Disturbed Flow Induces Endothelial Dysfunction by Regulating Thioredoxin-Interacting Protein-Mediated Mitochondrial Energy Homoeostasis

Yongshun Wang Department of Cardiology, Shenzhen People’s Hospital, Shenzhen, Guangdong, China Jingjin Liu Department of Cardiology, Shenzhen People’s Hospital, Shenzhen, Guangdong, China Xin Sun Department of Cardiology, Shenzhen People’s Hospital, Shenzhen, Guangdong, China Jie Yuan Department of Cardiology, Shenzhen People’s Hospital, Shenzhen, Guangdong, China Huadong Liu Department of Cardiology, Shenzhen People’s Hospital, Shenzhen, Guangdong, China Ruimian Chen Department of Cardiology, Shenzhen People’s Hospital, Shenzhen, Guangdong, China Bihong Liao Department of Cardiology, Shenzhen People’s Hospital, Shenzhen, Guangdong, China Haixia Gong Department of Cardiology, Shenzhen People’s Hospital, Shenzhen, Guangdong, China Zhengyuan Xia Department of Anesthesiology, The University of Hong Kong, Hong Kong SAR, China Shaohong Dong (  forres_dsh@163.com ) Shenzhen People's Hospital


Introduction
Shear stress is one of the primary mechanical forces large arteries are subjected under [1]. Mechanicallystimulated release of potent shear-responsive factors from endothelial cells regulates vessel tone and structure [2][3][4][5]. This process is facilitated by the endothelium being sensitive to hemodynamic shear stresses acting on the vessel luminal surface in the direction of blood ow. By extension, physiological and pathological variations of shear stress, caused by multiple pathophysiological conditions such as hyperlipidemia, hypertension, diabetes and in ammatory disorders, regulate endothelium-dependent changes in vascular diameter in an acute manner, and when sustained induce slowly-adaptive structural wall remodeling [6].
The varying spatiotemporal scales shear stress can be found in also contributes to regional and focal heterogeneity of endothelial gene expression. This process is important in the evolution of vascular pathology. Thioredoxin-interactive protein (TXNIP) is known to promote oxidative stress by binding and subsequently inhibiting thioredoxin activity [7,8]. In doing so, TXNIP is reported to in uence cardiac metabolism, including mitochondrial function and glucose uptake [9]. Researchers have also demonstrated that TXNIP modulates cellular glucose utilization and mitochondrial oxidation of metabolic fuels [5,[10][11][12]. On the other hand, TXNIP-null mice cannot survive prolonged fasting, exhibiting dysglycemia and dyslipidemia [11]. Besides its involvement in cellular redox and energy metabolism, increasing evidence points towards TXNIP having an important role in vascular function and in ammation process. Studies in endothelial cells showed that TXNIP promotes in ammatory response in response to disturbed ow [13] and arterial stiffness [14,15]. This pro-in ammatory effect is con rmed by the nding that it is required for NLRP3 in ammasome activation and IL-1β production in cultured THP-1 cells [16].
However, the role of TXNIP in endothelial dysfunction is not well addressed. Given the important role of TXNIP in redox homeostasis and in ammation, we hypothesized that its ablation would protect endothelial cells from oxidative stress-induced damage and reduce vascular in ammation. In the present study, we used a disturbed-ow model to investigate the effects of TXNIP deletion on cellular redox status and in ammatory response. Our data demonstrated that TXNIP plays an important role in the development of endothelial dysfunction.

Materials And Methods
Cell culture and disturbed ow 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 modi ed Eagle's medium containing 10% fetal bovine serum (FBS), 50 U/mL penicillin and 50 μg/mL streptomycin (Invitrogen, Carlsbad, CA), as speci ed by the manufacturer. To initiate disturbed ow treatment, con uent HUVECs, seeded onto collagen I-coated glass slides, were assembled into ow chambers and connected to the ow system for the shear experiments. HUVECs were exposed to steady laminar ow shear stress (12 dyn/cm 2 ), disturbed ow shear stress (0.5 ± 4 dyn/cm 2 ) 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 ow with negative control siRNA (NF+NC-siRNA), disturbed ow with negative control siRNA (DF+NC-siRNA) and disturbed ow 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 di uoride membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk at room temperature for 1 hour in Tris-buffered saline containing 0. Abcam Company]) was carried out overnight at 4°C. Afterwards, secondary antibody incubation with a peroxidase-conjugated A niPure 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 ow 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 quanti ed by calculating the number of junctions per eld. At least 5 different viewing elds 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'di uoro uorescein (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, uorescent intensity was determined at an excitation wavelength of 488 nm and an emission wavelength of 525 nm via a uorescent 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-tting glass Te on 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 clari ed 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. Brie y, 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).

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.

Electron microscopy
To determine the mitochondrial morphology, including number, size, and shape, HUVECs were sliced and xed in 2.5% glutaraldehyde in PBS at 4 °C overnight, then xed 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 acidi cation 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.

Statistical 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 signi cant 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 signi cant.

