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Abstract Background Patients with peripheral artery disease (PAD) are treated with preventive strategies to improve the cardiovascular risk. The incidence of cardiovascular events and mortality however remains high in PAD populations. We therefore aimed to better characterize PAD patients suffering from cardiovascular events and mortality in order to tailor preventive treatment. Methods Between 2018 and 2020, 246 PAD outpatients (17 newly diagnosed, 229 with known PAD) were prospectively enrolled in this observational cohort study. Patient data and blood samples were collected after inclusion, and the primary composite endpoint (myocardial infarction, elective coronary revascularization, ischemic stroke, acute limb ischemia, mortality) was evaluated after one year. Secondary outcomes included platelet reactivity, measured using the VerifyNow assay, and medication adherence, assessed using the Morisky Medication Adherence Scale-8 (MMAS-8). Logistic regression models were used to identify associations between characteristics and the occurrence of events. Results The cohort comprised 207 patients with claudication and 39 with chronic limb threatening ischemia. Twenty-six (10.6%) patients suffered from an event during follow-up. Prior myocardial infarction (OR 3.3 [1.4–7.7]), prior ischemic stroke (OR 4.5 [1.8–10.9]), higher levels of creatinine (OR 5.2 [2.2–12.6]), lower levels of high-density lipoprotein (OR 4.2 [1.5–10.6]) and lower haemoglobin levels (OR 3.1 [1.3–7.1]) were associated with events. Patients with events had more often high on-treatment platelet reactivity (HTPR) on aspirin (OR 5.9 [1.4–25.1]) or clopidogrel (OR 4.3 [1–19.3]). High adherence to medication was associated with the occurrence of events (OR 4.1 [1–18]). Conclusions Patients suffering from cardiovascular events and mortality were characterized by prior cardiovascular events as compared to patients who did not experience any events. Antiplatelet therapy was not optimally protective despite high medication adherence, and HTPR was independently associated with the occurrence of events. More research is needed on alternative treatment strategies such as dual antiplatelet therapy or combinations with anticoagulant drugs. Trial registration The Medical Ethics Committee (METC) of the MUMC+ approved the study (NL63235.068.17) and the study was registered in the Netherlands Trial Register ( NTR7250 ).

Abstract Peripheral artery disease (PAD) patients have an increased cardiovascular risk despite pharmacological treatment strategies. Biomarker research improving risk stratification only focused on known atherothrombotic pathways, but unexplored pathways might play more important roles. To explore the association between a broad cardiovascular biomarker set and cardiovascular risk in PAD. 120 PAD outpatients were enrolled in this observational cohort study. Patients were followed for one year in which the composite endpoint (myocardial infarction, coronary revascularization, stroke, acute limb ischemia and mortality) was assessed. Patient data and blood samples were collected upon inclusion, and citrated platelet-poor plasma was used to analyze 184 biomarkers in Olink Cardiovascular panel II and III using a proximity extension assay. Fifteen patients reached the composite endpoint. These patients had more prior strokes and higher serum creatinine levels. Multivariate analysis revealed increased plasma levels of protease-activated receptor 1 (PAR1), galectin-9 (Gal-9), tumor necrosis factor receptor superfamily member 11A (TNFRSF11A) and interleukin 6 (IL-6) to be most predictive for cardiovascular events and mortality. Positive regulation of acute inflammatory responses and leukocyte chemotaxis were identified as involved biological processes. This study identified IL-6, PAR1, Gal-9, TNFRSF11A as potent predictors for cardiovascular events and mortality in PAD, and potential drug development targets.

Abstract Hypoxia is prevalent in atherosclerotic plaques, promoting plaque aggravation and subsequent cardiovascular disease (CVD). Transmembrane protein carbonic anhydrase IX (CAIX) is hypoxia-induced and can be shed into the circulation as soluble CAIX (sCAIX). As plaque macrophages are hypoxic, we hypothesized a role for CAIX in macrophage function, and as biomarker of hypoxic plaque burden and CVD. As tumor patients with probable CVD are treated with CAIX inhibitors, this study will shed light on their safety profile. CAIX co-localized with macrophages (CD68) and hypoxia (pimonidazole), and correlated with lipid core size and pro-inflammatory iNOS+ macrophages in unstable human carotid artery plaques. Although elevated pH and reduced lactate levels in culture medium of CAIX knock-out (CAIXko) macrophages confirmed its role as pH-regulator, only spare respiratory capacity of CAIXko macrophages was reduced. Proliferation, apoptosis, lipid uptake and expression of pro- and anti-inflammatory genes were not altered. Plasma sCAIX levels and plaque-resident CAIX were below the detection threshold in 50 and 90% of asymptomatic and symptomatic cases, respectively, while detectable levels did not associate with primary or secondary events, or intraplaque hemorrhage. Initial findings show that CAIX deficiency interferes with macrophage metabolism. Despite a correlation with inflammatory macrophages, plaque-resident and sCAIX expression levels are too low to serve as biomarkers of future CVD.