Apolipoprotein M in High Density Lipoprotein Protects Against Astrocyte Apoptosis Induced By Ischemic Insult Via Sphingosine 1-Phosphate Signaling

Apolipoprotein M (ApoM) has been reported to be contained in high density lipoprotein (HDL) and bound with sphingosine-1-phosphate (S1P). ApoM-associated HDL exerts protective effects against cell death. The aims of the study were to evaluate the effects of ApoM-associated HDL on astrocytes following ischemic insult. Primary cultured mouse astrocytes were treated with oxygen-glucose deprivation (OGD) followed by recovery. The astrocytes underwent apoptosis after treatment with OGD for 4 h and recovery for 24 h. The addition of HDL with ApoM attenuated apoptotic cell death, but HDL without ApoM did not show any effect. Free S1P or ApoM-bound S1P promoted cell survival and inhibited apoptosis. Only S1P receptor 1 (S1PR1) expression was upregulated and blockage of S1PR1 with specic inhibitor or genetic knockdown of S1pr1 abolished the protective effects. In addition, administration of ApoM containing HDL or free S1P induced activation of Akt and Erk in the astrocytes, and pharmacological inhibition of Akt and Erk rescued cell death after OGD treatment. Taken together, ApoM is required for the protective effects of HDL, which depends on delivery of S1P to S1PR1 by ApoM in HDL, indicating ApoM may be a neuroprotective constituent in plasma. The goal of this study is to characterize the participation of ApoM and S1P in the regulation of astrocyte apoptosis and the signaling pathways involved. In the study, we investigated the effects of HDL particles on primary cultured mouse astrocytes. We demonstrated that ApoM-containing HDL attenuated astrocyte apoptosis induced by ischemic insult and recovery in vitro. The protective effects are mediated by ApoM-S1P complex and require the activation of Akt or extracellular-signal-regulated kinase (Erk) pathways. The results indicate that ApoM-HDL or ApoM-S1P may be a potential neuroprotective agent to counteract the cell death induced by brain ischemia.


Introduction
As the most abundant cell type in the brain, astrocytes are important for the maintenance of cerebral function in physiological conditions and homeostasis of blood-brain barrier. During cerebral ischemia and reoxygenation, neurons may undergo cell death resulting from accumulation of free oxygen radicals (Tajiri, Oyadomari et al. 2004, Peng, Zhao et al. 2015. Astrocytes can produce free radical scavenger substances and participate in the protection of neurons against oxidative injury (Papadopoulos, Koumenis et al. 1997, Murthy, Rao et al. 2001, Fulmer, VonDran et al. 2014). On the other hand, astrocytes can function as the defense of neurons by providing trophic support and physical barrier of glial scar (Huang, Li et al. 2014), preventing massive neuronal death. Astrocytes are also vulnerable to ischemic insults, thus, protection of astrocytes against cell death may be essential to prevent neuronal injuries as well as to maintain normal brain function. So far, little is known about the endogenous factors in blood that regulate glial cell survival.
Recently studies suggest that high density lipoproteins (HDL) in blood exhibits therapeutic e ciency for the treatment of cardiovascular disease due to the effects of healthy HDL in vitro ( . Among these bioactive lipids, only S1P is the physiological ligand for ApoM in vivo and most of S1P is carried by ApoM in blood (Liu, Allegood et al. 2015, Dusaban, Chun et al. 2017. Approximate 5% of HDL particles in blood carry ApoM-S1P complexes and are involved in lipid metabolism . Recently, some evidences show that ApoM participates in the homeostasis maintenance of endothelial monolayer (Zhu, Luo et al. 2018, Mathiesen Janiurek, Soylu-Kucharz et al. 2019). ApoM knockout mice exhibit reduced HDL-associated S1P in blood and increased vascular permeability (Zhu, Luo et al. 2018 S1P is a kind of sphingolipids and possesses several key physiological functions, including regulation of cell growth and survival. S1P exerts its effects via binding and activating its receptors, that is S1P receptors (S1PR, S1PR1-5), then activates speci c downstream signals (Van Doorn, Van Horssen et al. . Modulation of S1PR1 with chemical compound has shown e ciency to treat some disorders. For example, one of the S1PR1 modulators, FTY720 prevents lymphocyte egress from lymphoid organs and receives approval as an oral treatment for relapsing forms of multiple sclerosis (Kappos, Antel et al. 2006). Thus, S1PR1 may be a useful therapeutic target for metabolic diseases.
Recently, it has been proved that HDL particles protect against cell apoptosis in endothelial cells, which is related to the increased cholesterol e ux mediated by ApoM (Luscher, Landmesser et al. 2014, Galvani, Sanson et al. 2015. The goal of this study is to characterize the participation of ApoM and S1P in the regulation of astrocyte apoptosis and the signaling pathways involved. In the study, we investigated the effects of HDL particles on primary cultured mouse astrocytes. We demonstrated that ApoM-containing HDL attenuated astrocyte apoptosis induced by ischemic insult and recovery in vitro. The protective effects are mediated by ApoM-S1P complex and require the activation of Akt or extracellular-signalregulated kinase (Erk) pathways. The results indicate that ApoM-HDL or ApoM-S1P may be a potential neuroprotective agent to counteract the cell death induced by brain ischemia.

