Saskia Haitjema

Human Validation of Genes Associated With a Murine Atherosclerotic Phenotype

Objective— The genetically modified mouse is the most commonly used animal model for studying the pathogenesis of atherosclerotic disease. We aimed to assess if mice atherosclerosis-related genes could be validated in human disease through examination of results from genome-wide association studies. Approach and Results— We performed a systematic review to identify atherosclerosis-causing genes in mice and carried out gene-based association tests of their human orthologs for an association with human coronary artery disease and human large artery ischemic stroke. Moreover, we investigated the association of these genes with human atherosclerotic plaque characteristics. In addition, we assessed the presence of tissue-specific cis-acting expression quantitative trait loci for these genes in humans. Finally, using pathway analyses we show that the putative atherosclerosis-causing genes revealed few associations with human coronary artery disease, large artery ischemic stroke, or atherosclerotic plaque characteristics, despite the fact that the majority of these genes have cis-acting expression quantitative trait loci. Conclusions— A role for genes that has been observed in mice for atherosclerotic lesion development could scarcely be confirmed by studying associations of disease development with common human genetic variants. The value of murine atherosclerotic models for selection of therapeutic targets in human disease remains unclear.

Patients with diabetes differ in atherosclerotic plaque characteristics and have worse clinical outcome after iliofemoral endarterectomy compared with patients without diabetes

Objective: Diabetes mellitus (DM) is associated with peripheral arterial disease (PAD) and leads to worse clinical outcome compared with patients without DM. The objective of this study was to determine the impact of DM on iliofemoral artery plaque characteristics and to examine secondary clinical outcomes in patients with DM and PAD undergoing surgical revascularization. Methods: We analyzed 198 patients with and 453 patients without DM from the Athero‐Express biobank, a prospective ongoing biobank study, who underwent endarterectomy of the femoral or iliac artery between 2002 and 2013. Seven histologic plaque characteristics (calcification, collagen, lipid core, intraplaque hemorrhage, macrophages, microvessels, and smooth muscle cells) and secondary clinical outcome were compared. Composite outcome consisted of any of the following secondary manifestations of cardiovascular disease: stroke, myocardial infarction, cardiovascular death, or peripheral intervention. In addition, target vessel revascularization (TVR) was examined. The follow‐up period was standardized at 3 years after the procedure. Results: Patients with DM were more likely to have calcified plaques compared with patients without DM (odds ratio, 2.11; 95% confidence interval, 1.43‐3.12; P < .01). No other plaque characteristic differed significantly between the two groups. In total, 112 (57.1%) patients with DM and 198 (45.1%) patients without DM reached a composite end point during follow‐up, of whom 21 (10.7%) and 27 (6.2%) died of cardiovascular causes, respectively. DM was an independent predictor of composite cardiovascular events (hazard ratio, 1.36; 95% confidence interval, 1.020‐1.801; P = .01) during follow‐up. No difference in the incidence of TVR was observed between patients with and without DM (31.5% and 30%, respectively; difference in survival time, P = .86) or in longer duration of DM with composite event‐free survival (difference in survival time, P = .57). Conclusions: Patients with DM who undergo surgical revascularization for PAD with the use of thromboendarterectomy or remote endarterectomy have a more calcified atherosclerotic plaque and an increased incidence in composite cardiovascular events but no increase in TVR.

Growth Differentiation Factor 15 Is Associated With Major Amputation and Mortality in Patients With Peripheral Artery Disease.