Results
Increased expression of TXNIP in disturbed ow induced endothelial dysfunction To assess the functional role of ow disturbances on TXNIP expression, the cultured HUVECs were exposed to disturbed ow shear stress (0.4 dyn/cm2) or steady laminar ow (12 dyn/cm2) for 24 hours.
TXNIP expression levels in HUVECs were then examined by Western Blotting, which demonstrated that disturbed ow signi cantly enhanced TXNIP protein levels compared to laminar ow (Fig. 1A). 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 ow. In addition, to investigate whether ow velocity affects endothelial dysfunction, we tested the ability of HUVECs to form capillary-like structures. HUVECs were seeded in Matrigel after disturbed ow or laminar ow treatment, and tube formation was examined microscopically. Further con rmation of disturbed ow 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 ow conditions, whereas cells subjected to laminar ow exhibited a greater extent of capillary formation (Fig. 1B). However, TXNIP-siRNA treatment abrogated the formation of the disconnected capillary-like structures but increased the development of the proper capillary network under disturbed ow conditions (Fig. 1B). Collectively, these data suggest that disturbed ow led to a signi cant TXNIP expression increase, resulting in endothelial dysfunction.

Disturbed ow reducedproduction of NO in a TXNIP dependent manner
Both acute and chronic attenuation in NO production are major factors favouring endothelial dysfunction. To explore the possibility of disturbed ow regulating NO production, we measured NO and nitro-tyrosine levels in endothelial cells. The results showed that disturbed ow signi cantly reduced NO levels and increased nitro-tyrosine in HUVECs. By contrast, TXNIP-siRNA treatment attenuated all of these alterations in the DF group ( Fig. 2A and 2B). 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 ow with NC-siRNA group, compared with those in steady laminar ow group (Fig. 2C). 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 ow contributes to eNOS depression and activity.

Disturbed ow induced endothelial mitochondrial dysfunction through regulating TXNIP expression
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 ow inducing mitochondrial dysfunction, we detected mitochondrial ROS in isolated mitochondria from HUVECs, with or without TXNIP expression. The results showed that disturbed ow treatment exhibited higher levels of ROS compared to laminar ow, while TXNIP-siRNA signi cantly reduced mitochondrial ROS levels compared to the DF+NC-siRNA group (Fig. 3A). Similarly, disturbed ow also reduced ATP levels compared to laminar ow (Fig. 3B). By contrast, TXNIP-siRNA treatment in the disturbed ow group resulted in signi cantly increased ATP levels compared to the DF+NC-siRNA group (Fig. 3B). We also observed a remarkable reduction in mitochondrial membrane potential among the disturbed ow group, compared to mitochondria isolated from laminar ow-treated cells. Conversely, TXNIP-siRNA abrogated the reduction found in the disturbed ow group compared to the DF+NC-siRNA group (Fig. 3C). Mitochondrial morphology under electron microscopy in disturbed owtreated HUVECs showed bizarre shapes and poorly-de ned cristae. 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 ow may aggravate mitochondrial dysfunction by distorting mitochondrial morphological features and increasing ROS levels while silencing TXNIP partially reversed the pathological response in HUVECs.
Disturbed ow decreased glucose utilization by TXNIP-dependent activation As TXNIP has been identi ed as a key determinant of glucose utilization in cardiac metabolism, we investigated glucose uptake and lactate production in disturbed ow-treated HUVECs, with or without TXNIP. Glucose uptake and lactate production in disturbed ow-treated HUVECs was reduced to varying degrees compared to the laminar ow group (Fig. 3A and 3B). It is worth noting that TXNIP depletion completely abrogated the disturbed ow-induced reduction of those aforementioned metabolites compared to the DF+NC-siRNA treatment group (Fig. 4A and 4B). We further examined the impacts of TXNIP de ciency on aerobic metabolism within laminar or disturbed ow groups by measuring the oxygen consumption rate (OCR) using the XF24 extracellular ux analyze, indicating aerobic metabolism of glucose via tricarboxylic acid (TCA) cycle and mitochondrial oxidative phosphorylation. As illustrated in gure 4C and 4D, glucose or oligomycin (an ATP synthase inhibitor) addition triggered signi cant OCR increase in the laminar ow group, but only a moderate augmentation in the disturbed ow group. Conversely, TXNIP deletion triggered signi cant OCR increase in the disturbed ow group compared to the disturbed ow with NC-siRNA (DF+NC-siRNA) group. In addition, to determine the effects of different ow velocities on HUVEC glycolytic ux, varying levels of ow velocity were applied to cells, with or without TXNIP-siRNA present, and the glycolytic ux 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 nding, TXNIP deletion abolished the disturbed ow-mediated extracellular acidi cation rate (ECAR) reduction that would otherwise be present under such conditions (Fig. 4E). The quanti cation demonstrated that glycolysis and glycolytic capacity were signi cantly decreased in the disturbed ow group, compared to the laminar-ow group. Nevertheless, TXNIP depletion under disturbed ow conditions displayed similar bioenergetic pro les of glycolysis and glycolytic capacity as under laminar ow (Fig. 4F). Thus, to better understand the speci c 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 ow group compared to the laminar ow group, while TXNIP deletion exhibited higher levels of this protein than the DF+NC-siRNA group (Fig. 4G). This observed GLUT4 decrease was also associated with signi cantly-lessened expression of PDH E1α (Fig. 4G), 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.

