Healthy control subjects and patients with type 2 diabetes were recruited following informed consent. Human umbilical vein endothelial cells (HUVECs) were obtained by collagenase digestion of umbilical cords which were donated by the volunteers after written informed consent. The study was approved and supervised by the Institutional Review Board and Ethics Committee of Peking University First Hospital (Beijing, China).
S1P, C17-D-erythro-sphingosine-1-phosphate (C17-sph) and the antagonist of S1PR1 and S1PR3 were purchased from Avanti polar lipids (Alabama, USA). The antibodies for western blot against phospho-extracellular regulated kinase (ERK) 1/2 and cAMP-response element binding protein (CREB) were purchased from Cell Signal Technology, Danvers, MA. The antibodies of phospho-p38 mitogen-activated protein kinase (MAPK), p38 MAPK and β-actin were purchased from Santa Cruz Biotechnology, Santa Cruz, CA. Antibody against COX-2 and competitive enzyme immunoassay kit for 6-keto PGF1α were from Cayman Chemical (Michigan, IL). Horseradish peroxidase (HRP)-goat-anti-rabbit IgG and HRP-goat-anti-mouse IgG were purchased from MBL (Nagoya, Japan). Endothelial cell medium (ECM) was purchased from ScienCell Research Laboratories (Carlsbad, CA).
Healthy volunteers and patients underwent physical examination, laboratory tests and ultra-sound examination of the large arteries. The patients were all first diagnosed with T2DM in accordance with international standards; fasting plasma glucose (FPG) ≥ 7.0 mmol/L and glycated hemoglobin (HbA1c) greater than 6.5%. The patients were all first diagnosed in the hospital before initiation of treatment. The healthy subjects had no family history of diabetes, and they had normal FPG level and normal glucose tolerance. Allowing the criteria above, 15 T2DM and 15 healthy patients were recruited. The detailed information of the recruited patients and healthy volunteers has been described in a previous study .
Human umbilical vein endothelial cells (HUVECs) were obtained by collagenase treatment of umbilical cord vein as described previously . Cells were cultured on gelatin-coated dishes and propagated in endothelial cell medium supplemented with 5% FBS and endothelial cells were grown at 37°C in an incubator with humidified air containing 5% CO2. HUVECs were harvested when cells reached 70–80% confluent and passages three to fifty were used.
Blood collection and HDL isolation
The fresh blood from T2DM and healthy volunteers was drawn after overnight fasting into EDTA-Na2 vacuum tubes. Plasma was separated by centrifuging at 2,500 rpm at 4°C for 15 minutes. HDL (1.063-1.210 g/ml) was isolated by ultracentrifugation as described previously . HDL2 (1.063 < d < 1.125 g/ml) and HDL3 (1.125 < d < 1.21 g/ml) were isolated by sequential ultracentrifugation as previously described . HDL sub fractions were dialyzed against saline/EDTA (150 mM NaCl, 300 μM EDTA, pH 7.4), sterilized by filtering through a 0.22 μm membrane, and stored at 4°C until needed. The HDL was used within a month after isolation. The purity of the HDL was confirmed by SDS-PAGE and western blot using goat anti-apoA-I polyclonal antibody (DiaSorin, Stillwater, OK) and quantified through the measurement of apoA-I content by nephelometry (Dimension XPand, Dade Behring, Germany). Equal concentration of apoA-1 from isolated HDL was used for cell treatments and S1P level determination.
Modification, selected delipidation and reconstitution of HDL
HDL was incubated in glucose/phosphate-buffer saline (PBS) buffer with butylated hydroxytoluene (BHT) (the final concentration of glucose is 25 mmol/L) at 37°C for 7 days in vitro . During this procedure, the oxidation levels of modified HDL were measured (Additional file 1: Figure S1). After HDL was dialyzed with PBS, glycated HDL was used for cell experiments and reconstitution. Selective delipidation was achieved as described previously. HDL was agitated with di isopropyl ether in a ratio of 1:2 (vol/vol) at 4°C for 24 h . After extraction, the mixture was centrifuged at 2,000 rpm for 5 minutes to separate the aqueous and organic phase. Apo HDL&PL to (HDL containing the protein components and the phospholipids) was collected in the aqueous phase and PL-depleted HDL-lipids (the lipid component of HDL except for the phospholipids) in the organic phase. Alternatively, HDL was agitated with a mixture of butanol and di isopropyl ether (vol/vol, 40:60) in a ratio of 1:2 (vol/vol) for 30 minutes at the room temperature. After centrifugation we collected apoHDL (HDL containing the protein components only) in aqueous phase and HDL-lipids (all lipid component of HDL) in organic phase. ApoHDL&PL and apoHDL were filtered before use. Reconstituted HDL (rHDL) was made by adding S1P to glycated HDL at the desired concentration and then mixed by rotation overnight at 4°C for further usage . The levels of S1P used for reconstituting were nearly equal to the S1P we detected on reconstituted HDL (Additonal file 1: Figure S2).
