HDL-associated ApoM is anti-apoptotic by delivering sphingosine 1-phosphate to S1P1 & S1P3 receptors on vascular endothelium
© The Author(s). 2017
Received: 16 December 2016
Accepted: 1 February 2017
Published: 8 February 2017
High-density Lipoprotein (HDL) attenuates endothelial cell apoptosis induced by different cell-death stimuli such as oxidation or growth factor deprivation. HDL is the main plasma carrier of the bioactive lipid sphingosine 1-phosphate (S1P), which it is a signaling molecule that promotes cell survival in response to several apoptotic stimuli. In HDL, S1P is bound to Apolipoprotein M (ApoM), a Lipocalin that is only present in around 5% of the HDL particles. The goal of this study is to characterize ApoM-bound S1P role in endothelial apoptosis protection and the signaling pathways involved.
Human umbilical vein endothelial cells (HUVEC) cultures were switched to serum/grow factor deprivation medium to induce apoptosis and the effect caused by the addition of ApoM and S1P analyzed.
The addition of HDL+ApoM or recombinant ApoM-bound S1P promoted cell viability and blocked apoptosis, whereas HDL-ApoM had no protective effect. Remarkably, S1P exerted a more potent anti-apoptotic effect when carried by ApoM as compared to albumin, or when added as free molecule. Mechanistically, cooperation between S1P1 and S1P3 was required for the HDL/ApoM/S1P-mediated anti-apoptotic ability. Furthermore, AKT and ERK phosphorylation was also necessary to achieve the anti-apoptotic effect of the HDL/ApoM/S1P complex.
Altogether, our results indicate that ApoM and S1P are key elements of the anti-apoptotic activity of HDL and promote optimal endothelial function.
KeywordsApoM Apoptosis Endothelial cells HDL Lipocalins Sphingosine 1-phospate
ApoM-bound S1P and ApoM-containing HDL are anti-apoptotic.
HDL/ApoM/S1P complex signals through S1P1 and S1P3.
ApoM-bound S1P anti-apoptotic effect is more potent than albumin-bound S1P.
Apolipoprotein M (ApoM) is a member of the Lipocalin family and its structure is defined by an eight-stranded antiparallel β-barrel enclosing a hydrophobic binding pocket, where different ligands bind, e.g. retinol , oxidized phospholipids  and sphingosine 1-phosphate (S1P) . Out of these, S1P is the only ApoM-ligand known to bind in vivo. An unusual property of ApoM is that its signal peptide is not cleaved off during secretion and used by the mature ApoM protein to anchor the protein to the phospholipid bilayer of high-density lipoproteins (HDL) [4, 5]. The plasma concentration of ApoM is approximately 0.9 μM and around 5% of all HDL particles in circulation carry ApoM and S1P [6, 7]. ApoM is the major carrier of S1P in circulation (~65%), the remaining S1P in plasma being bound to albumin (~35%) .
Sphingolipids have multiple key physiological functions that are important for the regulation of cell growth and survival. Ceramide and sphingosine are inducers of growth arrest and apoptosis and many stress stimuli increase the cellular levels of these compounds. In contrast, S1P is associated with suppression of apoptosis [8, 9].Five different membrane-bound, G-protein coupled S1P receptors (S1PR, S1P1-5) are known and binding of S1P to these receptors activates multiple receptor-specific downstream signaling pathways. In this way, S1P is able to regulate several biologic processes, such as immune cell trafficking, angiogenesis, cell migration and cell survival . Indeed, S1PR represent important drug therapeutic targets. For instance, FTY720, also known as Fingolimod, is phosphorylated by endogenous kinases and works as a functional antagonist of S1P1 that has been approved for the treatment of multiple sclerosis .
The integrity of endothelial cells lining the vessels is crucial for vascular homeostasis and endothelial cell-death triggers vascular leakage and promotes inflammation in adjacent tissues . Additionally, apoptotic endothelial cells become pro-coagulant and may provoke formation of blood clots . Thus, increased endothelial cell apoptosis is associated with several cardiovascular pathologies, in particular with thrombosis and atherosclerosis .
HDL particles are potently anti-atherogenic and reduce endothelial cell apoptosis [15, 16]. Cholesterol efflux is one of the mechanisms underlying HDL protection of endothelium, and importantly, ApoM-containing HDL enhances cholesterol efflux [17, 18]. Likewise, it is known that free S1P attenuates apoptosis in endothelial cells [15, 19]. The goal of the present study was to further characterize the role of S1P in the regulation of human endothelial cell apoptosis and to define the signaling pathways involved. For that purpose, we took into account that HDL-associated S1P is bound to ApoM in plasma. We have used human ApoM-containing HDL (HDL+ApoM) and ApoM-lacking HDL (HDL-ApoM) to study regulation of apoptosis in human endothelial cells. Moreover, we have elucidated whether the anti-apoptotic properties of S1P are carrier dependent by comparing the anti-apoptotic effects of albumin-bound S1P, ApoM-bound S1P and S1P as a free molecule.
