Discovery of New Risk Markers for Ischemic Stroke Using a Novel Targeted Proteomics Chip

Abstract

Eligible subjects were 70 years of age and living in the city of Uppsala, Sweden. Subjects were chosen at random from the Total Background and Purpose-Emerging technologies have made it possible to simultaneously evaluate a large number of circulating proteins as potential new stroke risk markers. Methods-We explored associations between 85 cardiovascular proteins, assessed by a proteomics chip, and incident ischemic stroke in 2 independent cohorts of elderly (Prospective Investigation of the Vasculature in Uppsala Seniors [PIVUS]: n=977; 50% women, mean age=70.1 years, 71 fatal/nonfatal ischemic stroke events during 10.0 years; and Uppsala Longitudinal Study in Adult Men [ULSAM]: n=720, mean age=77.5 years, 75 ischemic stroke events during 9.5 years). The proteomics chip uses 2 antibodies for each protein and a polymerase chain reaction step to achieve a highspecific binding and the possibility to measure multiple proteins in parallel, but gives no absolute concentrations. Results-In PIVUS, 16 proteins were related to incident ischemic stroke using a false discovery rate of 5%. Of these, N-terminal pro-B-type natriuretic peptide (P=0.0032), adrenomedullin (P=0.018), and eosinophil cationic protein (P=0.0071) were replicated in ULSAM after adjustment for established stroke risk factors. In predefined secondary metaanalyses of individual data, interleukin-27 subunit α, growth/differentiation factor 15, urokinase plasminogen activator surface receptor, tumor necrosis factor receptor superfamily member 6, macrophage colony-stimulating factor 1, and matrix metalloproteinase-7 were also potential risk markers for ischemic stroke after adjustment for multiple comparisons (P<0.0006). The addition of N-terminal pro-B-type natriuretic peptide, adrenomedullin, and eosinophil cationic protein to a model with established risk factors increased the C-statistic from 0.629 to 0.689 (P=0.001). Conclusions-Our data suggest that large-scale proteomics analysis is a promising way of discovering novel biomarkers that could substantially improve the prediction of ischemic stroke. Lind et al New Risk Markers for Ischemic Stroke 3341Population Register. In total, 1016 individuals of 2025 invited took part in the investigation (50.1%) at age 70 years when also the blood sample for proteomics was collected. 12 A follow-up of incident ischemic stroke was performed after 10 years, without any loss of follow-up. After exclusion of the 39 individuals with stroke before the baseline investigation in Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS), 71 of remaining 977 subjects experienced an ischemic stroke during a median follow-up of 10.0 years (range, 0.04-10.9 years), resulting in an incidence rate of 7.9 per 1000 person years at risk. ULSAM StudyUppsala Longitudinal Study in Adult Men (ULSAM) is a longitudinal population-based study including men born between 1920 and 1924 in Uppsala County, Sweden, being invited for the first time at age 50 years (n=2322). 13 This study used the 77-year examination cycle as base...

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