Ethics statement
This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of Changzhou University. The mice were maintained in a room with 12 h light-dark cycle and had constant access to puri ed water and sterilized food. The neonatal mice were euthanized with carbon dioxide and the cerebral material was obtained 5 min after sacri ce of the animals. Both of male and female neonatal mice were used in this study.

Cell culture
Cortical astrocytes were isolated and cultured as previously described with modi cations (Huang, Li et al. 2014). The neonatal C57BL/6 mice born within 24 h were decapitated. Following removal of the meninges, the cerebral cortices were cut into small pieces and digested with 0.25% trypsin for 20 min at 37 °C. the dissociated cells were incubated in high glucose DMEM (Gibco, Grand Island, USA) supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 IU/ml of penicillin and 100 mg/ml streptomycin. The cultures were maintained in a humidi ed atmosphere (5% CO 2 and 95% air) at 37 °C.
After 24 h, the medium was replaced and thereafter twice a week. After 12 days, the con uent cultures were vigorously shaken to remove the microglial cells. The adherent cells were detached and reseeded at a density of 1.5 ´ 10 5 cells/ml.

Oxygen-glucose deprivation
OGD was performed as previously reported with modi cations (Huang, Zhang et al. 2008). The astrocytes were rinsed twice and incubated in Earle's solution without glucose. Then the cells were cultured in an anaerobic chamber lled with 95% N 2 and 5% CO 2 at 37 °C. At the end of the treatment, the cultures were returned to normal condition and the medium was changed with a culture medium for indicated time. Prior to OGD treatment, the cells were treated with or for 30 min, and the drugs were continuously applied until the end of recovery. The normoxia controls were washed and incubated with Earle's solution containing 5.6 mM glucose, then cultured under normal condition except OGD treatment.

siRNA treatment of cells
The astrocytes grown to 70% con uence in 6 well plate before transfection. Thereafter, 100 pmol of S1PR1 or S1PR3 siRNA (Thermo Fisher Scienti c Inc., USA) were diluted in 200 ml of Opti-MEM I (ThermoFisher Scienti c, USA) and 5 ml of P3000 reagent. Simultaneously, mix 8 ml of Lipofectamine 3000 (ThermoFisher Scienti c, USA) and 200 ml of Opti-MEM I solution. Then the duplex siRNA solution was added to the Lipofectamine 3000 solution and incubated for 20 min at room temperature. Thereafter, the siRNA complexes were added to 2 ml of complete medium and the cells were incubated for 16 h at 37°C in a 5% CO 2 incubator. Then replace the transfection medium with complete medium.

