Patients were enrolled with coronary slow-flow phenomenon (n = 16), defined via coronary angiography based on either a reduced Thrombolysis in Myocardial Infarction (TIMI) flow grade of 2, were randomly recruited at the Department of Cardiology, Shenzhen People’s Hospital. Exclusion criteria were: Coronary stenosis of more than 50%, Second/third-degree atrioventricular block, ventricular arrhythmia history, myocardial infarction or coronary revascularization, uncontrolled HF, significant renal/hepatic disease, severe COPD or aortic stenosis, acute pulmonary embolism/myocarditis, symptomatic cerebrovascular disease within 12 months, or expected mortality in ≤ 12 months. Control individuals were enrolled with atherosclerosis of coronary (n = 20) defined via coronary angiography and age- and sex-matched with coronary slow flow patients. In brief, in patients of slow flow, we collected 10 ml of blood from the coronary or peripheral artery from the same patients. To account for dilutions, all analyses were normalized to hematocrit. All patients provided written informed consent. The study was approved by the Committee for Medical and Health Ethics of Shenzhen People’s Hospital, Jinan University.

Human umbilical vein endothelial cells (HUVECs) were purchased from the American Type Culture Collection (Manassas, VA, USA), 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, USA), 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 h.

Small interfering RNAs (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′-ACAUCCAUAUAGCAGGGAGTT-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 (LF + NC-siRNA), disturbed flow with negative control siRNA (DF + NC-siRNA) and disturbed flow with TXNIP-siRNA (DF + TXNIP-siRNA).

Blood samples were collected from patients and centrifuged at 3000 rpm for 10 min at 4°C. Plasma TXNIP concentration was measured using human TXNIP and malondialdehyde ELISA kit (MyBioSource), according to the manufacturer’s instructions.

Protein samples with equal amount of total protein (20 μg) 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 h 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. Afterward, 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.

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, 5*10⁶ 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 at 72 h 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 analyzed.

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).

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 1000 × g for 5 min, followed by centrifugation twice more at 11000 × 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).

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 analyzed by CellQuest (ver. 5.2; DB CellQuest Pro).

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.

Human umbilical vein endothelial cells were seeded into culture plates and incubated for 5 h. The culture medium was then changed and cells were cultured for another 16 h. 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 h. Mitochondrial morphology was observed using an electron microscope, and the number of mitochondria was calculated using ImageJ software.

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.

To test our hypothesis that slow flow enhance oxidative metabolism, we collected plasma samples from coronary or peripheral artery in coronary slow flow patients or control individuals. Control individuals are age- and sex-matched with patients. Patients with coronary slow flow do not exhibit slow flow in peripheral artery. The baseline characteristics of the human subjects from whom plasma are depicted in Table 1. The plasma TXNIP levels were significantly increased in coronary of slow flow patients compared with control individuals or femoral artery from the same patients (Figure 1A). In addition, the malondialdehyde (MDA), known as a marker of oxidative stress, was significantly elevated in coronary of slow flow patients (Figure 1B). All these findings thus indicate that TXNIP may involve in oxidative disorder in slow flow patient’s coronary.

To assess the functional role of flow disturbances on TXNIP expression, the cultured HUVECs were exposed to disturbed flow shear stress (0.4 dyn/cm²) or steady laminar flow (12 dyn/cm²) for 24 h. TXNIP expression levels in HUVECs were then examined by Western Blotting, which demonstrated that disturbed flow significantly enhanced TXNIP protein levels compared to laminar flow (Figure 2A). The results also showed that the levels of Thioredoxin (TRX), referred to as a small redox protein acting as an electron donor to peroxidases and ribonucleotide reductase, had no changes under disturbed conditions compared to laminar flow. In addition, to investigate whether flow velocity affects endothelial dysfunction, we tested the ability of HUVECs to form capillary-like structures. HUVECs were seeded in Matrigel after disturbed flow or laminar flow treatment, and tube formation was examined microscopically. Further confirmation of disturbed flow inducing endothelial dysfunction through regulating TXNIP expression was queried through treating the cells with either TXNIP-siRNA or negative control siRNA (NC-siRNA). The results showed that the tube structures formed more slowly under disturbed flow conditions, whereas cells subjected to laminar flow exhibited a greater extent of capillary formation (Figure 2B). However, TXNIP-siRNA treatment abrogated the formation of the disconnected capillary-like structures but increased the development of the proper capillary network under disturbed flow conditions (Figure 2B). Collectively, these data suggest that disturbed flow led to a significant TXNIP expression increase, resulting in endothelial dysfunction.

Both acute and chronic attenuation in NO production are major factors favoring endothelial dysfunction. To explore the possibility of disturbed flow regulating NO production, we measured NO and nitrotyrosine levels in endothelial cells. The results showed that disturbed flow significantly reduced NO levels and increased nitrotyrosine in HUVECs. By contrast, TXNIP-siRNA treatment attenuated all of these alterations in the DF group (Figures 3A,B). We also measured the protein levels of endothelial nitric oxide synthase (eNOS), which is primarily responsible for vascular endothelial NO generation. Western Blot analysis showed that phosphorylation of eNOS at Ser1177 was downregulated in the disturbed flow with NC-siRNA group, compared with those in steady laminar flow group (Figure 3C). In comparison, the protein level of eNOS at Ser1177 in the DF + TXNIP-siRNA group was upregulated compared to the DF + NC-siRNA group. These results collectively suggested that elevated TXNIP during disturbed flow contributes to eNOS depression and activity.

