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Cardiovascular disease (CVD) is a leading cause of death worldwide. Approximately 60% of patients treated with low‐density lipoprotein (LDL)‐lowering drug treatments, with on‐target plasma cholesterol levels, are still suffering clinical acute ischemic events. Mechanisms, such as LDL aggregation, underlie extracellular and intracellular cholesterol accumulation in the vasculature. A peptide sequence (P3) of the low‐density lipoprotein receptor‐related protein 1 (LRP1) efficiently protects LDL from sphingomyelinase (SMase‐) and phospholipase A2 (PLA2)‐induced LDL aggregation. The aim is to design families of peptide derivatives from P3 with enhanced potency and proteolytic stability. New peptides are designed through in silico conformational sampling and ApoB‐100 molecular docking, and are tested in dual (biochemical‐cellular) screening assays. A total of 46 new peptides including linear, fragment, cyclic, and alanine scanning derivatives are generated through two consecutive optimization rounds. Structurally and functionally optimized peptides contain hotspot residues that are replaced by alanine. This strategy confers an increased capacity to form prone alpha‐helix conformations crucial for the electrostatic interaction with ApoB‐100. These new compounds are highly efficient at inhibiting LDL aggregation and human coronary vascular smooth muscle cell‐cholesteryl ester loading and should be studied in preclinical models of atherosclerosis., A.B.-A. and C.P. contributed equally to this work. The authors thank Dr. Ignasi Gich, Professor of Clinical Pharmacology and Therapeutics and Researcher of Sant Pau Biomedical Research Institute (IIB-SantPau) and CIBER Epidemiología y Salud Pública (CIBERESP), for this help in statistical analysis and graph representations. 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 No. RTC-2016-5078-1. Support was also received from the Fundació MARATÓ TV3 Project 201521-10 (to VLl-C), and FIS PI18/01584 (to VLl-C) from the Instituto de Salud Carlos III (ISCIII) and cofinanced with ERDFs. Support was also received from Ministerio de Economía y Competitividad to DdG-C (IJCI-2016-29393). CIBER Enfermedades Cardiovasculares (CIBERCV; CB16/1100403 (DdG-C, VLl-C) are projects run by the Instituto de Salud Carlos III (ISCIII). The authors also acknowledge the support from “Secretaria d’Universitats i Recerca del Departament d’Economia I Coneixement de la Generalitat de Catalunya” (2017SGR946 to VLl-C).

Background and aims Progranulin is a circulating protein that modulates inflammation and is found in atherosclerotic lesions. Here we determined whether inflammatory cell–derived progranulin impacts atherosclerosis development. Methods Ldlr–/– mice were transplanted with bone marrow from wild-type (WT) or Grn–/– (progranulin KO) mice (referred to as Tx-WT and Tx-KO, respectively). Results After 10 weeks of high-fat diet feeding, both groups displayed similarly elevated plasma levels of cholesterol and triglycerides. Despite abundant circulating levels of progranulin, the size of atherosclerotic lesions in Tx-KO mice was increased by 47% in aortic roots and by 62% in whole aortas. Aortic root lesions in Tx-KO mice had increased macrophage content and larger necrotic cores, consistent with more advanced lesions. Progranulin staining was markedly reduced in the lesions of Tx-KO mice, indicating little or no uptake of circulating progranulin. Mechanistically, cultured progranulin-deficient macrophages exhibited increased lysosome-mediated exophagy of aggregated low-density lipoproteins resulting in increased cholesterol uptake and foam cell formation. Conclusions We conclude that hematopoietic progranulin deficiency promotes diet-induced atherosclerosis in Ldlr–/– mice, possibly due to increased exophagy-mediated cholesterol uptake. Circulating progranulin was unable to prevent the increased lesion development, consistent with the importance of progranulin acting via cell-autonomous or local effects.

Monocyte-derived cells use an extracellular, acidic, lytic compartment (a lysosomal synapse) for initial degradation of large objects or species bound to the extracellular matrix. Akin to osteoclast degradation of bone, extracellular catabolism is used by macrophages to degrade aggregates of low density lipoprotein (LDL) similar to those encountered during atherogenesis. However, unlike osteoclast catabolism, the lysosomal synapse is a highly dynamic and intricate structure. In this study, we use high resolution three dimensional imaging to visualize compartments formed by macrophages to catabolize aggregated LDL. We show that these compartments are topologically complex, have a convoluted structure and contain sub-regions that are acidified. These sub-regions are characterized by a close apposition of the macrophage plasma membrane and aggregates of LDL that are still connected to the extracellular space. Compartment formation is dependent on local actin polymerization. However, once formed, compartments are able to maintain a pH gradient when actin is depolymerized. These observations explain how compartments are able to maintain a proton gradient while remaining outside the boundaries of the plasma membrane., Summary: Macrophages use extracellular compartments, termed lysosomal synapses, that are topologically complex, have a convoluted structure and contain sub-regions that are acidified to digest aggregated lipoproteins.

