Shear Stress Increases ICAM-1 and Decreases VCAM-1 and E-selectin Expressions Induced by Tumor Necrosis Factor-α in Endothelial Cells
Objective— Vascular endothelial cells (ECs) are subjected to shear stress and cytokine stimulation. We studied the interplay between shear stress and cytokine in modulating the expression of adhesion molecule genes in ECs.
Methods and Results— Shear stress (20 dynes/cm2) was applied to ECs prior to and/or following the addition of tumor necrosis factor (TNF)-α. Shear stress increased the TNF-α–induced expression of intercellular adhesion molecule-1 (ICAM-1) at both mRNA and surface protein levels, but decreased the TNF-α–induced expression of vascular adhesion molecule-1 (VCAM-1) and E-selectin. Transfection studies using promoter reporter gene constructs of ICAM-1, VCAM-1, and E-selectin demonstrated that these shear stress modulations of gene expression occur at the transcriptional levels. After 24-hour preshearing followed by 1 hour of static incubation, the effect of preshearing on TNF-α–induced ICAM-1 mRNA expression vanished. The recovery of the TNF-α–induced VCAM-1 and E-selectin mRNA expressions following preshearing, however, required a static incubation time of >6 hours (complete recovery at 24 hours). Pre- and postshearing caused a reduction in the nuclear factor-κB-DNA binding activity induced by TNF-α in the EC nucleus.
Conclusions— Our findings suggest that shear stress plays differential roles in modulating the TNF-α–induced expressions of ICAM-1 versus VCAM-1 and E-selectin genes in ECs.
- endothelial cell
- intercellular adhesion molecule-1
- shear stress
- vascular adhesion molecule-1
Vascular endothelial cells (ECs) are constantly exposed to fluid shear stress, a tangential force generated by the velocity gradient in viscous fluid flow. The nature and magnitude of shear stress play a significant role in the homeostasis of the structure and function of the blood vessel. Recent evidence suggests that physiological levels of laminar shear stress modulate cellular signaling and EC function and are protective against atherogenesis.1 In human carotid and coronary arteries, atherosclerotic plaques are found in the vicinity of arterial bifurcations and bends, where the local flow is disturbed.2 In contrast, regions of artery that experience laminar non-oscillatory shear stress were protected from atherosclerosis. The cytokine tumor necrosis factor-α (TNF-α) is an important mediator of the inflammatory processes that occur during the progression of atherosclerosis.3 Produced by macrophages that infiltrate the lesion, cytokines such as TNF-α are known to induce the expression of many endothelial genes that contribute to the complex processes involved in atherogenesis.3 Well know examples include the transcriptional regulation of various adhesion molecules, such as intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin.3 In contrast to laminar shear stress, cytokines are generally considered to be proatherogenic factors.
Despite the intensive studies on the effects of fluid shear stress on ECs, the interplay of shear stress and cytokines in modulating EC gene expression and function has not fully been clarified. It has been shown that physiological levels of laminar shear stress inhibit the apoptosis of ECs induced by TNF-α and H2O2.4,5 This inhibitory effect of shear stress is mediated by signaling processes involving the glutathione redox system and NO.5 Kawai et al6 found that the application of shear stress of 24 dynes/cm2 for 24 hours augments the expression of tissue plasminogen activator (t-PA) induced by interleukin-1β (IL-1β) or TNF-α, but attenuates the secretion of plasminogen activator inhibitor-1 (PAI-1) induced by these cytokines. They further demonstrated that shear stress inhibits the TNF-α–induced endothelial expression of tissue factor gene at both mRNA and antigen levels, in a shear-intensity and exposure-time dependent manner.7 Tsao et al8 reported that prior exposure to fluid flow for 4 hours decreases the EC adhesiveness induced by cytokines and lipoprotein and that this flow effect might be attributed in part to NO.8,9 A recent study by Surapisitchat et al10 demonstrated that pre-exposing ECs to shear stress of 12 dynes/cm2 for 10 minutes inhibits cytokine-mediated activation of c-Jun NH2-terminal kinase in ECs as a result of the activation of the extracellular signal-regulated kinase1/2 signaling pathway. Since atherosclerosis is a multifactorial disease involving a complex array of contributing factors, including shear stress and cytokines, it is of interest to investigate the effect of shear stress on endothelial function in the presence of cytokine stimulation.