Puri cations of HDL particles with or without ApoM
ApoM-containing HDL was separated by ultracentrifugation followed by immunoa nity chromatography. Firstly, HDL was isolated from mouse plasma as described with modi cations ( The total HDL obtained from ultracentrifugation was applied to the anti-ApoM column to isolated ApoMcontaining HDL. An anti-mouse polyclonal antibody against ApoM (PA5-92403, Thermo sher, USA) was coupled to 3 ml HiTrap N-hydroxy-succinimide (NHS)-activated columns (Amersham Biosciences) at 0.5 mg/ml gel, according to the manufacturer's instructions. The column was washed with 10 mM Tris-HCl, pH 7.5 with 500 mM NaCl and bound particles were eluted with glycine (0.1 M, pH 2.2). The elution was collected in 1 ml fraction before the ow-through was passed over the column again. The absorbance at 280 nm was determined in each fraction. The HDL preparations were subjected to ve rounds of anti-ApoM chromatography until all ApoM particles had been removed. S1P levels in HDL preparations were quanti ed by a Sphingosine 1-Phosphate ELISA Kit (Echelon Biosciences Inc., USA). S1P was 0.32 mM/mg of protein in HDL with ApoM. The S1P in HDL without ApoM could not be detected.

Loading of ApoM with S1P
The ApoM-bound S1P was produced according to the methods previously described (Christoffersen, Obinata et al. 2011). Firstly, S1P was dissolved in methanol followed by evaporization. Then same molar of recombinant murine ApoM (Uscn Life Science Inc., China) was added and sonicated for 3 min in 20 mM Tris-HCl with the pH value of 8.0.

Cell viability assay
Cell viability was assessed by MTT assay. Brie y, the cells were plated with 1.5 ´ 10 5 /ml in 96-well plates. After 24-h incubation, the cells were subject to OGD and recovery. After the treatment, 3-(4,5dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, USA) was added to each well with the nal concentration of 0.5 mg/ml. Following 4-h incubation at 37 °C, the medium was removed and 100 ml of dimethyl sulfoxide was added to each well. Then the absorbance at 570 nm was measured with a microplate reader (TECAN In nite F200, Tecan Trading AG, Switzerland). Results were expressed as the percentages of the control.

Cell death evaluation
The astrocyte death was assessed by the measurement of lactate dehydrogenase (LDH) released into the medium. After the treatments, 50 ml of supernatants was collected from each well and LDH activity was determined with a LDH assay kit (Roche, USA) according to the manufacturer's instructions.

Cell apoptosis assay
Cells grown on coverslips were washed with PBS and stained with Hoechst 33258 at 10 mg/mL for 10 min at 37 °C. Thereafter, the cells were observed under a uorescent microscope (Evos M5000 imaging systems, Thermo sher, USA). The cells with condensed or fragmented nuclei showing strong uorescence were identi ed as apoptotic cells. At least 10000 cells were counted in more 3 elds in each coverslip. The cell apoptosis was expressed as percentage of apoptotic cells.

Immunocytochemistry
Astrocytes seed on coverslips were xed with 4% paraformaldehyde for 30 min at room temperature and incubated with 5% normal goat serum for 1 h. Thereafter, the cells were exposed to mouse monoclonal anti-GFAP antibody (1: 400, EMD Millipore, USA) at 4 °C overnight. After rinsing with PBS, the cells were incubated with goat anti-mouse Alexa Fluo 488-conjugated secondary antibody (1: 600, Jackson ImmunoResearch Laboratories, USA). The coverslips were mounted with ProLong Gold Antifade Mountant with DAPI (Thermo sher Scienti c, USA) and observed under a uorescent microscope (Evos M5000 imaging systems, Thermo sher, USA).

Caspase 3 activity assay
The caspase 3 activity was measured with a colorimetric assay kit according to manufacturer's instructions (Abcam). The cell lysates containing 60 mg of protein were incubated with N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide at 200 mM for 2 h at 37 °C. the release of p-nitroanilide was quanti ed using a microtiter plate reader (Tecan In nity F50) at 405 nm.