Endothelial dysfunction is thought to be mediated mostly by reactive oxygen species (ROS). Mitochondria are the major cellular ROS producers, due to their crucial role in energy metabolism. To explore the possibility of disturbed flow inducing mitochondrial dysfunction, we detected mitochondrial ROS in isolated mitochondria from HUVECs, with or without TXNIP expression. The results showed that disturbed flow treatment exhibited higher levels of ROS compared to laminar flow, while TXNIP-siRNA significantly reduced mitochondrial ROS levels compared to the DF + NC-siRNA group (Figure 4A). Similarly, disturbed flow also reduced ATP levels compared to laminar flow (Figure 4B). By contrast, TXNIP-siRNA treatment in the disturbed flow group resulted in significantly increased ATP levels compared to the DF + NC-siRNA group (Figure 4B). We also observed a remarkable reduction in mitochondrial membrane potential among the disturbed flow group, compared to mitochondria isolated from laminar flow-treated cells. Conversely, TXNIP-siRNA abrogated the reduction found in the disturbed flow group compared to the DF + NC-siRNA group (Figure 4C). Mitochondrial morphology under electron microscopy in disturbed flow-treated HUVECs showed bizarre shapes and poorly defined cristae (Figure 4D). Conversely, TXNIP expression inhibition led to less disorganized mitochondrial morphology compared with that of the DF + NC-siRNA group. These results therefore demonstrated that disturbed flow may aggravate mitochondrial dysfunction by distorting mitochondrial morphological features and increasing ROS levels while silencing TXNIP partially reversed the pathological response in HUVECs.

As TXNIP has been identified as a key determinant of glucose utilization in cardiac metabolism, we investigated glucose uptake and lactate production in disturbed flow-treated HUVECs, with or without TXNIP. Glucose uptake and lactate production in disturbed flow-treated HUVECs was reduced to varying degrees compared to the laminar flow group (Figures 5A,B). It is worth noting that TXNIP depletion completely abrogated the disturbed flow-induced reduction of those aforementioned metabolites compared to the DF + NC-siRNA treatment group (Figures 5A,B). We further examined the impacts of TXNIP deficiency on aerobic metabolism within laminar or disturbed flow groups by measuring the OCR using the XF24 extracellular flux analyze, indicating aerobic metabolism of glucose via tricarboxylic acid (TCA) cycle and mitochondrial oxidative phosphorylation. As illustrated in Figures 5C,D glucose or oligomycin (an ATP synthase inhibitor) addition triggered significant OCR increase in the laminar flow group, but only a moderate augmentation in the disturbed flow group. Conversely, TXNIP deletion triggered significant OCR increase in the disturbed flow group compared to the disturbed flow with NC-siRNA (DF + NC-siRNA) group. In addition, to determine the effects of different flow velocities on HUVEC glycolytic flux, varying levels of flow velocity were applied to cells, with or without TXNIP-siRNA present, and the glycolytic flux was detected in DMEM assay medium following sequential addition of glucose, oligomycin and 2-deoxy-glucose (2-DG, a hexokinase inhibitor). Consistent with the OCR finding, TXNIP deletion abolished the disturbed flow-mediated ECAR reduction that would otherwise be present under such conditions (Figure 5E). The quantification demonstrated that glycolysis and glycolytic capacity were significantly decreased in the disturbed flow group, compared to the laminar-flow group. Nevertheless, TXNIP depletion under disturbed flow conditions displayed similar bioenergetic profiles of glycolysis and glycolytic capacity as under laminar flow (Figure 5F). Thus, to better understand the specific physiological role of TXNIP in glucose metabolism, we selectively measured glucose metabolism-related gene expression in endothelial cells. We found that the glucose transporter type 4 (GLUT4), a major mediator of glucose removal from the circulation, was downregulated in disturbed flow group compared to the laminar flow group, while TXNIP deletion exhibited higher levels of this protein than the DF + NC-siRNA group (Figure 5G). This observed GLUT4 decrease was also associated with significantly lessened expression of PDH E1α (Figure 5G), which provides the primary link between glycolysis and the TCA cycle. Together, these data indicate that elevation of TXNIP expression blunted glucose uptake, suggesting that it is the key regulator mediating glucose utilization.

To investigate whether TXNIP mediated HUVEC pro-inflammatory response under different flow velocities, NLRP3 and cytokine interleukin (IL)-1β expression were examined by Western Blotting. The expression of the cleaved form of NLRP3 and IL-1β was upregulated in disturbed flow-treated HUVECs, but decreased upon TXNIP siRNA treatment (Figure 6A). Application of disturbed flow, but not laminar flow, upregulated the levels of cell adhesion molecules VCAM1 and ICAM1 in HUVECs. By contrast, such increases were offset by TXNIP siRNA treatment (Figure 6B). Taken together, these results indicated that shear stress induced HUVEC inflammation, in turn contributing to endothelial dysfunction.