To investigate the interplay between shear stress and cytokine stimulation in modulating EC gene expression and function, we analyzed the TNF-α–induction of three adhesion molecules (ie, ICAM-1, VCAM-1, and E-selectin) in ECs that had been previously exposed to laminar shear stress for various periods of time (preshearing). In addition, shear stress was applied to ECs after the TNF-α administration to examine the modulating effects of postshearing on TNF-α–induced gene expression. We found that both preshearing and postshearing of ECs and their combination augment the TNF-α–induced ICAM-1 expression at mRNA and protein levels, but attenuate the TNF-α–induced VCAM-1 and E-selectin expressions at mRNA and protein levels. Our present findings clearly demonstrate that the interplays between laminar shear stress and cytokine lead to differential modulations of the EC expressions of various adhesion molecules that serve similar function in vascular biology.
Methods used in this study are available online at http://atvb.ahajournals.org.
Both Preshearing and Postshearing of ECs Augment the ICAM-1 Gene Expression and Attenuate the VCAM-1 and E-selectin Gene Expression Induced by TNF-α
Preshearing of ECs at 20 dynes/cm2, a physiological level found in arteries,11 augmented the TNF-α–induced ICAM-1 mRNA expression in a shear time-dependent manner (Figure 1 A). The normalized levels of the TNF-α–induced ICAM-1 mRNA in ECs that had been presheared for 4 and 24 hours were significantly increased as compared with the cells not presheared (P<0.05). In contrast to ICAM-1 expression, 4 hours or 24 hours of preshearing abolished the TNF-α–induced VCAM-1 mRNA expression to an extent similar to that in the control cells (P<0.01 versus ECs not presheared) (Figure 1B); however, preshearing had only a partial inhibitory effect on the TNF-α–induced E-selectin mRNA expression (P<0.05 versus ECs not presheared) (Figure 1C). These results indicate differential roles of laminar shear stress in modulating the TNF-α–induced ICAM-1 versus VCAM-1 and E-selectin gene expressions in ECs.
To further study the effects of shear stress on TNF-α–induced ICAM-1, VCAM-1, and E-selectin expressions, shear stress was applied to ECs either simultaneously with the addition of TNF-α or at 1, 2, or 3 hours after TNF-α addition. Shear stress application to ECs after the addition of TNF-α caused an augmentation of ICAM-1 mRNA expression (Figure 2 A). The longer the duration of shearing, the greater was the increase in ICAM-1 mRNA expression. When the shear stress application was started simultaneously with the TNF-α addition (ie, postshearing for 4 hours), a maximal increase in ICAM-1 mRNA level was found. In contrast to the results on ICAM-1, the TNF-α–induced VCAM-1 (Figure 2B) and E-selectin (Figure 2C) mRNA expressions in ECs were attenuated by postshearing. The largest decreases of VCAM-1 and E-selectin mRNA expressions were seen when shear stress was applied for 3 hours (starting at 1 hour after TNF-α) or 4 hours (simultaneous exposure of ECs to shear stress and TNF-α). These results demonstrate that postshearing exerts the same differential effects as preshearing on the TNF-α-induced ICAM-1 versus VCAM-1 and E-selectin gene expressions in ECs.