Statistical analysis
Statistical analyses were performed using SPSS 18.0 statistics Software. Data are expressed as means ± standard deviation (x̄ ± s). Difference between groups were examined using a one-way analysis of variance (ANOVA) or student's test (t test). When ANOVA test results for all data were signi cant, post hoc least signi cant difference (Bonferonni) was used to determine individual differences. If normality or variance homogeneity were not met, nonparametric tests were applied. A P value <0.05 was considered statistically signi cant.

Characterization of apoM-containing HDL particles in plasma
To determine the effects of ApoM in HDL on astrocyte cell death, we puri ed ApoM-containing HDL from plasma. After ultracentrifugation, the isolated HDL was puri ed on a HiTrap column with a monoclonal antibody against ApoM. Immunoblotting analysis showed that after 5 rounds of puri cation, ApoM could not be detected in HDL (Fig. 1A), indicating good preparations of ApoM-containing HDL and ApoM-free HDL. In addition, analysis using western blotting against ApoA-I, ApoB and ApoM demonstrated that HDL did not contain ApoB but contain rich ApoA-I (Fig.1B), consistent with the previous ndings (Christoffersen, Nielsen et al. 2006). ApoM-containing HDL and ApoM-free HDL contained similar levels of ApoA-I, while HDL contain higher level of ApoA-I, suggesting the partial removal of ApoA-I during the puri cation processes.

