biomolecules Article The Role of Distinctive Sphingolipids in the Inflammatory and Apoptotic Effects of Electronegative LDL on Monocytes Núria Puig 1,2 , Montserrat Estruch 1 , Lei Jin 1 , Jose Luis Sanchez-Quesada 1,3 and Sonia Benitez 1, * 1 Cardiovascular Biochemistry, Biomedical Research Institute Sant Pau (IIB-Sant Pau), Barcelona, Spain. C/Sant Quinti 77-79, 08041 Barcelona, Spain 2 Department of Biochemistry and Molecular Biology, Faculty of Medicine, Building M, Universitat Autònoma de Barcelona (UAB), Cerdanyola del Vallès, 08193 Barcelona, Spain 3 CIBER of Diabetes and Metabolic Diseases (CIBERDEM), 28029 Madrid, Spain * Correspondence: sbenitez@santpau.cat; Tel.: +34-935-537-595; Fax: +34-935-537-589  Received: 3 July 2019; Accepted: 20 July 2019; Published: 24 July 2019  Abstract: Electronegative low-density lipoprotein (LDL(−)) is a minor LDL subfraction that is present in blood with inflammatory and apoptotic effects. We aimed to evaluate the role of sphingolipids ceramide (Cer), sphingosine (Sph), and sphingosine-1-phosphate (S1P) in the LDL(−)-induced effect on monocytes. Total LDL was subfractioned into native LDL and LDL(−) by anion-exchange chromatography and their sphingolipid content evaluated by mass spectrometry. LDL subfractions were incubated with monocytes in the presence or absence of enzyme inhibitors: chlorpromazine (CPZ), d-erythro-2-(N-myristoyl amino)-1-phenyl-1-propanol (MAPP), and N,N-dimethylsphingosine (DMS), which inhibit Cer, Sph, and S1P generation, respectively. After incubation, we evaluated cytokine release by enzyme-linked immunosorbent assay (ELISA) and apoptosis by flow cytometry. LDL(−) had an increased content in Cer and Sph compared to LDL(+). LDL(−)-induced cytokine release from cultured monocytes was inhibited by CPZ and MAPP, whereas DMS had no effect. LDL(−) promoted monocyte apoptosis, which was inhibited by CPZ, but increased with the addition of DMS. LDL enriched with Sph increased cytokine release in monocytes, and when enriched with Cer, reproduced both the apoptotic and inflammatory effects of LDL(−). These observations indicate that Cer content contributes to the inflammatory and apoptotic effects of LDL(−) on monocytes, whereas Sph plays a more important role in LDL(−)-induced inflammation, and S1P counteracts apoptosis. Keywords: electronegative LDL; ceramide; sphingosine; inflammation; apoptosis; monocytes 1. Introduction Sphingolipids have an essential structural function in cell membranes. In addition, these lipid metabolites are emerging as important intracellular and intercellular signaling molecules that are involved in the progress of inflammatory diseases and atherosclerosis [1]. These bioactive lipids are present in atherosclerostic plaque, where they play inflammatory and apoptotic roles [2]. Sphingolipid metabolism is complex and highly interconnected, and each step is controlled by specific enzymes. The core component of sphingolipid metabolism is ceramide (Cer), which is generated by the hydrolysis of sphingomyelin (SM) by sphingomyelinase activity (SMase), and can be hydrolyzed by ceramidase (CDase) activity, yielding sphingosine (Sph). Sph can subsequently be phosphorylated by S1P kinase to generate sphingosine-1-phosphate (S1P) [3]. Cer is a widely studied molecule that regulates cell differentiation, inflammation, and apoptosis [4]. Its plasma concentration is elevated in patients who are at high cardiovascular risk [5,6]. The role of Biomolecules 2019, 9, 300; doi:10.3390/biom9080300 www.mdpi.com/journal/biomolecules Biomolecules 2019, 9, 300 2 of 14 Sph in atherosclerosis and inflammation is not clearly established, and its biological effects have been reported to overlap with those promoted by Cer [7]. Sph has been shown to promote inflammatory response [8] and apoptosis [9], although it has also been reported to induce an anti-apoptotic effect [10]. S1P is a pleiotropic bioactive metabolite, and is an important intercellular signaling molecule recognized by specific cell receptors whose subtype determines the effect on cells [11]. S1P is reported to promote cell proliferation, survival, and migration [12–14]. Regarding inflammation, some studies have described how it promotes inflammation [15–17], whereas others have reported an anti-inflammatory action, mainly when it is bound to its