S1P extraction and detection by UPLC-MS/MS
UPLC-MS/MS technique was employed to measure the levels of HDL-associated S1P as previously described. Serum samples mixed with internal standard C17-sph (50 μl of 1000 μg/L) was precipitated by methanol at the volume ratio of 1:4. After centrifugation at 12,000 rpm for 15 minutes, the supernatant was collected for UPLC-MS/MS analysis performed by a Waters ACQUITY UPLCTM system as described previously . Waters ACUITY UPLC BEH Phenyl column (1.8 μm; 2.1 mm × 100 mm) was selected for chromatographic separation. The injection volume was 5 μL. Methanol (A) and 0.5% formic acid in ultrapure water (B) were used as mobile phases. The gradient started at 10% A and then increased linearly to 60% in 6 minutes, and then to 100% at 6 minutes and kept for 2 minutes, followed by a decrease to initial conditions of 10% A and held for 2 minutes to allow for equilibration. The flow rate was 0.3 mL/min. The column was maintained at 40°C, and the sample room temperature was 10°C.
Real-time PCR assay for SPL and SGPP1/2
Real-time PCR was performed to determine mRNA levels of SPL and SGPP1/2 from peripheral blood of the patients and healthy volunteers. Total RNA was extracted using the TRIzol reagent (Invitrogen, USA) and reverse transcription was performed using an RT-PCR kit (TransGen Biotech, China). Real-time experiments were conducted on a DNA Engine Opticon System (MJ research Inc, USA) using SYBR Green PCR Master Mix kit in triplicate specific primers. The sequences of primers to determine the expression of the target gene were as follows: SPL [5’-CTTGATGCACTTCGGTGAGA-3’ (forward); 5’-TCCACCCCTTAGCAGTCATC-3’ (reverse)], SGPP1 [5’-ACCGCCATCCCCATTTCT-3’ (forward); 5’-AGGAATCCAGCAATAATATCCAG-3’ (reverse)], SGPP2 [5’-gTATTATACTCATGGTTCAAGGTG-3’ (forward); 5’-GTGTAGGTAACAAACTTGTAAGG-3’ (reverse)] and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [5’-CGGAGTCAACGGATTTGGTCGTAT-3’ (forward); 5’-AGCCTTCTCCATGGTGGTGAAGAC-3’ (reverse)]. The PCRs consisted of 5 min at 95°C followed by 40 cycles of denaturation for 30 sec at 95°C, annealing for 30 sec at 56°C and a primer extension for 30 sec at 72°C. The comparative CT method was used to quantify the expression of SPL, SGPP1 and SGPP2 using GAPDH as the normalized control.
ELISA: quantitation of 6-keto PGF1α
PGI-2 is non-enzymatically hydrated to 6-keto PGF1α and estimation of systemic PGI-2 production has often been assessed by measurement of 6-keto PGF1α . The cell culture supernatants were collected after centrifugation at 3000 rpm for 15 minutes. For accurate measurement of PGI-2 production, 6-keto PGF1α in cell culture supernatants were determined using enzyme immunoassay kit (Cayman Chemical). The final results were normalized to cell protein concentration.
Western blotting and EMSA
Western blot analysis was performed as described previously , cells were washed twice with cold 1X PBS and lysed on ice for 30 min in lysis buffer. The lysates were subjected to centrifugation at 12,000 rpm for 15 min at 4°C and the supernatant was utilized for analysis. Protein concentrations were determined using BCA method. The boiled samples were loaded on Ready SDS-10% PAGE gels and used for Western blot analysis with the protein- specific antibody. HRP- labeled secondary anti-body was used for detection of signal by electrochemiluminescence from Pierce (California, USA).
DNA-protein binding reactions were performed by incubating nuclear extracts with specific CREB-CIE (cis-inducible element) DNA binding. Polyclonal anti-CREB antibody was added to the reaction mixture containing the labeled probe. EMSA was performed as previously described .
For EMSA assay, 10 μg nuclear protein was used with 15 fmol of HRP-end-labeled double stranded oligonucleotides in mixed nuclear extraction from HUVEC after treated with HDL as described above. The sequence of oligonucleotide containing CREB was: 5’-AGA GAT TGC CTG ACG TCA GAC AGC TAG-3. Oligonucleotides were end-labeled with HRP polynucleotide kinase and purified on G-50 columns (Roche Diagnostics). The DNA binding reaction of HRP labeled double-stranded oligonucleotides was performed at room temperature for 20 min, according to the manufacturer's protocol. Electrophoretic mobility shift assays were performed with the Light Shift Chemiluminescent Kit from Pierce, according to the manufacturer's recommendations.
All experiments were repeated in triplicate if not mentioned. Data are presented as mean ± SEM unless indicated otherwise. Differences were compared with two-tailed Student’s t-test or one-way ANOVA using GraphPad Prism software. p < 0.05 was considered statistically significant.