Carbamylated LDL induces a pro-thrombotic state via the LOX-1 receptor and arterial thrombus formation: a novel mechanism of cardiovascular events in end-stage renal disease

Abstract

Background: Carbamylation of low-density lipoproteins (cLDL) alters protein structure and is thought to promote vascular inflammation and dysfunction during end-stage renal disease (ESRD). However, it remains unknown whether cLDL exerts prothrombotic effects in vascular cells and platelets and whether cLDL affects arterial thrombus formation in vivo. Methods: LDL was isolated from healthy subjects or patients with ESRD by sequential ultra-centrifugation. Ex vivo carbamylation of LDL (cLDL) from healthy subjects was induced with potassium cyanate. Arterial thrombus formation was analysed in a murine carotid artery photochemical injury model. Analysis of tissue factor (TF) and plasminogen activator inhibitor type-1 (PAI-1) expression was performed by Western blot and real-time PCR analysis. Platelet aggregation was analysed by impedance aggregometry. Results: Intravenous administration of cLDL (2mg/kg body weight) accelerated arterial thrombus formation in a murine photochemical carotid artery injury model as compared to treatment with native LDL (2mg/kg body weight) or vehicle (PBS) (n=8; p<0.05 versus nLDL and control). Analyses of tissue lysates demonstrated that cLDL induced the expression of TF and PAI-1 mRNA levels in carotid arteries (n=5, p<0.01). In human aortic smooth muscle cells (AoSMC) cLDL (10-300 μg/ml) induced TF and PAI-1 expression by 3.5- and 3-fold, as compared with nLDL (n=4; p<0.01). cLDL also enhanced basal and TNF-α induced expression of TF and PAI-1 in human aortic endothelial cells (n=4; p<0.02). In contrast, native LDL (nLDL) had no effect on endothelial expression or activity of TF and PAI-1. In both cell types, cLDL activated the mitogen-activated protein kinases p38 and ERK (n=3; p<0.05) as well as the transcription factor NFkB (n=4, p<0.05). Silencing of lectin-like oxidized LDL receptor-1 (LOX-1) using specific siRNA prevented the induction of TF and PAI-1 expression in vascular cells. In platelets cLDL activated the p38 MAP kinase and induced platelet aggregation. Finally, LDL isolated from patients with ESRD mimicked the prothrombotic effects of cLDL on vascular cells and platelets. Conclusions: These data demonstrate that cLDL at physiologically relevant concentrations,via activation of the LOX-1 receptor induces potent pro-thrombotic effects in vascular cells and platelets leading to enhanced thrombus formation in vivo. This observation may explain the markedly increased incidence of fatal acute thrombotic events in patients with end-stage renal disease and lead to the development of novel LDL targeting strategies in this population.