TXNIP activation promoted the disturbed ow-induced pro-in ammatory response
To investigate whether TXNIP mediated HUVEC pro-in ammatory response under different ow 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 ow-treated HUVECs but decreased upon TXNIP siRNA treatment (Fig. 5A). Application of disturbed ow, but not laminar ow, upregulated the levels of cell adhesion molecules VCAM1 and ICAM1 in HUVECs. By contrast, such increases were offset by TXNIP siRNA treatment (Fig. 5B). Taken together, these results indicated that shear stress induced HUVEC in ammation, in turn contributing to endothelial dysfunction.

Discussion
TXNIP is a key skeletal muscle regulator of glucose usage and metabolism, as well as a recently-found key in ammatory mediator [17][18][19] [16]. In light with the latter discovery, our ndings demonstrated it serving a signi cant role in endothelial redox and in ammatory responses, where its ablation led to prominent reduction in cellular ROS and restoration of mitochondrial function. This reduction was found to be associated with lowered in ammatory response and NLRP3 expression. Furthermore, eliminating TXNIP also yields bene cial effects in the forms of increased glucose uptake, lactate and ECAR levels in a glucose-utilization study, as well as lowered HUVEC cell adhesion through its lowering of VCAM and ICAM expression. All these ndings indicate TXNIP playing an important role in the development of HUVEC shear stress response and dysfunction. Figure 6 depicts our proposed mechanism for the dysfunctionality of endothelial cells under shear stress conditions. Diabetic individuals, who also are more prone to atherosclerosis, demonstrate higher TXNIP expression levels. Previous studies showed that lower TXNIP expression enhanced skeletal muscle glucose uptake and improved glycaemic control [17], suggesting TXNIP inhibition being a potential diabetic intervention strategy. It was reported that endogenous NO can suppress TXNIP expression and that TXNIP facilitates nitrosative stress. However, the direct effects of TXNIP on NO regulation in disturbed ow-induced endothelial dysfunction were not investigated. In this study, TXNIP/NO interaction under shear forces was demonstrated via eNOS coupling regulation. In addition, our results indicated lower ATP levels, along with increased ROS and membrane potential depolarization in mitochondria from disturbed ow-treated cells, which were all reversed under TXNIP deletion, demonstrating the latter's potential in preventing mitochondrial dysfunction. Overall, the results from this study have further clinical and therapeutic implications, where TXNIP ablation could be bene cial in diabetics via reducing vascular in ammation and dysfunction.
TXNIP expression has been previously demonstrated to be induced by a glucose-dependent signalling pathway [20]. This connection is of signi cant physiological relevance, owing to the nding that after its induction, TXNIP negatively regulates glucose uptake [18,21,22]. The operation of this regulatory pathway is as follows: Cells requiring more energy or "building blocks" for macromolecular synthesis demonstrate a higher glycolytic rate, and thus a decrease of levels for certain glycolytic metabolites. Both changes are sensed by the TXNIP transcriptional machinery to repress TXNIP expression. Based on our ndings, the data suggest that ectopic TXNIP expression blunts glucose uptake and lactate production.
Thus, we propose that TXNIP acts as a mediator to integrate cellular metabolic activity and energy requirements with cellular glucose supply, which may have important implications for endothelial cell glucose homeostasis regulation.
Several results suggest that TXNIP is a critical target for steady laminar ow-associated antiin ammatory effects [7,23]. Our results show that steady laminar ow increased the ability of HUVECs to form capillary like structures, as well as lowering TXNIP expression and NLRP3-mediated in ammation. Thus, it is likely steady laminar ow inhibits TXNIP expression, leading to the limitation of HUVEC in ammation and endothelial function improvement. This study also investigated the potential role of TXNIP in disturbed ow induced HUVEC in ammation response, where the results clearly demonstrated TXNIP ablation decreasing cellular ROS levels, as well as attenuating the stimulation of pro-in ammatory and pro-adhesion gene expression.
In summary, our data suggested TXNIP playing an important role in vascular in ammation and mitochondrial dysfunction, with respect to endothelial dysfunction development. TXNIP expression modulation could therefore be a potential therapeutic strategy for intervention in ow velocity-related vascular complications.

Declarations
Ethics approval and consent to participate All animal care was conducted in accordance with the "Principles of Animal Care" (Ethical and Animal Welfare Committee of Heilongjiang Province, China) and were approved by the ethics review board of Jinan University and Southern University of Science and Technology.
Authors' contributions YS W, JJ L and XS performed the experiments and were major contributors in writing the manuscript. HD L and SH D did data analysis and interpretation, as well as being responsible for the study design and manuscript drafting. JY was responsible for statistical analysis. YS W prepared the reagents and revised the manuscript. ZY X and SH D designed the entire study and provided funding. All authors read and approved the nal manuscript.