Apolipoprotein M and sphingosine-1-phosphate complex alleviates TNF-α-induced endothelial cell injury and inflammation through PI3K/AKT signaling pathway

Background In spite of the important role of Apolipoprotein-M (ApoM) and Sphingosine-1-Phosphate (S1P) played in atherosclerosis (AS), there was few related research reporting ApoM and S1P complex (ApoM-S1P) on biological activities of human umbilical vein endothelial cells (HUVECs). In this study, we explored the effect and mechanism of ApoM-S1P on TNF-α-induced inflammation in HUVECs. Methods TNF-α was utilized to induce HUVEC injury and inflammation. After HUVECs were treated with antagonists of ApoM, S1P, ApoM + S1P, and ApoM + S1P + S1PR, calcein-acetoxymethyl ester was employed for the assessment of the adhesion of HUVECs to THP-1, immunofluorescence for the observation of caspase-1expression in HUVECs, reactive oxygen species (ROS) kit for the detection of ROS level in HUVECs. The impact of TNF-α, ApoM, S1P and S1PR antagonists on inflammatory response, pyroptosis and adhesion of THP-1 monocytes to HUVECs were determined by detecting expressions of pyroptosis related proteins (IL-1β, IL-18, ASC, NLRP3 and caspase-1), inflammatory cytokines (IL-6 and IL-10), adhesion molecules (E-selectin, ICAM-1, and VCAM-1) and p-PI3K/p-AKT by qRT-PCR and Western blot, as well as by ELISA. Results TNF-α could increase adhesion of THP-1 monocytes to HUVECs and induce inflammatory response and pyroptosis in HUVECs, indicated by up-regulated expressions of E-selectin, ICAM-1, VCAM-1, IL-1β, IL-18, caspase-1, ASC, NLRP3, and IL-6, and down-regulated expression of IL-10. Co-treatment of ApoM-S1P on TNF-α treated HUVECs could protect HUVECs from injury and inflammation, evidenced by the attenuation of expressions of pyroptosis related proteins, inflammatory cytokines, and adhesion molecules, as well as the augment of PI3K and AKT phosphorylation. JTE-013, an antagonist of S1PR2, could reverse the amelioration of ApoM-S1P on pyroptosis and inflammation of HUVECs, indicating that ApoM-S1P could bind to S1PR2 to protect HUVECs from injury and inflammation through activating PI3K/AKT pathway. Conclusion ApoM-S1P could attenuate TNF-α induced injury and inflammatory response in HUVECs by binding to S1PR2 to activate PI3K/AKT pathway.


Background
Atherosclerosis (AS) is a widespread disease that causes morbidity and mortality, which could deteriorate into circulatory problems including cerebrovascular disease and coronary disease [1,2]. Despite the great advancement on both basic and clinical researches, the incidence of AS has been increased dramatically in recent decades and has resulted in millions of deaths around the world each year [3,4]. AS is characterized by the sedimentation of fibrous tissues and lipids in the intima of elastic arteries, with consequent formation of thrombus and structural damage caused by thickening and hardening of the vessel walls [5]. Previous study has unraveled that cell death and inflammation play critical roles in various stages of AS [6]. Typically, cell death is especially attributed to apoptosis and necrosis, nevertheless, other forms of cell death including pyroptosis has also been discovered [7]. Pyroptosis is a new type of pro-inflammatory programmed cell death different from apoptosis and necrosis, which can lead to cytokines release and activate pro-inflammatory immune cell mediators [8,9]. However, the roles of pyroptosis and its potential relationship with AS as well as the underlying mechanisms remained inadequately investigated.
Sphingosine-1-phosphate (S1P) is a membrane-derived sphingolipid that regulates a variety of cellular processes including migration, growth, inflammation, survival, and angiogenesis, and is considered as a key regulator of AS physiology and pathophysiology [10,11]. Up-regulated expression of pro-inflammatory cytokine IL-6 is proved to increase S1P at cellular level, which consequently results in elevated expression of S1P receptor 1 (S1PR1) in sickle cell disease mice models [12]. S1P signaling exerts a critical impact on lymphocyte transportation, blood vessel development, and neurogenesis and S1P receptor antagonist has been approved for the control of autoimmune neuroinflammation in patients with multiple sclerosis [13]. Inhibition of endothelial S1PR2 signaling attenuates endothelial senescence associated with functional impairment and endothelial activation induced by atherogenic stimulation [14]. As a carrier for S1P, apolipoprotein M (ApoM) is one of the apolipoprotein components of high-density lipoprotein (HDL), processing atheroprotective function by regulating cholesterol efflux [15,16]. The implication of ApoM and S1P in AS has been evidenced in rodent models [17]. Nevertheless, the impact of ApoM-S1P complex (ApoM-S1P) in AS is presently controversial [18]. Data in recent study demonstrated that pyroptosis may be associated with AS by affecting the stability of atherosclerotic lesion [5]. Considering the important role of ApoM-S1P in AS, it is possible that ApoM-S1P may regulate pyroptosis through certain pathway in AS, while no such studies verified our hypothesis. Here, we assessed the impact of ApoM-S1P on injury and inflammation of HUVECs and its possible mechanism. In this study, we discovered that ApoM-S1P could inhibit TNF-α-induced inflammation and pyroptosis of HUVECs via binding S1PR2 to activate the PI3K/AKT pathway.

