Preparation of AGE and sulfur compounds in AGE
AGE was prepared according to the previous report [38]. Briefly, raw garlic (Allium sativum L.) was sliced into pieces, which was followed by immersing in ethanol 20–50% (v/v) and aging for more than 10 months at room temperature. S1PC, SAC and SAMC were synthesized according to the previous methods [41,42], and the purity was >99.0%.
Antibodies and reagents
The following antibodies were used in this study: anti-TNFR1, anti-phospho-GEF-H1 (Ser886), anti-GEF-H1, anti-RhoA, anti-phospho-Cofilin (Ser3), anti-Cofilin, anti-phospho-MYPT1 (Thr696), anti-MYPT1, anti-phospho-MLC2 (Ser19), anti-MLC2, horseradish peroxidase (HRP)-conjugated rabbit IgG, HRP-conjugated mouse IgG and HRP-conjugated rat IgG from Cell Signaling Technology (Danvers, MA, USA); anti-VE-cadherin, anti-ZO-1 and Donkey anti-rabbit IgG conjugated to Alexa Fluor 594 from Thermo Fisher Scientific (Waltham, MA, USA); anti-Rac1/2/3, anti-phospho-ETS domain-containing transcription factor 1 (Elk1) (Ser383) and anti-Elk1 from Santa cruz Biotechnology (Dallas, TX, USA); anti-TNFR2 and anti-TNFR-associated factor 2 (TRAF2) from Funakoshi (Tokyo, Japan); anti-phospho-ERK1/2 (Thr202/Tyr204) and anti-ERK1/2 from BioLegend (San Diego, CA, USA); anti-GEF-H1 and anti-β-actin peroxidase conjugate from MBL life science (Nagoya, Japan); anti-Claudin-5 from Bioworld Technology (Nanjing, China); anti-MLCK from Abcam (Cambridge, UK); anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) peroxidase conjugate from WAKO pure chemical industries (Osaka, Japan); IgG from rabbit serum from Merck Millipore (Billerica, MA, USA); acti-stain 488 phalloidin from Cytoskeleton (Denver, CO, USA). Human recombinant TNF-α and human plasma-derived thrombin were purchased from WAKO pure chemical industries and Sigma Aldrich (St. Louis, MO, USA), respectively. Y-27632 and PD98059 were obtained from Merck Millipore and Thermo Fisher Scientific, respectively.
Cell culture
Human umbilical vein endothelial cells (HUVECs) were purchased from Lonza (Basel, Switzerland) and grown to be confluent in endothelial cell basal medium-2 (Lonza) containing 2% fetal bovine serum albumin (BSA) and supplements attached to the medium kit in the presence of 5% CO2 at 37°C.
In vitro endothelial permeability assay
Endothelial permeability was measured by using In vitro vascular permeability assay kit (Merck Millipore). HUVECs were seeded at 1.5 × 103 cells/well on transwell inserts and cultured in a receiver plate. After grown to be confluent, HUVECs were stimulated with 50 ng/mL TNF-α or 1 U/mL thrombin in the absence or presence of test substances (1-4 mg/mL AGE, 75-300 μM S1PC, 300 μM SAC or 300 μM SAMC) for 24 hours. In the case of the study using the inhibitors of RhoA and Rac signaling molecules, HUVECs were pre-treated with Y-27632 (a ROCK inhibitor, 10 μM) or PD98059 (a MEK1 inhibitor, 30 μM) for 1 hour before the TNF-α stimulation. Then, FITC-dextran was added on each transwell insert and the extent of permeability was determined by measuring the fluorescence of each receiver plate solution. The fluorescence intensity was quantified with EnVision plate reader (PerkinElmer, Waltham, MA, USA) at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.
Western blot analysis
After seeding on 24-well plates, HUVECs were stimulated with TNF-α 50 ng/mL in the absence or presence of test substances (75-300 μM S1PC, 300 μM SAC or 300 μM SAMC) for 10, 15, 20, 30, 40 minutes, 1, 3 or 24 hours. In the case of the study using the inhibitors of RhoA and Rac signaling molecules, HUVECs were pre-treated with 10 μM Y-27632 or 30 μM PD98059 for 1 hour before the TNF-α stimulation. Then, whole protein was extracted using Radioimmunoprecipitation assay (RIPA) buffer (WAKO pure chemical industries) containing protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific). After dilution with SDS sample buffer (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 0.005% bromophenol blue, 175 mM dithiothreitol), 10 μg of total protein was separated by SDS-PAGE and transferred onto nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA). The transferred proteins were incubated with antibodies and immunoreactive proteins were detected with ECL prime western blotting detection system (GE Healthcare, Little Chalfont, Buckinghamshire, UK) or Supersignal west femto maximum sensitivity substrate (Thermo Fisher Scientific). Chemiluminescent signals were visualized with ChemiDoc MP imaging system (Bio-Rad Laboratories) and quantified using ImageLab software (Bio-Rad Laboratories).
Extraction of membrane and cytoplasmic proteins
HUVECs were seeded on 12-well plates and stimulated with TNF-α 50 ng/mL in the absence or presence of 300 μM S1PC for 30 minutes or 24 hours. Then, cell membrane and cytoplasmic proteins were separately isolated by using Mem-PERTM plus membrane protein extraction kit (Thermo Fisher Scientific) according to the manufacturer’s protocol. The isolated membrane and cytoplasmic proteins were analyzed by western blotting. The total proteins were stained by using Quick-coomassie brilliant blue staining kit (WAKO pure chemical industries) according to the manufacturer’s instructions in order to normalize the membrane and cytoplasmic protein levels.