Atherosclerosis is a progressive disease characterized by modified low density lipoprotein (LDL) accumulation in the large artery walls, with subsequent phagocytosis by macrophages, transforming into foam cells, and vascular smooth muscle cells (VSMCs). In particular, native LDL (nLDL) can modify joining to each other giving rise to aggregated LDL (agLDL), which were reported to be internalized more avidly by VSMCs in in vitro conditions (1). Here, primarily cultured aortic valve interstitial cells (AVICs) were treated for 3 up to 21 days with 50 mg/ml blood-derived nLDL or agLDL, alone or combined with pro-calcific culture media, to ascertain whether (i) agLDL are taken up more rapidly than nLDL also by AVICs and (ii) treatment with LDL at low, normolipidemic-like concentration can influence AVIC mineralization. Ultrastructurally, LDL uptake was observed exclusively for agLDL-treated AVICs, which were characterized by a lot of endocytic vesicles and intracytoplasmic vacuoles entrapping agLDL particles. Consistently, treatment with agLDL resulted in a significant increase in intracellular amounts of both esterified cholesterol and triglycerides, as chromatographically assayed. Ultrastructural analyses also revealed pro-calcific AVIC degenerative process to occur, consisting in intracellular release of lipidic material and its layering at cell edges, there acting as major hydroxyapatite nucleator (2,3). The extent of pro-calcific effects resulted to be LDL-dependent, being mitigated in the presence of nLDL and exacerbated in the presence of agLDL. These findings were consistent with spectrophotometrical analyses showing decreased calcium amounts in AVIC cultures superstimulated with nLDL and increased mineral content in the counterpart superstimulated with agLDL. Since aortic valve stenosis is considered as a valve atherosclerotic lesion, these preliminary data suggest that avid uptake of agLDL by AVICs might strongly contribute to lipid accumulation within aortic valves, with agLDL possibly affecting subsequent valve tissue mineralization even at concentrations comprised within the normolipidemic range., Italian Journal of Anatomy and Embryology, Vol. 120, No. 1 (Supplement) 2015

Low density lipoprotein receptor-related protein 1 (LRP1) is a ubiquitously expressed cell surface receptor that mediates the internalization of more than 40 different ligands and participates in cell signaling. LRP1 plays a key pathophysiological role in the onset, development and thrombotic resolution of the atherosclerotic process due to its role in the intracellular lipid accumulation of vascular smooth muscle cells (VSMCs). During last years, LRP1 has also been proposed as a modulator of the inflammatory phenomenon in atherosclerosis. However, the investigations have obviated the function of LRP1 as a binding receptor of modified-lipids. In addition, the studies that analysed the relation between inflammation and LRP1 are focused on immunological cells from mice models. Whether LRP1 can respond to its modified-LDL ligands developing an inflammatory response in human VSMCs is unknown. Further investigations are, thus, necessary., Review | Biohelikon : Cell Biology

The transcription of the Low-density lipoprotein receptor-related protein (LRP1) is upregulated by aggregated LDL (agLDL) and angiotensin II (AngII) in human vascular smooth muscle cells (hVSMC). The polymorphism c.1-25C>G creates a new GC-box in the LRP1 promoter recognized by Sp1/Sp3 transcription factors. The aims of this study were 1) to evaluate the impact of c.1-25C>G polymorphism on LRP1 transcriptional activity and expression, and 2) to examine the response of c.1-25C>G LRP1 promoter to LDL and AngII. EMSA and Luciferase assays in HeLa cells showed that -25G promoter has enhanced basal transcriptional activity and specific Sp1/Sp3 binding. hVSMC with GG genotype (GG-hVSMC) had higher LRP1 mRNA and protein levels, respectively than CC genotype (CC-hVSMC). EMSA assays showed that the polymorphism determines scarce amount of SRE-B/SREBP-2 complex formation and the failure of agLDL to further reduce these SRE-B/SREBP-2 complexes. Taken together, these results suggest that c.1-25C>G, by difficulting SREBP-2 binding, prevents SREBP-2 displacement required for LRP1 promoter response to LDL. In contrast, c.1-25C>G strongly favours Sp1/Sp3 binding and AngII-induced activity in Sp1/Sp3 dependent manner in GG-hVSMC. This increase is functionally translated into a higher capacity of GG-hVSMC to become foam cells from agLDL in presence of AngII. These results suggest that c.1-25C>G determines a lack of response to agLDL and an exacerbated response to AngII. It is thus conceivable that the presence of the polymorphism would be easily translated to vascular alterations in the presence of the pro-hypertensive autacoid, AngII. (C) 2014 Elsevier Ireland Ltd. All rights reserved.

