NF-κB inhibition prevents acute shear stress-induced inflammation in the saphenous vein graft endothelium

The long saphenous vein (LSV) is commonly used as a conduit in coronary artery bypass grafting. However, long term patency remains limited by the development of vascular inflammation, intimal hyperplasia and accelerated atherosclerosis. The impact of acute exposure of venous endothelial cells (ECs) to acute arterial wall shear stress (WSS) in the arterial circulation, and the subsequent activation of inflammatory pathways, remain poorly defined. Here, we tested the hypothesis that acute exposure of venous ECs to high shear stress is associated with inflammatory responses that are regulated by NF-κB both in-vitro and ex-vivo. Analysis of the LSV endothelium revealed that activation of NF-κB occurred within 30 min after exposure to arterial rates of shear stress. Activation of NF-κB was associated with increased levels of CCL2 production and enhanced binding of monocytes in LSVECs exposed to 6 h acute arterial WSS. Consistent with this, ex vivo exposure of LSVs to acute arterial WSS promoted monocyte interactions with the vessel lumen. Inhibition of the NF-κB pathway prevented acute arterial WSS-induced CCL2 production and reduced monocyte adhesion, both in vitro and in human LSV ex vivo, demonstrating that this pathway is necessary for the induction of the acute arterial WSS-induced pro-inflammatory response. We have identified NF-κB as a critical regulator of acute endothelial inflammation in saphenous vein in response to acute arterial WSS. Localised endothelial-specific inhibition of the NF-κB pathway may be beneficial to prevent vein graft inflammation and consequent failure.


The long saphenous vein (LSV) is commonly used as a conduit in coronary artery bypass grafting. However, long term patency remains limited by the development of vascular inflammation, intimal hyperplasia and accelerated atherosclerosis. The impact of acute exposure of venous endothelial cells (ECs) to acute arterial wall shear stress (WSS) in the arterial circulation, and the subsequent activation of inflammatory pathways, remain poorly defined.
Here, we tested the hypothesis that acute exposure of venous ECs to high shear stress is associated with inflammatory responses that are regulated by NF-κB both in-vitro and ex-vivo. Analysis of the LSV endothelium revealed that activation of NF-κB occurred within 30 min after exposure to arterial rates of shear stress. Activation of NF-κB was associated with increased levels of CCL2 production and enhanced binding of monocytes in LSVECs exposed to 6 h acute arterial WSS. Consistent with this, ex vivo exposure of LSVs to acute arterial WSS promoted monocyte interactions with the vessel lumen. Inhibition of the NF-κB pathway prevented acute arterial WSS-induced CCL2 production and reduced monocyte adhesion, both in vitro and in human LSV ex vivo, demonstrating that this pathway is necessary for the induction of the acute arterial WSS-induced pro-inflammatory response. We have identified NF-κB as a critical regulator of acute endothelial inflammation in saphenous vein in response to acute arterial WSS. Localised endothelial-specific inhibition of the NF-κB pathway may be beneficial to prevent vein graft inflammation and consequent failure. Ischaemic heart disease (IHD) is one of the leading causes of mortality in the UK and worldwide 1 . Coronary artery bypass grafting (CABG) remains the gold standard intervention in the presence of complex coronary Acute arterial WSS activates pro-inflammatory responses and the NF-κB classical pathway in vitro. To validate our ex vivo findings and further investigate the molecular basis involved in the venous EC response to arterial rates of shear stress, we studied the effects of acute arterial WSS on HUVECs in vitro. Acute high shear stress significantly increased nuclear translocation of p65 (Fig. 3A,D) which was associated with reciprocally decreased IκBα expression (Fig. 3B,D) indicating the activation of the NF-κB classical pathway. Furthermore, NF-κB was phosphorylated at Serine residue 276 (Ser-276) following 30 min of acute arterial WSS (Fig. 3C,D), whilst also showing increased DNA binding to the NF-κB consensus oligonucleotide (Fig. 3E), both of which are indicative of transcriptional activation of NF-κB. This transcriptional activation was transient, with levels peaking at 30 min, which is likely indicative of the feed-forward mechanism of negative regulation of NF-κB by IκBα following 60 min acute arterial WSS exposure 13 . We observed that acute arterial WSS, but not low shear, was associated with up regulation of CCL2 ( Fig. 4A-D). The suppression of the NF-κB classical pathway using 20 μmol/L BAY pre-treatment resulted in reduced pro-inflammatory activation, similar to what was observed ex vivo (Fig. 5A).

