Materials and methods
Cell culture
The mouse aortic endothelial cell (MAEC) line was obtained from BEINA (Beijing, China) and cultured in Dulbecco’s Modified Eagle Medium (DMEM) with normal glucose (NG; D-glucose concentration, 5.6 mM) and containing 10% fetal bovine serum, added 1% penicillin and 1% streptomycin. MAECs were cultured at 37℃ in a humidity-controlled incubator with 5% CO2. Before the experiment began, the cells were typically divided into two groups. The experimental group was cultured with DMEM having high glucose (HG; D-glucose concentration, 25 mM). The control group was cultured with DMEM having NG (D-glucose concentration, 19.5 mM) plus α-mannitol (5.6 mM) to balance the osmotic pressure so that it was consistent with the experimental group. The specific conditions for each experiment are described in the figure legends.
Calcium imaging
MAECs were loaded with Fura-8-AM in DMEM with NG or HG and 0.02% Pluronic F127 (Invitrogen) for 30 min at room temperature in the dark. Excess Fura-8-AM was washed out by superfusion with a Ca2+-free solution containing (in mM) 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 25 NaHCO3 and 11.1 glucose at pH 7.4. Thereafter, MAECs were placed on the stage of an inverted fluorescence microscope (Nikon T200; Tokyo, Japan) and perfused with a Ca2+-free solution. Fura-8-AM was alternatingly excited at a wavelength of 488 nm using a Xenon lamp. Fluorescence emission was acquired at a wavelength of 510 nm. For each excitation wavelength, background fluorescence was subtracted, and the ratio of the emitted fluorescence (F1/F0) was calculated as a measure of the cytosolic Ca2+ concentration. For SOCE measurements, intracellular Ca2+ stores were emptied by rapid application of an inhibitor of Ca2+ reuptake, thapsigargin (TG; 2 µM) or by incubation with ATP (100 µM) for 10 min in a Ca2+-free solution. Thereafter, SOCE was measured by the application of 2 mM extracellular Ca2+ as previously described.11
Co-immunoprecipitation assay
After MAECs were lysed on ice with protein lysis buffer (1% Nonidet P-40, 150 mM NaCl, 20 mM Tris-HCl, pH 8.0, with the addition of a protease inhibitor cocktail), they were centrifuged at 12 000× g and 4°C for 20 min. Orai1–3 or VE-cadherin proteins were immunoprecipitated by incubating 800 μg of extracted protein with 5 µg of anti-Orai1–3 antibodies (ProteinTech Group, Chicago, Illinois, USA) or anti-VE-cadherin antibody (Affinity Biosciences, Ohio, USA), respectively, on a rocking platform overnight at 4℃. Protein A agarose was then added and incubated for an additional 3 hours at 4℃. The immunoprecipitates were washed with cell lysis solution, and cell lysates (100 µL) and loading buffer (25 µL) were added. Next, the sample was boiled at 100°C for 10 min. The resulting obtained supernatant was used for protein electrophoresis. For the immunoblots, all of the samples were fractionated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis using 15% agarose gels, transferred to polyvinylidene fluoride membranes, and probed with the indicated primary antibodies at a dilution of 1:200 in a phosphate-buffered saline (PBS; in mM: 137 NaCl, 2.7 KCl, 10 Na2HPO4, 1.8 KH2PO4, pH 7.4) containing 0.1% Tween-20 and 5% non-fat dry milk. Immunodetection was accomplished using horseradish peroxidase (HRP)-conjugated secondary antibody (1:5000, Affinity Biosciences) followed by an enhanced chemiluminescence plus detection system (Peiqing, Shanghai, China).
Extraction of membrane proteins and western blot analysis
Membrane proteins, specifically VE-cadherin and p-VE-cadherin, were isolated using a Membrane and Cytosol Protein Extraction Kit (Beyotime Biotechnology, China) following the manufacturer’s instructions. Cell lysates from MAECs were extracted with lysis buffer (in mM: 20 Tris, 150 NaCl, 1 EDTA, 1 EGTA, 1% Triton X-100, 0.1% SDS and 1% protease inhibitor cocktail; pH 7.4), and the protein concentration was determined using a BCA assay (Rayto, Shandong, China). For the immunoblots, the polyvinylidene difluoride membranes carrying the transferred proteins were incubated at 4℃ overnight with anti-Orai1–3, anti-VE-cadherin and anti-p-VE-cadherin Y731 antibodies (Affinity Biosciences) (all diluted 1:250) or with β-tubulin as a control (1:50 000, Affinity Biosciences). Immunodetection was conducted using HRP-conjugated antirabbit secondary antibodies (1:5000). Densitometry was performed using ImageJ analysis software, and intensities were normalized to those of β-tubulin.
