15824934, 2021, 16, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16723 by Readcube (Labtiva Inc.), Wiley Online Library on [03/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License Received: 26 December 2020 | Revised: 19 May 2021 | Accepted: 29 May 2021 DOI: 10.1111/jcmm.16723 ORIGINAL ARTICLE Microvesicles carrying LRP5 induce macrophage polarization to an anti-­inflammatory phenotype Aureli Luquero1 | Gemma Vilahur1,2 | Javier Crespo1 | Lina Badimon1,2,3 | Maria Borrell-­Pages1,2 1 Cardiovascular Program ICCC, IR-­Hospital de la Santa Creu i Sant Pau, IIB-­Sant Pau, Abstract Barcelona, Spain Microvesicles (MV) contribute to cell-­to-­cell communication through their trans- 2 CIBER-­C V, Instituto de Salud Carlos III, ported proteins and nucleic acids. MV, released into the extracellular space, exert Madrid, Spain 3 Cardiovascular Research Chair, UAB, paracrine regulation by modulating cellular responses after interaction with near and Barcelona, Spain far target cells. MV are released at high concentrations by activated inflammatory Correspondence cells. Different subtypes of human macrophages have been characterized based on Maria Borrell-­Pages, Cardiovascular surface epitopes being CD16+ macrophages associated with anti-­inflammatory phe- Research Program, IR-­Hospital de la Santa Creu i Sant Pau, C/Sant Antoni Maria Claret notypes. We have previously shown that low-­density lipoprotein receptor-­related 167, 08025 Barcelona, Spain. protein 5 (LRP5), a member of the LDLR family that participates in lipid homeostasis, Email: mborrellpa@santpau.cat is expressed in macrophage CD16+ with repair and survival functions. The goal of our Funding information study was to characterize the cargo and tentative function of macrophage-­derived Instituto de Salud Carlos III, Grant/Award Number: CIBERCV CN16/11/00411, MV, whether LRP5 is delivered into MV and whether these MV are able to induce FIS2019-­01282, TERCEL RD16/0011/018, inflammatory cell differentiation to a specific CD16− or CD16+ phenotype. We show, CIBERCV CB16/11/00411 and FIS2020-­01282; Generalitat of Catalunya-­ for the first time, that lipid-­loaded macrophages release MV containing LRP5. LDL Secretaria d’Universitats i Recerca del loading induces increased expression of macrophage pro-­inflammatory markers and Departament d’Economia i Coneixement de la Generalitat, Grant/Award Number: increased release of MV containing pro-­inflammatory markers. Conditioning of fresh 2014SGR1303, PERIS SLT006 and macrophages with MV released by Lrp5-­silenced macrophages induced the transcrip- 2017SGR1480; Spanish Ministry of Science and Innovation, Grant/Award tion of inflammatory genes and reduced the transcription of anti-­inflammatory genes. Number: SAF2016-­76819-­R and PID2019-­ Thus, MV containing LRP5 induce anti-­inflammatory phenotypes in macrophages. 107160RB-­I 00; Spanish Society of Cardiology, Grant/Award Number: FEC2019 KEYWORDS inflammation, lipids, LRP5, macrophages, MV 1 | I NTRO D U C TI O N the procoagulant anionic phosphatidylserine making MV Annexin V positive.11,12 Their cargo defines their shape, size and function. Extracellular microvesicles (MV) are cell shed particles (diame- Because MV reflect the condition of their parental cells, they repre- ter ranging from 0.1 to 1 μm) released when cells are stimulated, sent a potential diagnostic tool to identify diverse diseases, including damaged or undergoing apoptosis.1-­9 They can also be released by cancer, metabolic and cardiovascular diseases. When MV are shed healthy cells.10 MV are formed by direct budding of small cytoplas- from their cells of origin, they circulate in blood carrying messengers matic protrusions that are detached from the cell surface into the for recipient cells that will receive the signal and regulate their cel- extracellular space. They are characterized by the externalization of lular growth, differentiation and transformation.4-­10,13-­16 Indeed, the This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd. J Cell Mol Med. 2021;25:7935–7947.  wileyonlinelibrary.com/journal/jcmm | 7935 | 15824934, 2021, 16, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16723 by Readcube (Labtiva Inc.), Wiley Online Library on [03/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 7936 LUQUERO et al. interaction of MV with target cells and the release of their content investigation conformed to the principles outlined in the Declaration 17 modulate cell responses. MV have been described in inflammatory of Helsinki with informed consent given by donors. Briefly, blood processes and associated with several cardiovascular risk factors18 was applied on 15 mL of Ficoll-­Hypaque and centrifuged at 300 g contributing to the initiation and progression of cardiovascular dis- for 1 hour at 22°C, with no brake. Mononuclear cells were obtained eases, including atherosclerosis. from the central white band of the gradient, exhaustively washed in Atherosclerosis is characterized by chronic inflammation induced Dulbecco's phosphate buffer saline, and suspended in RPMI medium by increasing accumulation of low-­density lipoproteins (LDL) and (Gibco) supplemented with 10% human serum AB (Sigma). Isolated apoptotic cells in the intima layer of the arteries.19 The low-­density monocytes (Mo) were left overnight in culture, washed and treated lipoprotein receptor-­related protein 5 (LRP5) is a multifunctional re- with 100 μg/mL nLDL (native LDL) or agLDL (aggregated LDL) for the ceptor involved in both endocytosis of lipids and the canonical Wnt described times. A second set of isolated Mo were left 7 days in cul- signalling pathway. 20 LRP5 is a single-­pass transmembrane receptor ture and allowed to differentiate into macrophages (Mac) by chang- that participates in the Wnt/β-­catenin signalling pathway. LRP5 acti- ing the cell culture media (RPMI supplemented with 10% human vation causes the stabilization of β-­catenin that translocates into the serum AB, 100 units/mL penicillin and 100 µg/mL streptomycin) nucleus, binds to the transcription factor TCF/LEF1 and starts the every 3 days. After several washings with PBS to completely remove transcription of Wnt target genes that regulate fundamental aspects serum, human macrophages were incubated with 100 μg/mL nLDL of embryonic cell development 21 and adult cell function. 