Abbreviations
We further validated these findings using adenoviral-mediated over-expression of WT-IκBα (Supplementary Figure 9), which demonstrated that the overexpression of WT-IκBα prevented induction of CCL2 following exposure to acute arterial WSS (Fig. 5B). Moreover, immunoanalysis showed a significant reduction in the expression of CCL2 following 4 h of acute arterial WSS, as compared with static controls (Fig. 5C,D). Whilst, overexpression of WT-IκBα prevented the loss of VE-Cadherin cell-cell contacts following acute arterial WSS exposure (Fig. 5E,F), indicative of an intact endothelial barrier.
We next looked at the impact of NF-κB inhibition on monocyte-EC interactions in vitro, dynamically and in real-time, following exposure to acute arterial WSS. We noted that the pre-treatment of HUVECs with 20 μmol/L BAY significantly reduced acute arterial WSS-induced dynamic monocyte adhesion to the vEC monolayer (Fig. 5G,H).

NF-κB pathway-related gene expression was promoted by acute arterial WSS in vivo.
Having observed a significant increase in CCL2 mRNA and protein in the LSV endothelium, we then analysed a publicly available gene expression microarray dataset, generated from a rabbit bilateral interposition vein graft model 14  www.nature.com/scientificreports/ Change (LogFC)) of differentially expressed genes (1602 genes) significantly different (p < 0.01) from baseline, in any of the 4 graft conditions were hierarchically clustered and plotted (Supplementary Figure 8A). Interestingly, gene set enrichment analysis between the two acute groups (high and low shear) revealed highly significant enrichment of biological process GO-terms involved in the inflammatory response, cytokine-mediated signalling and neutrophil activity, in grafts exposed to high shear, but not low (Supplementary Figure 8B,C). In addition, the high shear-exposed grafts showed the least overlap with any other graft condition, in terms of both gene overlap and GO-terms, suggesting that grafts exposed to high shear had the largest functionally unique change of any other condition (Supplementary Figure 8D,E). Within grafts exposed to high shear stress for 2 h, significantly up-regulated genes with a LogFC > 1.5 (a total of 114 genes) were analysed further for functional pathway and motif enrichment. Transcriptional regulatory inference, performed using LISA 15 , ranked NF-κB as www.nature.com/scientificreports/ the most probable transcriptional regulator of this gene-set, which was further supported by the observation that the NF-κB motif was highly enriched within the promoter regions of these genes (Supplementary Figure 8F,G). Finally, KEGG and REACTOME pathway analysis revealed that this gene list was strongly associated with the TNFα-and NF-kappaB signalling pathways, further supporting the role for NF-κB activation in vein grafts following acute shear stress exposure (Supplementary Figure 8H).

