Abstract 216: Profiling of Atherosclerotic Lesions by Tissue Microarrays Reveals Novel Proteins Mediating Disease Progression and Plaque Vulnerability
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.
- © 2013 by American Heart Association, Inc.