Sterol regulatory element-binding proteins (SREBPs) negatively modulate the expression of the CD91/low-density lipoprotein receptor-related protein (LRP1), a carrier and signaling receptor that mediates the endocytosis of more than 40 structurally and functionally distinct ligands. The aim of this work was to analyze whether lipopolysaccharide (LPS) can regulate LRP1 expression through SREBPs in human monocyte-derived macrophages (HMDM). LPS led to LRP1 mRNA and protein inhibition in a dose-and time-dependent manner. Concomitantly, a strong upregulation of SREBP-1 mRNA and SREBP-1 nuclear protein levels was observed in LPS-treated HMDM. The specific silencing of SREBP-1 efficiently prevented LRP1 reduction caused by LPS. SREBP-1 mRNA and nuclear protein levels remained high in HMDM treated with LPS unexposed or exposed to LDL. Native (nLDL) or aggregated LDL (agLDL) per se downregulated SREBP-2 expression levels and increased LRP1 expression. However, lipoproteins did not significantly alter the effect of LPS on SREBP-1 and LRP1 expression. Collectively, these data support that lipoproteins and LPS exert their modulatory effect on LRP1 expression through different SREBP isoforms, SREBP-2 and SREBP-1, respectively. These results highlight a crucial role of SREBP1 as a mediator of the downregulatory effects of LPS on LRP1 expression in human macrophages, independently of the absence or presence of modified lipoproteins. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Objective— Low density lipoprotein retention and aggregation in the arterial intima are key processes in atherogenesis. Aggregated LDL (agLDL) is taken up through low-density lipoprotein receptor-related protein 1 (LRP1) by human vascular smooth muscle cells (VSMC). AgLDL increases LRP1 expression, at least in part, by downregulation of sterol regulatory element-binding proteins. It is unknown whether agLDL has some effect on the ubiquitin-proteasome system, and therefore on the LRP1 receptor turnover. The objective of this study was to analyze the effect of agLDL on the degradation of LRP1 by the ubiquitin-proteasome system in human VSMC. Methods and Results— Human VSMC were isolated from the media of human coronary arteries. Ubiquitinylated LRP1 protein levels were significantly reduced in human VSMC exposed to agLDL (100 μg/mL) for 20 hours (agLDL: 3.70±0.44 a.u. versus control: 9.68±0.55 a.u). Studies performed with cycloheximide showed that agLDL prolongs the LRP1 protein half life. Pulse-chase analysis showed that LRP1 turnover rate is reduced in agLDL-exposed VSMC. Two-dimensional electrophoresis shows an alteration in the proteomic profile of a RING type E3 ubiquitin ligase, CHFR. Real-time PCR and Western blot analysis showed that agLDL (100 μg/mL) decreased the transcriptional and protein expression of CHFR. CHFR silencing increased VSMC, but not macrophage, LRP1 expression. However, CHFR silencing did not exert any effect on the classical low-density lipoprotein receptor protein levels. Furthermore, immunoprecipitation experiments demonstrated that the physical interaction between CHFR and LRP1 decreased in the presence of agLDL. Conclusion— Our results demonstrate that agLDL prolongs the half life of LRP1 by preventing the receptor ubiquitinylation, at least in part, through CHFR targeting. This mechanism seems to be specific for LRP1 and VSMC.