Background Peripheral artery disease (PAD) is one of the most common clinical presentations of atherosclerosis, and its prevalence is still increasing. Despite improvement of health care, morbidity and mortality risks remain high, including the risk of amputation. GDF15 (growth differentiation factor 15) is a member of the transforming growth factor family that is involved in apoptosis and inflammation; therefore, GDF15 is a potential biomarker to identify patients at high risk of adverse clinical outcomes. Methods and Results Circulating GDF15 levels were measured using a multiplex immunoassay in patients with critical limb ischemia and PAD from 2 different patient cohorts that included patients with clinically manifest PAD: the JUVENTAS (Rejuvenating Endothelial Progenitor Cells via Transcutaneous Intra‐Arterial Supplementation) trial (n=160, 67 major events; critical limb ischemia) and the Athero‐Express Biobank (n=386, 64 major events; PAD). Kaplan–Meier curves demonstrated that high levels of GDF15 were associated with increased risk of major events, defined as major amputation (at or above the ankle joint) and all‐cause mortality, in both cohorts (highest versus lowest, JUVENTAS: hazard ratio: 4.01 [95% confidence interval, 2.05–7.84; P<0.0001]; Athero‐Express: hazard ratio: 3.27 [95% confidence interval, 1.64–6.54; P=0.0008]). In the JUVENTAS trial, this was more pronounced in women. Cox proportional multivariable regression models with median follow‐up of 3 years, corrected for common confounders, showed hazard ratios of 1.70 (95% confidence interval, 1.18–2.69; P=0.0053) and 1.57 (95% confidence interval, 1.02–2.41; P=0.041) per 2.78‐fold increase of GDF15 in JUVENTAS and Athero‐Express, respectively. Conclusions High GDF15 levels are associated with increased risk of major amputation and/or death in PAD patients. GDF15 levels could be of additive value to identify patients who are at high risk of amputation or death and could help guide treatment choices.

Circulating GDF-15 levels predict future secondary manifestations of cardiovascular disease explicitly in women but not men with atherosclerosis

BACKGROUND Elevated serum levels of growth differentiation factor-15 (GDF-15), is an established risk factor for a range of cardiovascular diseases. We aimed to evaluate the predictive value of plasma GDF-15 as a biomarker for secondary cardiovascular events (CVE) in patients with atherosclerosis undergoing carotid endarterectomy (CEA). Secondly, we determined whether plasma GDF-15 was associated with carotid plaque characteristics. METHODS Circulating GDF-15 levels were determined by Luminex assay in a cohort of 1056 patients from the Athero-Express biobank. Composite endpoint was defined as major CVE, death and peripheral vascular interventions. Findings were validated in 473 patients from the independent Carotid Plaque Imaging Project biobank. RESULTS GDF-15 levels did not associate with secondary CVE in the total cohort. However, following a significant interaction with sex, it was found to be strongly, independently predictive of secondary CVE in women but not men (quartile 4 vs. quartile 1: HR 3.04 [95% CI 1.35-6.86], p=0.007 in women vs. HR 0.96 [95% CI 0.66-1.40], p=0.845 in men). This was also observed in the validation cohort (women: HR 2.28 [95% CI 1.04-5.05], p=0.041), albeit dependent upon renal function. In addition, GDF-15 was associated with the presence of plaque smooth muscle cells and calcification. CONCLUSION High circulating GDF-15 levels are predictive of secondary CVE in women but not in men with carotid atherosclerotic disease undergoing CEA, suggesting a potential use for GDF-15 as a biomarker for secondary prevention in women. Sex differences in the role of GDF-15 in atherosclerotic disease deserve further interest.

Loss of Y Chromosome in Blood Is Associated With Major Cardiovascular Events During Follow-Up in Men After Carotid Endarterectomy