Endothelial permeability assay
VE permeability was assessed using a transwell assay conducted as previously reported by Huang et al.12 In brief, MAECs were seeded on polyester membranes (pore size, 0.4 µm; Millipore, USA) in transwell inserts, placed in 24-well plates, and incubated for 24 hours to allow the growth of a confluent monolayer. The medium on the apical side of the transwell chamber was replaced with 200 μL of fluorescein isothiocyanate-dextran, 20 kDa (FD-20; Sigma, USA) solution (25 mg/mL in DMEM NG or HG medium). The transwell inserts were then moved into new wells with 500 µL of control medium containing the SOCE agonist ATP (100 μM) or the SOCE inhibitor BTP2 (10 μM). Thirty minutes after incubation, 30 μL of medium was aspirated from the apical or basolateral compartments to determine the FD-20 concentration. Samples were diluted with 270 μL PBS, and fluorescence was measured using a fluorescence microplate reader (Flexstation 3, Molecular Devices, USA) with an excitation/emission wavelength of 492/515 nm, a slit width of 1.5 nm for excitation and a slit width of 10 nm for emission. The permeability of the confluent monolayer is expressed as the fluorescence intensity of FD-20.
Transendothelial electrical resistance in vitro
The permeability of the MAECs was determined using confluent monolayers after the cells were cultured in NG or HG medium for 7 days. Measurements of transendothelial electrical resistance (TER) were performed using a Millicell-ERS (Electrical Resistance System) voltmeter (Millipore). The MAEC monolayers were treated with ATP (100 μM) or BTP2 (10 μM). The TER of the MAEC monolayers was calculated using the following equation: TER (Ω cm2 monolayer)=(average resistance of well–average resistance of blank well in Ω)×monolayer area (in cm2).13
Mouse model of type 2 diabetes
Sixteen male C57BL/6J mice (18–20 g, 4 weeks of age) were obtained from the Experimental Animal Center of Anhui Medical University and used in compliance with the US National Institutes of Health guideline (publication no. 8523). The mice were randomly placed in one of two groups: a normal control group (n=8) and a mouse model of type 2 diabetes group (n=8). Mice in the experimental group received a single intraperitoneal injection of streptozotocin (STZ, 40 mg/kg, dissolved in pH 4.5 citrate buffer; Biosharp). Control mice received an equivalent volume of citrate buffer. After the STZ injection, both groups were fed a standard mouse chow diet and were housed in a standard temperature-controlled (25±2℃) environment. Mice were considered a successful model of diabetes when their fasting blood glucose levels reached 16.7 mM, approximately 1 week after the STZ injection. Immunohistochemical assays were performed 8 weeks after the STZ injection.
Immunofluorescence analysis
MAECs (4×104 cells) were seeded on cover glass. After treatment, the cells were washed three times with PBS and fixed using 4% paraformaldehyde (Ebiogo, Hefei, China). Cell membranes were permeabilized with 0.2% Triton X-100 (Beyotime Biotechnology, China) for 15 min and blocked with 3% bovine serum albumin (Beyotime Biotechnology) for 2 hours at room temperature. Primary rabbit anti-Orai1–3, anti-VE-cadherin or anti-p-VE-cadherin Y731 antibodies (diluted 1:200) were incubated with the cells overnight. Then Orai1–3, VE-cadherin and p-VE-cadherin Y731 were labeled using FITC-conjugated secondary antibodies (diluted 1:100; Invitrogen) via incubation for 2 hours. The nucleus of each cell was labeled with 4',6-diamidino-2-phenylindole (DAPI). The immunofluorescence signals were detected and images were captured using an LSM 880 confocal microscopy system (Zeiss, Germany).
Immunohistochemistry assay
Mouse thoracic aortas were perfused with PBS and fixed for 24 hours with 4% paraformaldehyde and embedded in paraffin. Paraffin sections of aortic ring segments were sliced to a thickness of 5 µm, dewaxed and washed with PBS three times. The sections were incubated with 3% hydrogen peroxide solution at 37℃ for 30 min, a citrate antigen retrieval solution for 20 min and 5% bovine serum albumin in PBS for 1 hour at room temperature. The sections were then incubated with anti-Orai1–3, anti-VE-cadherin or anti-p-VE-cadherin Y731 antibodies (diluted 1:250) overnight at 4℃. After undergoing three washes with PBS, the sections were incubated with a HRP-labeled secondary antibody. Sufficient DAPI to cover the section was added to label cell nuclei. The rinsed sections were counterstained with hematoxylin for 15 s and then mounted onto glass slides. Histological images were obtained using a digital camera mounted on an Olympus BX51 microscope.
Statistical analysis
Summary data are reported as means±SEM. Statistical analyses were performed using GraphPad Prism V.5.0. Experimental results were compared using t-tests to correct for comparison. Two-sided p values <0.05 were considered statistically significant.