20,22,23 or 100 µg/mL agLDL in serum-­free medium.20,26-­28 At the end of the LDL loading induces high LRP5 expression in human macro- experiments, human Mo and Mac were exhaustively washed (twice 20 phages. Macrophages can be classified into classical activated with PBS, twice with PBS/1% BSA, once with PBS/1%BSA/heparin CD14+CD16−, pro-­ inflammatory macrophages and alternatively 100 U/mL, twice with PBS/1% BSA and twice with PBS) and prepared activated CD14−CD16+, anti-­inflammatory macrophages. 24,25 LRP5 for the collection of mRNA and protein detection as described below. participates in inflammation and macrophage polarization by asso- ciation with the anti-­inflammatory macrophage subtype CD16+ de- rived from CD14+CD16+ patrolling circulating monocytes. 26 LRP5 2.2 | LDL isolation and modification confers the motile function to CD16+ macrophages by triggering the canonical Wnt signalling. Furthermore, CD16+LRP5+ macrophages, Human LDL (d1.019-­d1.063 g/mL) were obtained as previously de- found in advanced atherosclerotic human plaques, trigger an anti-­ scribed. 28 Briefly, human LDLs were obtained from pooled sera of 26 inflammatory, defensive and repair response. normocholesterolemic volunteers and isolated by sequential ultra- The in-­depth understanding of the formation, cargo and function centrifugation. LDLs were dialyzed three times against 200 volumes of MV is an ongoing task in the field. The objectives of this study were of 150 mmol/L NaCl, 1 mmol/L EDTA, and 20 mmol/L Tris-­HCl, pH (a) to characterize the cargo and function of macrophage-­derived 7.4, overnight and once against 150 mmol/L NaCl. LDL protein con- MV and their ability to induce inflammatory cell differentiation to a centration was determined by the bicinchoninic acid, and vortexing CD16− or a CD16+ phenotype, and (b) to investigate whether LRP5 was monitored by measuring the turbidity (absorbance at 680 nm). is delivered into MV and whether it can exert paracrine functions. The model system of agLDL was generated by vortexing LDL (1 mg/ We show that LDL-­ loaded macrophages release MV carry- mL) for 4 minutes at room temperature at maximal speed. The per- ing LRP5 and exert paracrine and/or autocrine regulation. LDL centage of LDL in aggregated form was calculated by measuring loading induces increased expression of macrophage cellular pro-­ the fraction of protein recovered in the pellet obtained after cen- inflammatory markers and increased release of MV. Interestingly, trifugation at 10 000 g for 10 minutes. The different fractions were LRP5 is released in MV that contain both pro-­and anti-­inflammatory analysed by agarose electrophoresis, and the precipitated fraction markers. Conditioning of recipient macrophages with MV released composed of 100% agLDL was added to cell cultures. by Lrp5-­ silenced macrophages induced pro-­ inflammatory gene transcription and a reduced expression of anti-­inflammatory genes indicating that LRP5 induces macrophage differentiation into the 2.3 | MV isolation and quantification anti-­inflammatory phenotype. LDL-­loaded or non-­loaded human Mo and Mac were cultured for 24 or 48 hours and the MV released into the supernatants collected. 2 | M E TH O DS MV were isolated by five-­step high-­speed centrifugations. Briefly, 2 mL of fresh supernatant aliquots were centrifuged at 3200 g for 2.1 | Isolation of human monocytes and human 20 minutes to guarantee complete cell and debris removal. The macrophages primary cultures and LDL loading recovered supernatants were centrifuged at room temperature at 300 g, (10 minutes); at 1200 g, (20 minutes); and at 12 500 g (5 min- Human monocytes were obtained by standard protocols from utes) in two repeated processes to ensure the elimination of nLDL buffy coats of healthy blood donors. 20,26-­28 All procedures were ap- or agLDL. The cleared supernatants were transferred to another vial proved by the Institutional Review and Ethics Committee, and the and centrifuged at 20 500 g for 150 minutes at RT to pellet the MV. | 15824934, 2021, 16, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16723 by Readcube (Labtiva Inc.), Wiley Online Library on [03/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License LUQUERO et al. 7937 Supernatants were removed and the MV-­enriched pellets (MVp) (Table S2) for 30 minutes in 100 µL 0.5%BSA/PBS. The reaction were suspended in 100 μL citrate-­PBS. was stopped by adding 4 volumes of 0.5%BSA/PBS to the mix. MVp (5 μL) in combination with 2-­3 specific monoclonal antibod- Cells were sorted using a FACSAria-­I (BD Biosciences) operated ies (1-­5 μL each) labelled with phycoerythrin, 488 or the isotype-­ using a 100 µm nozzle with the 488 nm and 633 nm laser lines. matched control antibodies were added in a final volume of 50 μL After positive selection of CD11b+CD14+ cells, two populations annexin binding buffer with 5 μL of Annexin V (AV) to label and were sorted: CD11b+CD14+CD206+/CD11b+CD14+CD206− or + characterize AV MV with bioactive and biomarker molecules from CD11b CD14 CD80+/CD11b+CD14+CD80−. After sorting, mac- + + their parental cells. Table S1 shows the different antibodies and the rophage populations were centrifuged separately for 10 minutes at concentrations used for microvesicle identification and characteri- 200 g. Then, cells were suspended in RPMI GlutaMax medium sup- zation. Samples were incubated 20 minutes at room temperature in plemented with 10% AB human serum with 1% penicillin/streptomy- the dark and diluted with annexin binding buffer before being imme- cin and seeded into 6-­well plates for 24 hours. Flow cytometry data diately analysed and counted on a FACSCanto II flow cytometer. The acquisition, analysis and image preparation were performed using number of monocytes or macrophage per well were counted using the FACSDiva software (BD Bioscience). Neubauer chambers, and the number of MV/cell type was obtained. AV binding level was corrected for autofluorescence using flu- orescence signals obtained with MV in a calcium-­free buffer PBS. 2.6 | Supernatant collection MV were identified and quantified based on their forward scatter/ side scatter characteristics according to their size, binding or not to Cell sorted macrophages were cultured in serum-­ free RPMI AV and reactivity to specific monoclonal antibodies. Figure S1 shows GlutaMax medium for 2 days when supernatants were collected and representative plots for MV identification and characterization by centrifuged at 15 000 