Figure 1: Sampling at Surgery and Workflow of the Study

Carotid plaques and local and peripheral blood were collected at carotid endarterectomy (CEA) surgery. Sampling of local blood during test occlusion, after 5 min of vessel clamping and prior to arteriotomy, is shown in the photograph. The internal carotid artery (ICA), external carotid artery (ECA), common carotid artery (CCA), and positions of vessel clamps are indicated. Discovery analyses comprised carotid plaques (cp) from 127 patients and 10 normal arteries (na) used for microarray profiling; plaques and adjacent peripheral tissue from 18 patients for liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS); and matched local and peripheral plasma samples from 43 patients for screening by antibody bead arrays. Results from these 3 analyses were overlapped and revealed biliverdin reductase B (BLVRB) as the only significant candidate. Venn diagram shows numbers of significantly dysregulated candidates in each analysis. Both BLVRB and the intraplaque hemorrhage (IPH) pathway to which it functionally belongs were explored further in omics datasets. BLVRB was validated in plasma by sandwich immunoassays and in plaques utilizing additional microarrays and quantitative polymerase chain reaction (qPCR) from both same and nonoverlapping subcohorts of Biobank of Karolinska Endarterectomies patients and normal individuals. Functional investigations were conducted in plaques in situ and in vitro. IHC = immunohistochemistry; IFL = immunofluorescence; PLA =proximity ligation assay; TMA = tissue microarray.