major carrier, high-density lipoprotein (HDL) [18–20]. Although LDL may contain low quantities of S1P, it mainly carries Cer and Sph [21]. Some studies have shown that LDL with an increased Cer content induces inflammation [22,23], whereas the effect of an increased content of Sph and S1P in LDL is unknown. Electronegative low-density lipoprotein (LDL(−)) is a minor LDL subfraction found in blood circulation that has various atherogenic effects, such as the induction of inflammation in cultured cells. It differs from native LDL (LDL(+)) in several physicochemical properties [24], including the presence of phospholipolytic activities. Indeed, LDL(−) has a fivefold to sixfold increased concentration of the enzyme platelet-activating factor acetylhydrolase (PAF-AH) compared with LDL(+) [25]. In addition, a phospholipase C (PLC)-like activity is believed to be associated with LDL(–), having a high avidity to degrade lysophosphatidylcholine (LPC) by lysoPLC activity and sphingomyelin (SM) by SMase activity [26]. These enzymatic activities seem to be involved in the increased concentrations in LDL(−) of Cer and non-esterified fatty acid (NEFA), which are responsible for LDL(–)-induced cytokine release in monocytes [22,27,28]. However, other compounds, such as those resulting from Cer hydrolysis—Sph and S1P—could also be increased in LDL(−) and be involved, not only in the inflammatory effect of LDL(–), but also in a putative apoptotic effect on monocytes. LDL(−) exerts an apoptotic effect in macrophages [29] and in endothelial cells [30], being attributed in the latter to the increased Cer content in LDL(−) [30]. In fact, Cer and NEFA are inductors of apoptosis [31,32], and an increased content of these molecules confers apoptotic properties on LDL [31,33,34]. Moreover, modified LDL can promote apoptosis by increasing the Cer content in cells [31,35]. In the current study, we sought to determine the specific involvement of the sphingolipids Cer, Sph, and S1P in the inflammatory and apoptotic effects of LDL(−) in human monocytes isolated from plasma. For this purpose, we used specific inhibitors of SMase, CDase, and Sph kinase activities, as summarized in the Methods section. 2. Material and Methods 2.1. LDL Isolation and Modification Plasma samples from healthy normolipemic subjects (total cholesterol <5.2 mmol/L, triglyceride <1 mmol/L) were obtained in EDTA-containing Vacutainer tubes. All the subjects gave their written informed consent, and the study was conducted after approval from the Institutional Ethics Committee of the Hospital Sant Pau (IIBSP-APO-2013-105, 25 February 2015). All the LDL preparations were performed in conditions that prevented oxidation and endotoxin contamination [36]. LDL (1.019–1.050 kg/L) was isolated from plasma by sequential flotation ultracentrifugation at 4 ◦ C. In some experiments, LDL was modified by different treatments: by incubation with SMase (10 U/g apoB) for 2 h at 37 ◦ C to increase its Cer concentration (SMase-LDL); by incubation with NEFA (NEFA-LDL) [28]; and by incubation with Sph-enriched liposomes (10 µM) (Sph-LDL), as described for Cer-enriched liposomes [22]. The enrichment in these compounds was assessed by thin-layer chromatography, and the aggregation level of modified LDL was evaluated by gradient gel electrophoresis, as previously described [22]. Total LDL was fractionated in LDL(+) and LDL(−) by preparative anion-exchange chromatography in an ÄKTA-FPLC system (GE Healthcare) [37]. LDL fractions were concentrated with Amicon Biomolecules 2019, 9, 300 3 of 14 centrifugal filters (Merck Millipore). LDL(+) and LDL(−) compositions were determined in a Cobas® 501 autoanalyzer, including total cholesterol, triglyceride, apoB (Roche, Basel, Switzerland), NEFA, and phospholipid (Wako, Neuss, Germany). 