Cell culture and apoptosis induction
Human Umbilical Vein Endothelial Cells (HUVEC) were obtained from Gibco, grown in 1% gelatin pre-coated plates in M200 medium containing 1% penicillin and streptomycin and low serum growth supplement (LSGS) (all from Gibco) at 37 °C in a humidified 5% CO2 incubator. The culture medium was replaced every 2 days, and cells were subcultured at 90–95% confluence. Cells were studied between passages 2–8.
LSGS contains fetal bovine serum (FBS), human epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), heparin and hydrocortisone. Removal of all these components was used to induce apoptosis in HUVEC. This treatment will be referred as serum/GF deprivation. For that, cells were washed twice with M200 medium without LSGS. The absence of S1P in M200 medium without LSGS was verified by mass spectrometry as it was previously described in [7, 20].
Purifications (ApoM and HDL)
Recombinant soluble human ApoM (residues 22–188, without the signal peptide, Swiss-Prot entry O95445) was expressed in E. coli, purified from inclusion bodies and refolded as described in Ahnström et al. . ApoM binding to S1P was confirmed by intrinsic fluorescence quenching and isoelectric focusing as described in Sevvana et al. . ApoM loading with S1P was performed as in Ruiz et al..
HDL was isolated from human plasma obtained from the Blood Bank at Växjö Hospital, Sweden, as described in Ruiz et al. . Briefly, HDL were separated by ultracentrifugation followed by size exclusion chromatography. HDL+ApoM and HDL-ApoM were isolated by immunoaffinity chromatography with M23 and M58 monoclonal antibodies against ApoM.
S1P levels in HDL preparations were quantified by mass spectrometry as it was previously described [7, 20]. S1P was ~0.146 μM/mg protein in total HDL, ~0.417 μM/mg of protein in HDL+ApoM and ~0.008 μM/mg protein in HDL-ApoM.
Protein quantification, protein electrophoresis and western blot
Sample protein concentration was quantified using BCA protein assay kit (Pierce) according manufacturer’s instructions.
Electrophoresis was done in 4–15% gradient pre-casted SDS-gels (Bio-Rad) under reducing conditions. Western blotting was done after separation in a Trans-Blot Turbo transfer system (Bio-Rad). An Antibody against phospho-ERK1 (T202/Y204) / phospho-ERK2 (T185/Y187) ERK1/2 was from R&D systems; antibodies against total ERK (#9102), pSer473 AKT (D9E), total AKT (C67E7) were from Cell Signaling and an antibody against GAPDH was from Santa Cruz Biotechnology (#20357).
Annexin V staining and flow cytometry
Cells were detached with TrypLE Express (Gibco), washed and resuspended in Annexin V binding buffer (BD Bioscience). Then, cells were stained with PE Annexin V and 7-ADD according manufacturer’s instructions (BD Bioscience) and analyzed in a Cytomics FC500 (Beckman Coulter) flow cytometer. Data were analyzed with FlowJo X v.10.0 7r2. Early apoptotic cells were defined by Annexin V+ and 7-ADD−.
Measurement of caspase-3 activity
Caspase-3 activity was measured using a colorimetric assay kit according to manufacturer’s instructions (Abcam). Briefly, cell lysates (50 μg total protein) were incubated in the presence of N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide (Ac-DEVD-pNA, 200 μM) and the release of pNA was measured using a plate reader (TECAN Infinite F200) at 405 nm.
Cell viability assay
Cell viability was evaluated by the MTT assay following manufacturer’s instructions (Roche). Briefly, viable cells are defined by their ability to reduce MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) to formazan, which is a measure of an active metabolism. The conversion was quantified using a plate reader (TECAN Infinite F200) at 570 nm and optical density value was utilized as an indicator of cell viability.
Quantitative real-time PCR (qPCR)
Total cellular RNA was isolated using RNeasy Kit according to the manufacturer’s instructions (Qiagen) and quantified using a NanoDrop spectrophotometer (ND2000, Thermo Scientific). qPCR were performed with a CFX384 C1000 thermal cycler (Bio-Rad) using the Super Scrip III Platinum One Step qRT-PCR kit (Invitrogen) and TaqMan probes (Applied Biosystems): 4326317E (GAPDH), Hs00173499_m1 (S1P1), AJ39RQ5 (S1P2), Hs00245464_s1 (S1P3), Hs02330084_s1 (S1P4) and Hs00928195_s1 (S1P5) according manufacturer’s instructions. Samples were measured as quadruplicates. The relative expression of each gene was calculated according to the ΔΔCT method . Expression of the housekeeping gene GAPDH was used to normalize for variations in RNA input.