Combinatory Effects of Preshearing and Postshearing on TNF-α–Induced ICAM-1, VCAM-1, and E-selectin Gene Expressions in ECs
To investigate whether preshearing and postshearing exert additive effects on modulating TNF-α–induced gene expression in ECs, we also examined the mRNA expressions of ICAM-1, VCAM-1, and E-selectin under conditions where shear stress application was started 24 hours before TNF-α stimulation and continued for 4 hours until RNA sampling. In these experiments, shear stress significantly increased the TNF-α–induced ICAM-1 mRNA expression (Figure IA; available online at http://atvb.ahajournals.org), whereas the TNF-α–induced VCAM-1 (Figure IB) and E-selectin (Figure IC) expressions were inhibited. These results are similar to those of preshearing, suggesting that the effects of preshearing and postshearing on the TNF-α–induced gene expressions were not additive.
To further determine whether the shear stress modulations of TNF-α–induced ICAM-1, VCAM-1, and E-selectin expressions are transcriptional events, ECs were transiently transfected with the promoter constructs containing the promoter regions of ICAM-1 (–850 bp), VCAM-1 (–2296 bp), or E-selectin (–540) and the reporter gene luciferase, and then exposed to flow for 4 hours before and 4 hours after TNF-α addition. As shown in Figure 3, ECs treated with TNF-α for 4 hours under static condition significantly increased ICAM-1, VCAM-1, and E-selectin promoter activities by approximately 3.1-, 4.2-, and 3.2-folds compared with static control cells, respectively. Shear stress applied to ECs 4 hours before and 4 hours after TNF-α addition significantly enhanced the ICAM-1 promoter activities, but attenuated the VCAM-1 and E-selectin promoter activities induced by TNF-α (P<0.05 versus TNF-α-treated ECs without shearing for each measurement). In contrast, ECs that were transfected with the empty vector pGL2 revealed no responses to TNF-α and shear stress in comparison to the control cells (P>1). This indicates that transcription of the luciferase gene itself is not influenced by the TNF-α and shear stress treatments. These results clearly demonstrated that shear stress modulation of ICAM-1, VCAM-1, and E-selectin induction by TNF-α involves transcriptional regulation.
Effects of Shear Stress on Stability of ICAM-1, VCAM-1, and E-selectin mRNA
Results are available online at http://atvb.ahajournals.org.
Effects of Shear Stress on TNF-α–Induced Adhesion Molecule Expression on ECs
Since both preshearing and postshearing of ECs and their combination altered the TNF-α–induced ICAM-1, VCAM-1, and E-selectin gene expressions, we investigated whether these shearing conditions also resulted in changes in the TNF-α–induced surface expressions of these adhesive proteins on ECs. Flow cytometric analysis was performed with the monoclonal antibodies against ICAM-1, VCAM-1, and E-selectin, and representative data are shown in Figure 4. Under static conditions, stimulation of ECs by TNF-α (100 U/mL) for 4 hours resulted in an increase in ICAM-1, VCAM-1, and E-selectin surface expressions, with a mean fluorescence intensity of 230, 188, and 201, respectively, as compared with 97, 22, and 26 in untreated cells. Preshearing of ECs (20 dynes/cm2) for 24 hours augmented this TNF-α–induced ICAM-1 expression, with a mean fluorescence intensity of 307, but reduced the TNF-α–induced VCAM-1 and E-selectin expressions, with a mean fluorescence intensity of 19 and 93, respectively. When ECs treated with TNF-α were subjected to postshearing, augmented ICAM-1 and reduced VCAM-1 and E-selectin surface expressions were also found, with mean fluorescence intensities of 287, 68, and 112, respectively. As expected, the combination of preshearing and postshearing of ECs also enhanced ICAM-1 and attenuated VCAM-1 and E-selectin protein expressions induced by TNF-α on ECs. These results show that the differential effects of shearing (both pre- and postshearing and their combination) on TNF-α–induced ICAM-1 versus VCAM-1 and E-selectin gene expressions are accompanied by corresponding changes in their protein expressions on EC surface.