2.2. Lipidomic Analysis Baseline levels of sphingolipids in LDL(+) and LDL(−) were evaluated by liquid chromatography- mass spectrometry (LC-SM) in the CIBERDEM-Metabolomics Platform of Universitat Rovira i Virgili. First, a lipid extraction of LDL samples (32 µg apoB) was performed. LDLs were lyophilized and resuspended in 220 µL of methanol (MeOH) followed by vortexing. Then, 440 µL of dichloromethane were added followed by vortexing. Afterwards, 140 µL of ultrapure water was added, vortexed, and stored at room temperature for 20 min. The solution was centrifuged for 10 min at 14,500 rpm (4 ◦ C). Next, 400 µL of the organic phase was collected and evaporated to dryness with N . Samples 2 were reconstituted in 150 µL of MeOH:toluene (9:1) and transferred to LC-MS vials. Lipid extracts (5 µL) were injected in a Ultra High Perfomance Liquid Chromatography system (1290 Agilent, Santa Clara, CA, USA) coupled to a triple quadrupole (QqQ) mass spectrometer (6490 Agilent Technologies) operated in positive electrospray ionization (ESI+) mode. The instrument was set to acquire in multiple reaction monitoring mode. Lipids were separated using C18-RP (ACQUITY UPLC BEH 2.1 × 150 mm, 1.7 µm, Waters) chromatography at 65 ◦ C and at a flow rate of 0.4 mL/min. The solvent system was A = acetonitrile:water (60:40) in 10 mM of ammonium formate, and B = isopropanol:acetonitrile (90:10) in 10 mM of ammonium formate. The gradient elution started at 15% B and went to 30% from minute 0 to 2, 48% B from minute 2 to 2.5, 82% B from minute 2.5 to 11, and 99% B from minute 11 to 11.5. Quality control samples (QC) consisting of pooled samples were injected before the first study sample and then periodically after four study samples. Cer 14:0, Cer 16:0, Cer 18:0, Cer 20:0, Cer 22:0, Cer 24:0, Sph 18:1, and S1P 18:1 relative concentrations were analyzed by this method. Total Cer was considered to be the sum of all the Cer species evaluated. 2.3. Inflammatory Action on Monocytes Peripheral blood from volunteers was collected, and mononuclear cells were isolated by density gradient centrifugation using a density solution of 1.077 kg/L (Lympholyte Cedarlane) [37]. Cells were seeded on 12-well plates (2 × 106 cells/mL), and monocytes were separated from lymphocytes according to their adhesive properties. All the subjects gave their written informed consent, and the study was conducted after approval from the Institutional Ethics Committee of the Hospital Sant Pau. To evaluate the role of sphingolipids in the inflammatory and apoptotic effects of LDL(−), several inhibitors were tested: chlorpromazine (CPZ) (Sigma) is an inhibitor of SMase activity; d-erythro-2-(N-myristoyl amino)-1-phenyl-1-propanol (MAPP) (Cayman) is an inhibitor of neutral/alkaline CDase activity; and N,N-dimethylsphingosine (DMS) (Cayman) is an inhibitor of Sph kinase. The effect of these inhibitors is summarized in Table 1. Table 1. Inhibitors used in the study. Inhibitor Abbreviation Enzyme Substrate Product Cer and Chlorpromazine CPZ SMase SM phosphorylcholine d-erythro-2-(N-myristoyl MAPP CDase Cer Sph and NEFA amino)-1-phenyl-1-propanol N,N-dimethylsphingosine DMS Sph kinase Sph S1P All the inhibitors were used at 10 µM. LDL(+) and LDL(−) were incubated in the presence or absence of these inhibitors for 20 h; afterwards, they were dialyzed against RPMI 1640 medium (supplemented with 1% fetal calf serum, 2 mM of L-glutamine, 0.1 U/L of streptomycin, and 0.1 U/L of penicillin), and filtered in sterile conditions. Then, monocytes were incubated for 20 h with Biomolecules 2019, 9, 300 4 of 14 these LDL subfractions (60 mg apoB/L) (pre-incubation conditions). Monocytes were also incubated simultaneously with inhibitors plus LDL(+) or LDL(−) (60 mg apoB/L) (co-incubation), and without LDL (blank or basal condition) in the presence or absence of inhibitors. All the incubations were performed in the above-mentioned RPMI 1640 medium. Cell supernatants were collected after 20 h to evaluate the release of IL1β, IL6, IL10, and MCP1, using ELISA kits (eBioscience for all, except Diaclone for IL1β), as described [38], and following the manufacturer’s recommendations. Moreover, monocytes were collected to analyze apoptosis by flow cytometry. 2.4. Apoptosis Assay Apoptosis assays were performed in monocytes incubated with LDL(−) plus MAPP or DMS, and with LDL(−) pre-incubated with CPZ (all inhibitors at 10 µM). Apoptosis was determined by an Annexin V-FITC, Apoptosis detection kit (eBioscience-Invitrogen, Carlsbad, CA, USA). Briefly, monocytes were collected and resuspended in 200 µL of binding buffer (200,000 cells). Cell suspension was stained with Annexin V-FITC, which binds to phosphatidylserine, for 10 min in darkness at room temperature. Afterwards, cells were washed and resuspended in 190 µL of binding buffer. After the addition of 10 µL of propidium iodide (PI), the fluorescence on stained cells was measured by flow cytometry in a MACSQuant® Analyzer MQ10 (Milteny Biotech GmbH, Bergisch Gladbach, Germany) and analyzed using MACSQuant software (Milteny Biotech GmbH). Non-stained cells (A- PI- ) were considered to be viable cells, A+ PI- was considered to indicate early apoptosis, double staining (A+ PI+ ) was considered to indicate late apoptosis, and A- PI+ was considered to indicate necrotic cells. Total apoptosis is the sum of early and late apoptosis. 