Common Variants Associated With OSMR Expression Contribute to Carotid Plaque Vulnerability, but Not to Cardiovascular Disease in Humans.

Background and Aims: Oncostatin M (OSM) signaling is implicated in atherosclerosis, however the mechanism remains unclear. We investigated the impact of common genetic variants in OSM and its receptors, OSMR and LIFR, on overall plaque vulnerability, plaque phenotype, intraplaque OSMR and LIFR expression, coronary artery calcification burden and cardiovascular disease susceptibility. Methods and Results: We queried Genotype-Tissue Expression data and found that rs13168867 (C allele) was associated with decreased OSMR expression and that rs10491509 (A allele) was associated with increased LIFR expression in arterial tissues. No variant was significantly associated with OSM expression. We associated these two variants with plaque characteristics from 1,443 genotyped carotid endarterectomy patients in the Athero-Express Biobank Study. After correction for multiple testing, rs13168867 was significantly associated with an increased overall plaque vulnerability (ß = 0.118 ± s.e. = 0.040, p = 3.00 × 10-3, C allele). Looking at individual plaque characteristics, rs13168867 showed strongest associations with intraplaque fat (ß = 0.248 ± s.e. = 0.088, p = 4.66 × 10-3, C allele) and collagen content (ß = -0.259 ± s.e. = 0.095, p = 6.22 × 10-3, C allele), but these associations were not significant after correction for multiple testing. rs13168867 was not associated with intraplaque OSMR expression. Neither was intraplaque OSMR expression associated with plaque vulnerability and no known OSMR eQTLs were associated with coronary artery calcification burden, or cardiovascular disease susceptibility. No associations were found for rs10491509 in the LIFR locus. Conclusions: Our study suggests that rs1316887 in the OSMR locus is associated with increased plaque vulnerability, but not with coronary calcification or cardiovascular disease risk. It remains unclear through which precise biological mechanisms OSM signaling exerts its effects on plaque morphology. However, the OSM-OSMR/LIFR pathway is unlikely to be causally involved in lifetime cardiovascular disease susceptibility.

The age- and sex-specific composition of atherosclerotic plaques in vascular surgery patients.

BACKGROUND AND AIMS: The sex- and age-related differences in the composition of iliofemoral atherosclerotic plaques are largely unknown. Therefore, the aim of the current study is to gain insight into plaque composition across strata of age and sex in a large cohort of vascular surgery patients. METHODS: Peripheral atherosclerotic plaques of patients who underwent iliofemoral endarterectomy (n = 790) were harvested between 2002 and 2014. The plaques were semi-quantitatively analyzed for the presence of lipid cores, calcifications, plaque hemorrhages (PH), collagen, macrophage and smooth muscle cell (SMC) content, and quantitatively for microvessel density. Patients were stratified by age tertiles and sex. RESULTS: Ageing was independently associated with rupture-prone iliofemoral plaque characteristics, such as higher prevalence of plaque calcifications (OR 1.52 (95%CI:1.03-2.24) p = 0.035) and PH (OR 1.46 (95%CI:1.01-2.09) p = 0.042), and lower prevalence of collagen (OR 0.52 (95%CI:0.31-0.86) p = 0.012) and SMCs (OR 0.59 (95%CI:0.39-0.90) p = 0.015). Sex-stratified data showed that men had a higher prevalence of lipid cores (OR 1.62 (95%CI:1.06-2.45) p = 0.025) and PH (OR 1.62 (95%CI:1.16-2.54) p = 0.004) compared to women. These sex-differences attenuated with increasing age, with women showing an age-related increase in calcifications (p = 0.002), PH (p = 0.015) and decrease in macrophages (p = 0.005). In contrast, men only showed a decrease in collagen (p = 0.043). CONCLUSIONS: Atherosclerotic iliofemoral plaques derived from men display more rupture-prone characteristics compared to women. Yet, this difference is attenuated with an increase in age, with older women having more rupture-prone characteristics compared to younger women.

Cerebral Small Vessel Disease in Standard Pre-operative Imaging Reports Is Independently Associated with Increased Risk of Cardiovascular Death Following Carotid Endarterectomy.