Reprogramming of ICAM-1, VCAM-1, and E-selectin Expressions in Presheared and TNF-α–Treated ECs After the Removal of Shear Stress
To analyze the reprogramming of the TNF-α–induced ICAM-1, VCAM-1, and E-selectin expressions in presheared ECs after the removal of shear stress, ECs sheared for 24 hours were either immediately treated with TNF-α for 4 hours under static conditions or subjected to static incubation for 1, 6, or 24 hours before the TNF-α treatment. As shown in Figure 5 A, TNF-α treatment given immediately at the end of 24-hour preshearing caused an increase in ICAM-1 expression. When the 24 hour-presheared ECs were subjected to 1 hour of static incubation, the TNF-α induction of ICAM-1 expression returned to the level seen in the cells not presheared (P>1), ie, the preshearing effect subsided during the 1-hour static incubation. The same result was also found in the presheared ECs subjected to static incubation for 6 or 24 hours prior to the TNF-α treatment. VCAM-1 or E-selectin expression in presheared ECs remained quiescent or downregulated in response to TNF-α after subjecting the cells to static incubation for 1 or 6 hours, as compared with the cells not presheared (Figures 5B and 5C). When the static incubation was extended to 24 hours, these cells completely regained their responsiveness of VCAM-1 and E-selectin expression to TNF-α. These results indicate that the regulatory effects of shear stress on the TNF-α–induced gene expression are time-dependent, with the subsidence of the preshearing effects occurring more rapidly for ICAM-1 than VCAM-1 and E-selectin.
Shear Stress Attenuates NF-κB-DNA Binding Activity in the Nucleus Induced by TNF-α
Since the promoter regions of the ICAM-1, VCAM-1, and E-selectin genes contain the NF-κB binding domain that has been shown to be activated by TNF-α,12–14 we investigated whether the TNF-α–induced NF-κB-DNA binding activity was influenced by shear stress. Nuclear protein extracts from ECs that had been either (1) presheared for 24 hours prior to 1 or 4 hours of TNF-α treatment or (2) treated with TNF-α under flow conditions for 1 or 4 hours were prepared for electrophoretic mobility-shift assay (EMSA). The EMSA results obtained from incubating EC nuclear protein extracts with oligonucleotides corresponding to the NF-κB binding sequences showed that TNF-α treatment caused an increase in binding activity (Figure 6). Preshearing of ECs before TNF-α treatment or the simultaneous exposure of ECs to shear stress and TNF-α caused a decrease in NF-κB-DNA binding activities. This binding was specific for NF-κB, because it was abolished by coincubation of nuclear proteins with 20-fold unlabeled oligonucleotides. This specificity was further substantiated by the supershifting in gel mobility of the NF-κB-oligonucleotide complex after preincubation of nuclear proteins with an antibody to p65.
Our present study provides several lines of evidence that the physiological level of laminar shear stress plays differential roles in mediating the TNF-α–induced ICAM-1 and VCAM-1 and E-selectin expressions in ECs. First, both preshearing and postshearing of ECs and their combination augmented the TNF-α–induced ICAM-1 mRNA expression, but decreased the TNF-α–induced VCAM-1 and E-selectin expressions. Second, shear stress increased the ICAM-1 and decreased the VCAM-1 and E-selectin promoter activities induced by TNF-α, whereas the mRNA stability of these adhesion molecules was not affected by shear stress (Figures II and III, available online at http://atvb.ahajournals.org). Third, these differential effects of shear stress on the TNF-α–induced ICAM-1 versus VCAM-1 and E-selectin protein expressions were shown by flow cytometric analysis. Fourth, when ECs that had been presheared for 24 hours were subjected to 1 hour of static incubation, their TNF-α–induced ICAM-1 mRNA expression returned to the same level as in the cells not presheared. The recovery of the TNF-α–induced VCAM-1 and E-selectin mRNA expressions, however, required a longer static incubation time (complete recovery at 24 hours). These results suggest that the regulatory effects of shear stress on the TNF-α–induced gene expressions are transient and that the effect lasts for a shorter period for the augmentation of ICAM-1 than the suppression of VCAM-1 and E-selectin. Finally, the TNF-α-induced NF-κB-DNA binding activity was reduced in the nucleus of ECs subjected to either preshearing or postshearing. This finding suggests that shear stress plays a role in modulating the cytokine-induced signal transduction, especially at the transcriptional level.