2.5. Statistical Analyses Differences between groups were tested using the Wilcoxon signed-rank test for paired data. Results were expressed as mean ± SD. A value of p < 0.05 was considered to be statistically significant. 3. Results 3.1. LDL(−) Has an Increased Content in Sph and Cer We aimed to evaluate the content of Cer, Sph, and S1P in LDL(+) and LDL(−) by mass spectrometry. Figure 1 shows that LDL(−) had a much higher content in Cer than LDL(+), as previously described by a less sensitive method [22]. In addition, LDL(−) showed a twofold content in Sph compared to Biomolecules 2019, 9, x FOR PEER REVIEW 3 of 15 LDL(+), whereas S1P was undetectable in both LDL subfractions. Figure 1. Sphingosine (Sph) and ceramide (Cer) basal content in low-density lipoprotein (LDL) Figure 1. Sphingosine subfractions. (Sph) (blue Native LDL(+) and ceramide bars) and(Cer) basal negative content LDL(−) in bars) (red low-density (32 µg oflipoprotein (LDL)B apolipoprotein subfractions. Native LDL(+) (blue bars) and negative LDL(−) (red bars) (32 µg of apolipoprotein (apoB) were analyzed by mass spectrometry to evaluate the baseline levels of sphingolipids. Cer includes B (apoB) were analyzed by mass spectrometry to evaluate the baseline levels of sphingolipids. all the Cer species that were evaluated. Results are expressed as a relative increase versus LDL(+), Cer includes mean ± SD all (n the= Cer 4), *species that were vs. LDL(+), evaluated. Results are expressed as a relative increase versus p < 0.05. LDL(+), mean ± SD (n = 4), *vs. LDL(+), p < 0.05. 2.2. Sph and Cer are Involved in the Cytokine Release Promoted by LDL(−) in Monocytes The role of Cer, Sph, and S1P in the cytokine release promoted by LDL(−) in monocytes was evaluated. For this purpose, specific inhibitors involved in the generation of the above-mentioned sphingolipids were used, respectively: chlorpromazine (CPZ) (SMase inhibitor), D-erythro-2-(N- myristoyl amino)-1-phenyl-1-propanol (MAPP) (CDase inhibitor), and N,N-dimethylsphingosine Figure 1. Sphingosine (Sph) and ceramide (Cer) basal content in low-density lipoprotein (LDL) subfractions. Native LDL(+) (blue bars) and negative LDL(−) (red bars) (32 µg of apolipoprotein B (apoB) were analyzed by mass spectrometry to evaluate the baseline levels of sphingolipids. Cer includes Biomolecules all9,the 2019, 300Cer species that were evaluated. Results are expressed as a relative increase versus5 of 14 LDL(+), mean ± SD (n = 4), *vs. LDL(+), p < 0.05. 3.2. Sph 2.2. Sphand andCer Cerare AreInvolved Involvedininthe theCytokine CytokineRelease ReleasePromoted Promotedby byLDL(−) LDL(−)ininMonocytes Monocytes The role The role of of Cer, Cer, Sph, and S1P in the cytokine release promoted by LDL(−) LDL(−) in in monocytes monocytes was was evaluated. For evaluated. Forthis thispurpose, purpose,specific specificinhibitors inhibitorsinvolved involvedin in the the generation generation of of the the above-mentioned above-mentioned sphingolipids were sphingolipids were used, used,respectively: respectively:chlorpromazine chlorpromazine (CPZ)(CPZ) (SMase (SMase inhibitor), inhibitor), D-erythro-2-(N- d-erythro-2- (N-myristoyl myristoyl amino)-1-phenyl-1-propanol amino)-1-phenyl-1-propanol (MAPP) (MAPP) (CDaseinhibitor), (CDase inhibitor),and N,N-dimethylsphingosine and N,N-dimethylsphingosine (DMS) (DMS) (Sph (Sph kinase kinase inhibitor). inhibitor). Inhibitors Inhibitors alone alone did did not not modify modify the therelease release of of cytokines cytokines compared compared to to basal basal values values in in the theabsence absence of of LDLs LDLs (data (data not not shown). shown). In In the the presence presence of of LDL(−), LDL(−), thethe addition addition of of DMS DMS did did not not alter alter the the release release ofof monocyte monocyte chemoattractant protein 11(MCP1) chemoattractant protein (MCP1)and andinterleukin interleukin (IL)-6 (IL)-6 promoted promoted by by LDL(−), LDL(−), thereby thereby suggesting suggesting that that S1P generation S1P generation wasinvolved was not not involved in the in the inflammatory inflammatory action ofaction of this lipoprotein this lipoprotein (Figure (Figure 2). 