OBJECTIVE: Cerebral white matter lesions (WMLs) and lacunar infarcts are surrogates of cerebral small vessel disease (SVD). WML severity as determined by trained radiologists predicts post-operative stroke or death in patients undergoing carotid endarterectomy (CEA). It is unknown whether routine pre-operative brain imaging reports as part of standard clinical practice also predict short and long term risk of stroke and death after CEA. METHODS: Consecutive patients from the Athero-Express biobank study that underwent CEA for symptomatic high degree stenosis between March 2002 and November 2014 were included. Pre-operative brain imaging (computed tomography [CT] or magnetic resonance imaging [MRI]) reports were reviewed for reporting of SVD, defined as WMLs or any lacunar infarcts. The primary outcome was defined as any stroke or any cardiovascular death over three year follow up. The secondary outcome was defined as the 30 day peri-operative risk of stroke or cardiovascular death. RESULTS: A total of 1038 patients were included (34% women), of whom 659 (63.5%) had CT images and 379 (36.5%) MRI images available. Of all patients, 697 (67%) had SVD reported by radiologists. Patients with SVD had a higher three year risk of cardiovascular death than those without (6.5% vs. 2.1%, adjusted HR 2.52 [95% CI 1.12-5.67]; p = .026) but no association was observed for the three year risk of stroke (9.0% vs. 6.7%, for patients with SVD vs. those without, adjusted HR 1.24 [95% CI 0.76-2.02]; p = .395). No differences in 30 day peri-operative risk were observed for stroke (4.4% vs. 2.9%, for patients with vs. those without SVD; adjusted HR 1.49 [95% CI 0.73-3.05]; p = .28), and for the combined stroke/cardiovascular death risk (4.4% vs. 3.5%, adjusted HR 1.20 [95% CI 0.61-2.35]; p = .59). CONCLUSION: Presence of SVD in pre-operative brain imaging reports can serve as a predictor for the three year risk of cardiovascular death in symptomatic patients undergoing CEA but does not predict peri-operative or long term risk of stroke.

Validation of randomized controlled trial-derived models for the prediction of postintervention outcomes in chronic limb-threatening ischemia.

BACKGROUND: Chronic limb-threatening ischemia (CLTI) represents the most severe form of peripheral artery disease and has a large impact on quality of life, morbidity, and mortality. Interventions are aimed at improving tissue perfusion and averting amputation and secondary cardiovascular complications with an optimal risk-benefit ratio. Several prediction models regarding postprocedural outcomes in CLTI patients have been developed on the basis of randomized controlled trials to improve clinical decision-making. We aimed to determine model performance in predicting clinical outcomes in selected CLTI cohorts. METHODS: This study validated the Bypass versus Angioplasty in Severe Ischaemia of the Leg (BASIL), Finland National Vascular registry (FINNVASC), and Prevention of Infrainguinal Vein Graft Failure (PREVENT III) models in data sets from a peripheral artery disease registry study (Athero-Express) and two randomized controlled trials of CLTI in The Netherlands, Rejuvenating Endothelial Progenitor Cells via Transcutaneous Intra-arterial Supplementation (JUVENTAS) and Percutaneous Transluminal Angioplasty and Drug-eluting Stents for Infrapopliteal Lesions in Critical Limb Ischemia (PADI). Receiver operating characteristic (ROC) curve analysis was used to calculate their predictive capacity. The primary outcome was amputation-free survival (AFS); secondary outcomes were all-cause mortality and amputation at 12 months after intervention. RESULTS: The BASIL and PREVENT III models showed predictive values regarding postintervention mortality in the JUVENTAS cohort with an area under the ROC curve (AUC) of 81% and 70%, respectively. Prediction of AFS was poor to fair (AUC, 0.60-0.71) for all models in each population, with the highest predictive value of 71% for the BASIL model in the JUVENTAS population. The FINNVASC model showed the highest predictive value regarding amputation risk in the PADI population with AUC of 78% at 12 months. CONCLUSIONS: In general, all models performed poor to fair in predicting mortality and amputation. Because the BASIL model performed best in predicting AFS, we propose use of the BASIL model to aid in the clinical decision-making process in CLTI. However, improvements in performance have to be made for any of these models to be of real additional value in clinical practice.

Exercise reduces inflammatory cell production and cardiovascular inflammation via instruction of hematopoietic progenitor cells.