g, 15 minutes, 4°C. Pellets were discharged flow cytometry analysis. and supernatants were precipitated using a methanol/chloroform Acquisition was performed at 1 minute per sample and flow rate protocol. Briefly, one volume of supernatant was mixed with three was measured before each experiment. Forward scatter, side scatter volumes of cold methanol and one volume of chloroform, vortexed and fluorescence data were obtained with the settings in the loga- vigorously for 30 seconds, and then, three volumes of H2O were rithmic scale. The lower detection limit was placed as a threshold added to the sample to induce phase separation. The mix was cen- above the electronic noise of the flow cytometer. To identify posi- trifuged at 10 000 g for 5 minutes, and the upper phase was elimi- tive marked events, thresholds were also set based on samples incu- nated without disturbing the interphase. Three volumes of methanol bated with the same final concentration of isotype-­matched control were added to the mix, and samples were centrifuged at 10 000 g antibodies after titration experiments. Data were analysed with the for 5 minutes. Supernatants were discharged and the precipitated FACSDivaTM software (version 6.1.3; Becton Dickinson). To reduce proteins (pellet) were let to air-­dry. Finally, samples were suspended background noise, buffers were prepared on the same day and fil- in 100 µL of lysis protein buffer solution and frozen at −20°C until tered through 0.2 μm pore size filters under vacuum. western blots were performed. 2.4 | Macrophages isolation by flow cytometry 2.7 | Western blot Cellular protein expression was assessed in primary cultures of Protein extracts (50 µL) were resolved by SDS-­PAGE and transferred human macrophages by flow cytometry. Cell suspensions in flow cy- to nitrocellulose membranes, blocked with 5% bovine serum albumin tometry buffer (0.1% sodium azide/1%BSA/PBS) were gently centri- and probed for monoclonal primary antibodies against IL-­1β, TNFα fuged at 200 g, 10 minutes, RT. Pellet samples were then suspended and TGF-­β from Cell Signalling. Membranes were then washed and in flow cytometry buffer and stained for 20 minutes with specific blotted with antimouse secondary antibodies (Dako). Band densi- antibodies as described in Table S2. Figure S2 shows the gating ties were determined with the ChemiDoc XRS system (Bio-­Rad) in strategy for live macrophages by flow cytometry analysis. Samples chemiluminescence detection modus and Quantity-­One software were diluted with 400 μL flow cytometry buffer prior to being im- (Bio-­Rad). mediately analysed. For each sample, at least 10 000 events were acquired on a FACSCantoII (Beckton Dickinson). Data was analysed with the FACSDiva 6.1.3 software. 2.8 | LRP5 silencing Human macrophages were transfected with 100 nmol/L of siRNA-­ 2.5 | Macrophages isolation by cell sorter Random (siR) or siRNA-­LRP5 (si5) using HiPerfect® as recommended by the manufacturer. Small anti-­LRP5 interfering RNAs (si5, s8293) Lipid loaded macrophages were gently detached from culture dishes were synthesized by Applied Biotechnologies and Silencer Selective and stained with CD11b, CD14 and CD206 or CD80 antibodies Negative Control #1 (siR, 4390843) by Ambion. | 15824934, 2021, 16, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16723 by Readcube (Labtiva Inc.), Wiley Online Library on [03/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 7938 LUQUERO et al. 2.9 | RNA isolation and Real time PCR hypothesized that the LDL loading would increase the generation of MV carrying LRP5. Primary cultures of human monocytes and mac- Total RNA was isolated from cultured human monocytes and mac- rophages were treated with 100 μg/mL nLDL or agLDL for 24 hours or rophages using the total RNA extraction kit (Qiagen). Total RNA 48 hours and, indeed, agLDL loading induced a massive generation of concentration was determined by NanoDrop ND-­1000 spectropho- AV+MV from monocytes while a modest amount of AV+MV were re- tometer (NanoDrop Technologies, Inc), and purity was checked by leased by macrophages (Figure 1D). LDL loading induced the release of the A260/A280 ratio (ratios between 1.8 and 2.1 were considered ac- AV+LRP5+MV in larger quantities in monocytes than in macrophages ceptable), in addition, an agarose gel was run to assess quality. cDNA (Figure 1E). However, the relative release of AV+LRP5+MV (normal- was synthesized from 1 μg RNA with cDNA reverse transcription kit ized by total AV+MV) was significantly induced by LDL loading in mac- (Qiagen) The resulting cDNA samples were amplified by polymerase rophages both after 24 hours and 48 hours incubation (Figure 1F). chain reaction (PCR) using a DNA thermal cycler (MJ Research) and We then estimated the amount of MV produced by each mono- the following specific human probes from Applied Biotechnologies: cyte or macrophage (MV/Mo and MV/Mac). Lipid-­loaded Mo release LRP5, iNOS, CD80, CD163 and IL1Ra. Normalization was performed more AV+ MV than Mac after 24 hours (175 ± 21 AV+MV/Mo vs against r18S. 6 ± 0.8 AV+MV/Mac, Figure S3A) and 48 hours agLDL incubation (108 ± 15 AV+MV/Mo vs 6 ± 0.4 AV+MV/Mac, Figure S3A). AgLDL treatments induced more LRP5+MV release in individual monocytes 2.10 | Statistical analysis than in macrophages both after 24 hours and 48 hours incubation (Figure S3B). Finally, the relative amount of AV+LRP5+MV/cell type A StatView statistical package was used for all the analysis. Results (normalized by AV+MV/cell type) released by macrophages was are expressed as mean ± SD or n (%) when indicated. When pos- higher than that released by monocytes after 24 hours and 48 hours sible, comparisons among groups were performed by parametric agLDL incubation (Figure S3C). (one factor ANOVA) analysis. Statistical significance was consid- ered when P < .05. All the experiments were performed at least three times. 