Immunization with the Gly 1127 -Cys 1140 amino acid sequence of the LRP1 receptor reduces atherosclerosis in rabbits. Molecular, immunohistochemical and nuclear imaging studies

Abstract

Altres ajuts: The main support to develop this project was received from Fundació MARATÓ TV3 Project 201521-10 (to VLl-C) and co-financed with ERDFs. Support was received from Fundació Marató TV3 (Project 201521-10) for post-doctoral funding to OB. DdG-C, VL-C and JCE-G are members of the Group of Vascular Biology of the Spanish Society of Atherosclerosis (SEA).

Background : The LRP1 (CR9) domain and, in particular, the sequence Gly 1127 -Cys 1140 (P3) plays a critical role in the binding and internalization of aggregated LDL (agLDL). We aimed to evaluate whether immunization with P3 reduces high-fat diet (HFD)-induced atherosclerosis. Methods : Female New Zealand White (NZW) rabbits were immunized with a primary injection and four reminder doses (R1-R4) of IrP (irrelevant peptide) or P3 conjugated to the carrier. IrP and P3-immunized rabbits were randomly divided into a normal diet group and a HFD-fed group. Anti-P3 antibody levels were determined by ELISA. Lipoprotein profile, circulating and tissue lipids, and vascular pro-inflammatory mediators were determined using standardized methods while atherosclerosis was determined by confocal microscopy studies and non-invasive imaging (PET/CT and Doppler ultrasonography). Studies treating human macrophages (hMΦ) and coronary vascular smooth muscle cells (hcVSMC) with rabbit serums were performed to ascertain the potential impact of anti-P3 Abs on the functionality of these crucial cells. Results : P3 immunization specifically induced the production of anti-P3 antibodies (Abs) and did not alter the lipoprotein profile. HFD strongly induced cholesteryl ester (CE) accumulation in the aorta of both the control and IrP groups, and their serum dose-dependently raised the intracellular CE of hMΦ and hcVSMC, promoting TNFR1 and phospho-NF-kB (p65) overexpression. These HFD pro-inflammatory effects were dramatically decreased in the aorta of P3-immunized rabbits and in hMΦ and hcVSMC exposed to the P3 rabbit serums. Microscopy studies revealed that P3 immunization reduced the percentage of lipids, macrophages, and SMCs in the arterial intima, as well as the atherosclerotic extent and lesion area in the aorta. PET/CT and Doppler ultrasonography studies showed that the average standardized uptake value (SUV) of the aorta and the arterial resistance index (ARI) of the carotids were more upregulated by HFD in the control and IrP groups than the P3 group. Conclusions : P3 immunization counteracts HFD-induced fatty streak formation in rabbits. The specific blockade of the LRP1 (CR9) domain with Anti-P3 Abs dramatically reduces HFD-induced intracellular CE loading and harmful coupling to pro-inflammatory signaling in the vasculature.