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Aggregated LDL is the first ligand reported to interact with the cluster II CR9 domain of low-density lipoprotein receptor-related protein 1 (LRP1). In particular, the C-terminal half of domain CR9, comprising the region Gly1127-Cys1140 exclusively recognizes aggregated LDL and it is crucial for aggregated LDL binding. Our aim was to study the effect of the sequence Gly1127-Cys1140 (named peptide LP3 and its retro-enantio version, named peptide DP3) on the structural characteristics of sphingomyelinase- (SMase) and phospholipase 2 (PLA2)-modified LDL particles. Turbidimetry, gel filtration chromatography (GFC) and transmission electronic microscopy (TEM) analysis showed that LP3 and DP3 peptides strongly inhibited SMase- and PLA2-induced LDL aggregation. Nondenaturing polyacrylamide gradient gel electrophoresis (GGE), agarose gel electrophoresis and high-performance thin-layer chromatography (HPTLC) indicated that LP3 and DP3 prevented SMase-induced alterations in LDL particle size, electric charge and phospholipid content, respectively, but not those induced by PLA2. Western blot analysis showed that LP3 and DP3 counteracted changes in ApoB-100 conformation induced by the two enzymes. LDL proteomics (LDL trypsin digestion followed by mass spectroscopy) and computational modeling methods evidenced that peptides preserve ApoB-100 conformation due to their electrostatic interactions with a basic region of ApoB-100. These results demonstrate that LRP1-derived peptides are protective against LDL aggregation, even in conditions of extreme lipolysis, through their capacity to bind to ApoB-100 regions critical for ApoB-100 conformational preservation. These results suggests that these LRP1(CR9) derived peptides could be promising tools to prevent LDL aggregation induced by the main proteolytic enzymes acting in the arterial intima., The Ministry of Science and Innovation of Spain, in the framework of the State Plan of Scientific and Technical Innovation Investigation 2013–2016, awarded funding to the project “DEVELOPMENT OF AN INNOVATIVE THERAPY FOR THE TREATMENT OF THE ATHEROSCLEROSIS THROUGH INHIBITION OF CHOLESTEROL VASCULAR ACCUMULATION”, led by IPROTEOS SL with file number RTC-2016-5078-1. This project was also financed by the Ministry of Economy, Industry and Competitiveness (MINECO) in the framework of the Subprogram RETOS-COLABORACIÓN, 2016 call. The project is also co-financed by the European Union with the objective to promoting the technological development, innovation and quality research. Support was also received from SAF2017-89613R (to SV), co-financed by the European Regional Development Fund (ERDF), the Fundació la Marató de TV3 Project 201521-10 (to VLlC), FIS PI13/00364 and PI16/00471 (to JSQ) and FIS PI18/01584 (to VLlC) from the Instituto de Salud Carlos III (ISCIII) and co-financed with ERDF. Support was also received from Ministerio de Economía y Competitividad to DdG-C (IJCI-2016-29393). CIBER Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM; CB07/08/0016 (JSQ) and CIBER de Enfermedades Cardiovasculares (CIBERCV; CB16/1100403 (DdG-C, VLlC) are projects run by the Instituto de Salud Carlos III (ISCIII). The CRG/UPF Proteomics Unit is member of ProteoRed PRB3 consortium which is supported by grant PT17/0019 of the PE I+D+i 2013–2016 from the Instituto de Salud Carlos III (ISCIII) and ERDF. We also acknowledge support from the Spanish Ministry of Economy and Competitiveness, “Centro de Excelencia Severo Ochoa 2013–2017”, SEV-2012-0208, and “Secretaria d'Universitats i Recerca del Departament d'Economia I Coneixement de la Generalitat de Catalunya” (2017SGR595 to ES and 2017SGR946 to VLl-C). SB, JLS-Q, AR-U, DdG-C and VL-C are members of the Group of Vascular Biology of the Spanish Society of Atherosclerosis (SEA).

Dyslipemia has a direct impact on cardiac remodeling by altering extracellular matrix (ECM) components. One of the main ECM components is elastin, a proteic three-dimensional network that can be efficiently degraded by cysteine proteases or cathepsins. Dyslipemic status in insulin resistance and combined hyperlipoproteinemia diseases include raised levels of very low density lipoproteins (VLDL), triglyceride (TG)-cholesteryl ester (CE)-rich lipoproteins. Enhanced VLDL concentration promotes cardiomyocyte intracellular cholesteryl ester (CE) accumulation in a LRP1-dependent manner. The aim of this work was to analyze the effect of cardiomyocyte intracellular CE accumulation on tropoelastin (TE) characteristics and to investigate the role of LRP1 and cathepsin S (CatS) on these effects. Molecular studies showed that LRP1 deficiency impaired CE selective uptake and accumulation from TG-CE-rich lipoproteins (VLDL+IDL) and CE-rich lipoproteins (aggregated LDL, agLDL). Biochemical and confocal microscopic studies showed that LRP1-mediated intracellular CE accumulation increased CatS mature protein levels and induced an altered intracellular TE globule structure. Biophysical studies evidenced that LRP1-mediated intracellular CE accumulation caused a significant drop of Tg2 glass transition temperature of cardiomyocyte secreted TE. Moreover, CatS deficiency prevented the alterations in TE intracellular globule structure and on TE glass transition temperature. These results demonstrate that LRP1-mediated cardiomyocyte intracellular CE accumulation alters the structural and physical characteristics of secreted TE through an increase in CatS mature protein levels. Therefore, the modulation of LRP1-mediated intracellular CE accumulation in cardiomyocytes could impact pathological ventricular remodeling associated with insulin-resistance and combined hyperlipoproteinemia, pathologies characterized by enhanced concentrations of TG-CE-rich lipoproteins.