Objective— Carotid plaque instability is a major cause of ischemic stroke, but detailed knowledge about underlying molecular pathways is still lacking. Here, we evaluated large-scale transcriptomic and protein expression profiling in a biobank of carotid endarterectomies followed by characterization of identified candidates, as a platform for discovery of novel proteins differentially regulated in unstable carotid lesions. Approach and Results— Genes highly upregulated in symptomatic versus asymptomatic plaques were selected from Affymetrix microarray analyses (n=127 plaques), and tissue microarrays constructed from 34 lesions were assayed for 21 corresponding proteins by immunohistochemistry. Quantification of stainings demonstrated differential expression of CD36, CD137, and DOCK7 ( P <0.05) in unstable versus stable lesions and the most significant upregulation of a proprotein convertase, PCSK6 ( P <0.0001). Increased expression of PCSK6 in symptomatic lesions was verified by quantitative real-time polymerase chain reaction (n=233), and the protein was localized to smooth muscle α-actin positive cells and extracellular matrix of the fibrous cap by immunohistochemistry. PCSK6 expression positively correlated to genes associated with inflammation, matrix degradation, and mitogens in microarrays. Stimulation of human carotid smooth muscle cells in vitro with cytokines caused rapid induction of PCSK6 mRNA. Conclusions— Using a combination of transcriptomic and tissue microarray profiling, we demonstrate a novel approach to identify proteins differentially expressed in unstable carotid atherosclerosis. The proprotein convertase PCSK6 was detected at increased levels in the fibrous cap of symptomatic carotid plaques, possibly associated with key processes in plaque rupture such as inflammation and extracellular matrix remodeling. Further studies are needed to clarify the role of PCSK6 in atherosclerosis.

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Proprotein convertases (PCSKs) are conserved among species and involved in processing of MMPs and growth factors, but poorly characterised in atherosclerosis. Previously we demonstrated upregulation of PCSK6 in a large cohort of plaques from symptomatic vs. asymptomatic patients. This protease localized to smooth muscle cells (SMCs) and showed a positive correlation to markers of inflammation, extracellular matrix remodeling and cytokines in plaques. Here we aimed at elucidating its role in vascular development and disease. In zebrafish embryos PCSK6 localized to heart and vasculature and its ablation caused defective peripheral vascular patterning with cerebral and myocardial hemorrhage. Increased expression of PCSK6 in vascular pathologies was validated by microarrays from carotid plaques vs. undiseased arteries (n=32 patients, p<0.0001), abdominal (AAA, n=14, p<0.0001) and thoracic aortic aneurysms (TAA, n=244, p=0.012). By immunohistochemistry, PCSK6 localized mainly to SMCs in the fibrous cap and neo-vessels in plaques, AAA and TAA tissues. Investigation of mouse carotid ligation, rat artery balloon injury and human restenosis tissues with pronounced intimal hyperplasia confirmed induction of PCSK6 in proliferating SMCs. By microarrays following progression of rat carotid injury, PCSK6 was downregulated in early phases defined by inflammatory pathways, while upregulated in later phases with SMCs activation and localized in SMCs. Expression of PCSK6 in this model was positively correlated to PDGFB and IGF1 (Spearman r>0.7, p<0.0001). PCSK6 overexpression in SMCs in vitro increased their migration in a wound-healing assay, especially upon PDGFBB stimulation. By eQTL analyses several polymorphisms in the PCSK6 gene were found to influence its expression in plaques and aneurysm tissue. Among these, rs6598465 showed association with maximum progression of arterial intima-media thickness in high-risk coronary artery disease subjects (n=3400, p=0.037). We identified a functional link between elevated expression of PCSK6 and vascular remodeling characterized by SMC activation. The present study establishes PCSK6 as one of the key modulators of pathological processes in relation to plaque vulnerability.