Background— Recent studies found an immune regulatory role for Y chromosome and a relationship between loss of Y chromosome (LOY) in blood cells and a higher risk of cancer and mortality. Given involvement of immune cells in atherosclerosis, we hypothesized that LOY is associated with the severity of atherosclerotic plaque characteristics and outcome in men undergoing carotid endarterectomy. Methods and Results— LOY was quantified in blood and plaque from raw intensity genotyping data in men within the Athero-Express biobank study. Plaques were dissected, and the culprit lesions used for histology and the measurement of inflammatory proteins. We tested LOY for association with (inflammatory) atherosclerotic plaque phenotypes and cytokines and assessed the association of LOY with secondary events during 3-year follow-up. Of 366 patients with carotid endarterectomy, 61 exhibited some degree of LOY in blood. LOY was also present in atherosclerotic plaque lesions (n=8/242, 3%). LOY in blood was negatively associated with age (&bgr;=−0.03/10 y; r2=0.07; P=1.6×10–7) but not with cardiovascular disease severity at baseline. LOY in blood was associated with a larger atheroma size (odds ratio, 2.15; 95% confidence interval, 1.06–4.76; P=0.04); however, this association was not significant after correction for multiple testing. LOY was independently associated with secondary major cardiovascular events (hazard ratio=2.28; 95% confidence interval, 1.11–4.67; P=0.02) in blood when corrected for confounders. Conclusions— In this hypothesis-generating study, LOY in blood is independently associated with secondary major cardiovascular events in a severely atherosclerotic population. Our data could indicate that LOY affects secondary outcome via other mechanisms than inflammation in the atherosclerotic plaque.

Additional Candidate Genes for Human Atherosclerotic Disease Identified Through Annotation Based on Chromatin OrganizationCLINICAL PERSPECTIVE

Background— As genome-wide association efforts, such as CARDIoGRAM and METASTROKE, are ongoing to reveal susceptibility loci for their underlying disease—atherosclerotic disease—identification of candidate genes explaining the associations of these loci has proven the main challenge. Many disease susceptibility loci colocalize with DNA regulatory elements, which influence gene expression through chromatin interactions. Therefore, the target genes of these regulatory elements can be considered candidate genes. Applying these biological principles, we used an alternative approach to annotate susceptibility loci and identify candidate genes for human atherosclerotic disease based on circular chromosome conformation capture followed by sequencing. Methods and Results— In human monocytes and coronary endothelial cells, we generated 63 chromatin interaction data sets for 37 active DNA regulatory elements that colocalize with known susceptibility loci for coronary artery disease (CARDIoGRAMplusC4D) and large artery stroke (METASTROKE). By circular chromosome conformation capture followed by sequencing, we identified a physical 3-dimensional interaction with 326 candidate genes expressed in at least 1 of these cell types, of which 294 have not been reported before. We highlight 16 genes based on expression quantitative trait loci. Conclusions— Our findings provide additional candidate-gene annotation for 37 disease susceptibility loci for human atherosclerotic disease that are of potential interest to better understand the complex pathophysiology of cardiovascular diseases.

Time-dependent differences in femoral artery plaque characteristics of peripheral arterial disease patients

BACKGROUND AND AIMS Peripheral arterial disease (PAD) is a common manifestation of atherosclerosis with an increasing incidence worldwide. The disease is still associated with high morbidity and mortality risks. Previous research in carotid arteries indicates that atherosclerotic plaque characteristics have stabilized over time in patients considered for surgery. It is currently unknown whether this time-dependent stabilization occurs in ilio-femoral arteries as well. Our objective was to analyze whether local ilio-femoral atherosclerotic plaque characteristics have changed over time. METHODS 497 patients within the Athero-Express biobank who underwent primary endarterectomy of the iliac or femoral artery between 2002 and 2013 were analyzed. We investigated six histological plaque characteristics: calcification, collagen, fat content, intraplaque haemorrhage, macrophages and smooth muscle cells. RESULTS Over the course of 10 years, we observed a lower percentage of all plaque characteristics that are considered indicators of a vulnerable plaque, such as: plaques with a large lipid core from 37.9% to 14.9% and plaques with intraplaque haemorrhage from 69.0% to 34.8% when the two-year cohorts 2003-2004 and 2011-2012 were compared, respectively. Multivariable analyses showed that time-dependent changes occurred independently of changing procedural and patient characteristics. CONCLUSIONS In this cohort of peripheral arterial disease patients undergoing primary endarterectomy, we observed a time dependent shift of plaque characteristics towards a less lipid rich lesion with less intraplaque haemorrhage. These findings indicate research in cardiovascular disease would benefit from contemporary patient characteristics and plaque specimens to optimize translational potential.