Load ApoM with S1P
An eight-folds of S1P (Cayman, Ann Arbor, MI, USA) was added to ApoM (Abnova, Taiwan, China) and incubated for 30 min at room temperature to load ApoM with S1P. Unbound S1P was removed using a desalting PD10 column (GE Healthcare, Beijing, China) after the mixture was incubated with serum-free M200 medium. Then ApoM-S1P was diluted to 0.5 μM and stored at -20°C.

THP-1-HUVECs adhesion assays
Before adhesion assays, HUVECs were pretreated with 50 ng/mL of TNF-α for 6 h. Then HUVECs were treated with ApoM (1.0 μM), S1P (1.0 μM), or ApoM-S1P for 24 h [21] and grouped into TNF-α group, Control group, TNF-α + ApoM group, TNF-α + S1P group, and TNF-α + ApoM + S1P group. Antagonists for S1P receptors (S1PR1, S1PR2 and S1PR3) were added in TNF-α stimulated HUVECs for 30 min before cells were subjected to ApoM-S1P treatment. According to antagonist treatment, cells were grouped into TNF-α + ApoM + S1P + W146 group, TNF-α + ApoM + S1P + JTE-013 group, and TNFα + ApoM+S1P + CAY10444 group, respectively. THP-1 monocytes were labeled with 5 μmol/L calceinacetoxymethyl ester (Thermo Fisher Scientific, Waltham, MA, USA) and cultured in the incubator for 30 min. The labeled cells were washed twice with PBS and resuspended in serum-free medium. Then culture medium of HUVECs was removed. The resuspensions of labeled THP-1 monocytes were added onto monolayers of HUVECs. After 1 h incubation, the plates were rinsed twice with medium without serum. The adherent THP-1 monocytes on HUVECs were counted with a fluorescence microscope (Olympus IX71, Tokyo, Japan). The culture dish was divided into four quadrants according to its diameter. For each quadrant, one 200 × visual field in the middle of the quadrant was chosen for analysis. The average number of the four quadrants was identified as the number of adherent THP-1 monocytes.
Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA of HUVECs was extracted by RNeasyPlus Micro (Qiagen, dusseldorf, Germany). The ratio of OD 260 nm to OD 280 nm was measured to quantify and test the purity of RNA. Reverse Transcription of RNA into cDNA was conducted by Reverse Transcription System (Promega, Madison, WI, USA). The mRNA expression levels of E-selectin, ICAM-1, VCAM-1, IL-1β, IL-18, caspase-1, ASC, NLRP3, IL-6, and IL-10 were detected by qRT-PCR using SYBR Premix ExTaq (Takara Bio, Inc., Otsu, Japan). GAPDH was regarded as internal reference. All tests were conducted in triplicates. All data and figures were depicted as the mean ± standard deviation (SD), and data analysis was employed the 2 -ΔΔCt method [22]. The amplified primer sequences of each gene and its primers are shown in Table 1.