Recently we demonstrated upregulation of the proprotein convertase PCSK6 in a large cohort of human carotid atherosclerotic plaques (n=127) compared to normal arteries and in symptomatic vs. asymptomatic lesions. PCSK6 was localized to smooth muscle cells (SMCs) in the fibrous cap and showed a positive correlation to markers of inflammation, extracellular matrix remodeling and cytokines. Here, we aimed at elucidating the role of this protease in vascular disease by examining its expression in different human pathologies and in animal models. Increased expression of PCSK6 in vascular diseases was validated in public microarray datasets and other available human cohorts. PCSK6 was upregulated in carotid atherosclerotic plaques vs. controls (n=32 patients, p<0.0001), as well as in abdominal aortic aneurysm (AAA) vs. normal tissue (n=14, p<0.0001) and in thoracic aortic aneurysm (TAA) tissue from bicuspid vs. tricuspid patients (n=244, p=0.012). By eQTL analyses, several SNPs in the PCSK6 genomic region were shown to influence its expression in carotid plaques and TAA tissue. Among these, rs6598465 showed a mild association to progression of maximum intima-media thickness in the left and right arteries in a separate cohort of high-risk coronary artery disease subjects (n=3400, p=0.037). By immunohistochemistry, PCSK6 localized mainly to SMCs in carotid plaques, AAA and TAA tissue, but was also found to be expressed by CD68 and CD163+ macrophages. Investigation of mouse, rat and human artery tissues with pronounced intimal hyperplasia revealed strong expression of PCSK6 in proliferating SMCs. In rat carotid artery balloon injury, PCSK6 was downregulated in the early phases after injury mostly defined by inflammatory response, while upregulated in later phases with prominent SMC activation, and consistently localized in SMCs. Expression of PCSK6 in this model was strongly positively correlated solely to PDGFB and IGF1 (p<0.0001), cytokines known to induce SMC proliferation. We established a functional link between elevated expression of PCSK6 and vascular diseases characterized by inflammation and SMC proliferation. Further investigations in vitro are necessary to provide mechanistic insight into the role of this protease in vascular disease.

Objective High-throughput immunohistochemistry (IHC) techniques are not commonly applied in studies of vascular diseases. Tissue microarrays (TMAs) are collections of multiple tissue cores placed in parallel in a single acceptor paraffin block, thereby enabling large-scale expression analyses of many samples concomitantly. Our aim was to evaluate this approach in qualitative and quantitative studies of candidate proteins implicated in atherosclerotic carotid stenosis. Methods and Results We constructed TMAs from a collection of 34 carotid plaques (6 asymptomatic/28 symptomatic patients) and assayed them for expression of 21 proteins by IHC. Majority of proteins chosen for the study were previously identified through microarray profiling of symptomatic vs. asymptomatic plaques (n=150) from the Biobank of Karolinska Endarterectomies. Quantification of stainings highlighted differential expression of several proteins in unstable compared to stable lesions (ADIPOR1, CD36, CD137, DOCK7), the most significantly upregulated being a secreted member of the proprotein convertase family, PCSK6 (p<0.0001). We localized this protein to smooth muscle actin positive cells and extracellular matrix of the fibrous cap in situ, overlapping with fibronectin and collagen IV. Endogenous PCSK6 localizes to cytosol and peripheral membrane extensions of human carotid smooth muscle cells (hcSMCs) in vitro. Increased expression of PCSK6 positively correlates to macrophages and lymphocytes markers in gene arrays, matrix degrading proteins (MMP9, ADAM9, Heparanase) and growth factors (PDGFB, TGF). Interestingly, stimulation of hcSMCs with several cytokines caused rapid upregulation of PCSK6 mRNA levels. Conclusions Here we demonstrate the feasibility of a scaled-up method for simultaneous and standardized expression studies in a large collection of human atherosclerotic lesions. Our results show that PCSK6, a novel protease previously not associated with atherosclerosis, is detected at increased levels in symptomatic carotid plaques, possibly in association with inflammation and extracellular matrix remodeling leading to plaque rupture. Further mechanistic studies are needed to dissect the role of PCSK6 in progression of atherosclerosis.