Time‐dependent trends in cardiovascular adverse events during follow‐up after carotid or iliofemoral endarterectomy

Recent observations have suggested a decline in vulnerable carotid artery and iliofemoral atherosclerotic plaque characteristics over the past decade. The aim of this study was to determine whether, in the presence of clinically manifest carotid or peripheral artery disease, secondary adverse cardiovascular events decreased over this period.

High Levels of (Un)Switched Memory B Cells Are Associated With Better Outcome in Patients With Advanced Atherosclerotic Disease

Background Atherosclerosis is an inflammatory lipid disorder and the main underlying pathology of acute ischemic events. Despite a vast amount of data from murine atherosclerosis models, evidence of B‐cell involvement in human atherosclerotic disease is limited. We therefore investigated the association of circulating B‐cell subtypes with the occurrence of secondary cardiovascular events in advanced atherosclerotic disease. Methods and Results This cohort study consists of 168 patients who were included in the Athero‐Express biobank between 2009 and 2011. Before surgery, peripheral blood mononuclear cells were isolated and stored in liquid nitrogen. After gentle thawing of the peripheral blood mononuclear cells, different B‐cell subtypes including naïve, (un)switched memory, and CD27+ CD43+ B1‐like B cells, were analyzed by flow cytometry. Univariable and multivariable Cox proportional hazard models were used to analyze associations between B‐cell subtypes, circulating antibodies and secondary cardiovascular manifestations during the 3‐year follow‐up period. Mean age was 70.1±9.6 years, males represented 62.8% of the population, and 54 patients had secondary manifestations during follow‐up. High numbers of unswitched memory cells were protective against secondary outcome (hazard ratio, 0.30 [95% CI, 0.13–0.69]; P<0.01). Similar results were obtained for the switched memory cells that also showed to be protective against secondary outcome (hazard ratio, 0.33 [95% CI, 0.14–0.77]; P=0.01). Conclusions A high number of (un)switched memory B cells is associated with better outcome following carotid artery endarterectomy. These findings suggest a potential role for B‐cell subsets in prediction and prevention of secondary cardiovascular events in patients with atherosclerosis.

The impact of female sex on long-term survival of patients with severe atherosclerosis undergoing endarterectomy.

OBJECTIVES Long-term age- and sex-specific mortality data in patients undergoing carotid endarterectomy (CEA) and iliac/femoral endarterectomy (FEA) are scarce. We examined long-term mortality in these patient groups, stratified by age and sex. METHODS Between 2002 and 2012, 1771 patients (1200 men, 571 women) treated by CEA, and 685 patients (495 men, 190 women) who underwent FEA, were included and linked to the national mortality registry of the Netherlands. Absolute mortality risks during follow-up were analyzed by life-table and Kaplan Meier survival analyses in two age groups and stratified by sex, and compared to a matched sample from the general population. In addition, multivariable Cox regression analyses were performed. RESULTS After CEA, with a median follow-up duration of 4.3 years (interquartile range 2.0-7.1), 298 all-cause deaths had occurred in men (25%) and 105 (18%) in women. As in the general population, cumulative survival after CEA was significantly better in women compared to men (P = 0.002) and absolute CEA-associated mortality risk in women was similar to that of the general population. For FEA patients, mortality risk was worse than for CEA patients and the general population in both sexes and surprisingly, female sex did not have a favorable effect on survival. Following FEA, 130 men (26%) and 51 women (27%) died after a median follow-up time of 3.0 years (interquartile range 1.5-5.9). Stratifying by age, and adjusting for cardiovascular risk factors did not change these trends. CONCLUSIONS Long-term mortality after CEA is higher in men than in women, and in women mortality risk is similar to the general population. After FEA, the benefit of women as seen after CEA is lost.