3.3 | agLDL loading induces macrophage polarization 3 | R E S U LT S We previously observed that LRP5 is mainly expressed in CD16+ macrophages and lipid loading induces LRP5 expression in these 3.1 | Monocytes and macrophages induce cells20,26; therefore we investigated whether macrophage polariza- LRP5+MV secretion tion could be induced by agLDL. Lipid-­loaded macrophages were gently detached from the culture dish and these live macrophages To characterize the released MV, supernatants of primary cultures were counted by flow cytometry showing that the agLDL loading of monocytes and macrophages were collected after 24 hours and did not affect cell survival (Figure 2A). Macrophage population was 48 hours (Figure 1A). Mo release around 200 000 MV/mL after defined by size, with specific pro-­and anti-­inflammatory antibod- 24 hours and around 250 000 MV/mL after 48 hours in culture ies and with the well-­known macrophage markers CD11b and CD14. (Figure 1A). Mac release around 52 000 MV/mL after 24 hours Figure S2 shows the gating strategy for live macrophages by flow cy- and almost 100 000 MV/mL after 48 hours in culture (Figure 1A). tometry analysis. Results show that lipid loading induces the expres- These time differences in MV release did not reach statistical sig- sion of CD80+ and CD83+ and reduces the expression of cell surface nificance. However, there was a statistically significant difference in CD16 (CD80+: 2.16% expression in control conditions to 15.81% MV release between Mo and Mac both at 24 hours and at 48 hours expression after agLDL loading; CD83+: 2.06% expression in con- (Figure 1A). trol conditions to 8.65% expression after lipid loading and CD16−: + positive MV (AV MV) We then analysed released Annexin V-­ 77.3% expression in control conditions and 86.3% expression in lipid and no significant differences were found between MV release loaded cells). The expression levels of the anti-­inflammatory marker at 24 hours and 48 hours neither in Mo nor in Mac. Mo released CD16+ was reduced in lipid loaded macrophages while the expres- higher number of AV+MV than Mac both at 24 hours and at 48 hours sion levels of the anti-­inflammatory proteins, CD206+ and CD163+ + (Figure 1B). AV MV released by monocytes and by macrophages at did not vary with respect to control conditions. Therefore, lipid load- 24 hours and 48 hours contained LRP5 (Figure 1C). ing induces a pro-­inflammatory polarization in macrophages with in- creased CD80+, CD83+ and CD16− expression in cells (Figure 2B,C). Macrophage pro-­ inflammatory phenotype after lipid load- + 3.2 | Lipid loading increases LRP5 MV secretion ing was confirmed by pro and anti-­inflammatory protein secretion analyses. Cell sorting was performed on lipid loaded macrophages We have previously shown that lipid loading with modified lipopro- to obtain CD11b+CD14+CD206− and CD11b+CD14+CD206+ or teins (agLDL) increases LRP5 expression in macrophages.20,26 We CD11b+CD14+CD80− and CD11b+CD14+CD80+ macrophage | 15824934, 2021, 16, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16723 by Readcube (Labtiva Inc.), Wiley Online Library on [03/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License LUQUERO et al. 7939 F I G U R E 1 AgLDL treatments in macrophages induce LRP5+MV secretion. 24 hours or 48 hours supernatants from undifferentiated monocytes (Mo) or from 7 to 10 days fully differentiated macrophages (Mac) were collected and the amount of (A) microvesicles/mL; (B) Annexin V+ microvesicles/mL and (C) Annexin V+ LRP5+ microvesicles/mL were analysed. (D) Monocytes (Mo) or macrophages (Mac) were treated with 100 μg/mL nLDL or 100 μg/mL agLDL for 24 hours or 48 hours and the amount of AV+ MV/mL and of (E) AV+ LRP5+ MV/mL was analysed. (F) The ratio between MV that are AV+LRP5+/AV+ in control and lipid-­loaded monocytes (Mo) and macrophages (Mac). All experiments were performed at least four times in duplicates or triplicates. *P < .05, **P < .01, ***P < .005 subpopulations. The different macrophage subpopulations were pro-­inflammatory polarized phenotype in macrophages observed by seeded in culture dishes and supernatants were collected after cell surface markers expression after lipid loading. 48 hours. Increased release of the pro-­ inflammatory proteins TNFα and IL1β was observed in the pro-­inflammatory CD80+ sub- population while the levels remained low in the anti-­inflammatory 3.4 | Lipid loading induces LRP5 expression in + CD206 subpopulation (Figure 2D). Conversely, the release of the macrophages anti-­inflammatory protein TGFβ was higher in the CD206+ macro- phage subpopulation than in the CD80+ macrophage subpopulation We next examined the expression levels of LRP5 in the differ- (Figure 2D). Therefore, inflammatory protein release confirms the ent macrophage subpopulations by staining macrophages with a | 15824934, 2021, 16, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16723 by Readcube (Labtiva Inc.), Wiley Online Library on [03/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 7940 LUQUERO et al. F I G U R E 2 AgLDL treatments induce LRP5 expression in pro-­and anti-­inflammatory macrophages. (A) Flow cytometry was used to quantify live macrophages after treatment or not with 100 μg/mL agLDL. (B) Cellular expression of CD80, CD83, CD16, CD206 and CD163 in control and agLDL-­treated macrophages. (C) Quantification of the graphs depicted in (B). (D) TNFα, IL1β and TGFβ expression in supernatants of agLDL-­treated and cell sorted macrophage subpopulations. (E) LRP5 expression levels by flow cytometry in CD16−, CD80+ and CD83+ pro-­inflammatory macrophages in control and after agLDL treatment. (F) Same in anti-­inflammatory CD16+, CD2016+ and CD163+ expressing macrophages. All experiments were performed at least four times in duplicates or triplicates. *P < .05, **P < .01 | 15824934, 2021, 16, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16723 by Readcube (Labtiva Inc.), Wiley Online Library on [03/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License LUQUERO et al. 7941 F I G U R E 3 Macrophages treated with agLDL release LRP5+MV. (A) Flow cytometry was used to quantify AV+ MV secreted by control and 100 μg/mL AgLDL-­treated macrophages. (B) CD16− MV/mL, CD80+ MV/mL, CD16+ MV/mL and CD206+ MV/mL released by control and agLDL-­treated macrophages (C) Same as in (B) but performing the ratio against AV+MV. (D) Quantification of the graphs shown in (C). (E) Flow cytometry detection of LRP5 expression in CD16−MV, CD80+MV, CD16+MV and CD206+MV released by control and agLDL-­treated macrophages. All experiments were performed at least four times in duplicates or triplicates. *P < .05, **P < .01, ***P < .005 specific antibody for LRP5. Pro-­inflammatory CD16− macrophages Similarly, CD80 + and CD83+ macrophages showed increased levels show increased LRP5 cellular expression after agLDL loading of cellular LRP5 after lipid loading compared to controls (CD80 +: compared to controls (12.3% and 2.6% respectively; Figure 2E). 