A sedentary lifestyle, chronic inflammation and leukocytosis increase atherosclerosis; however, it remains unclear whether regular physical activity influences leukocyte production. Here we show that voluntary running decreases hematopoietic activity in mice. Exercise protects mice and humans with atherosclerosis from chronic leukocytosis but does not compromise emergency hematopoiesis in mice. Mechanistically, exercise diminishes leptin production in adipose tissue, augmenting quiescence-promoting hematopoietic niche factors in leptin-receptor-positive stromal bone marrow cells. Induced deletion of the leptin receptor in Prrx1-creERT2; Leprfl/fl mice reveals that leptin's effect on bone marrow niche cells regulates hematopoietic stem and progenitor cell (HSPC) proliferation and leukocyte production, as well as cardiovascular inflammation and outcomes. Whereas running wheel withdrawal quickly reverses leptin levels, the impact of exercise on leukocyte production and on the HSPC epigenome and transcriptome persists for several weeks. Together, these data show that physical activity alters HSPCs via modulation of their niche, reducing hematopoietic output of inflammatory leukocytes.

Preoperative hypertension is associated with atherosclerotic intraplaque hemorrhage in patients undergoing carotid endarterectomy.

BACKGROUND AND AIMS: Both hypertension and atherosclerotic plaque characteristics such as intraplaque hemorrhage (IPH) are associated with cardiovascular events (CVE). It is unknown if hypertension is associated with IPH. Therefore, we studied if hypertension is associated with unstable atherosclerotic plaque characteristics in patients undergoing carotid endarterectomy (CEA). METHODS: Prospectively collected data of CEA-patients (2002-2014) were retrospectively analyzed. Blood pressure (BP) was the mean of 3 preoperative measurements. Preoperative hypertension was defined as systolic BP ≥ 160 mmHg. Post-CEA, carotid atherosclerotic plaques were analyzed for the presence of calcifications, collagen, smooth muscle cells, macrophages, lipid core, IPH and microvessel density. Associations between BP (systolic and diastolic), patient characteristics and carotid plaque characteristics were assessed with univariate and multivariate analyses with correction for potential confounders. Results were replicated in a cohort of patients that underwent iliofemoral endarterectomy. RESULTS: Within CEA-patients (n = 1684), 708 (42%) had preoperative hypertension. Increased systolic BP was associated with the presence of plaque calcifications (adjusted OR1.11 [95% CI 1.01-1.22], p = 0.03), macrophages (adjusted OR1.12 [1.04-1.21], p < 0.01), lipid core >10% of plaque area (adjusted OR1.15 [1.05-1.25], p < 0.01), IPH (adjusted OR1.12 [1.03-1.21], p = 0.01) and microvessels (adjusted beta 0.04 [0.00-0.08], p = 0.03). Increased diastolic BP was associated with macrophages (adjusted OR1.36 [1.17-1.58], p < 0.01), lipid core (adjusted OR1.29 [1.10-1.53], p < 0.01) and IPH (adjusted OR1.25 [1.07-1.46], p < 0.01) but not with microvessels nor plaque calcifications. Replication in an iliofemoral-cohort (n = 657) showed that increased diastolic BP was associated with the presence of macrophages (adjusted OR1.78 [1.13-2.91], p = 0.01), lipid core (adjusted OR1.45 [1.06-1.98], p = 0.02) and IPH (adjusted OR1.48 [1.14-1.93], p < 0.01). CONCLUSIONS: Preoperative hypertension in severely atherosclerotic patients is associated with the presence of carotid plaque macrophages, lipid core and IPH. IPH, as a plaque marker for CVE, is associated with increased systolic and diastolic BP in both the CEA and iliofemoral population.

The Impact of Diabetes and Time on the Atherosclerotic Plaque and Cardiovascular Outcome in Patients Undergoing Iliofemoral Endarterectomy.