Astrocytes undergo cell death following ischemic insult in vitro
When astrocytes were treated with deprivation of glucose and oxygen, cells underwent cell death (Huang, Zhang et al. 2008). As shown in Fig. 1C, the cell viability was reduced after 4-h OGD treatment, while 2-h OGD did not affect the viability [F (5, 12) = 85.205, P < 0.001], suggesting the time-dependent effects of OGD on cell death. To assess the cell viability after OGD and recovery, the cells were treated with 4-h OGD and recovery for 24 h, 48 h and 72 h. The results showed that 24-h recovery after 4-h OGD resulted in signi cant cell death [F (4, 35) = 66.317, P < 0.001; F (4, 35) = 118.808, P < 0.001 respectively], as evidenced by MTT reduction assay and LDH assay ( Fig. 1D and 1E). As shown in Fig. 1F. after staining with hematoxylin, the astrocytes exposed to 4-h OGD and 24-h recovery exhibited cell injury. Then we choose 4-h OGD/24-h recovery as the treatment for astrocytes in the following study.
During cerebral ischemia in mouse brain, astrocytes in the ischemic core undergo apoptosis and dysfunction resulted from oxidative stress, leading to the consequent neuronal death (Liu, Chen et al. 2014, Amri, Ghouili et al. 2017). In order to clarify the role ApoM in the protection by HDL against cell apoptosis, the astrocytes were stained with Hoechst 33258. The apoptotic astrocytes showed condensed nuclei and enhanced blue uorescence. As shown in Fig. 4A and 4B, the astrocytes exhibited a weak apoptotic ratio but a signi cantly high apoptotic ration after OGD treatment. HDL + decreased the percentage of apoptotic cells in a dose-dependent manner [F(6, 35) = 81.542, P < 0.001; F(4, 25) = 32.879, P < 0.001 respectively]. HDL + at 10 ~ 20 mg/ml signi cantly protected against cell apoptosis compared to OGD only, while 10 ~20 mg/ml of HDLtreatment did not confer protection against cell apoptosis.
Under apoptosis, caspase 3 in cells will be cleaved, leading to the increased expression ratio of Bax and Bcl-2 (Petrache, Fijalkowska et al. 2006). As shown in Fig. 4C and 4D, OGD reduced the expression of Bcl-2 and induced the expression of Bax. Thus, the ratio of Bax/Bcl-2 was enhanced after OGD treatment, and administration of HDL + , not HDL -, reduced the expression ratio. In addition, the expression of cleaved caspase 3 (p17) was determined by immunoblotting in the astrocytes. The results showed that caspase 3 was cleaved after OGD treatment, showing the activation of caspase 3. Those inhibitors were added to the astrocytes in the presence of HDL + for 10 min (Akt activation) or 1 h (Erk activation). Then the astrocytes were lysed and the phosphorylation of Akt or Erk protein was analyzed by western blotting. As shown in Fig. 5A   It has been reported that ApoM functions as a carrier of S1P in HDL, leading to the activation of downstream signaling of S1P after activating its receptors (Liu, Seo et al. 2014. In order to directly compare the effects of free S1P and ApoM-bound S1P, we loaded soluble mouse ApoM with S1P to obtain ApoM-bound S1P. Thereafter, we tested the Akt and Erk phosphorylations in the cells after treated with free S1P or ApoM-bound S1P. We found that ApoM-bound S1P induced expression of phosphorylated Akt or ERK comparable to that of free S1P, suggesting the similar effects of S1P bound or unbound to ApoM. However, we did not observe any effects of ApoM on cell apoptosis or Akt activation (data not shown).
3.7. S1pr1 but not S1pr3 is induced in astrocytes after OGD treatment Upon activation, S1P exerts its biological effects through a group of G-protein coupled receptors, including S1P receptors (S1PR) 1-5. Thus, to determine the participation of S1P receptors in the antiapoptotic effects of HDL + on astrocytes, the expression of S1prs in astrocytes after OGD were assessed.
It has been shown that S1pr1 and S1pr3 are main S1P receptors expressed in astrocytes (Pebay, Toutant et al. 2001), so only these two receptors were analyzed in this study.
After the cells were treated with OGD and recovery, the mRNA levels of S1pr1 and S1pr3 were assessed by real-time quantitative RT-PCR. The data showed that the mRNA expression of S1pr1 in the astrocytes was up-regulated after OGD treatment, while the S1pr3 level was not changed after OGD (Fig. 6A) [t(2) = 5.426, P < 0.001; t(2) = 10, P = 0.771 respectively]. To con rm the results, the protein levels of S1pr1 and S1pr3 were determined by immunoblotting. As shown in Fig. 6B, only S1pr1 protein expression was markedly induced in the astrocytes after OGD treatment [t(2) = 7.531, P < 0.001; t(2) = 10, P = 0.926]. These data suggest that S1pr1 may be required for the anti-apoptotic property of HDL + .
3.8. Activation of S1pr1 mediates protective effects of ApoM-associated HDL Next, we determined which S1P subtype receptor is responsible for HDL anti-apoptotic property. Thus, we employed pharmacological antagonists of S1P receptors in the study. In the presence of HDL + , a S1pr1 antagonist W146 at 1 mM or a S1pr3 antagonist CAY10444 at 10 mM was added to the culture medium . These results suggest that S1pr1 but not S1pr3 activation is required for the anti-apoptotic effect of HDL + , which involves the activation of Akt/Erk1/2 signaling pathways.
3.9. Knockdown of S1pr1 abolishes the protection of ApoM-associated HDL To exclude the possibility of unspeci c effects of S1pr inhibitors, speci c siRNAs were added to the astrocytes to silence the expression of S1pr1 and S1pr3. After the cells were treated with siRNAs for 3 days, the cell protein extracts were used for analysis of S1pr1 and S1pr3 by immunoblotting. As shown in Fig. 8A, immunoblotting results showed that protein expression of S1pr1 or S1pr3 was reduced by their respective siRNA. Thereafter, the apoptotic astrocytes after treatment with OGD were determined by Hoechst staining. We found that only S1pr1 silencing mitigated the anti-apoptotic effect of HDL + in astrocytes (Fig. 8B) [F(3, 20) = 21.869, P < 0.001]. In addition, the Akt and Erk activation were determined by immunoblotting analysis. As shown in Fig. 8C, HDL + or ApoM-bound S1P induced Akt and Erk phosphorylation, while the upregulation of phosphorylated Akt or Erk were blocked after S1pr1 silencing.