3.89% expression in control conditions to 11.8% expression after | 15824934, 2021, 16, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16723 by Readcube (Labtiva Inc.), Wiley Online Library on [03/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 7942 LUQUERO et al. lipid loading; CD83+: 1.27% expression in control conditions to not (Figure 4A). Analysis of donor macrophages mRNA expres- 6.85% expression after agLDL loading; Figure 2E). Interestingly, sion by RT-­P CR showed a 92 ± 3% LRP5 reduction in siRNA-­LRP5 macrophages that express the anti-­inflammatory markers CD16+, control cells and a 90 ± 2% LRP5 reduction in siRNA-­LRP5 lipid-­ CD206+ or CD163+ also showed significantly increased LRP5 ex- loaded macrophages. LRP5 mRNA expression was increased in pression after agLDL loading (CD16+: 16.7% in control conditions lipid-­loaded macrophages (Figure 4B). LRP5 silencing did not mod- + to 28.3% in lipid-­loaded macrophages, CD206 : 9.8% in controls to ify the number of AV+MV/mL released by macrophages neither in + 21.8% in lipid-­loaded macrophages and CD163 : 12.84% in con- control nor in lipid-­loaded conditions (Figure 4C). However, a con- trols to 27.8% in agLDL-­t reated macrophages) indicating that lipid sistent reduction in LRP5+AV+MV release by siRNA-­LRP5-­t reated loading induce LRP5 expression in both pro-­and anti-­inflammatory macrophages was observed both in untreated and agLDL-­loaded macrophages but with a higher expression in anti-­inflammatory macrophages (Figure 4D). macrophages (Figure 2F). 3.8 | Gene expression levels in conditioned 3.5 | Inflammatory profile of MV after lipid loading macrophages MV release was investigated in supernatants from LDL-­ loaded MV released by the different sets of donor macrophages were isolated macrophage (100 μg/mL agLDL). Lipid loading induced significantly and used to condition naive macrophages and monocytes. Treatment + higher release of AV MV (Figure 3A). LDL loading induced a signifi- of naive macrophages with macrophage-­ derived MV released by cant increase in the release of CD16−, CD80 + and CD83+ MV but control macrophages or with MV released by siRNA-­LRP5-­treated not CD16+, CD206+ and CD163+ MV (Figure 3B). Normalization macrophages did not modify their LRP5 cellular mRNA expression by total AV+ MV showed that only CD16−, CD80 + and CD83+ (Figure 5A). Similarly, treatment with MV released by control or lipid-­ − MV levels were increased after agLDL loading (CD16 : 65.82% in loaded macrophages did not modify LRP5 gene expression levels in control conditions to 86.81% after lipid loading, CD80 +: 9.87% in recipient macrophages indicating that LRP5 contained in MV does not control conditions to 19.22% after lipid loading and CD83+: 6.31% affect LRP5 gene transcription (Figure 5A). in control conditions to 12.92% after lipid loading, Figure 3C,D). However, macrophage gene transcription of the pro-­ + + + Macrophage-­derived MV containing CD16 , CD206 and CD163 inflammatory molecules iNOS and CD80 was increased by MV anti-­ inflammatory markers remained constant before and after devoid of LRP5 indicating that LRP5 blocks the expression of macrophage lipid loading (CD16+: 2.99% in control conditions vs pro-­inflammatory genes in recipient macrophages (Figure 5B,C). loaded macrophages, CD206+: 2.09% in control 1.09% in lipid-­ Treatment with MV released by lipid-­loaded macrophages induced + conditions vs 2.31% after agLDL loading and CD163 : 1.30% in iNOS and CD80 gene transcription in recipient macrophages inde- control conditions to 1.16% after lipid loading Figure 3C,D) indi- pendent of LRP5 expression (Figure 5B,C). The anti-­inflammatory cating that lipid loading induces the release of MV containing pro-­ genes CD163 and IL1Ra showed decreased expression levels inflammatory markers. in macrophages conditioned with MV released by siRNA-­ LRP5 macrophages, indicating that LRP5+MV induce higher levels of anti-­ inflammatory genes expression in recipient macrophages 3.6 | LRP5+MV contain pro-­inflammatory and anti-­ (Figure 5D,E). Anti-­ inflammatory gene transcription in macro- inflammatory proteins phages conditioned with MV released by lipid-­loaded macrophages remained constant independently of LRP5 expression in donor MV released from lipid-­loaded and non-­loaded macrophages were macrophages (Figure 5D,E). isolated and stained for pro-­inflammatory and anti-­inflammatory markers and for LRP5. Interestingly, LRP5 was delivered into MV containing both pro-­inflammatory and anti-­inflammatory proteins, 3.9 | Gene transcription in conditioned monocytes indicating that the delivery of LRP5 into MV is independent of the inflammatory proteins delivered into the MV (Figure 3E). LRP5 gene expression levels remained constant in monocytes treated with MV released by both LRP5-­ expressing and LRP5-­ silenced macrophages. Interestingly LRP5 gene levels were in- 3.7 | Characterization of donor creased in monocytes conditioned with MV released by lipid-­loaded macrophages and their released MV macrophages (Figure 5F). However, iNOS and CD80 expression lev- els in monocytes conditioned with MV released by untreated and Macrophage specific inhibition of LRP5 expression (with siRNA) lipid-­loaded macrophages in the presence or absence of LRP5 were was used to identify whether LRP5 was playing a role in mac- not significantly modified (Figure 5G,H) as did the expression levels rophage differentiation towards a CD16− or a CD16+ phenotype. of the anti-­inflammatory genes CD163 and IL1Ra (Figure 5I,J) indi- Macrophages were silenced or not for LRP5 and agLDL-­loaded or cating that monocyte gene expression is unaffected by LRP5+MVs. | 15824934, 2021, 16, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16723 by Readcube (Labtiva Inc.), Wiley Online Library on [03/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License LUQUERO et al. 7943 F I G U R E 4 Characterization of donor macrophages and their secreted MV. (A) Schematic of experiment. Donor macrophages were silenced or not for LRP5 (siLRP5) and treated or not with agLDL. After 48 hours, macrophage secreted MV were isolated and suspended to treat receptor macrophages or receptor monocytes. (B) LRP5 gene expression in donor macrophages after LRP5 silencing and agLDL treatments. (C) Macrophage-­derived AV+MV/mL secreted by donor macrophages. (D) Macrophage-­derived LRP5+MV released by donor macrophages. Experiments were performed four times in triplicates. *P < .05, **P < .01, ***P < .005 4 | D I S CU S S I O N is a 2.6% expression of cell surface LRP5 in CD16− macrophages as compared to 16.7% in CD16+ macrophages. These results are Microvesicles can stimulate targets cells by direct interaction with in line with our previous findings where LRP5 immunofluores- target receptors and the transfer of the bioactive molecules they cent staining was increased in CD16+ macrophages as compared contain. 