OBJECTIVE: The incidence of diabetes is rapidly increasing and diabetes is associated with an increased risk of peripheral artery disease. Recent studies have shown a time dependent decline in vulnerable plaque features and secondary cardiovascular events in iliofemoral endarterectomy (IFE) patients. IFE patients with diabetes have a high risk of cardiovascular events. It is not known, however, whether vulnerable plaque features and cardiovascular events reduce over time in IFE patients with diabetes. METHODS: Between 2003 and 2014, 691 atherosclerotic plaques were obtained by IFE, from 212 patients with and 479 patients without diabetes. Plaques were immunohistochemically stained and analysed for the presence of intraplaque haemorrhage, lipid core, calcification, collagen, smooth muscle cells, and macrophages. Patients were stratified according to their diabetic status and year of inclusion. All patients had a follow up of three years in which cardiovascular adverse events were recorded. RESULTS: A time dependent decrease was observed in intraplaque haemorrhage, plaque lipid core, and percentage of macrophages in IFE patients with diabetes. After multivariable correction for changes in risk factors over time, intraplaque haemorrhage (64.2% [2002-2005] vs. 39.6% [2012-2014], p = .01) became significantly less prevalent. Interestingly, the percentage of severely calcified plaques remained high over time. The number of secondary events decreased over time in patients without diabetes (HR 1.80, 95% CI 1.15-2.81 (p = .010) for 2002-2005 vs. 2012-2014), but remained high and unchanged in patients with diabetes. CONCLUSION: In patients with diabetes undergoing IFE, a time dependent stabilisation of atherosclerotic plaque features was found in line with previous observations in patients with severe atherosclerosis. The presence of severely calcified lesions remained high and unchanged. The secondary event rate remained high in patients with diabetes in contrast to a significant decrease in patients without diabetes. These findings stress the need for improvement of care in IFE patients with diabetes.

Circulating CD14+CD16- classical monocytes do not associate with a vulnerable plaque phenotype, and do not predict secondary events in severe atherosclerotic patients.

AIMS: Mouse studies have established distinct monocyte subtypes that participate in the process of atherosclerotic lesion formation. The pro-inflammatory Ly6Chigh monocyte subtype actively contributes to murine plaque progression and destabilization. Also in humans, different peripheral monocyte subtypes have been identified, of which the CD14+CD16- classical monocyte is suggested to display similar pro-atherosclerotic properties as the murine Ly6Chigh subtype. We aimed to investigate if circulating CD14+CD16- classical monocytes associate with characteristics of a vulnerable carotid atherosclerotic plaque and if they associate with the risk of secondary adverse manifestations of atherosclerotic disease. METHODS AND RESULTS: We enrolled 175 carotid endarterectomy patients of the Athero-Express biobank in our study. Just prior to surgical procedure, blood was collected and peripheral blood mononuclear cells were isolated. Characterization of monocyte subsets was performed by flow cytometry. Plaque characteristics were semi-quantitatively scored for the presence of fat, collagen, intraplaque hemorrhage and calcification. Vessel density, smooth muscle cells and macrophages were assessed quantitatively on a continuous scale. All features of a vulnerable plaque phenotype, including low amounts of collagen and smooth muscle cells, and increased fat content, vessel density, intraplaque hemorrhage and plaque macrophages were not significantly associated with differential levels of peripheral classical CD14+CD16- monocytes or other monocyte subsets. Using Cox regression models to evaluate the prognostic value of circulating monocyte subtypes, we found that total counts of peripheral monocytes, as well as CD14+CD16- classical and other monocyte subtypes were not associated with the risk of secondary cardiovascular events during 3 years follow-up. CONCLUSION: Circulating classical CD14+CD16- monocytes do not associate with specific vulnerable plaque characteristics. In addition, they do not predict secondary adverse manifestations. This suggests that in patients with established carotid artery disease, the circulating monocytes do not reflect plaque characteristics and have no value in identifying patients at risk for future cardiovascular events.

Testosterone to oestradiol ratio reflects systemic and plaque inflammation and predicts future cardiovascular events in men with severe atherosclerosis.

