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Low shear stress upregulates the expression of fractalkine through the activation of mitogen-activated protein kinases in endothelial cells

Fractalkine (FKN) is a cytokine which plays an important role in atherosclerosis and other inflammatory diseases. Studies have shown that FKN induces integrin-independent leukocyte adhesion to primary endothelial cells under physiological flow conditions. Further, increased expression of FKN has been demonstrated in atherosclerotic lesions induced by low shear stress. However, the signal transduction mechanisms involved in low shear stress-induced FKN upregulation are not well characterized. In this study, EA.hy926 cells were subjected to varying intensity of fluid shear stress for different time durations. Further, mRNA and protein expressions of FKN were assessed by quantitative real-time PCR and Western blotting, respectively. Upregulation of FKN expression, which was induced via activation of mitogen-activated protein kinases signaling pathway under conditions of low shear stress, was studied both in the presence and absence of inhibitors. Low shear stress (∼4.58 dyne/cm2) for more than 1 h promoted FKN expression and activated the extracellular signal-regulated kinase (ERK)1/2, p38, and Jun N-terminal kinase (JNK) mitogen-activated protein kinases signaling pathways by their phosphorylation. Inhibitors of ERK1/2, p38, and JNK pathways downregulated the FKN expression. In this study, fluid shear stress affected FKN expression in endothelial cells via activation of ERK1/2, p38, and JNK in a time-dependent manner. Our findings serve to advance the theoretical basis for prevention and treatment of atherosclerosis.

Fractalkine (FKN) is a cytokine which plays an important role in atherosclerosis and other inflammatory diseases. Studies have shown that FKN induces integrin-independent leukocyte adhesion to primary endothelial cells under physiological flow conditions. Further, increased expression of FKN has been demonstrated in atherosclerotic lesions induced by low shear stress. However, the signal transduction mechanisms involved in low shear stressinduced FKN upregulation are not well characterized. In this study, EA.hy926 cells were subjected to varying intensity of fluid shear stress for different time durations. Further, mRNA and protein expressions of FKN were assessed by quantitative real-time PCR and Western blotting, respectively. Upregulation of FKN expression, which was induced via activation of mitogen-activated protein kinases signaling pathway under conditions of low shear stress, was studied both in the presence and absence of inhibitors. Low shear stress ($4.58 dyne/cm 2 ) for more than 1 h promoted FKN expression and activated the extracellular signalregulated kinase (ERK)1/2, p38, and Jun N-terminal kinase (JNK) mitogen-activated protein kinases signaling pathways by their phosphorylation. Inhibitors of ERK1/2, p38, and JNK pathways downregulated the FKN expression.
In this study, fluid shear stress affected FKN expression in endothelial cells via activation of ERK1/2, p38, and JNK in a time-dependent manner. Our findings serve to advance the theoretical basis for prevention and treatment of atherosclerosis. Blood Coagul Fibrinolysis 29:361-368 Copyright ß 2018 Wolters Kluwer Health, Inc. All rights reserved.

Introduction
Atherosclerosis is a common underlying disorder in several cardiovascular disorders, which accounts for a large proportion of overall morbidity and mortality burden. Inflammation plays a role in all phases of the atherosclerosis [1][2][3][4]. Chemokines and adhesive molecules are the main proinflammatory factors that mediate vascular inflammation. Fractalkine (FKN), an identified chemokine with both chemoattractant and adhesive functions, participates in the atherosclerotic pathological process by mediating the activation of inflammatory cells and their aggregation or adhesion to the vascular walls [5][6][7][8]. Normal vascular endothelial cells (VECs) have a negligible or nil expression of FKN, as against that in atherosclerotic lesions, in which a high expression of FKN has been reported [9][10][11]. Knockout of the FKN gene in CCR2 À/À mice resulted in a phenotypemanifesting reduced atherosclerosis [12].Endothelial injury is the initial step in the pathogenesis of atherosclerosis [13][14][15]. VECs form an interface between the blood and vessel wall, the VECs and are subjected to constant blood-flow-induced shear stress. A shear stress of 5-12 dyne/cm 2 is essential to maintain the structural and functional integrity of VECs [16]. However, fluid shear stress outside this range injures the VECs, which initiates the atherosclerosis [17]. Atheromas typically occur at bends and bifurcations in which shear stress is low and uneven. Low shear stress and oscillatory shear stress have been shown to promote atherosclerosis in animal models [18]. The alteration in hemodynamics and its effects on cell rheology may be important in the pathogenesis of atherosclerosis [19].
Mitogen-activated protein kinase (MAPK) signaling pathway is ubiquitous in mammals and is widely involved in several physiological and pathological processes [20]. There are four subfamilies of MAPKs; the most studied members are extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 proteins [21]. Many extracellular stimuli such as physical stress, inflammatory cytokines, growth factors, and bacterial complexes can activate MAPK signaling pathway [22]. Activated MAPK regulates cell proliferation, differentiation, and apoptosis through phosphorylation of nuclear factor (NF)-kB family proteins [23]. In-vitro studies have shown that MAPK signaling mediates low shear-stressinduced oxidative damage, which is crucial in promoting atherosclerosis in human VECs [24]. Furthermore, FKN is an efficient cell-adhesion protein that adheres to endothelial cells under physiological flow conditions and can be upregulated in atherosclerotic lesions induced by low shear stress [25][26][27]. However, not much is known about the signal transduction mechanisms underlying the low shear-stress-induced FKN upregulation.
In this study, EA.hy926 cells were cultured and exposed to a certain shear stress for varying durations. Expression levels of FKN mRNA and protein under shear stress were measured. Further, we also assessed the effect of specific inhibitors of ERK1/2, p38, and JNK pathways on the expression of FKN under low shear stress conditions.