Immunoprecipitation
Immunoprecipitation was performed to investigate the interaction between GEF-H1 and Rho family proteins. HUVECs were seeded on 10 cm dishes and stimulated with TNF-α 50 ng/mL in the absence or presence of 300 μM S1PC for 1 hour. Then, whole proteins were isolated using RIPA buffer containing protease and phosphatase inhibitor cocktail. Cell lysates were immunoprecipitated using GEF-H1 antibody and protein G magnetic beads (Merck Millipore). The magnetic beads were washed 5 times by RIPA buffer and boiled in SDS sample buffer at 95°C for 5 minutes. Finally, the released proteins from beads were analyzed by western blotting.
Immunofluorescent staining
To investigate the effect of S1PC on cellular localization of the junctional proteins and F-actin, these proteins were visualized by immunofluorescent staining. HUVECs were seeded on 8-well slide chambers (WATSON, Tokyo, Japan) and stimulated with TNF-α 50 ng/mL in the absence or presence of 300 μM S1PC for 24 hours. Then, the cells were fixed in 4% paraformaldehyde (WAKO pure chemical industries), permeabilizated with 0.3% TritonX-100 (Sigma Aldrich) in phosphate buffered saline (PBS, NISSUI, Tokyo, Japan) and blocked with 3% BSA (Sigma Aldrich) in PBS followed by the incubation with primary antibodies in 3% BSA/PBS. Then, the cells were treated with an Alexa Fluor 594-conjugated secondary antibody to detect junctional proteins. F-actin was labeled by the incubation with acti-stain 488 phalloidin. Finally, the cells were mounted using Vectashield medium-containing 4’, 6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Berlingame, CA, USA). The fluorescent staining was visualized with Biorevo Bz9000 fluorescence microscope (Keyence, Osaka, Japan) and analyzed using Bz-II analyzer software (Keyence).
RhoA and Rac activation assay
The effects of S1PC on the activities of Rho family proteins were evaluated by using RhoA and Rac activation assay kits (Merck Millipore). HUVECs were seeded on 10 cm dishes and stimulated with TNF-α 50 ng/mL in the absence or presence of 300 μM S1PC for 1 hour. After being detached using Mg2+ lysis/wash buffer (MLB) containing protease and phosphatase inhibitor cocktail, the cells were incubated with glutathione-agarose beads (Sigma Aldrich) for 30 minutes at 4°C. Then, the supernatant collected from the incubation medium was added with Rho or Rac assay reagent containing glutathione-agarose beads bound to fusion proteins that include Rho or Rac binding domain of each effector protein, and then gently agitated. After centrifugation, the precipitated bead pellets were washed 3 times using MLB and boiled in SDS sample buffer. Finally, the supernatants were analyzed by western blotting.
Quantitative real-time PCR
HUVECs were seeded on 48-well plates and stimulated with TNF-α 50 ng/mL in the absence or presence of 300 μM S1PC for 9 hours. Then, total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific) and used for the synthesis of the first-strand cDNA by using Primescript RT reagent kit with gDNA Eraser (Takara, Shiga, Japan). The quantitative real-time PCR was performed with Piko Real Real-time PCR system (Life technologies, Carlsbad, CA, USA) using SYBR premix Ex Taq II (Takara). The primer sequences used for PCR were as follows: human MLCK forward, 5ʹ-AGCCCGCTCAATGCAGAAAA-3ʹ; human MLCK reverse, 5ʹ-AGCAGCACTTCCCTCCACAA-3ʹ; human β-Actin forward, 5ʹ-CGCGAGAAGATGACCCAGAT-3ʹ; human β-Actin reverse, 5ʹ-GGTGAGGATCTTCATGAGGTAGTC-3ʹ. The relative mRNA expression level of MLCK to β-Actin was calculated using the comparative CT (ΔΔCT) method.
Knockdown of GEF-H1 gene with small interfering RNA (siRNA)
To examine whether GEF-H1 is a key molecule of TNF-α-activated RhoA and Rac signaling pathways, we prepared GEF-H1 knockdown HUVECs with siRNA. HUVECs seeded on 12-well plates were transfected with 9.6 pmol control siRNA (Silencer select negative control No. 1 siRNA, Thermo Fisher Scientific) or GEF-H1-targeting siRNA (Silencer select pre-designed hARFGEF2 siRNA, ID: s17546, Thermo Fisher Scientific) using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific) according to the manufacturer’s instructions. At 48 hours after the transfection, the cells were stimulated with TNF-α 50 ng/mL for 15, 30 minutes, 1 or 24 hours, and the levels of protein expressions were evaluated by western blotting.
Cell proliferation assay
Cell proliferation assay was performed to investigate the effect of S1PC on the proliferation of HUVECs by using Cell counting kit-8 (Dojindo Molecular Technologies, Kumamoto, Japan). Briefly, HUVECs seeded on 96-well plates were treated with 75-300 μM S1PC for 48 or 72 hours, and each well of the plates was added with CCK-8 regent and incubated for 1 hour. The colorimetric absorbance of the wells was measured at 450 nm with Multiskan FC microplate reader (Thermo Fisher Scientific).
Statistical analysis
Data were expressed as means ± standard deviation (SD) after outlier data were detected and removed using Thompson's rejection test. Bonferroni’s or Dunnett’s multiple comparison test was performed to assess statistically significant differences between groups by using WinSTAT statistics software (M. Sato, Japan) or KyPlot software (KyensLab, Tokyo, Japan), respectively. Differences at P-values less than 0.05 were considered to be statistically significant.