Aims: The effects of testosterone on cardiovascular disease (CVD) as reported in literature have been ambiguous. Recently, the interplay between testosterone and oestradiol as assessed by testosterone/oestradiol (T/E2) ratio was suggested to be better informative on the normal physiological balance. Considering the role in CVD, we hypothesized that a low T/E2 ratio in men with CVD is associated with increased inflammation, a more unstable plaque and a worse cardiovascular outcome. Methods and results: Testosterone and oestradiol concentrations were determined in blood samples of 611 male carotid endarterectomy patients included in the Athero-Express Biobank Study. T/E2 ratio was associated with baseline characteristics, atherosclerotic plaque specimens, inflammatory biomarkers, and 3 year follow-up information. Patients with low T/E2 ratio had more unfavourable inflammatory profiles compared with patients with high T/E2 as observed by higher levels of C-reactive protein [2.81 µg/mL vs. 1.22 µg/mL (P < 0.001)] and higher leucocyte counts [8.98*109/L vs. 7.75*109/L (P = 0.001)] in blood. In atherosclerotic plaques, a negative association between T/E2 ratio and number of neutrophils [B = -0.366 (P = 0.012)], plaque calcifications [OR: 0.816 (P = 0.044)], interleukin-6 (IL-6) [B = -0.15 (P = 0.009)], and IL-6 receptor [B = -0.13 (P = 0.024)] was found. Furthermore, in multivariate Cox regression analysis, low T/E2 ratio was independently associated with an increased risk for major cardiovascular events (MACE) during 3 year follow-up [hazard ratio 1.67 (95% confidence interval 1.02-2.76), P = 0.043]. In men with elevated body mass index (BMI), these effects were strongest. Conclusion: In male patients with manifest atherosclerotic disease, low T/E2 ratio was associated with increased systemic inflammation, increased inflammatory plaque proteins, and an increased risk of future MACE as compared to men with normal T/E2 ratio. These effects are strongest in men with elevated BMI and are expected to be affected by aromatase activity in white fat tissues. Normalization of T/E2 ratio may be considered as target for the secondary prevention of CVD in men.

A single preoperative blood test predicts postoperative sepsis and pneumonia after coronary bypass or open aneurysm surgery.

BACKGROUND: Major surgery comes with a high risk for postoperative inflammatory complications. Preoperative risk scores predict mortality risk but fail to identify patients at risk for complications following cardiovascular surgery. We therefore assessed the value of preoperative red cell distribution width (RDW) as a predictor for pneumonia and sepsis after cardiovascular surgery and studied the relation of RDW with hematopoietic tissue activity. METHODS: RDW is an easily accessible, yet seldomly used parameter from routine haematology measurements. RDW was extracted from the Utrecht Patient Orientated Database (UPOD) for preoperative measurements in patients undergoing open abdominal aortic anuerysm repair (AAA)(N = 136) or coronary artery bypass grafting (CABG)(N = 2193). The cohorts were stratified in tertiles to assess effects over the different groups. Generalized Linear Models were used to determine associations between RDW and postoperative inflammatory complications. Hematopoietic tissue activity was scored using fluor-18-(18F)-deoxyglucose positron emission tomography and associated with RDW using linear regression models. RESULTS: In total, 43(31.6%) and 73 patients (3.3%) suffered from inflammatory complications after AAA-repair or CABG, respectively; the majority being pneumonia in both cohorts. Postoperative inflammatory outcome incidence increased from 19.6% in the lowest to 48.9% in the highest RDW tertile with a corresponding risk ratio (RR) of 2.35 ([95%CI:1.08-5.14] P = 0.032) in AAA patients. In the CABG cohort, the incidence of postoperative inflammatory outcomes increased from 1.8% to 5.3% with an adjusted RR of 1.95 ([95%CI:1.02-3.75] P = 0.044) for the highest RDW tertile compared with the lowest RDW tertile. FDG-PET scans showed associations of RDW with tissue activity in the spleen (B = 0.517 [P = 0.001]) and the lumbar bone marrow (B = 0.480 [P = 0.004]). CONCLUSION: Elevated RDW associates with increased risk for postoperative inflammatory complications and hematopoietic tissue activity. RDW likely reflects chronic low-grade inflammation and should be considered to identify patients at risk for postoperative inflammatory complications following cardiovascular surgery.

Smoking is Associated to DNA Methylation in Atherosclerotic Carotid Lesions.