Methods
Cell culture EA.hy926 cell line was purchased from Shanghai Cell Bank (Shanghai, China) and was cultured in HyClone Medium 199 (Thermo Fisher Scientific, Waltham, Massachusetts, USA), including 10% fetal bovine serum, penicillin (100 IU/ml), and streptomycin (100 mg/ml); the medium was changed every 48 h until the cells reached confluence. Then the cells were detached with 0.25% trypsin-ethylenediamine tetraacetic acid (Gibco-BRL, Grand Island, New York, USA), and the cell suspension was transferred onto Fibronectin (BD, Franklin Lakes, New Jersey, USA)-coated sterile glass slides and incubated in 37 8C humidifed incubator until the cells adhered to the wall. Subsequently, cells were serum starved for 8 h before every single experiment. For cell signaling pathway experiments, cells were pretreated with the MAPK pathway inhibitors PD98059, SB203580, and SP600125 (Sigma, Ronkonkoma, New York, USA) for 30 min before exerting the shear stress.

Quantitative real-time PCR analysis
Total RNA was extracted from EA.hy926 cells using Trizol reagent (Invitrogen, Waltham, Massachusetts, USA), and its quality was confirmed by Nanodrop 2000 (Thermo Scientific, USA). Template complementary DNA (cDNA) was synthesized from 1 mg of RNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, USA). Two microliters of the template cDNA was added to PCR reaction mixture (25 ml) containing 2 Â SYBR Green PCR master mix (12.5 ml) (QIAGEN, Germantown Maryland, USA) and the primers (10 mmol/ l) for PCR. The following primers were used (Shanghai Biological Engineering Company, Shanghai, China): FKN, The PCR conditions were as follows: one cycle of 10 min at 95 8C, followed by 40 cycles of 95 8C for 15 s, 60 8C for 30 s, and 72 8C for 30 s. Relative gene expression levels were calculated using the 2 ÀDDCt method. Experiments were performed as triplicates, including non-Reverse Transcriptase and nontemplate controls. Dissociation analysis confirmed the specificity of the reaction.

ELISA
After each experiment, the perfusate (in serum-free HyClone Medium 199) was harvested, kept at À80 8C for 48 h, and placed in a vacuum freeze-drier to reduce its volume. The whole-dried solid powder was weighed, placed in a tube, and dissolved in 1 ml distilled water and a diluted solution (200 ng/ml) of which was prepared before detection. FKN concentration was determined using a human chemotactic factor CX3CL1 (FKN) ELISA kit (USCN Life Science Inc., Wuhan, China) according to the manufacturer's instructions. The microplate reader was used at an optical density of 450 nm to measure the absorbance (Thermo Scientific Multiskan Spectrum, Vantaa, Finland).

Statistical analysis
All results were expressed as mean AE SD. Data were analyzed by one-way analysis of variance (ANOVA), least significant difference test, or by multiple ANOVA using SPSS software version 17.0 (SPSS Inc., Chicago, Illinois, USA). A value of P less than 0.05 was considered as statistically significant.

