotype by analyzing 211 carotid plaques for COMP expression using immunohistochemistry. Results— Plaque area that stained positive for COMP was significantly larger in atherosclerotic plaques associated with symptoms (n=110) compared with asymptomatic plaques (n=101; 9.7% [4.7–14.3] versus 5.6% [2.8–9.8]; P =0.0002). COMP was positively associated with plaque lipids ( r =0.32; P =0.000002) and CD68 cells ( r =0.15; P =0.036) but was negatively associated with collagen ( r =−0.16; P =0.024), elastin ( r =−0.14; P =0.041), and smooth muscle cells ( r =−0.25; P =0.0002). COMP was positively associated with CD163 ( r =0.37; P =0.00000006), a scavenger receptor for hemoglobin/haptoglobin and a marker of Mhem macrophages, and with intraplaque hemorrhage, measured as glycophorin A staining ( r =0.28; P =0.00006). Conclusions— The present study shows that COMP is associated to symptomatic carotid atherosclerosis, CD163-expressing cells, and a vulnerable atherosclerotic plaque phenotype in humans.

Background Cadmium is a toxic metal with multiple adverse health effects, including risk of cardiovascular disease (CVD). The mechanistic link between cadmium and CVD is unclear. Our aim was to examine the associations between blood cadmium (B-Cd) and 88 potential protein biomarkers of CVD. Methods B-Cd and 88 plasma proteins were measured in a community-based prospective cohort, the Malmö Diet and Cancer study. The primary analysis was performed in never smokers (n = 1725). Multiple linear regression was used with adjustments for age and sex, and correction for multiple comparisons using the false discovery rate method. Proteins significantly associated with B-Cd were replicated in long-term former smokers (n = 782). Significant proteins were then studied in relation to incidence of CVD (i.e., coronary events or ischemic stroke) in never smokers. Results Fifteen proteins were associated with B-Cd in never smokers. Eight of them were replicated in long-term former smokers. Kidney injury molecule-1, fibroblast growth factor-23 (FGF23), tumor necrosis factor receptor-2, matrix metalloproteinase-12, cathepsin L1, urokinase plasminogen activator receptor, C-C motif chemokine-3 (CCL3), and chemokine (C-X3-C motif) ligand-1 were associated with B-Cd both in never smokers and long-term former smokers. Except for CCL3 and FGF23, these proteins were also significantly associated with incidence of CVD. Conclusions B-Cd in non-smokers was associated with eight potential plasma biomarkers of CVD and kidney injury. The results suggest pathways for the associations between B-Cd and CVD and kidney injury.