BACKGROUND: Tobacco smoking is a major risk factor for atherosclerotic disease and has been associated with DNA methylation (DNAm) changes in blood cells. However, whether smoking influences DNAm in the diseased vascular wall is unknown but may prove crucial in understanding the pathophysiology of atherosclerosis. In this study, we associated current tobacco smoking to epigenome-wide DNAm in atherosclerotic plaques from patients undergoing carotid endarterectomy. METHODS: DNAm at commonly methylated sites (cytosine-guanine nucleotide pairs separated by a phospho-group [CpGs]) was assessed in atherosclerotic plaque samples and peripheral blood samples from 485 carotid endarterectomy patients. We tested the association of current tobacco smoking with DNAm corrected for age and sex. To control for bias and inflation because of cellular heterogeneity, we applied a Bayesian method to estimate an empirical null distribution as implemented by the R package bacon. Replication of the smoking-associated methylated CpGs in atherosclerotic plaques was executed in the second sample of 190 carotid endarterectomy patients, and results were meta-analyzed using a fixed-effects model. RESULTS: Tobacco smoking was significantly associated to differential DNAm in atherosclerotic lesions of 4 CpGs (false discovery rate <0.05) mapped to 2 different genes ( AHRR, ITPK1) and 17 CpGs mapped to 8 genes and RNAs in blood. The strongest associations were found for CpGs mapped to the gene AHRR, a repressor of the aryl hydrocarbon receptor transcription factor involved in xenobiotic detoxification. One of these methylated CpGs were found to be regulated by local genetic variation. CONCLUSIONS: The risk factor tobacco smoking associates with DNAm at multiple loci in carotid atherosclerotic lesions. These observations support further investigation of the relationship between risk factors and epigenetic regulation in atherosclerotic disease.

Impaired kidney function is associated with intraplaque hemorrhage in patients undergoing carotid endarterectomy.

BACKGROUND AND AIMS: Previously, we showed that patients undergoing carotid endarterectomy have an increased risk for major atherosclerotic events in the presence of moderate or poor kidney function. Acceleration of vascular inflammatory responses is considered to be causally involved in progression of atherogenesis and poor outcome in chronic kidney disease patients. The association between kidney function and plaque composition has not been thoroughly investigated yet. The aim of this study was to investigate the association between kidney function and atherosclerotic plaque composition in patients undergoing carotid endarterectomy. METHODS: Atherosclerotic plaques, harvested from 1796 patients who underwent carotid endarterectomy, were immunohistochemically stained for macrophages, smooth muscle cells, calcifications, collagen, microvessels, lipid core size and intraplaque hemorrhage. Cytokines were measured in plaque and plasma and associated with kidney function. Quantitative proteomics were performed on 40 carotid plaques and associated with kidney function. RESULTS: Decreased kidney function was associated with increased odds ratio of intraplaque hemorrhage, OR 1.15 (95% CI; 1.02-1.29 (p = 0.024)) and increased odds ratio of fibrous-atheromatous plaques (plaques with lipid core presenting more than 10% of total plaque surface) OR 1.21 (95% CI; 1.07-1.38 (p = 0.003)) per decrease of 20 points in eGFR. Proteomics revealed that decreased kidney function was associated with upregulation of the classical pathway of the complement system and the intrinsic pathway of the coagulation system. CONCLUSIONS: Decreased kidney function was associated with plaque hemorrhage but not with inflammatory plaque characteristics. Our data suggests that other pathways than the inflammation-pathway are involved in plaque vulnerability and poor outcome in patients with decreased kidney function.

Biobanking in carotid artery disease: translation to clinical practice.

Biobanking of atherosclerotic tissue samples has contributed to our understanding of vascular occlusive disease. The careful examination of atherosclerotic plaques derived during vascular surgery or autopsies helped shape our minds in understanding the underlying substrate of arterial thrombosis. This review will outline concepts of progression of atherosclerotic disease that have been based on descriptions of human plaque pathology. In addition, we will discuss the current shift in clinical presentation and underlying pathology of acute cerebral and coronary events that asks for a careful consideration of the currently widely applied description of the "vulnerable plaque". The shift in atherosclerotic plaque characteristics that associate with a thrombotic event reflects the treatment and risk factor management that has undergone major changes in recent times. These changes may influence the value of past biobanking efforts in the current era: many inferences are being made upon sample data from cohorts that have been assembled in previous decades while large shifts in patient demographics and disease substrates over time occurred raises the question if biomarkers validated in historical biobanks can be extrapolated to the current era. As an example of altering profiles of biomarkers in the last decade, a panel of twelve selected plasma proteins was measured in the Athero-express cohort, showing time-dependent trends in serum biomarkers over the last decade. These findings strengthen our hypothesis that the pathogenesis of cardiovascular disease (CVD) is changing and future biobanking is required to successfully keep track of the mechanisms involved in CVD pathogenesis today.