2). By By contrast, contrast, the addition the addition of CPZ inhibited of CPZ inhibited theofrelease the release of these cytokines these cytokines induced by induced LDL(−). by LDL(−). Figure Figure 2. 2. MCP1 MCP1 and and interleukin-6 interleukin-6 (IL-6) (IL-6) release release promoted promoted byby LDL(−) LDL(−)inin human human monocytes monocytes in in the the presence presence or or absence absence of of DMS DMS and and chlorpromazine chlorpromazine CPZ. CPZ. Monocytes Monocytes were incubated incubated with LDL(−) LDL(−) (60(60 mg mgapoB/L) apoB/L) in in the the absence absence (red (red bars) bars) oror presence presence ofof DMS DMS (green (green bars) bars) and and CPZ CPZ (purple (purple bars), bars), both both at at 10 10 µM. µM. After 20 hours of incubation, incubation,cytokine cytokinerelease releasewas wasmeasured measuredby byELISA. ELISA.Results Results are are expressed expressed as mean as ± SD mean ± SD = 5), (n(n = 5*),vs. absence *vs. absenceofofinhibitor, p< inhibitor, < 0 .05. p 0.05. Finally, the Finally, the addition additionof ofMAPP MAPPdecreased decreasedMCP1 MCP1 and andIL-6 release IL-6 induced release by LDL(−) induced as well by LDL(−) as that as well as of IL-1β that and IL-10, of IL-1β in a concentration-dependent and IL-10, mannermanner in a concentration-dependent (Figure 3). However, (Figure the effect the 3). However, promoted effect by LDL(+)by promoted was not modified LDL(+) was notbymodified the addition of any by the inhibitor addition of (in anythe case of MAPP, inhibitor (in the inhibition lower case of MAPP, than 5%). lower inhibition Takenthantogether, 5%). the results Taken indicate together, thethat both results Sph and indicate Cerboth that contribute Sph andtoCer LDL(−)-induced contribute to inflammation in human monocytes. LDL(−)-induced inflammation in human monocytes. To confirm the role of increased Sph and Cer content in LDL on the inflammatory response of monocytes, we conducted experiments in which LDL(+) was enriched “in vitro” with these compounds by incubation with liposomes containing Sph or by SMase lipolysis, respectively. Supplementary Figure S1 shows the enrichment of Sph and Cer in these “in vitro”-modified LDLs and the effect of such modification in the aggregation of LDL particles. The increase in Sph and Cer was 1.5-fold and sixfold, respectively, as calculated by densitometry versus unmodified LDL. Figure 4 shows that compared to unmodified LDL, LDL enriched with Sph (Sph-LDL), and LDL treated with SMase (SMase-LDL) induced the release of IL-6, as a representative cytokine induced by LDL(−). Biomolecules 2019, 9, 300 6 of 14 Biomolecules 2019, 9, x FOR PEER REVIEW 4 of 15 Figure 3. MCP1, IL-1β, IL-6, and IL-10 release promoted by LDL(−) in human monocytes in the presence or absence of MAPP. Monocytes were incubated with LDL(−) (60 mg apoB/L) in the absence of MAPP (red bars) or in the presence of MAPP at 5 µM (yellow bars) and 10 µM (light yellow bars). After 20 hours of incubation, cytokine release was measured by ELISA. Results are expressed as mean ± SD (n = 5), *vs. the absence of inhibitor, p < 0 .05. To confirm the role of increased Sph and Cer content in LDL on the inflammatory response of monocytes, we conducted experiments in which LDL(+) was enriched “in vitro” with these compounds by incubation with liposomes containing Sph or by SMase lipolysis, respectively. Supplemental Figure 1 shows the enrichment of Sph and Cer in these “in vitro”-modified LDLs and the effect of such modification in the aggregation of LDL particles. The increase in Sph and Cer was 1.5-fold and sixfold, Figure Figure 3. respectively, 3. MCP1, IL-1β, MCP1, IL-6, IL-1β, and IL-6,as andcalculated IL-10 IL-10release by densitometry releasepromoted promoted by LDL(−) by versus LDL(−)ininhuman human unmodified monocytes monocytes LDL. Figure 4 in the in the presence shows thatpresence compared or to absence or absence of MAPP. unmodified of MAPP. Monocytes LDL, were Monocytes were incubated