Novel Multiomics Profiling of Human Carotid Atherosclerotic Plaques and Plasma Reveals Biliverdin Reductase B as a Marker of Intraplaque Hemorrhage

Figures

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  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…

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  Figure 2. BLVRB Is Enriched in Plaques and Plasma From Carotid Atherosclerosis Patients BLVRB messenger ribonucleic acid (mRNA) was highly up-regulated in comparison between plaques and normal arteries, both in the discovery (n = 127 cp + 10 na) and validation microarray dataset (n = 50 cp + 5 na) (A), and these results were confirmed by qPCR from 2 sets of plaques compared with normal arteries (B). Plots show log2 mean ± SD for microarrays and mean fold change for qPCR. By proteomic analysis of plaques compared with matched adjacent arterial tissue (n = 18), BLVRB was also strongly up-regulated (C). Immunohistochemistry confirms the lack of signal for BLVRB in normal artery, but strong widespread staining in carotid plaque (red signal, negative control [ctrl] subjects shown in insets) (D). BLVRB was detected with 2 antibodies in the bead array–based plasma screening as strongly up-regulated in local versus peripheral plasma (E). This enrichment was confirmed by sandwich immunoassay (SIA) in the same samples as well as in another set of 66 patients (F). Plots show median of fluorescence intensity (MFI) with mean ± SD. Bonferroni corrected p values shown in A and C, nominal values in E…

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  Figure 3. BLVRB Is Up-Regulated in Patients With Symptomatic Carotid Atherosclerosis Stratification of patients based on clinical symptoms of atherosclerosis showed that BLVRB mRNA (A) and protein (B) expression was induced in comparison between symptomatic (S) and asymptomatic (AS) patients in plaques by microarrays, qPCR, and LC-MS/MS, as well as in local plasma by antibody bead arrays using both human protein atlas antibodies targeting BLVRB (HPA041937 and HPA041698) (C). Abbreviations as in Figures 1 and 2.

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  Figure 4. BLVRB Localizes to CD68+ Cells in Plaques In plaque transcriptomic (A) and proteomic (B) datasets, BLVRB showed strong significant correlations to markers of macrophages (CD163, CD36). By double immunohistochemistry, BLVRB (red signal) localized primarily to CD68+ cells in the necrotic core of plaques (green signal, enlarged insets). No signal was observed in adventitia of normal arteries (C). By single immunohistochemistry, BLVRB was also detected in necrotic core regions positive for CD163 (D). neg = negative; other abbreviations as in Figures 1 and 2.

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  Figure 5. BLVRB May Be Associated With Heme-Catabolism in Plaques via HMOX1 Heme oxygenase 1 (HMOX1) mRNA was highly up-regulated in Biobank of Karolinska Endarterectomies microarrays comparing plaques and normal arteries and strongly correlated to that of BLVRB. Plots showing log2 mean ± SD and Spearman correlation (A). Induction of HMOX1 and correlation with BLVRB could be verified on the protein level by LC-MS/MS from plaques compared with adjacent tissue (B). No staining for HMOX1 was observed in the normal carotid artery, whereas weak signal for BLVRB could occasionally be seen in adventitia. Using consecutive sections of plaques, HMOX1 was localized to the sites of hemorrhage with infiltrated erythrocytes, where it overlapped with BLVRB and Perls blue staining for iron deposits (C, enlarged insets). The correlation between BLVRB and HMOX1 content and intraplaque hemorrhage was also assessed quantitatively, using histological staining on TMA constructed from 106 plaques (D, plot to the right). Abbreviations as in Figures 1, 2, and 3.

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  Figure 6. BLVRB and HMOX1 Colocalize and Cointeract in Plaques By PLA, BLVRB and HMOX1 proteins cointeracted directly in plaques in regions with hemorrhage (arrows and enlarged insets). PLA probe detecting HMOX1 was used as a positive control. Abbreviations as in Figures 1, 2, and 5.

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  Figure 7. BLVRB and HMOX1 Are Induced in Macrophages on Iron Exposure THP-1 human monocytic cell line–derived human macrophages were stimulated with ferric ammonium citrate (FAC) and cellular iron uptake quantified over time using the ferrozine assay (top panel). qPCR shows a rapid induction of HMOX1 expression already after 4 h of stimulation, whereas BLVRB was up-regulated after 24 h. Plots show mean fold change ± SEM of 3 independent experiments. p values calculated with analysis of variance (ANOVA) across all groups and Student's t-test in comparison to 2 h time point. All results normalized to the nonstimulated control cells (A). Perls staining confirms presence of cellular iron deposits after 24 h of stimulation. By immunofluorescence, BLVRB protein is induced and localized to subcellular vesicles in FAC-treated cells, whereas HMOX1 shows perinuclear and diffused cytoplasmic localization (arrows). Bottom panels show higher magnification (B). DAPI = 4,6-diamino-phenylindole; other abbreviations as in Figures 1, 2, and 5.

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  Figure 8. Increased BLVRB and HMOX1 Levels Associate With Adverse Cardiovascular Events BLVRB showed significant enrichment by SIA comparing peripheral plasma from both discovery (n = 43) and validation (n = 66) subsets of patients with carotid atherosclerosis compared with peripheral plasma from normal individuals (n = 61) (A). BLVRB was also enriched in an independent population cohort stratified by coronary artery calcium (CAC) score (n = 599 individuals with score 0, n = 281 score 0 to 100, n = 151 score >100), as shown with 2 different antibodies. p values calculated with ANOVA across all groups and Student t-test in comparison to score 0 (B). Plots show MFI with SD. BLVRB and HMOX1 transcripts were also significantly up-regulated in plaques from patients that experienced a previous infarction (n = 27) compared with those without previous events (n = 98). Plots showing mRNA expression log2 mean ± SD (C). Plot illustrating major adverse cardiovascular and cerebrovascular events–free survival of patients during the 2,000-day follow-up period after surgery, based on HMOX1 expression in Biobank of Karolinska Endarterectomies plaques above (black) and below (red) the median values (x-axis,…