Discussion
Vein graft disease following CABG remains a problem of serious clinical significance despite decades of surgical advances 16,17 . Haemodynamics influence the inflammatory process by controlling leukocyte margination and adhesive interactions and by generating shear stress, which in turn alters EC physiology 18,19 . Increased shear stress rates in the LSV graft have long been considered to directly affect graft patency and survival; however, at present, there is no clinical consensus as to the extent of this effect, perhaps due to the diverse range of rates of graft WSS exposure, postoperatively 20,21 . Furthermore, the molecular cascades that occur after acute increases in WSS rates in LSV graft ECs remain poorly understood. We, and others, have previously shown that arterial and venous EC respond differently to acute high shear stress, in both in vivo and ex vivo graft models 13,[22][23][24] . Here, we show that the NF-κB classical pathway is activated in the endothelium of LSV in response to acute exposure to arterial rates of shear stress and is a critical regulator of vascular inflammation in veins. In vitro experiments further demonstrated that targeting NF-κB prior to acute flow induction was sufficient to prevent endothelial CCL2 expression and monocyte recruitment, as well as EC cell-cell contact disruption, another hallmark of the dysfunctional endothelium. These findings in the LSV were further substantiated by evidence from analysis of a previously published 14 in vivo rabbit vein graft model and DNA microarray. Differential expression analysis revealed significantly www.nature.com/scientificreports/ up-regulated NF-κB and inflammatory pathway-related genes between acute high and low shear-exposed vein grafts. Together, these data provide a possible mechanism for the regulation of acute vein graft inflammation, a factor which is inextricably linked to downstream pathologies. Furthermore, owing to the unique window of opportunity to locally pre-treat the autologous vein graft prior to implantation, inhibition of NF-κB may represent an exciting possibility in the resolution of vascular inflammation within the graft. Acute onset of increased rates of shear stress is known to promote inflammatory transcriptional responses in ECs in vitro, a process mediated through NF-κB, p38-MAPK and activating protein 1 (AP-1) 17,24-27 . However, no studies so far have directly addressed the role of acute shear stress on NF-κB pathway activation in the vein graft endothelium.
Our findings with regards to activation of CCL2 are in concordance with previously published small animal in vivo data 28,29 . Several previous studies have shown that in vivo, local NF-κB inhibition, using either a decoy cis-element (κB-oligodeoxynucleotide), siRNA-mediated knockdown or an alternate gene therapy approach to overexpress IκBα, limits IH development in multiple vein graft models [30][31][32] . However, all such previous models propose the same conclusions for their observed results; that it is due to the actions of NF-κB inhibition on reduction of medial VSMC proliferation and migration, and not the role of NF-κB in the acute adaptive response of the vein graft to its new haemodynamic environment. Following this acute haemodynamic adaptation, ECs typically Figure 3. Acute high shear stress and activation of the NF-κB classical pathway-in vitro. The NF-κB classical pathway was activated under acute high shear stress as indicated firstly by the nuclear translocation of NF-κB and reciprocal reduction in cytosolic levels of IκB⍺, as well as phosphorylation and increased DNA binding affinity. HUVECs were exposed to acute arterial WSS for 30 and 90 min or maintained in static conditions, then cellular fractions were collected to be analysed by Western blotting. Total amounts of protein were quantified relative to stain-free loading controls and expressed as a fold change of static control. (A) Nuclear NF-κB, (B) cytosolic levels of IκB⍺ and (C) Phospho-NF-κB (Serine residue 276 (Ser276)) from whole cell lysates were analysed by WB, and normalised to stain-free loading controls from nuclear, cytosolic and whole cell lysates, respectively. Western blots shown are representative of 5 or 6 independent experiments showing, in addition to NF-κB and IκB⍺, markers of nuclear and cytosolic fractions, Lamin A/C and GAPDH respectively (D). The transcriptional activation of NF-κB under acute arterial WSS, for both 30 and 90 min, was assessed by phosphorylation at Serine residue 276 (C) and DNA binding activity (E) in whole cell lysates. ELISA based DNA binding activity assay was performed using total cell lysates and was expressed relative to static controls and were representative of 3 independent experiments. One-way ANOVA followed by post-hoc pairwise comparisons with Bonferroni correction was used to calculate significance, where, ** indicates p < 0.01 and * indicates p < 0.05.

Scientific RepoRtS
| (2020) 10:15133 | https://doi.org/10.1038/s41598-020-71781-6 www.nature.com/scientificreports/ promote endogenous mediators that promote quiescence and dampen inflammation, through the activation of KLF2 and Nrf2 and repression of NF-κB, within 12 h of high WSS exposure 33 . Our novel findings demonstrate a potential role of acute NF-κB inhibition in maintaining this quiescent, anti-inflammatory endothelium prior to and under conditions of arteriovenous transposition that may further help to ameliorate the pathogenic progression to late-stage graft failure.
In conclusion, we identified that acute arterial WSS is responsible for early pro-inflammatory activation of the LVG EC, a process regulated by NF-κB (p65) activation, resulting in upregulation of pro-inflammatory mediators and increased monocyte recruitment, thus, providing the first evidence for the mechanistic involvement of NF-κB in shear-induced inflammation in the vein graft endothelium.

Methods
Detailed descriptions of all materials and methods can be found in the Supplementary Methods.  . NF-κB target gene response to acute high shear stress-in vitro. Pro-inflammatory, NF-κB target genes were increased in ECs exposed to acute arterial WSS, in vitro. (C,D) HUVECs were exposed to AASS at 12 dyn/cm 2 (A) and acute low shear stress (ALSS) at 0.5 dyn/cm 2 (B) for 90 and 120 min in order to compare pro-inflammatory gene transcript levels by RT-qPCR. Data represent 6 independent experiments. (C,D) HUVECs were exposed to AASS at 12 dyn/cm 2 for 4 h or maintained in static conditions, then immunostained for quantification of total CCL2 using CellProfiler. (C) Mean arbitrary fluorescence intensity of CCL2 staining was evaluated in whole cells. (D) Images are representative of 3 independent experiments and scale bar represents 15 µm. One-way ANOVA followed by post-hoc pairwise comparisons with Bonferroni correction (A,B) and two-tailed two-sample t-test (C) were used to calculate significance, where, * indicates p < 0.05. www.nature.com/scientificreports/ coronary artery bypass graft surgery, from anonymised consenting patients, were exposed to acute arterial WSS, at 12 ± 0.2 dyn/cm 2 , for between 30 min to 6 h using an in-house designed bioreactor system.