The effect of preshearing on the TNF-α–induced ICAM-1 expression is a function of the duration of preshearing. This time dependence might explain the difference between our results on the enhanced TNF-α–induced ICAM-1 expression by 24 hours of preshearing (Figure 4) and the results from the study by Taso et al9 that preshearing of ECs for 4 hours did not increase the TNF-α–induced ICAM-1 protein expression on ECs. Our previous studies15,16 demonstrated that ECs exposed to shear stress for 24 hours induced their ICAM-1 surface protein expression. Thus, 24-hour preshearing exerts an additive effect to enhance the TNF-α–induced ICAM-1 expression (Figure 4). The inhibitory effect of preshearing on the TNF-α–induced VCAM-1 expression seems to be less dependent on the duration of preshearing. Thus, the TNF-α–induced VCAM-1 expression is inhibited by preshearing for either 4 hours9 or 24 hours (Figure 4).
The augmentation of the TNF-α–induced ICAM-1 expression by shear stress was not persistent, as indicated by the rapid return of the TNF-α–induced ICAM-1 expression to that of the static level within 1 hour after stoppage of the preshearing. Thus, a continuous application of shear stress to ECs is needed to sustain its regulatory effect on the TNF-α–induced gene expression in ECs. The recovery of the shear stress inhibition of the TNF-α–induced VCAM-1 and E-selectin expressions was slower than that of ICAM-1 after stoppage of the preshearing.
If such shear-induced modulation of EC gene expression in vitro is also operative in the arterial tree in vivo, the VCAM-1 and E-selectin genes may be rendered quiescent in the arterial ECs exposed to a constant shear flow, thus the shear stress has anti-inflammatory and antiatherogenic functions.
Our current study shows that postshearing also augmented the TNF-α–induced ICAM-1 and attenuated the TNF-α–induced VCAM-1 and E-selectin expressions in ECs. This suggests that shear stress exerts regulatory effects on the TNF-α–induced gene expressions regardless of whether it is applied before or after the addition of TNF-α. Whether the preshearing and postshearing share identical signaling pathways in ECs to modulate the TNF-α–induced gene expressions remains unclear. Our present study demonstrated that the combination of preshearing and postshearing did not exert more regulatory effect than that of preshearing alone on the TNF-α–induced ICAM-1, VCAM-1, and E-selectin mRNA expressions (Online Figure I versus Figure 1), suggesting that the effects of preshearing and postshearing on the TNF-α–induced gene expressions are not additive.
The mechanisms by which shear stress exerts differential effects on the TNF-α–induced ICAM-1 versus VCAM-1 and E-selectin expressions in ECs remain unclear. Cytokines and mechanical forces are well known mediators of gene expression in cells via activation of transcriptional factors, including NF-κB.8,17–19 Molecular cloning of ICAM-1, VCAM-1, and E-selectin has provided evidence for the presence of NF-κB-binding domains in the promoter regions that are responsible for TNF-α activation. However, the modulation of ICAM-1, VCAM-1, and E-selectin by NF-κB may differ in terms of DNA binding affinity or transcriptional activation.20 De Caterina et al17 and Khan et al21 demonstrated that NO can inhibit cytokine-induced expression of VCAM-1, but not ICAM-1, in ECs via the inhibition of NF-κB activation. Weber et al22 indicated that the antioxidants pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC) reduce the TNF-α–induced VCAM-1, but not ICAM-1, expression in ECs by preventing the TNF-α–induced NF-κB mobilization and that these treatments also inhibits the subsequent monocyte adhesion to TNF-α–treated ECs. They concluded that the induction of VCAM-1, but not ICAM-1, by TNF-α was controlled by NF-κB. Stangl et al23 reported that homocysteine, an atherogenic agent, significantly inhibits the TNF-α–induced VCAM-1 and E-selectin expressions in ECs by reducing the TNF-α–induced NF-κB activity, but caused much less pronounced inhibition of the TNF-α–induced ICAM-1 expression. These observations suggest differential effects of NF-κB activation in mediating the cytokine-induced expression of adhesion molecules (ICAM-1 versus VCAM-1) and perhaps in ECs.