LDL incubated enrichedwith LDL(−) withwith LDL(−) (60 Sph mg (60 apoB/L) (Sph-LDL), mg apoB/L)in the absence and in the LDL treated with absence of MAPP (red bars) or in the presence of MAPP at 5 µM (yellow bars) and 10 µM (light of MAPP (red bars) or in the presence of MAPP at 5 µM (yellow bars) and 10 µM (light yellow bars).yellow bars). SMase (SMase-LDL) induced After 20 hours the release of incubation, of IL-6, cytokine release as a representative was measured by ELISA. Resultscytokine are expressedinduced as mean by LDL(−). After 20 hours of incubation, cytokine release was measured by ELISA. Results are expressed as mean ± SD (n = 5), * vs. the absence of inhibitor, p < 0.05. ± SD (n = 5), *vs. the absence of inhibitor, p < 0 .05. To confirm the role of increased Sph and Cer content in LDL on the inflammatory response of monocytes, we conducted experiments in which LDL(+) was enriched “in vitro” with these compounds by incubation with liposomes containing Sph or by SMase lipolysis, respectively. Supplemental Figure 1 shows the enrichment of Sph and Cer in these “in vitro”-modified LDLs and the effect of such modification in the aggregation of LDL particles. The increase in Sph and Cer was 1.5-fold and sixfold, respectively, as calculated by densitometry versus unmodified LDL. Figure 4 shows that compared to unmodified LDL, LDL enriched with Sph (Sph-LDL), and LDL treated with SMase (SMase-LDL) induced the release of IL-6, as a representative cytokine induced by LDL(−). Figure 4. IL-6 release promoted by “in vitro” modified LDL. Monocytes were incubated with unmodified Figure 4. LDL IL-6(blue release bars) orpromoted modified LDLby “inbars), (yellow vitro” modified both at LDL. 60 mg apoB/L. LDL Monocytes was modified bywere incubated with incubation with Sph-enriched liposomes (at 0 and 10 µM) and by sphingomyelinase (SMase) treatment (at 0 and unmodified LDL (blue bars) or modified LDL (yellow bars), both at 60 mg apoB/L. LDL was modified 10 U enzyme/g apoB). After 20 hours of incubation, cytokine release was measured by ELISA. Results by incubation with Sph-enriched are expressed liposomes as µg/L and as mean ± SD (n = 6),(at 0 unmodified * vs. and 10 µM)LDL, pand by sphingomyelinase (SMase) < 0.05. treatment (at 0 and 10 U enzyme/g apoB). After 20 hours of incubation, cytokine release was measured Next, we sought to determine whether the inhibition induced by MAPP and CPZ on cytokine by ELISA. Results are expressed as μg/L and as mean ± SD (n = 6 ), *vs. unmodified LDL, p < 0 .05. release was due to a direct effect on LDL(−) or was a consequence of an effect on cell enzymes. For this purpose, LDL(−) was pre-incubated with MAPP or CPZ, and then dyalized to remove excess inhibitor Next,prior we to sought to determine its addition to monocytes.whether This effect the inhibition was compared induced to that promotedbywhen MAPP and CPZ monocytes were on cytokine incubated simultaneously with LDL(−) and an inhibitor (co-incubation). release was due to a direct effect on LDL(−) or was a consequence of an effect on cell enzymes. ForFigure 5 shows IL-6 release, Figure 4. IL-6 release as a representative promoted cytokine inducedbyby“inLDL(−). vitro” Nomodified LDL.difference significant Monocytes waswere incubated observed with when using this purpose, LDL(−) unmodified MAPP between was pre-incubated LDLpre-incubation (blue bars) or modified with MAPP LDL (yellow and co-incubation. or CPZ, bars), both By contrast, CPZat 60 and then mg apoB/L. exerted dyalized a moreLDL to was modified effective remove excess inhibitory inhibitor prior by into incubation effect its with addition co-incubation topre-incubation Sph-enriched than in monocytes. (at This 0 and effect liposomes conditions. µM) was 10These and by compared indicateto thatthat sphingomyelinase observations promoted (SMase) CPZ has a when monocytestreatment were direct incubated (aton effect 0 and cells, Usimultaneously 10perhaps enzyme/g apoB). After by inhibiting with cell 20 LDL(−) hours SMase. and an of incubation, inhibitor cytokine release(co-incubation). was measured Figure 5 by ELISA. Results are expressed as μg/L and as mean ± SD (n = 6 ), vs. unmodified LDL, p < 0 .05. * shows IL-6 release, as a representative cytokine induced by LDL(−). No significant difference was Next, we sought to determine