Low density lipoprotein receptor-related protein (LRP1) is upregulated in vascular smooth muscle cells by intravascular aggregated LDL (agLDL) - LDL trapped in the arterial intima and systemic LDL. LRP1 upregulation in hypercholesterolemic aortas is concomitant with SREBP downregulation. However, the specific role of SREBP isoforms in LRP1 transcription and LDL-induced LRP1 upregulation in human vascular smooth muscle cells (VSMC) is unknown. In the present study were port that specific silencing of either SREBP-1 or SREBP-2 enhanced LRP1 whereas overexpression of the active SREBP isoforms decreased LRP1 expression. Gel mobility shift and ChIP assays demonstrated that SREBP-1a, SREBP-1c and SREBP-2 were able to bind to three putative SRE sequences; SRE-A (-1042 to -1028), SRE-B (-115 to -101) and SREC (+226 to +234). ChIP assays demonstrated that agLDL (100 mu g/mL, 24 h) significantly and specifically decreased SREBP-2 binding to the LRP1 promoter. Luciferase assays demonstrated that agLDL increased the transcriptional activity of A/B or A/C double mutants but failed to increase that of the double B/C mutant. Our results show that both SREBP-1 and SREBP-2 negatively modulated LRP1 transcription. Furthermore, agLDL exerted an upregulatory effect on LRP1 expression by decreasing SREBP-2 binding to LRP1 promoter. Two SRE-like sequences control the response of LRP1 to agLDL. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Background: Our previous results demonstrated that aggregated low density lipoprotein (agLDL) induces tissue factor (TF) expression and activation through Rho A translocation in human vascular smooth muscle cells (VSMC). We also previously demonstrated that membrane sphingomyelin (SM) content is higher in agLDL-exposed VSMC than in control cells. The main enzymes regulating cellular SM content are the family of sphingomyelinases (Smases) that hydrolize SM to phosphorylcholine and ceramide (CER). Objectives: We wished to investigate whether agLDL has the ability to modulate acidic- (A-) and neutral (N-) Smase activity and whether or not this effect is related to the upregulatory effect of agLDL on Rho A translocation and TF activation in human VSMC. Methods and Results: By measuring generated [14C]-phosphorylcholine, we found that agLDL significantly decreased A-Smase and specially N-Smase activity. Pharmacological Smase inhibitors increased Rho A and TF. Specific loss-of-function of A-Smase or N-Smase 1 (N1-Smase) by siRNA treatment (500 nmol L-1, 12 hours) dramatically increased membrane Rho A protein levels (5- and 3-fold, respectively). Concomitantly, TF protein expression and TF procoagulant activity were also increased. Inhibition of A-Smase or N-Smase activity by agLDL, siRNA-anti A- or N1-Smase or pharmacological treatment significantly increased the SM content of vascular cells. The inhibition of SM synthesis by fumonisin B-1 (FB1) prevented the upregulatory effect of agLDL on TF. Conclusions: These results demonstrate that inhibition of both A- and N1-Smase might explain the upregulatory effect of agLDL on TF activation, and suggest that this effect is related, at least in part, to membrane SM enrichment.

Aggregated LDL (agLDL) is internalized by LDL receptor-related protein (LRP1) in vascular smooth muscle cells (VSMCs) and human monocyte-derived macrophages (HMDMs). AgLDL is, therefore, a potent inducer of massive intracellular cholesteryl ester accumulation in lipid droplets. The adipocyte differentiation-related protein (ADRP) has been found on the surface of lipid droplets. The objectives of this work were to analyze whether agLDL uptake modulates ADRP expression levels and whether the effect of agLDL internalization on ADRP expression depends on LRP1 in human VSMCs and HMDMs. AgLDL strongly upregulates ADRP mRNA (real-time PCR) and protein expression (Western blot) in human VSMCs (mRNA: by 3.06-fold; protein: 8.58-fold) and HMDMs (mRNA: by 3.5-fold; protein: by 3.71-fold). Treatment of VSMCs and HMDMs with small anti-LRP1-interfering RNA (siRNA-LRP1) leads to specific inhibition of LRP1 expression. siRNA-LRP1 treatment significantly reduced agLDL-induced ADRP overexpression in HMDMs (by 69%) and in VSMCs (by 53%). Immunohystochemical studies evidence a colocolocalization between ADRP/macrophages and ADRP/VSMCs in advanced lipid-enriched atherosclerotic plaques. These results demonstrate that agLDL-LRP1 engagement induces ADRP overexpression in both HMDMs and human VSMCs and that ADRP is highly expressed in advanced lipid-enriched human atherosclerotic plaques. Therefore, LRP1-mediated agLDL uptake might play a pivotal role in vascular foam cell formation.