Synthetic protein scaffold-mediated manipulation of LOX-1 scavenger receptor in cell function, vascular physiology and disease

Binding and internalization of oxidized low-density lipoprotein (oxLDL) by immune cells within the arterial sub-intimal layer is a key step in the initiation and progression of atherosclerosis. Scavenger receptors (SRs) are a “super-family” of cell surface receptors expressed by immune cells which promote the removal of harmful non-self or altered-self targets, including oxidatively modified ligands such as oxLDL. Lectin-like oxidized LDL receptor-1 (LOX-1) is a member of the SR-E family of scavenger receptors. LOX-1 – oxLDL binding has been shown to exert significant pro-atherogenic effects. In this study, we used in vitro and in vivo models of atherosclerosis to explore the effectiveness of LOX-1 targeting in the inhibition of oxLDL binding and the amelioration of atherosclerosis and neointimal hyperplasia. LOX-1 was targeted using novel non-antibody artificial binding proteins, known as Affimers. Immunofluorescence studies using a tetracycline- inducible cell line expressing LOX-1 demonstrate the binding efficiency of fluorescent-labelled LOX-1 Affimers to LOX-1 in live cells. In same cell line, we co-incubated fluorescent-labelled oxLDL with increasing concentrations of LOX- 1 Affimers. The findings show that LOX-1 Affimers inhibit LOX-1 – oxLDL binding. ELISA studies were carried out to identify LOX-1 affimers with comparable affinity for both human and murine LOX-1. The most cross-reactive LOX-1 Affimer was taken forward in to a transgenic mouse model of diet-induced atherosclerosis. This model used APO-E null and APO-E/LOX-1 null mice to compare the effects of LOX-1 knockout with LOX-1 targeting using LOX-1 affimers. LOX-1 knockout resulted in a reduction in aortic and peripheral arterial atherosclerosis, and neointimal hyperplasia. LOX-1 targeting with affimers produced promising results. Treatment with LOX-1 affimers reduced atherosclerosis and NIH, but to a lesser extent compared with LOX-1 knockout. Treatment with LOX-1 affimer exerted additional metabolic benefits including protective effects against diet- induced obesity and insulin resistance. The results from these studies suggest that LOX-1 is an attractive target in the development of novel therapeutics in atherosclerosis. Targeted LOX-1 inhibition potentially offers protective benefits in associated metabolic disorders such as obesity and insulin resistance