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 worldwide1. Coronary artery bypass grafting (CABG) remains the gold standard intervention in the presence of complex coronary disease, diabetes and poor ventricular function2,3,4,5,6. The long saphenous vein (LSV) is the most used conduit in many patients7,8, however, its use is complicated by considerable rates of late stenosis or occlusion due to the development of intimal hyperplasia (IH) and superimposed atherosclerosis9. IH is a chronic inflammatory process that starts with endothelial cell (EC) activation, followed by the migration and proliferation of smooth muscle cells (SMC) to the intima, which is then accompanied by alterations in extracellular matrix10 (ECM). Vascular inflammation is regulated by signalling intermediaries, including Nuclear Factor-κB, NF-κB (p65), that trigger EC expression of chemokines such as monocyte chemotactic protein-1 (CCL2) and other pro-inflammatory molecules.
The susceptibility of vessels to inflammatory processes may, in part, be related to the vascular bed from which they originate and the types of haemodynamic environment to which they are chronically exposed11. Venous ECs in situ, are adapted to chronic levels of low shear stress and, following grafting, they become exposed to shear stresses (defined as force/wall area and measured in dyn/cm2) up to tenfold higher in the arterial system12. Acute onset of increased rates of shear stress is known to alter the phenotype of venous EC; however, the mechanisms that underpin this response have not been fully elucidated. To gain better insight into the intrinsic molecular mechanisms underlying pro-inflammatory responses of veins to grafting, we investigated the impact of acute shear changes on NF-κB activation and associated vECs response in vitro, ex vivo and in vivo.
Acute arterial WSS induces pro-inflammatory responses in LSV ECs ex vivo
Freshly harvested segments of human LSV were acutely exposed to arterial shear stress, ex vivo, at 12 dyn/cm2, a rate which has previously been associated with EC activation and vascular remodelling in ex vivo venous bypass graft models12,13. We noted a significant increase in CCL2 mRNA levels in LSV ECs following exposure to acute high shear stress (acute arterial WSS) (Fig. 1A). En face immunofluorescent staining revealed that exposure of veins to acute arterial WSS increased CCL2 expression in ECs at the protein level (Fig. 1B,C and Supplementary videos 2 and 3).
NF-κB inhibition prevented a pro-inflammatory response to acute arterial WSS in LSV endothelium
To investigate the possible role of NF-κB, we analysed the expression of p65 NF-kB sub-units in LSVs exposed to arterial shear stress. acute arterial WSS enhanced p65 nuclear translocation in ECs (Fig. 1D,E and Supplementary video 1), indicating that acute arterial shear stress activates NF-kB in LSV endothelium. To investigate the function of NF-κB on pro-inflammatory responses to acute arterial WSS, a selective small-molecule NF-kB inhibitor, BAY11-7085 (BAY) was used (Supplementary Figure 9). The pre-treatment of LSV with 50 µmol/L of BAY modulated EC responses to acute arterial WSS by reducing the levels of CCL2 (Fig. 2A,B). Furthermore, NF-κB inhibition reduced acute arterial WSS induced monocyte adhesion to the LSV endothelium (Fig. 2C,D). Thus, we conclude that NF-κB inhibition can suppress the initiation of monocyte adhesion to ECs in veins under acute arterial WSS.
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 exposure13. 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).
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 model14. Grafts were maintained under low or high shear stress, for 2 and 24 h each. Log expression values (Log Fold 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 LISA15, ranked NF-κB as 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).
Vein graft disease following CABG remains a problem of serious clinical significance despite decades of surgical advances16,17. Haemodynamics influence the inflammatory process by controlling leukocyte margination and adhesive interactions and by generating shear stress, which in turn alters EC physiology18,19. 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, postoperatively20,21. 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 models13,22,23,24. 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 published14 in vivo rabbit vein graft model and DNA microarray. Differential expression analysis revealed significantly 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)17,24,25,26,27. 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 data28,29. 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 models30,31,32. 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 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 exposure33. 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.
Detailed descriptions of all materials and methods can be found in the Supplementary Methods.
Ex vivo perfusion of veins
The study was approved by the NRES Committee East of England—Norfolk ethics number (REC14/EE/1097) and all use of human tissue conformed to the principles outlined in the Declaration of Helsinki. Surplus segments of surgically prepared human long saphenous vein (LSV), resected during coronary artery bypass graft surgery, from anonymised consenting patients, were exposed to acute arterial WSS, at 12 ± 0.2 dyn/cm2, for between 30 min to 6 h using an in-house designed bioreactor system.
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 × 106 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 ECs34. 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/cm2 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 previously35, 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/cm2 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/cm2), for a further 10 min, and imaged in real time (for detailed description, see Supplementary methods). Total number of adhered monocytes were then enumerated.
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.
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.
Coronary artery bypass grafting
Ischaemic heart disease
Venous endothelial cells
Long saphenous vein
Monocyte chemoattractant protein 1
Human umbilical vein endothelial cells
Intercellular adhesion molecule 1
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This research was supported by the National Institute for Health Research Bristol Cardiovascular Biomedical Research Unit at the University Hospitals Bristol NHS Foundation Trust, NIHR Biomedical Centre at the University Hospitals Bristol NHS Foundation Trust, Above and Beyond charitable fund and the University of Bristol. The views expressed in this publication are those of the author(s) and not necessarily those of the NHS, the National Institute for Health Research or the Department of Health.
The authors declare no competing interests.
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Ward, A.O., Angelini, G.D., Caputo, M. et al. NF-κB inhibition prevents acute shear stress-induced inflammation in the saphenous vein graft endothelium. Sci Rep 10, 15133 (2020). https://doi.org/10.1038/s41598-020-71781-6
Frontiers in Physiology (2021)