All animals had food and water 0.05. Results P3- Immunization Ginkgolide B induces the production of anti-P3 antibodies in rabbits ELISA analyses showed the absence of specific antibodies against P3 in the serum of both the control (Figure ?(Figure1A)1A) and IrP-injected groups (Figure ?(Figure1B)1B) as well as its presence in P3-immunized rabbits serum (Figure ?(Figure1C).1C). Ginkgolide B 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 hM 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 hM 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 (SUVmean) 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. model, similar to humans in the cholesterol-carried lipoprotein profile. In the rabbit model of HFD-induced atherosclerosis, cholesteryl esters are mainly carried by ApoB-100 lipoproteins and there is an elevated participation of Ginkgolide B SMCs in fatty streak lesions 26. In addition, this model has been previously validated to study the effects of HDL on fatty streak formation and evolution 27 as well as to study vascular inflammation by the mainstay imaging technique 18F-FDG/PET 28. The aim of this work was to study the potential therapeutic relevance of a LRP1 (CR9)-specific blockade with anti-P3 Ab to counteract HFD-induced atherosclerosis. Our results showed that anti-P3 antibodies reduced HFD-induced cholesteryl ester accumulation and pro-inflammatory signaling in the aorta. The potent anti-inflammatory efficacy of anti-P3 Abs allowed for the corroboration of the treatment’s efficacy via non-invasive imaging techniques, such as 18F-FDG/PET and Doppler ultrasonography, which provided a high translational impulse to this innovative, anti-atherosclerotic, potentially therapeutic tool. Methods Peptide Synthesis and Ginkgolide B conjugation The P3 peptide used to immunize rabbits contained the following sequence GDNDSEDNSDEENC corresponding to the amino acids 1127 to 1140 located in LRP1 cluster II (domain CR9) 24. The P3 sequence corresponds to an area of high homology between human and rabbit LRP1, with the difference that the asparagine (N) in humans was replaced by a serine (S) in the rabbit protein. In addition, the amino acid C1148 in the rabbit sequence (GDNDCEDNSDEENC) was replaced by S to achieve greater peptide immunogenic effectiveness. The irrelevant peptide (IrP) has the same sequence than P3 but with amino acids in D-enantiomer configuration. Both peptides were synthesized by the Laboratory of Proteomics & Protein Chemistry, Department of Experimental & Health Sciences, Pompeu Fabra University, by the solid-phase method using a Prelude peptide synthesizer (Protein Technologies, Inc.). Peptides were purified by high-performance liquid chromatography (HPLC, Waters 600) using UV detection at 254 nanometers (Waters 2487) and characterized by mass spectrometry (Applied Biosystems 4700 Proteomics Analyser). Peptides were conjugated to the transporter molecule Keyhole limpet haemocyanin (KLH) for immunizations and with bovine serum albumin (BSA) for ELISAs. The conjugation of peptide to KLH and BSA (Sigma, St. Louis, MO) was performed as previously described 29. Peptide-KLH conjugates were used for rabbit immunization and peptide-BSA conjugates for substrate in Pde2a the immunoassay ELISA to detect specific anti-P3 Abs in the rabbit serum. Animal model Thirty New Zealand White (NZW) rabbits from the San Bernardo Farm animal centre (Navarra, Spain) weighing 1.8-2 kg (6-7 months-age) were used in this study. Rabbits were housed in a Tecniplast R-Suite cage with a surface area of 4.264 cm2. Housing temperature was maintained at 21C, relative humidity ranged between 40-60%, and the light period was 12 hours a day. All animals had food and water 0.05. Results P3- Immunization induces the production of anti-P3 antibodies in rabbits ELISA analyses showed the absence of specific antibodies against P3 in the serum of both the control (Figure ?(Figure1A)1A) and IrP-injected groups (Figure ?(Figure1B)1B) as well as its presence in P3-immunized rabbits serum (Figure ?(Figure1C).1C). Anti-P3 Abs levels were maintained in P3-immunized rabbits serum throughout the entire diet period. Previous studies focusing on the functional evaluation of anti-P3 Abs showed their efficacy in reducing foam cell formation through the blockade of the LRP1/agLDL interaction 24. Here, confocal microscopy studies revealed that Abs in the P3-immunized rabbits serum.