Enzyme-linked Immunosorbent assay (ELISA)
The supernatants from untreated and treated HUVECs were centrifuged (300 g, 5 min) and cell-free supernatants were frozen at -80°C until usage. The assays were conducted utilizing commercial ELISA kits (abcam, san francsico, USA) in duplicates, according to the manufacturers' instructions. The microplate absorbance reader (Tecan, Groening, Austria) was employed to measure the absorbance at 450 nm. Standard curves were applied to calculate the concentrations of analytes.

Immunofluorescence
HUVECs were seeded in 24-well plates at the density of 5 × 10 3 cells/well, followed by fixation in 4% paraformaldehyde for 15 min, permeation with 0.5% triton-X-100 for 20 min, and incubation with appropriate concentration of primary antibody of caspase-1 (1:500, ab1872, abcam, san francsico, USA) overnight at 4°C. After that, sections were incubated with secondary antibody of Alexa Fluor 488-labeled goat anti-

Detection of reactive oxygen species (ROS)
The ROS was measured using DCF-DA kit (BestBio, Shanghai, China) according to the manufacturer's instructions. Cells (1 × 10 7 /mL) were labeled with 10 μM of fluorescent probe DCFH-DA, followed by incubation in an incubator for 20 min at 37°C, and serumfree cell culture solution washing for three times. The fluorescence activity was analyzed by fluorescence microscopy with an excitation wavelength of 488 nm and an emission wavelength of 520 nm. The amount of intracellular ROS was proportional to the fluorescence intensity of DCF, and the fluorescence intensity indicated the ROS level.

Statistical analysis
All data were analyzed using SPSS 17.0 software (SPSS, Chicago, IL, USA) and GraphPad Prism 6.0. T test was used for comparison between two groups, and differences among groups were analyzed using One-way analysis of variance. P < 0.05 was regarded as a statistically significant difference.

TNF-α enhances the adhesion of HUVECs and induces pyroptosis
In the early stages of AS, endothelial cell function is impaired, and the expression of adhesion molecules is significantly increased, thereby recruiting pro-inflammatory immune cells to adhere to the vascular wall [23]. In this study, we discovered that the number of THP-1 monocytes adhering to the surface of HUVECs in the TNF-α group was significantly increased compared with the Control group (Fig. 1a), and up-regulate the mRNA and protein levels of adhesion molecules Eselectin, ICAM-1, VCAM-1 (P < 0.001) (Fig. 1b-c). ELISA illustrated that the contents of three adhesion molecules in TNF-α group were higher than those in the Control group (P < 0.05) (Fig. 1d). All these stated that TNF-α could enhance the adhesion function of HUVECs and induce the generation of adhesion molecules.
Pyroptosis is a more recently identified pathway of programmed cell death and accompanied with the severe inflammation response [24]. The detection on effect of TNF-α treatment on pyroptosis of HUVECs The effect of TNF-α on pyroptosis was determined by qRT-PCR (e), western blot (f) and ELISA (g) to detect the expression levels of IL-1β, IL-18, caspase-1, ASC, NLRP3, IL-6, and IL-10; The expression of caspase-1 was performed by immunofluorescence (h), and ROS level was measured by DCF-DA kit (I); Data were presented as mean ± SD, ** P < 0.01 *** P < 0.001 compared to the Control group; HUVECs, human umbilical vein endothelial cells; SD, standard deviation displayed that the mRNA levels of caspase-1, ASC, NLRP3, IL-1β and IL-18 in TNF-α group were higher than those in Control group, while antiinflammatory cytokine were lower than Control group (P < 0.01) (Fig. 1e). Consistent expression pattern was found on protein expressions of pyroptosis and inflammation related factors (P < 0.05) (Fig. 1f). ELISA results manifested that compared with Control group, the contents of IL-1β, IL-18, and proinflammatory cytokine IL-6 in TNF-α group were increased, while the content of anti-inflammatory cytokine IL-10 was decreased (P < 0.01) (Fig. 1g). Subsequently, immunofluorescence was utilized to inspect the expression of caspase-1 in HUVECs. The results manifested that TNF-α group had higher caspase-1 expression than the Control group (Fig. 1h). Meanwhile, DCF-DA kit detection results described that there was elevated ROS level in TNF-α group in comparison to the Control group (P < 0.001) (Fig. 1i). These indicated that TNF-α could enhance inflammation and promote pyroptosis of HUVECs.
ApoM-S1P plays a protective role through PI3K/AKT signaling pathway Previous evidence has demonstrated that PI3K/AKT is a downstream signaling pathway of S1P [26]. Therefore, we hypothesized that ApoM+S1P may exert its protective role in AS though PI3K/AKT signaling pathway. In this regards, we detected the effect of ApoM, S1P or/and SIPR2 antagonist JTE-013 on phosphorylation level of PI3K and AKT with the application of Western blot. The analysis revealed that TNF-α group had a lower phosphorylation level of PI3K and AKT than Control group. TNF-α + ApoM + S1P could partially restore the phosphorylation level of PI3K and AKT when compared with TNF-α group, while SIPR2 antagonist JTE-013 could inhibit the phosphorylation level of PI3K and AKT in comparison to ApoM-S1P treatment alone (P < 0.001). The phosphorylation level of PI3K and AKT in TNF-α + ApoM + S1P group was substantially higher than those in TNF-α + ApoM group and TNF-α + S1P group (P < 0.001) (Fig. 4). These indicated that ApoM-S1P could reverse the inhibitory effect of TNF-α on PI3K/AKT signaling pathway via mediating SIPR2.