whether the inhibition induced by MAPP and CPZ on cytokine release was due to a direct effect on LDL(−) or was a consequence of an effect on cell enzymes. For this purpose, LDL(−) was pre-incubated with MAPP or CPZ, and then dyalized to remove excess inhibitor prior to its addition to monocytes. This effect was compared to that promoted when Biomolecules 2019, 9, x FOR PEER REVIEW 5 of 15 shows IL-6 release, as a representative cytokine induced by LDL(−). No significant difference was observed when using MAPP between pre-incubation and co-incubation. By contrast, CPZ exerted a observed when using MAPP more effective between inhibitory effect inpre-incubation co-incubation than andinco-incubation. By contrast, pre-incubation conditions. CPZ exerted a These observations more effective indicate that inhibitory CPZ has effect ina direct effect on cells, perhaps co-incubation than in by inhibiting cell SMase. conditions. These pre-incubation Biomolecules 2019, 9, 300 observations indicate that CPZ has a direct effect on cells, perhaps by inhibiting cell7 SMase. of 14 Figure 5. IL-6 release promoted by LDL(−) in pre-incubation or co-incubation with MAPP and CPZ . Monocytes were incubated with LDL(−) (60 mg apoB/L), which was pre-incubated (green bars) or Figure 5. IL-6 (yellow co-incubated release promoted withby bars) by LDL(−) the in pre-incubation inhibitors MAPP and or co-incubation CPZ, both at 10with µM.MAPP After and CPZ. 20 MAPP h of Figure 5. IL-6 release Monocytes were promoted incubated with LDL(−) LDL(−) in (60 pre-incubation mg apoB/L), which or was co-incubation pre-incubated with (green bars) or and CPZ . incubation, cytokine release was measured by ELISA. Results are expressed as % of inhibition Monocytesversus wereLDL(−) incubated co-incubated with (yellow bars) LDL(−) with in the absence ofthe (60 mg an inhibitors inhibitor, MAPP apoB/L), mean ±andSD CPZ, which (n = both at was 5 ), *vs. 10 µM.pre-incubated (green bars) or co- After 20 h of incubation, pre-incubation conditions, p < cytokine incubated 0(yellow release bars) with the inhibitors MAPP and CPZ, both at 10 µM. After 20in htheof incubation, was measured by ELISA. Results are expressed as % of inhibition versus LDL(−) .05. absence of an inhibitor, mean ± SD (n = 5), * vs. pre-incubation conditions, p < 0.05. cytokine release was measured by ELISA. Results are expressed as % of inhibition versus LDL(−) in 3.3. 3.3. Cer is Is Involved in in LDL(−)-Induced Apoptosis * the absenceCerof Involved an LDL(−)-Induced inhibitor, (n = 5 ), vs. pre-incubation conditions, p < 0 .05. mean ± SDApoptosis Figure Figure 66shows shows that that LDL(−) LDL(−) displayed displayed a twofold a twofold increase increase in the in the total total apoptotic apoptotic effect on effect on monocytes monocytes compared to compared LDL(+), to LDL(+), which which did did notApoptosis promote not promote apoptosis. apoptosis. LDL(−) did not induceLDL(−) did not statistically induce significant 2.3. Cer is Involved statistically insignificant LDL(−)-Induced differences in necrosis compared to LDL and the blank of cells. differences in necrosis compared to LDL and the blank of cells. Figure 6 shows that LDL(−) displayed a twofold increase in the total apoptotic effect on monocytes compared to LDL(+), which did not promote apoptosis. LDL(−) did not induce statistically significant differences in necrosis compared to LDL and the blank of cells. Biomolecules 2019, 9, x FOR PEER REVIEW 8 of 14 B) LDL(+) LDL(-) Figure 6. Apoptosis Figure induced 6. Apoptosis by LDL(+) induced and LDL(−) by LDL(+) in human and LDL(−) monocytes. in human (A) Early, monocytes. late, and (A) Early, late, total and total apoptosis, apoptosis, and necrosis induced in basal conditions (blank) or by LDL(+) (blue bars) LDL(−) and necrosis induced in basal conditions (blank) or by LDL(+) (blue bars) and and LDL(−) (red bars) (60 mg (red bars) (60apoB/L) were were mg apoB/L) evaluated by flow evaluated by cytometry. Results flow cytometry. are expressed Results as percentage are expressed of of as percentage apoptotic cells, mean ± SD (n = 6), * vs. blank cells, and # vs. LDL(+), p < 0.05 (B). Representative apoptotic cells, mean ± SD (n = 6 ), vs. blank cells, and # vs. LDL(+), p < 0 .05 (B). Representative * experiment showing experiment the effect showing of