Cell culture and disturbed flow treatment
Human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection (Manassas, VA), and were grown under culturing condition with Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (FBS), 50 U/mL penicillin and 50 μg/mL streptomycin (Invitrogen, Carlsbad, CA), as specified by the manufacturer. To initiate disturbed flow treatment, confluent HUVECs, seeded onto collagen I-coated glass slides, were assembled into flow chambers and connected to the flow system for the shear experiments. HUVECs were exposed to steady laminar flow shear stress (12 dyn/cm2), disturbed flow shear stress (0.5 ± 4 dyn/cm2) for 24 hours.
Small interfering RNA (siRNA) transfection
SiRNAs were used to silence the TXNIP expression. The TXNIP-siRNA duplex was synthesized by Shanghai GenePharma Co., Ltd. (sense: 5’CUCCCUGCUAUAUGGAUGUTT-3’; anti-sense: 5’-ACAUCCAUAUAGCAG
GGAGTT-3’). The cells, treated with either the transfection reagents (vehicle) or non-targeting siRNA (sense: 5’- UUCUCCGAACGUGUCACGUTT-3’; anti-sense: 5’-ACGUGACACGUUC GGAGGAGAATT-3’), served as controls. The cells were transfected with 200 nM siRNA using the X-treme siRNA Transfection Reagent (Roche Applied Science, Penzberg, Germany), following the manufacturer’s instructions. Three experimental groups were conducted: the treatment group of laminar flow with negative control siRNA (NF+NC-siRNA), disturbed flow with negative control siRNA (DF+NC-siRNA) and disturbed flow with TXNIP-siRNA (DF+TXNIP-siRNA)
Western Blot analysis
Protein samples with equal amount of total protein were separated on SDS-PAGE (8-15%). The separated protein gel was then transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk at room temperature for 1 hour in Tris-buffered saline containing 0.1% Tween-20, primary antibody incubation (TXNIP, Cat#: 14715; Thioredoxin 1, Cat#: 2285; GLUT4, Cat#: 2213; pyruvate dehydrogenase E1-alpha [PDH E1α], Cat#: 31866; p-eNOS, Cat#: 9571; Total-eNOS, Cat#: 5880; NLRP3, Cat#: 13158; VCAM-1, Cat#: 13662; ICAM-1, Cat#: 4915; Cleaved-IL-1β, Cat#: 83186 and GAPDH, Cat#: 5174 were all from Cell Signaling Technology; Anti-Nitro tyrosine antibody [Cat#: ab42789, Abcam Company]) was carried out overnight at 4°C. Afterwards, secondary antibody incubation with a peroxidase-conjugated AffiniPure goat anti-rabbit or anti-mouse IgG was conducted for 90 min at room temperature. After washing 3 times, the membranes were subjected to ECL detection. Densitometric analysis was performed using the Tanon Gel Imaging System (Shanghai Tanon, Shanghai, China). The housekeeping gene GAPDH served as a loading control.
Tube Formation Assay
Two hundred µl of Biocoat Matrigel (Becton Dickinson) was added into each well in the 24-well plate and incubated at 37°C for 30 min to solidify. The same batch of Matrigel was used for all the experiments. After flow velocity treatment, cells were suspended in culture medium and plated on the Matrigel-coated plate. Gels were examined using a phase-contrast microscope equipped with a digital camera after plating. Capillary-like structures were assessed and quantified by calculating the number of junctions per field. At least 5 different viewing fields per well were analysed.
Measurement of NO production
The generation of intracellular nitric oxide (NO) was monitored using the 4-amino-5-methylamino-2,7’-difluorofluorescein (DAF-FM DA) reagent (Beyotime Institute of Biotechnology). HUVECs were incubated with DAF-FM DA solution at 37 °C for 30 min. After washing cells three times with PBS, fluorescent intensity was determined at an excitation wavelength of 488 nm and an emission wavelength of 525 nm via a fluorescent microplate reader (SpectraMax M2, Molecular Devices Corp., USA)
Mitochondrial isolation and measurement of ATP levels
For mitochondrial isolation, HUVECs were manually homogenized using a medium-fitting glass Teflon Potter-Elvehjem homogenizer in isolation buffer (mitochondrial isolation buffer: 250 mM sucrose, 0.5 mM EDTA, 10 mM Tris, and 0.1% BSA at pH 7.4). The homogenate was then clarified through centrifuging two times at 1000x g for 5 min, followed by centrifugation twice more at 11000x g for 10 min. The resulting supernatant and mitochondrial pellets were collected and diluted with mitochondrial isolation buffer three times of the original volume.
Mitochondrial ATP was measured by the mitochondrial ToxGlo™ assay according to the manufacturer’s protocol. Briefly, isolated HUVECs mitochondria were plated at 1 mg/well in both white and clear bottomed 96-well culture plates. The assay solution (100 µL/well) was then added, and the plate was incubated at room temperature for 30 min. Luminescence was measured using a luminometer (Molecular Devices).
Mitochondrial ROS levels
Isolated mitochondria were doubly-stained with MitoTracker Red (0.5 μM; excitation/emission 550/590 nm,Invitrogen company) and dichlorodihydrofluorescein (DCF) diacetate (10 μM; excitation/emission 488/535 nm,Invitrogen company). The superoxide levels were examined according to the change in MitoSOX Red fluorescence using a confocal microscopy (Zeiss LSM 780). Mean values were analysed by CellQuest (ver. 5.2; DB CellQuest™ Pro)
Mitochondria membrane potential
Isolated mitochondria were stained for 30 min with 0.1 mM tetramethylrhodamine ethyl ester (Invitrogen company excitation/emission 564/580 nm) at room temperature and measured by flow cytometry to detect the mitochondrial membrane potential.
Glucose Consumption and Lactate Secretion
HUVECs were seeded into culture plates and incubated for 5 hours. The culture medium was then changed and cells were cultured for another 16 hours. The levels of glucose in the culture medium were measured using an assay kit from Nanjing Jiancheng Bioengineering Institute (Nanjing, China), following the manufacturer's recommendations. Lactate concentration was measured with a Lactate Assay Kit (Biovision Inc.), in accordance with the manufacturer's instructions. The glucose consumption and lactate secretion were normalized to the cell number.
To determine the mitochondrial morphology, including number, size, and shape, HUVECs were sliced and fixed in 2.5% glutaraldehyde in PBS at 4 °C overnight, then fixed under 1% osmium tetroxide in PBS for 2 hours. The sliced hearts were double-stained with uranyl acetate and lead citrate. Mitochondrial morphology was observed using an electron microscope, and the number of mitochondria was calculated using ImageJ software.
Assays for glucose metabolism
Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of HUVECs were measured using the Seahorse XF Glycolysis Stress Test Kit on an XF24 Extracellular Flux Analyzer (Agilent Technologies), following the manufacturer’s instructions. Cells were grown under standard growth conditions for 1 day prior to the metabolic analysis.
GraphPad Prism 6.0 software is used to perform statistical analyses. Data are presented as mean ± SD. All pairs were compared to each other via either Student’s t-test or least significant difference (LSD) test, as appropriate. Experimental mice groups were subject to correlation analyses through Bonferroni’s post hoc test. One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons, were utilized for comparing multiple groups among each other. P-values <0.05 were considered significant.