2,3,7,8,10,13,15 Here, we show, for the first time, that LRP5 to CD16− macrophages. 26 Here, we show that LRP5 expression is delivered into MV released by macrophages and monocytes. levels in control macrophages expressing anti-­inflammatory pro- However, the release of LRP5+MV is only significantly increased in teins are higher than in macrophages expressing pro-­inflammatory fully differentiated lipid-­loaded macrophages. markers (CD16−, CD80 + and CD83+) indicating that in control In general, macrophages are classified into two main phe- conditions, there is more LRP5 expressed in macrophages with − notypes, classical M1 CD16 activated macrophages and al- anti-­inflammatory phenotype. Interestingly, LRP5 cell surface ex- ternative M2 CD16+ activated macrophages, which regulate pression is increased in all lipid-­l oaded macrophages. Indeed, mac- pro-­ inflammatory and anti-­ inflammatory responses, respec- rophages expression of pro-­ inflammatory or anti-­ inflammatory tively. 29 Regulation of lipid-­induced macrophage polarization is a markers on their cell surface is independent of LRP5 expression very new field of investigation. A recent study showed that satu- levels, indicating that LRP5 expression is upregulated in lipid-­ rated fatty acid treatment induced M1-­p redominant macrophages, loaded macrophages irrespective of the macrophage inflammatory while polyunsaturated fatty acid induced M2-­p redominant mac- phenotype. rophages. 30 Treatment of hepatocytes with conditioned media In the presence of extracellular lipids, there is increased release from M1-­ p olarized macrophages promoted lipid synthesis and of MV. This is in line with previous studies where statin treatment accumulation indicating that lipid-­induced macrophage M1 po- (a lipid lowering agent) reduced MV shedding from platelets, en- larization stimulates hepatic lipid metabolism. 30 In this study, we dothelial cells and leukocytes carrying markers of cell activation.31 show that lipid-­loaded macrophages show high cell surface ex- Similarly, decreased MV release and decreased cargo of cell activa- pression of pro-­inflammatory proteins while cell surface expres- tion markers after statin treatment in different cell lineages have also sion of anti-­inflammatory proteins (CD16+, CD163+ and CD206+) been described.32-­36 We have explored whether MV show different remains constantly low indicating that lipid loading induces M1 inflammatory phenotypes if they are released by untreated or lipid-­ polarization. This results are supported by cell sorting experi- loaded macrophages. Because MV are released from the cell sur- ments where isolated specific pro-­inflammatory subpopulations face of their cells of origin, we used the same pro-­inflammatory and of macrophages release pro-­inflammatory proteins. LRP5 expres- anti-­inflammatory markers used to characterize CD16− and CD16+ + sion levels are higher in CD16 expressing macrophages as com- macrophages to map their released MV. Lipid-­loaded macrophages pared to CD16− macrophages. Indeed, in control conditions there show increased release of CD16−MV, CD80+MV and CD83+MV, 15824934, 2021, 16, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16723 by Readcube (Labtiva Inc.), Wiley Online Library on [03/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License | LUQUERO et al. 7944 | 15824934, 2021, 16, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16723 by Readcube (Labtiva Inc.), Wiley Online Library on [03/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License LUQUERO et al. 7945 F I G U R E 5 Gene expression levels in receptor macrophages and monocytes. Receptor macrophages were treated with macrophage-­ derived MV released by Control macrophages, siRNA-­LRP5-­treated macrophages, 100 μg/mL agLDL-­treated macrophages or siRNA-­ LRP5+AgLDL-­treated macrophages and mRNA expression levels of (A) LRP5, (B) iNOS, (C) CD80, (D) CD163 and (E) IL1Ra were analysed. Receptor monocytes were treated with macrophage-­derived MV released by Control macrophages, siRNA-­LRP5-­treated macrophages, 100 μg/mL agLDL-­treated macrophages or siRNA-­LRP5+AgLDL-­treated macrophages and mRNA expression levels of (F) LRP5, (G) iNOS, (H) CD80, (I) CD163 and (J) IL1Ra were analysed. Experiments were performed four times in triplicates. *P < .05, ***P < .005 while the release of MV containing anti-­ inflammatory markers unable to induce high expression of pro-­inflammatory genes. Also, (CD16+, CD163+ and CD206+) remained similar to untreated mac- MV released by lipid-­loaded macrophages induced increased ex- rophages indicating that lipid-­loaded macrophages release MV with pression of pro-­inflammatory genes independent of LRP5 expres- a pro-­inflammatory phenotype. The molecular mechanisms behind sion further supporting that different stimulus in the cells of origin the preferential incorporation of different proteins into budding MV generates MV with different cargoes that will have different func- remain to be elucidated, but it has been suggested that it could be tions in the target cells. mediated by the cytoplasmic domains of the protein to be included In conclusion, here we demonstrate for the first time that a li- into the MV.37 poprotein receptor, LRP5, is delivered into MV. MV released by Similar to the increased cellular expression of LRP5 in lipid-­ lipid-­ loaded macrophages contain mainly pro-­ inflammatory pro- loaded CD16− and CD16+ macrophages, lipid loading induced teins and LRP5. LRP5+MV induce an anti-­inflammatory genotype in + − + increased release of LRP5 MV in both CD16 MV and CD16 MV naive macrophages. Therefore, a systematic blockade of monocyte/ indicating that the expression of inflammatory markers in MV is macrophage infiltration in the prevention of atherosclerosis may be independent of the delivery of LRP5 into MV. However, after