Scientific
En face immunostaining. Briefly, segments of LSV were assessed for the cellular localisation of NF-κB and quantity of CCL2 after exposure to acute arterial WSS by immunostaining of en face prepared vessels (for detailed description, see Supplementary Methods). The LSV endothelium was imaged en face, using confocal microscopy (Leica, Germany) and image analysis was all performed in 3D, either using Imaris (Supplementary Figure 3) or ImageJ (Supplementary Figure 4).
Ex vivo monocyte adhesion. Following a 3-h incubation with 50 µmol/L NF-κB inhibitor, BAY11-7085, and exposure to acute arterial WSS for 6 h, LSV segments were dissected longitudinally, pinned with the luminal surface facing upwards and co-cultured, in static conditions, with 1 × 10 6 Calcein AM-labelled (10 µmol/L) THP-1 cells for 15 min. Immediately after co-culture and washing, in situ adhered monocytes and LSV segments were imaged using fluorescence microscopy (Zeiss, Germany).
Intimal RNA extraction. For extraction of RNA from LSV ECs, a technique was used which involves the flushing of the lumen of the vessel with a severe lysis buffer, in this case, Qiazol (Qiagen), to disrupt cells in the intimal layer, which were predominantly ECs 34 . Briefly, segments were cut transversely into 2-3 cm lengths and washed in ice-cold DPBS. Using a 1 mL syringe and 18-gauge unbevelled needle with the tip inserted a into the vessel lumen, the vessel was flushed with ice-cold DPBS. Finally, the lumen was quickly flushed (3-6 s) with 350 μL of ice-cold Qiazol and the eluate was collected in a 1.5 mL Eppendorf tube. This eluate was subjected to the RNA isolation protocol described later.
EC culture and shear stress. HUVECs were exposed to laminar, unidirectional shear stress (at 0.5 or 12 dyn/cm 2 to simulate venous and arterial rates of shear stress, respectively) for varying times, using parallel plate flow chambers designed in-house (Supplementary Figure 2), as described previously 35 , or maintained in static conditions. Following HUVEC dynamic culture for variable time-points, cells were subjected to analysis for immunocytochemistry, qPCR or Western Blotting.
In vitro real-time monocyte adhesion. HUVECs were exposed to acute arterial WSS using the microfluidic capillary Bioflux 200 system, which allowed for dynamic co-culture with THP-1 cells and visualisation in real-time (Supplementary video 4). Briefly, following a 3 h incubation with 20 µmol/L NF-κB inhibitor, BAY11-7085, CmDiI-labelled HUVECs were exposed to acute arterial WSS at 12 dyn/cm 2 for 4 h, after which point, Calcein-labelled THP-1 cells were introduced into the system and co-cultured, under shear stress (at 1 dyn/ cm 2 ), for a further 10 min, and imaged in real time (for detailed description, see Supplementary methods). Total number of adhered monocytes were then enumerated.
Immunocytochemistry. Briefly, HUVECs cultured on glass slides and subsequently exposed to shear stress, or maintained in static conditions, were fixed in 3% Paraformaldehyde (PFA). Following fixation, HUVECs were stained with primary antibodies against either NF-κB, CCL2 or VE-Cadherin, followed by appropriate secondary antibodies. Slides were then imaged using the Zeiss AxioObserver Z1 fluorescent microscope and automated quantification was performed with CellProfiler analysis software (for detailed description, see supplementary methods).
NF-κB activity assay. Total HUVEC lysates were used to detect NF-κB binding activity using a TransAM p65 DNA-binding ELISA kit (Active Motif, USA). The manufacturer's instructions were followed and the colourimetric reaction endpoint was read at 450 nm.

Statistical analyses.
For experiments where only two groups were analysed, data were subjected to a paired, two-tailed t-test. For experiments where more than two groups were analysed, a one-or two-way ANOVA was used depending on the number of independent variables, followed by post-hoc pairwise comparisons with Bonferroni correction for multiple comparisons. If datasets were large enough (for example for immocytochemical analyses, where 20 images per sample were analysed), normal distribution was assessed with the D' Agostino-Pearson test; all data assessed passed normality tests, as such, parametric analyses were appropriate. The cut-off value for statistical significance was 0.05. Data are presented as mean ± SEM. All statistical analysis was performed with GraphPad Prism 7.0. www.nature.com/scientificreports/