LDL(+) the effect (left panel) of LDL(+) and LDL(−) (left panel) (right(right and LDL(−) panel) on human panel) monocytes. on human monocytes. The effect of enzyme inhibitors was also assayed in LDL(−)-induced apoptosis. Figure 7 shows that DMS enhanced the apoptotic effect of LDL(−), whereas CPZ decreased this effect, both in early and late apoptosis. These observations suggest that Cer is involved in LDL(−)-induced apoptosis, whereas S1P would tend to counteract this action. On the other hand, the presence of MAPP did not promote any significant effect, despite a slight tendency to increase late apoptosis. Biomolecules 2019, 9, 300 8 of 14 The effect of enzyme inhibitors was also assayed in LDL(−)-induced apoptosis. Figure 7 shows that DMS enhanced the apoptotic effect of LDL(−), whereas CPZ decreased this effect, both in early Biomolecules 2019, 9, x FOR PEER REVIEW 7 of 15 and late apoptosis. These observations suggest that Cer is involved in LDL(−)-induced apoptosis, whereas S1P would tend to counteract this action. On the other hand, the presence of MAPP did not Biomolecules 2019, 9, x FOR PEER REVIEW 7 of 15 promote any significant effect, despite a slight tendency to increase late apoptosis. Figure 7. Apoptosis induced by LDL(−) in the presence of inhibitors. Early, late, and total apoptosis induced Figure 7.byApoptosis LDL(−) (60induced mg apoB/L) were evaluated by LDL(−) in the absence in the presence or presence of inhibitors. of MAPP Early, late, (yellow and total bars), apoptosis Figure DMS induced7. Apoptosis (green by bars), LDL(−) induced or(60 CPZ by (purple mg LDL(−) apoB/L)bars), were in the presence allevaluated at 10 µM. in of Results inhibitors. Early, are expressed the absence late, as fold or presence and total change(yellow of MAPP in apoptosis bars), induced versus by LDL(−) LDL(−) DMS (green in the bars), (60 or mg (purple absence CPZ apoB/L) were all of inhibitors, bars), evaluated mean inResults ± SD at 10 µM. the (n =absence or presence *vs. non-inhibitor, 6),are expressed as fold < 0 .05. of pMAPP change (yellow bars), in apoptosis DMS (green versus LDL(−)bars), or CPZ in the (purple absence bars), all atmean of inhibitors, 10 µM. ± SD (n = 6), Results are*expressed p < 0.05. as fold change vs. non-inhibitor, in apoptosis However, versus LDL(−)MAPP in theand CPZofalone absence did not inhibitors, meanpromote ± SD (napoptosis in the absence = 6), *vs. non-inhibitor, p < of LDL(−), whereas 0 .05. DMS However, MAPP andTable did (Supplementary CPZ S1). alone did not promote apoptosis in the absence of LDL(−), whereas However, DMSLDL(+) MAPP and did (Supplementary CPZ Table alone S1). did was modified to increase not promote its content in Cer,apoptosis Sph, andinNEFA,the absence in orderof to LDL(−), whereas reproduce the DMS LDL(+) did (Supplementary apoptotic was modified behavior Table of LDL(−). S1). 8 its to increase Figure content shows thatinalthough Cer, Sph,NEFA-LDL and NEFA,and in order to reproduce Sph-LDL promotedthe a LDL(+) was modified behavior of to LDL(−). increase Figure its 8 content shows in that Cer, Sph, although and NEFA, NEFA-LDL in and order to Sph-LDL reproduce slight non-significant apoptotic effect, the highest and statistically significant effect on total apoptosisa apoptotic promotedthe apoptotic slight was behavior when Cerofcontent non-significant found LDL(−).inFigure apoptotic 8the shows effect,increased LDL that highest and after although NEFA-LDL statistically SMase andeffect significant treatment. Sph-LDL promoted on total a apoptosis slight non-significant was found when Cer apoptotic content ineffect, the highest LDL increased andSMase after statistically significant effect on total apoptosis treatment. was found when Cer content in LDL increased after SMase treatment. Figure 8. Total apoptosis induced by modified LDL(–). Total apoptosis induced by LDL modified by Figure SMase 8. Total apoptosis treatment (yellow induced by(green bars), Sph modified LDL(–). bars), Total apoptosis or non-esterified fatty induced by LDL acid (NEFA) modified enrichment by (blue SMase treatment (yellow bars), Sph (green bars), or non-esterified fatty acid (NEFA) enrichment bars), as described in the Methods section. Results are expressed as mean ± SD (n = 5), * vs. unmodified (blue Figure bars), p8. LDL, as Total 0.05. apoptosis