Discussion
TNF-α is a critical inflammatory factor that has been displayed to cause the interaction between vascular endothelial cells and invading monocytes [20]. TNF-α also plays a crucial role in atherosclerotic inflammatory cascade through up-regulation expression of adhesion molecule, activation of endothelial cell, and remodeling of vascular [27]. In the current study, HUVECs was firstly stimulated by TNF-α and then subjected to ApoM, SIP, or ApoM-S1P treatment to observe the changes on expression levels of cell adhesion, inflammation, and pyroptosis related factors. Our study displayed that ApoM-S1P could markedly down-regulate the expression of the pro-inflammatory cytokine, adhesion molecules, and pyroptosis related proteins by binding to S1PR2 so as to active the PI3K/AKT signaling pathway, thereby inhibiting the inflammatory response and pyroptosis of TNF-α-induced HUVECs.
ApoM-S1P has recently been proved to possess unique anti-inflammatory effects [18]. In this study, we discovered that ApoM-S1P exerts a crucial prestige on inflammatory response and pyroptosis of TNF-α induced HUVECs. Previous results support the implication of ApoM-S1P in various diseases. For example, ApoM-S1P has a major influence on diet-induced obesity and postprandial triglyceride metabolism [28]. Furthermore, ApoM-S1P is a pivotal element of antiapoptotic activity of HDLs, which can promote the optimization of endothelial function [29]. Another research illustrated that ApoM-S1P is a therapeutic target for endothelial barrier dysfunction [30]. In addition, AS is reported to be associated with inflammatory processes in the endothelial cells of the vessel wall [31]. Consistent with results from previous study, we found ApoM-S1P could attenuate cell adhesion, inflammation and pyroptosis of HUVECs, therefore may also beneficial to attenuate AS progression. To further explore the mechanism herein, S1PR antagonist was added in TNF-α induced HUVECs before ApoM-S1P treatment. S1P regulates various biological activities by binding to different G-protein-coupled receptors known as S1PR1-S1PR5 [32]. Here, we observed that the S1PR2 antagonist reverse the protective effect of ApoM-S1P on pyroptosis and inflammation, presented by increased expressions of IL-1β, IL-18, IL-6 and IL-10. Therefore, we hypothesized that S1PR2 may have certain role to play in ApoM-S1P treatment to Fig. 3 ApoM-S1P inhibits TNF-α induced adhesion and pyroptosis through binding to S1PR2. Notes: To explore the downstream mechanism of ApoM-S1P on attenuation of pyroptosis and inflammatory response of HUVECs, S1PR2 antagonist was added in TNF-α stimulated HUVECs before cells were subjected to ApoM-S1P treatment. Fluorescence microscope was used to count the number of adhesion of THP-1 monocytes (200 ×) (a); The mRNA and protein levels of E-selectin, ICAM-1, and VCAM-1in HUVECs were detected by qRT-PCR (b) and Western blot (c); ELISA was utilized to test the contents of IL-1β, IL-18, IL-6, and IL-10 (d); DCF-DA kit for inspection of ROS level (e); Data are displayed as mean ± SD, ** P < 0.01 *** P < 0.001 compared to the TNF-α + ApoM + S1P + JTE-013 group; HUVECs, human umbilical vein endothelial cells; SD, standard deviation HUVECs. S1PR2 has potential utility in the drug targeted treatment of vascular inflammatory disorders [33]. Consistent with our hypothesis, data in previous study manifested that the pro-inflammatory impacts of S1P on HUVECs are largely mediated by S1PR2 [34]. The anti-inflammatory function of S1PR2 was also discovered in inflammatory bowel disease, as data showed that miR-126 could down-regulate S1PR2 expression and thus inhibit the activation of its downstream pathway PI3K/AKT signal pathway [35].