lipid less effective than originally expected if the levels of macrophage-­ loading, only pro-­inflammatory MV were released. Therefore, only derived LRP5+MV are affected and reduced. + − + + + + LRP5 CD16 MV, LRP5 CD80 MV and LRP5 CD83 MV were re- leased. This raises the very interesting question of how is LRP5 AC K N OW L E D G E M E N T S delivered and released with MV containing pro-­ inflammatory We thank S. Huertas for excellent technical assistance. This work markers. Notably, MV production and release are stimuli and signal was supported by the Spanish Ministry of Science and Innovation 38,39 dependent. For example, cytokine IL1β induces MV shedding and FEDER funds [PID2019-­ 107160RB-­ I 00 to LB]; the Instituto from circulating monocytes.40 Accordingly, here we show that lipid de Salud Carlos III [CIBERCV CB16/11/00411 to LB, TERCEL stimuli induce pro-­inflammatory MV release. As lipid loaded macro- RD16/0011/018 to LB and FIS2020-­01282 to MBP]; the Generalitat phages show increased expression of LRP5 at the cell surface, it is of Catalunya-­Secretaria d'Universitats i Recerca del Departament plausible that this LRP5 will be delivered to their MV and released d'Economia i Coneixement de la Generalitat [2017SGR1480 to LB; as LRP5+MV. Human macrophages expressing LRP5 have been PERIS SLT006 to LB]; the Spanish Society of Cardiology [FEC2019 shown to provide survival and repair to damaged tissues. 26 It is our to MBP] and the Fundación Investigación Cardiovascular-­Fundación hypothesis that this is the function of LRP5+MV but further work Jesus Serra for their continuous support. needs to be performed to prove it. We also explored the function of macrophage-­derived LRP5+MV C O N FL I C T O F I N T E R E S T in the polarization fate of macrophages. Lipid-­loaded macrophages The authors confirm that there are no conflicts of interest. that did not express LRP5 showed similar MV release than lipid-­ loaded LRP5+ macrophages indicating that LRP5 does not partici- AU T H O R C O N T R I B U T I O N pate in the MV release pathway. A reduction in LRP5+MV release Aureli Luquero: Data curation (lead); Formal analysis (equal); from macrophages without LRP5 was observed. Investigation (equal); Methodology (lead); Software (equal); Classically activated CD16− macrophages are characterized review & editing (equal). Gemma Visualization (equal); Writing-­ by the expression of several pro-­inflammatory markers, including Vilahur: Conceptualization (equal); Formal analysis (equal); iNOS and CD80 41,42 while alternatively activated anti-­inflammatory review & editing (equal). Javier Methodology (equal); Writing-­ CD16+ macrophages express CD163 and IL1Ra.42,43 Treatment with Crespo: Formal analysis (supporting); Methodology (supporting); MV released by macrophages devoid of LRP5 induced iNOS and Software (equal); Writing-­review & editing (equal). Lina Badimon: CD80 expression and reduced CD163 and IL1Ra expression in naive Conceptualization (lead); Funding acquisition (lead); Methodology macrophages indicating that LRP5+MV induce macrophages to dif- (equal); Resources (equal); Supervision (equal); Writing-­review & ferentiate towards an anti-­inflammatory phenotype. A limitation of editing (equal). Maria Borrell-­Pages: Conceptualization (support- this study is that the size of LRP5+MV was not assessed; therefore, ing); Data curation (equal); Formal analysis (equal); Funding ac- + we could not determine if LRP5 MV have a different size than MV quisition (supporting); Investigation (lead); Methodology (equal); devoid of LRP5. However, always the same procedure was followed Supervision (lead); Writing-­original draft (lead); Writing-­review & to prepare MV and only MV released by control macrophages were editing (lead). | 15824934, 2021, 16, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16723 by Readcube (Labtiva Inc.), Wiley Online Library on [03/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License 7946 LUQUERO et al. DATA AVA I L A B I L I T Y S TAT E M E N T postischemic collateral vessel formation. Arterioscler Thromb Vasc Biol. 2015;35:348-­357. The data underlying this article are available in the article and in its 18. Boulanger CM, Loyer X, Rautou PE, Amabile N. Extracellular vesi- online supplementary material. cles in coronary artery disease. Nat Rev Cardiol. 2017;14:259-­272. 19. Badimon L, Vilahur G. Thrombosis formation on atherosclerotic le- ORCID sions and plaque rupture. J Intern Med. 2014;276:618-­632. Maria Borrell-­Pages https://orcid.org/0000-0002-1759-9756 20. Borrell-­Pages M, Romero JC, Juan-­Babot O, Badimon L. Wnt path- way activation, cell migration, and lipid uptake is regulated by low-­density lipoprotein receptor-­related protein 5 in human macro- REFERENCES phages. Eur Heart J. 2011;32:2841-­2850. 1. Ridger VC, Boulanger CM, Angelillo-­Scherrer A, et al. Microvesicles 21. Clevers H, Nusse R. Wnt/β-­ c atenin signaling and disease. Cell. in vascular homeostasis and diseases position paper of the European 2012;149:1192-­1205. society of cardiology (ESC) working group on atherosclerosis and 22. Borrell-­Pages M, Vilahur G, Romero JC, Casaní L, Bejar MT, vascular biology. Thromb Haemost. 2017;117:1296-­1316. Badimon L. LRP5/canonical Wnt signalling and healing of ischemic 2. Mathivanan S, Ji H, Simpson RJ. Exosomes: extracellular or- myocardium. Basic Res Cardiol. 2016;111:67. ganelles important in intercellular communication. J Proteomics. 23. Badimon L, Casaní L, Camino-­Lopez S, Juan-­Babot O, Borrell-­Pages 2010;73:1907-­1920. M. GSK3β inhibition and canonical Wnt signaling in mice hearts 3. Camussi G, Deregibus MC, Bruno S, Cantaluppi V, Biancone L. after myocardial ischemic damage. PLoS One. 2019;14:e0218098. Exosomes/microvesicles as a mechanism of cell-­to-­cell communica- 24. Mosser DM, Edwards JP. Exploring the full spectrum of macro- tion. Kidney Int. 2010;78:838-­8 48. phage activation. Nat Rev Immunol. 2008;8:958-­969. 4. Taylor DD, Gercel-­Taylor C. Exosomes/microvesicles: mediators of 25. Murray PJ, Wynn TA. Protective and pathogenic functions of mac- cancer-­associated immunosuppressive microenvironments. Semin rophage subsets. Nat Rev Immunol. 2011;11:723-­737. Immunopathol. 2011;33:441-­454. 26. Borrell-­Pages M, Romero JC, Crespo J, Juan-­Babot O, Badimon L. 5. Cocucci E, Racchetti G, Meldolesi J. Shedding microvesicles: arte- LRP5 associates with specific subsets of macrophages: molecular facts no more. Trends Cell Biol. 2009;19:43-­51. and functional effects. J Mol Cell Cardiol. 