Upregulation and localisation of apelin in human atherosclerosis

Sarah Pitkin, Rhoda Kuc, Janet Maguire, Anthony Davenport

University of Cambridge, Cambridge, UK

Apelins are endogenous ligands for the previously designated ‘orphan’ G-protein coupled receptor APJ. We have shown APJ localised to human cardiomyocytes, vascular endothelial and smooth muscle cells (Kleinz et al., 2005) with apelin peptides restricted to the endothelium (Kleinz et al., 2004). Apelins have been shown to constrict human saphenous vein (Katugampola et al., 2001) and act as vasodilators in man in vitro (Salcedo et al., 2007) and in vivo (Japp et al., 2008). APJ deficiency is protective against oxidative stress-linked atherosclerosis in mice (Hashimoto et al., 2007). Our aim was to investigate whether the apelin-APJ system is altered in atherosclerosis in man.

Human coronary artery samples were extracted using SepPac C18 cartridges. Apelin peptide levels were determined by radioimmunoassay (RIA) using anti-apelin-36 serum and [125I](Pyr1)apelin-13. Levels were expressed as mean pg/g wet mass ± s.e. mean. n-Values are the number of patients from whom tissues were obtained. Immunohistochemistry (IHC) was carried out on normal and atherosclerotic coronary artery (CA) using apelin- and APJ-specific antibodies (Phoenix Pharmaceuticals, USA) with detection by peroxidase/anti-peroxidase method. Dual-labelling fluorescence IHC was carried out using antibodies against smooth muscle α-actin, von-Willebrand factor (endothelial cell marker), CD3 (T-cell marker) and CD68 (macrophage marker), and immunoreactivity was visualised using confocal microscopy.

RIA revealed a significant increase in apelin peptide levels in human atherosclerotic CA (73.2±14.9 pg/g, n=6) compared to histologically normal CA (24.1±3.5 pg/g, n=6, P<0.01). IHC revealed apelin peptides restricted to the endothelium in normal CA, whereas intense staining of the atherosclerotic plaque was observed in diseased CA. APJ-like immunoreactivity (-LI) was present on the endothelium and smooth muscle of normal and atherosclerotic CA; additionally staining of the atherosclerotic plaque was observed. Fluorescent dual-labelling IHC revealed colocalisation of both apelin-LI and APJ-LI, with cell markers for smooth muscle and macrophages, but not T-cells, within the atherosclerotic plaque.

These data suggest that apelin peptides are produced within the atherosclerotic plaque in man, possibly by smooth muscle cells and macrophages. Since apelins have been shown to be mediators of oxidative stress in vascular tissue (Hashimoto et al., 2007), the increase in apelin levels may be detrimental in atherosclerosis.

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