Sphingosine-1-Phosphate (d18:1; Lipid Maps LMSP01050001) was purchased from Avanti Polar Lipids and Sigma; bovine fatty acid free albumin was from Sigma; W146, CAY10444 and ML-031 were from Cayman Chemical; SEW2871 and CYM5541 were from Tocris Bioscience; LY294002, U0126 and PD98059 were from R&D systems.
Statistical analyses were performed with SigmaPlot 11.0 software (Systat Software Inc.). A value of p < 0.05 was defined as threshold for significant changes. Student t-test and Mann-Whitney U-test were used for two-sample comparisons and ANOVA was used when assaying for multiple comparisons. The particular tests used for post hoc analyses depended on homoscedasticity, and are stated in the figure legends.
HDL+ApoM protects endothelial cells against apoptosis and promotes cell survival
Thus, we conclude that the HDL anti-apoptotic effect in serum/GF deprived endothelial cells is primary mediated by HDL containing ApoM and S1P.
S1P1 and S1P3 activation mediate the protective effect of ApoM-associated HDL
In conclusion, HDL required S1P1 and S1P3 signaling to achieve their anti-apoptotic effect in serum/GF deprived HUVEC. However, pharmacological activation of S1P1 or S1P3 was sufficient to mimic HDL protection.
ApoM-bound S1P confers longer protection to endothelial cells against serum/GF deprivation
Since anti-apoptotic and pro-survival effects of S1P were carrier dependent, we investigated if differences can be due to particular activation of S1PR. To study this, we performed Caspase-3 assays as in Fig. 5b, but in the presence of the S1P1 antagonist W146 or the S1P3 antagonist CAY10444. Interestingly, all three alternative ways to supply S1P to endothelial cells required S1P1 and S1P3 signaling to become anti-apoptotic (Fig. 5e and f).
Thus, we concluded that the anti-apoptotic effect of S1P in serum/GF deprived endothelial cells was carrier dependent, ApoM-bound S1P being the most powerful of all three carriers. Furthermore, anti-apoptotic activity of S1P was mediated by S1P1 and S1P3 with independence of which S1P carrier was used.
PI3K/AKT and ERK1/2 signaling pathways are implicated in the anti-apoptotic effect of S1P in serum/GF deprived cells
In conclusion, S1P anti-apoptotic effect on serum/GF deprived endothelial cells went via S1P1 and S1P3 and required the phosphorylation of AKT and ERK1/2 (Additional file 1: Fig. S1).
Previous studies have pointed out the protective role of HDL on endothelial cells upon different cell-death stimuli, including oxidized LDL [29, 30] and serum/GF deprivation [15, 16]. Likewise, anti-apoptotic properties of free S1P have been demonstrated [9, 25–27, 30, 31]. Here we connect previous findings and show that ApoM-containing HDL, and therefore S1P, have anti-apoptotic and pro-survival properties in serum/GF deprived endothelial cells (Figs. 1 and 2). Importantly, S1P also promotes survival in cardiomyocytes , macrophages  and other cell types [33–36]. Now, it would be relevant to study S1P protection in other human cell types taking in account ApoM. It is important to highlight that HDL particles are highly heterogenic in protein and lipid composition and additional cytoprotective mechanisms are possible . Which ones are relevant may depend on the cell-death stimulus, time and concentration used.
de Souza et al.  isolated HDL subpopulations and found that small and dense HDL3, which are enriched in S1P [and ApoM, ], have cytoprotective activity superior to that of large and light HDL2. Interestingly, reconstituted HDL (rHDL) with added S1P did not enhance the anti-apoptotic effect achieved by rHDL without S1P . Similarly, S1P-fortified HDL subfractions did not to significantly improve the anti-apoptotic effect of non-S1P-fortified HDL. Both scenarios could be explained by the fact that the exogenous S1P was not bound to ApoM and therefore may not properly interact with S1PR. This explanation concurs with Fig. 5a-d, where ApoM-S1P displayed significantly elevated anti-apoptotic activity as compared to free S1P or albumin-S1P. In agreement, apoptosis was not inhibited when albumin-S1P was used at 1–100 nM . Likewise, rHDL anti-apoptotic ability is enhanced when plasmalogens are incorporated to rHDL , but the molecular mechanism behind has not been described yet. Several endothelial cell types express ApoM  and the S1P transporter Spns2 . Possibly, rHDL including plasmalogens are better acceptors for ApoM and S1P than plasmalogen-free rHDL.