Discussion
In the study, we found that ApoM associated HDL exerts protective effects on astrocyte cell death induced by ischemic insult in vitro. The protective effects by HDLs is mediated by the ApoM-S1P complexes. Thus, we connect previous ndings and demonstrate that ApoM-containing HDL or S1P in HDLs have anti-apoptotic properties. In addition, we found that S1PR1 activation by S1P and the downstream Akt/Erk activation participate in the anti-apoptotic effects of HDLs, pharmacological inhibition or genetically knockdown of S1pr1 abolish the protection of HDLs. Accumulating evidence shows that HDL particles protect cells against cell death induced by endoplasmic reticulum (ER) stress and serum starvation, which is related to its anti-oxidative or anti-in ammatory properties , Nagao, Toh et al. 2017, Durham, Chathely et al. 2018). Therefore, our results con rmed the protective of HDL particles on astrocytes death after ischemic insults. S1P is generated through phosphorylation of sphingosine and involved in various processes ( Here, we only determined the cell apoptosis that is representative of the pattern of cell death. This is one of the limitations of our work and should be investigated in future work.
In addition, he downregulation of GFAP of the astrocytes was restored by addition of ApoM-bound HDL. It has been reported that S1P bound to albumin also show low activity against endothelial apoptosis (Ruiz, Okada et al. 2017), suggesting the S1P-ApoM complex have the highest a nity to interact with S1PRs. In contrast to the previous report that ApoM-S1P shows higher protective effects (Ruiz, Okada et al. 2017), our data showed that ApoM-S1P complex exhibited similar anti-apoptotic properties compared to free S1P in astrocytes. The results may be explained by the similar activation of S1prs after exposure to ApoM-S1P or free S1P due to the upregulation of S1pr1, although free S1P may activate S1PRs less properly than ApoM-S1P complex (Ruiz, Okada et al. 2017). In addition, S1P will be internalized and degraded in cells after binding with S1PRs, which could be mediated by different S1P carrier ( In this study, we found that S1pr1, but not S1pr3, was responsible for the protective effects of HDL particles. S1pr1 expression after OGD was up-regulated while the S1pr3 expression was comparable before and after OGD. Only inhibitor of S1pr1 could abolish the protection by the HDL, while the inhibitor of S1pr3 could not. This suggests that the pattern of S1PR activation to achieve protection may be different depending on tissues.
Akt-Erk signaling plays an important role in mediation of cell injury or cell death. Akt has been found as an oncogene and is activated by many growth factors. Activation of Akt has been proved to inhibit apoptosis induced by serum deprivation, UV irradiation and chemical agents ( In our study, the phosphorylation level of Akt was strongly reduced after OGD treatment. ApoM associated HDL or free S1P phosphorylated Akt, while HDL without ApoM did not show any effects on Akt phosphorylation. After Akt phosphorylation, Erk phosphorylation was also observed, indicating the implication of Akt and Erk signaling pathways in the protection of total HDL. Activation of Akt by ApoM associated HDL was strongly suppressed by speci c Akt inhibitor which also attenuated the protective effects of ApoM-HDL. Taken together, Akt/Erk signaling may mediate the antiapoptotic effect of ApoM associated HDL in astrocytes.

Conclusions
In summary, HDL containing ApoM induced the activation of Akt/Erk signaling, which conferred protection against the OGD-induced apoptotic death in astrocytes. Thus, pharmacological of genetic activation of Akt or Erk may induce the anti-apoptotic effects after ischemic insults in neuronal cells.
These results imply the potential involvement of these pathways by HDL containing ApoM and the complete molecular mechanisms needs to be elucidated. Continued attempts to identify novel target responsible for the Akt activation and to clarify the downstream signaling will pave the way to exploiting therapeutic strategies for the management of cerebral ischemic disorders.

Declarations
CRediT authorship contribution statement Xiaojia Huang and Linhong Deng performed the research design. Xiaojia Huang, Zhiqi Zhai, Ting Zhou, Chengju Sheng and Chao Zhou conducted the experiments. Xiaojia Huang collected the data and wrote the manuscript. All authors read and approved the manuscript.

Con ict of Interest
The authors declare that there are no con icts of interest.