2016;90:146-­156. 6. Février B, Raposo G. Exosomes: endosomal-­derived vesicles ship- 27. Borrell-­Pagès M, Romero JC, Badimon L. LRP5 negatively regulates ping extracellular messages. Curr Opin Cell Biol. 2004;16:415-­421. differentiation of monocytes through abrogation of Wnt signalling. 7. Hunter MP, Ismail N, Zhang X, et al. Detection of microRNA ex- J Cell Mol Med. 2014;18:314-­325. pression in human peripheral blood microvesicles. PLoS One. 28. Badimon L, Luquero A, Crespo J, Peña E, Borrell-­Pages M. PCSK9 and 2008;3:e3694. LRP5 in macrophage lipid internalization and inflammation. Cardiovasc 8. Ratajczak J, Wysoczynski M, Hayek F, Janowska-­ Wieczorek A, Res. 2020;cvaa254. https://doi.org/10.1093/cvr/cvaa254 Ratajczak MZ. Membrane-­derived microvesicles: important and un- 29. Tacke F, Zimmermann HW. Macrophage heterogeneity in liver in- derappreciated mediators of cell-­to-­cell communication. Leukemia. jury and fibrosis. J Hepatol. 2014;60:1090-­1096. 2006;20:1487-­1495. 30. Wu HM, Ni XX, Xu QY, Wang Q, Li XY, Hua J. Regulation of lipid-­ 9. Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. induced macrophage polarization through modulating peroxisome Exosome-­mediated transfer of mRNAs and microRNAs is a novel proliferator-­activated receptor-­gamma activity affects hepatic lipid mechanism of genetic exchange between cells. Nat Cell Biol. metabolism via a Toll-­ like receptor 4/NF-­ κB signaling pathway. 2007;9:654-­659. J Gastroenterol Hepatol. 2020;35:1998-­2008. 10. Baj-­Krzyworzeka M, Szatanek R, Węglarczyk K, et al. Tumour-­ 31. Suades R, Padró T, Alonso R, Mata P, Badimon L. Lipid-­lowering derived microvesicles carry several surface determinants and therapy with statins reduces microparticle shedding from en- mRNA of tumour cells and transfer some of these determinants to dothelium, platelets and inflammatory cells. Thromb Haemost. monocytes. Cancer Immunol Immunother. 2006;55:808-­818. 2013;110:366-­377. 11. MacKenzie A, Wilson HL, Kiss-­ Toth E, Dower SK, North RA, 32. Nomura S, Shouzu A, Omoto S, et al. Effects of eicosapentaenoic Surprenant A. Rapid secretion of interleukin-­1β by microvesicle acid on endothelial cell-­derived microparticles, angiopoietins and shedding. Immunity. 2001;15:825-­835. adiponectin in patients with type 2 diabetes. J Atheroscler Thromb. 12. Frey B, Munoz LE, Pausch F, et al. The immune reaction against 2009;16:83-­90. allogeneic necrotic cells is reduced in Annexin A5 knock out mice 33. Nomura S, Inami N, Shouzu A, Rase F, Maeda Y. The effects of whose macrophages display an anti-­inflammatory phenotype. J Cell pitavastatin, eicosapentaenoic acid and combined therapy on Mol Med. 2009;13:1391-­1399. platelet-­derived microparticles and adiponectin in hyperlipidemic, 13. Martínez MC, Larbret F, Zobairi F, et al. Transfer of differentiation diabetic patients. Platelets. 2009;20:406-­414. signal by membrane microvesicles harboring hedgehog morpho- 34. Tramontano AF, O’Leary J, Black AD, Muniyappa R, Cutaia MV, El-­ gens. Blood. 2006;108:3012-­3 020. Sherif N. Statin decreases endothelial microparticle release from 14. Ratajczak J, Miekus K, Kucia M, et al. Embryonic stem cell-­derived human coronary artery endothelial cells: implication for the Rho-­ microvesicles reprogram hematopoietic progenitors: evidence kinase pathway. Biochem Biophys Res Commun. 2004;320:34-­38. for horizontal transfer of mRNA and protein delivery. Leukemia. 35. Sommeijer DW, Joop K, Leyte A, Reitsma PH, Ten Cate H. 2006;20:847-­856. Pravastatin reduces fibrinogen receptor gpIIIa on platelet-­derived 15. Collino F, Deregibus MC, Bruno S, et al. Microvesicles derived microparticles in patients with type 2 diabetes. J Thromb Haemost. from adult human bone marrow and tissue specific mesenchy- 2005;3:1168-­1171. mal stem cells shuttle selected pattern of miRNAs. PLoS One. 36. Mobarrez F, He S, Bröijersen A, et al. Atorvastatin reduces throm- 2010;5:e11803. bin generation and expression of tissue factor, p-­selectin and GPIIIa 16. Montecalvo A, Larregina AT, Shufesky WJ, et al. Mechanism of on platelet-­derived microparticles in patients with peripheral arte- transfer of functional microRNAs between mouse dendritic cells rial occlusive disease. Thromb Haemost. 2011;106:344-­352. via exosomes. Blood. 2012;119:756-­766. 37. Barclay AN, Brown M, Law SKA, McKnight A, Tomlinson M, Van der 17. Arderiu G, Peña E, Badimon L. Angiogenic microvascular endothe- Merwe P. The Leucocyte Antigen Factsbook. Oxford, UK: Elsevier, lial cells release microparticles rich in tissue factor that promotes Academic Press; 1997:231. | 15824934, 2021, 16, Downloaded from https://onlinelibrary.wiley.com/doi/10.1111/jcmm.16723 by Readcube (Labtiva Inc.), Wiley Online Library on [03/01/2023]. See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License LUQUERO et al. 7947 38. Williams C, Palviainen M, Reichardt NC, Siljander PRM, Falcón-­ macrophages results in an M2 phenotype under inflammatory con- Pérez JM. Metabolomics applied to the study of extracellular vesi- ditions. Immunobiology. 2017;222:900-­912. cles. Metabolites. 2019;9:276. 39. Turchinovich A, Drapkina O, Tonevitsky A. Transcriptome of extra- cellular vesicles: state-­of-­the-­art. Front Immunol. 2019;10:202. S U P P O R T I N G I N FO R M AT I O N 40. Ward JR, West PW, Ariaans MP, et al. Temporal interleukin-­ 1β Additional supporting information may be found online in the secretion from primary human peripheral blood monocytes by Supporting Information section. P2X7-­independent and P2X7-­dependent mechanisms. J Biol Chem. 2010;285:23147-­23158. 41. Xue Q, Yan Y, Zhang R, Xiong H. Regulation of iNOS on immune cells and its role in diseases. Int J Mol Sci. 2018;19:3805. How to cite this article: Luquero A, Vilahur G, Crespo J, 42. Bertani FR, Mozetic P, Fioramonti M, et al. Classification of M1/ Badimon L, Borrell-­Pages M. Microvesicles carrying LRP5 M2-­polarized human macrophages by label-­free hyperspectral re- induce macrophage polarization to an anti-­inflammatory flectance confocal microscopy and multivariate analysis. Sci Rep. phenotype. J Cell Mol Med. 2021;25:7935–­7947. https://doi. 2017;7:8965. 43. Alvarado-­ Vazquez PA, Bernal L, Paige CA, et al. Macrophage-­ org/10.1111/jcmm.16723 specific nanotechnology-­driven CD163 overexpression in human