Riwanto et al.  demonstrated that ApoJ enhances HDL anti-apoptotic effect on endothelial cells. However, ApoJ is absent in our HDL+ApoM preparations  and, therefore, ApoM-S1P anti-apoptotic effect cannot be ascribed to ApoJ. In contrast, HDL anti-apoptotic activity is impaired in HDL enriched in ApoC-III , which it is less abundant in HDL+ApoM than in HDL-ApoM . Thus, the poor anti-apoptotic capacity of ApoC-III containing HDL can be explained by the low content in ApoM-S1P.
Endothelial-cell survival is enhanced by free S1P via S1P1 and S1P3 . We corroborated this finding and demonstrated that parallel activation of both S1P1 and S1P3 by HDL+ApoM is required to achieve S1P anti-apoptotic and pro-survival effects (Figs. 4 and 5). Furthermore, we show that S1P1 and S1P3 activation requirement is independent of the S1P carrier (Fig. 5e-f). However, activation by ApoM-S1P renders a longer protection than albumin-S1P. These apparently conflicting data can be explained by S1P carrier specific degradation of S1P1 [43, 44]. Following activation of S1P1 by albumin-S1P, S1P1 is internalized and degraded by the proteasome, whereas S1P1 is internalized and recycled to the plasma membrane after ApoM-S1P activation. Unfortunately, no data on S1P-carrier dependent biology of S1P3 are available. However, an analogous situation to S1P1 may be plausible for S1P3.
Beyond S1PR, other plasma membrane receptors connect apoptosis, HDL and its major component, ApoA1. First, HDL3 acts via Scavenger Receptor Class B Type I (SR-BI) to inhibit apoptosis on endothelial cells . Indeed, Li et al.  over-expressed SR-BI in CHO cells and elaborated an attractive model in which SR-BI is a pro-apoptotic receptor in absence of HDL. This model needs to be validated in endothelial cells, but the fact that HDL+ApoM are more efficient than HDL-ApoM in stimulating cholesterol efflux suggests that HDL+ApoM may have higher affinity for SR-BI than in HDL-ApoM. Additionally, stimulation of F1-ATPase by lipid-free ApoA1 inhibits endothelial cell apoptosis , but interactions between HDL and F1-ATPase have not been reported.
AKT and ERK1/2 phosphorylation mediate HDL and S1P cytoprotective actions [15, 16, 27, 28]. Moreover, HDL+ApoM and albumin-S1P, but not HDL-ApoM, phosphorylate AKT and ERK . Here, we confirmed S1P-dependent phosphorylation of ERK and AKT and demonstrated that blockage of AKT and ERK signaling abolishes S1P anti-apoptotic effects (Fig. 6). Importantly, activation of S1P1 and S1P3 by ApoM-S1P or albumin-S1P phosphorylate AKT and ERK (Fig. 7). Interestingly, S1P induces AKT activation and protects against ischemia/reperfusion in mouse cardiomyocytes via S1P2 and S1P3 [47, 48]. This suggests that the pattern of S1PR activated by S1P to achieved cytoprotection may be tissue/organism dependent.
Retinol binding protein (RBP) is another member of the Lipocalin family and transports retinol in plasma. Interestingly, apo-RBP is pro-apoptotic, whereas holo-RBP is anti-apoptotic . We did not identify any pro-apoptotic activity of apo-ApoM per se in endothelial cells. However, ApoM over-expression promotes apoptosis in the human hepatoma derived cell line HepG2 . Moreover, two other plasma Lipocalins: Lipocalin-type prostaglandin D2 synthase (L-PGDS) and Apolipoprotein D (ApoD) mitigate cell-death and promote viability [51, 52]. Interestingly, ApoM and ApoD are HDL enriched in HDL3 . Thus, abundance of Lipocalins in HDL3 could explain the high cytoprotective ability of HDL3.
Taken together, our results demonstrate that the HDL/ApoM/S1P-complex plays an essential role in vascular biology and protects endothelial cells from apoptosis. This is especially relevant in pathologies where endothelial cell apoptosis is altered such as in thrombosis and atherosclerosis.
- HDL+ApoM :
- HDL-ApoM :
Human umbilical vein endothelial cells
We thank Li J. Guo for technical assistance throughout the project.
This work was supported by the Swedish Research Council (#07143), the Swedish Heart and Lung Foundation, Söderberg’s Foundation, and Österlund’s Foundation. Funding agencies did not participate in the experimental design of the study, collection, analysis, interpretation of data, decision to publish or manuscript preparation.
Availability of data and materials
Data available on request from the authors.
BD conceived the original project. MR, HO and BD designed the experiments. MR and HO performed the experiments. MR, HO and BD wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
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