In addition, TNF-α may induce NF-κB activation and adhesion molecule expression in ECs by increasing their intracellular reactive oxygen species (ROS) generation.24,25 TNF-α and ROS may exert differential effects on ICAM-1, VCAM-1, and E-selectin expressions in ECs through distinct signaling mechanisms. For example, Rahman et al26 indicated that VCAM-1 and E-selectin share a common redox-sensitive mechanism for their expressions in response to TNF-α stimulation. They suggested that TNF-α stimulation to ECs increases their intracellular ROS generation, which subsequently induces the VCAM-1 and E-selectin expressions via the activation of NF-κB. Our recent study demonstrated that both preshearing and postshearing and their combination significantly reduced the intracellular ROS levels in ECs induced by TNF-α, as indicated by the reduction of TNF-α–induced superoxide production in ECs subjected to these shearing treatments (data not shown). It is very likely that the reduction in TNF-α–induced ROS generation by shearing may cause the inhibition of NF-κB activation and consequently contribute to the inhibition of TNF-α–induced VCAM-1 and E-selection expressions in ECs. However, the TNF-α–induced ICAM-1 expression was augmented, rather than inhibited, by shear stress. Whether this shear stress augmentation of TNF-α–induced ICAM-1 expression resulted from the shear-reduced ROS generation in TNF-α–treated ECs remains unclear. Munoz et al27 demonstrated that the antioxidant PDTC, which can specifically inhibit oxidant-induced NF-κB activity, increases the ICAM-1 expression in ECs by activation of AP-1. Chen et al28 demonstrated that the TNF-α–induced expression of VCAM-1 and E-selectin was markedly enhanced by the mitochondrial glutathione depletion, whereas the TNF-α–induced ICAM-1 expression was not affected by this depletion. Roebuck et al29 found that H2O2 activates ICAM-1 transcription through AP-1/Ets element within the promoter, which is different from the mechanisms involved in the TNF-α–induced ICAM-1 expression. These observations suggest that (1) ROS may play differential roles in modulating ICAM-1 and VCAM-1 and E-selectin expressions in ECs and (2) TNF-α and ROS may exert effects on ICAM-1 expression through distinct mechanisms. The results from these studies may be used to explain the distinct role of shear stress in modulating TNF-α–induced ICAM-1 versus VCAM-1 and E-selectin expressions in ECs. The precise molecular mechanisms by which shear stress regulate cytokine-induced gene expression in ECs warrant further investigation.
In summary, our present study clearly demonstrates that shear stress increases the TNF-α–induced ICAM-1 expression in ECs at both mRNA and surface protein levels, but it decreases the TNF-α–induced VCAM-1 and E-selectin expressions at mRNA and surface protein levels. Furthermore, the effect of shear stress on VCAM-1 and E-selectin expressions has a much longer persistence than that on ICAM-1. Our findings suggest differential roles of shear stress in modulating cytokine-induced adhesion molecule expression in ECs.
This work was supported in part by grants ME-090-PP-I3, ME-091-PP-I3, and ME-092-PP-02 from the National Health Research Institutes, Taiwan, R.O.C.
- Received October 1, 2003.
- Accepted October 30, 2003.
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