Although we found the implication of S1PR2 in pyroptosis of HUVECs, the possible downstream mechanism of S1PR2 in regulating pyroptosis of HUVECs remains to be clarified. PI3K/AKT is a downstream signaling pathway of S1P receptors, which is essential for vascular inflammation [21]. PI3K/AKT pathway-related proteins are the most frequently altered in human cancers, which associated with tumorigenesis, cellular transformation, drug resistance, and cancer progression [36]. Previous research has discovered that the alteration of PI3K/AKT pathway plays very important prognostic and predictive roles in colorectal cancer [37]. What's more, growing evidences found that S1PR2 have a great impact on PI3K/ AKT pathway. For instance, S1PR2 mediates endothelial cells dysfunction through PI3K/AKT pathway under high glucose condition [38]. The regulation of S1P/S1PR on AS progression is complicated and the protective role of ApoM-S1P on pyroptosis of HUVECs may have certain association with cellular signal pathway. S1PR2 has been found to associate with the G proteins Gα12/13, Gαq, or Gαi, while the coupling of S1PR2 with G proteins could further trigger the activation of PI3K/AKT pathway [39]. Although this study found the regulatory role of S1PR2 on p-PI3K and p-AKT levels, considering the complication of pathological courses of HUVEC injury and AS, we can only speculate a possible hypothesis and more studies are required to explore the possible mechanism herein. Due to time and budget limitations, we failed to set up an in vivo model to verify our speculation. But this indicates a promising future direction for our future studies.

Conclusion
Collectively, in this study, the results concluded from above findings indicated that ApoM-S1P inhibits TNFα-induced inflammation response in HUVECs through binding to S1PR2 to activate PI3K/AKT pathway. In this regards, we supported the beneficial role of ApoM-S1P in attenuating HUVECs injury. Nonetheless, these results must be interpreted with caution, and more studies are required to validate the results of current study.

Acknowledgements
Thanks for all the supports and contributions of participators.
Authors' contributions YL had full access to all of the data in the study and takes responsibility for the integrity of the data, the accuracy of the data analysis and wrote the manuscript draft. YL performed research. YL and LT contributed substantially to the study design and the writing of the manuscript. All authors read and approved the final version of the manuscript.

Funding
This work was supported by the Natural Science Foundation of Hunan Province (14JJ7006), the Science and Technology Innovation Planning Project of Hunan Province (2017SK50104).These funding bodies didn't play the role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Availability of data and materials
The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate Not applicable.