Effect of shear stress intensity on fractalkine expression
To assess the effect of shear stress intensity on FKN gene expression, a flow rate of 0-19.64 dyne/cm 2 was used for 2 h. Each slide was exposed to shear stress separately, and each experiment was repeated by using five separate slides. After each experiment, fluid was collected to measure the concentration of FKN in the perfusate. The cells were harvested and total RNA was extracted for quantitative real-time PCR (qRT-PCR). FKN mRNA expression (Fig. 1a) was significantly higher at 4.58 dyne/ cm 2 than that at other intensities of shear stress (P < 0.05, n ¼ 3) and FKN protein expression (Fig. 1b) was significantly higher at 4.58 dyne/cm 2 than that at other intensities except 15.71 dyne/cm 2 . These results indicated that FKN gene was more sensitive to a shear stress of 4.58 dyne/cm 2 .

The effect of shear stress duration on fractalkine expression
To assess the effect of the duration of exposure to shear stress on FKN expression, we exposed the cells to a fixed shear stress of 4.58 dyne/cm 2 for a range of time durations (0, 0.5, 1, 2, 3, 5, 7, 9, and 11 h). Each experiment was repeated using five separate slides. After each  experiment, fluid was collected to measure the concentration of FKN in the perfusate, and mRNA was extracted from cells for qRT-PCR. At a shear stress of 4.58 dyne/cm 2 , the FKN mRNA expression in the cells increased and reached a peak at 1 h of application of the shear stress (Fig. 2a). The FKN secreted in the perfusate increased over time, and the FKN protein level peaked at 2 h (Fig. 2b). The results of one-way ANOVA were significant (P < 0.05, n ¼ 3), which indicated that expression of FKN gene was sensitive to a shear stress of 4.58 dyne/cm 2 and that the effect also depends on the duration of exposure to shear stress.

Low shear stress activates mitogen-activated protein kinases signaling
To determine the relationship between low shear-stressinduced FKN expression and MAPK signal transduction pathways, levels of phosphorylated ERK1/2, p38, and JNK were measured in the cells exposed to low shear stress for different time intervals (0, 5, 10, 30, and 60 min). As shown in Figs. 3-5, low shear stress promoted ERK1/ 2, p38, and JNK phosphorylation in a time-dependent manner. Phosphorylation of ERK1/2 reached a peak level at 5 min and began to decline thereafter (Fig. 3a). When pretreated with PD98059 before low shear stress (4.58 dyne/cm 2 ) intervention, ERK1/2 activation was inhibited and decreased significantly when compared with control group (Fig. 3b). Highest phosphorylation of p38 was observed at 30 min (Fig. 4a) under low shear stress (4.58 dyne/cm 2 ), and this p38 activation process was inhibited when pretreated with SB203580 (Fig. 4b). JNK phosphorylation was enhanced with the extending of low shear stress exposure time and reached a peak level at 60 min (Fig. 5a); this progression was suppressed by JNKspecific inhibitor SP600125 (Fig. 5b).

Mitogen-activated protein kinase inhibition deregulated low shear-stress-induced fractalkine expression
Different MAPK inhibitors were used before exposure to shear stress to assess their effect on the mRNA expressions of FKN induced by low shear stress. Cellular RNA was extracted for qRT-PCR analysis, respectively. qRT-PCR results showed that PD98059, SB203580, and SP600125 downregulated the activation of ERK1/2, p38, and JNK in EA.hy926 cells subjected to shear stress of 4.58 dyne/cm 2 . In addition, downregulation of FKN expression was seen in stress-exposed cells as compared with that observed in the control without shear stress (Fig. 6a-c). In other words, FKN expression in the PD98059, SB203580, and SP600125 groups was considerably lower than that in the low shear stress group. The change in FKN mRNA expressions after treatment with different MAPK inhibitors indicated the involvement of MAPK signaling pathways in low shear-stress-induced FKN upregulation.

Discussion
Endothelial cell dysfunction was shown to be caused by disordered shear stress. Shear stress refers to the frictional force exerted by the flowing blood onto the vascular wall, which is thought to play a critical role in angiogenesis, vascular remodeling [29], and atherosclerosis [30].
In the current study, low shear stress (4.58 dyne/cm 2 ) was shown to affect FKN expression in endothelial cells; further, low shear stress increased ERK1/2, p38, and JNK activation in a time-dependent manner. Furthermore, this activation was inhibited by specific inhibitors, which resulted in a downregulation of the FKN expression induced by low shear stress.
VEGF, IL-8, MCP-1, and lipocalin-type prostaglandin D2. Low shear stress may also increase oxidative stress and induce subendothelial accumulation of LDL [34][35][36]. Recent studies have shown that FKN, a unique chemotactic factor, has both chemoattractant and adhesive functions. It is upregulated in low shear-stressinduced atherosclerotic lesions [25]. However, not much is known about the underlying molecular mechanisms.
FKN is the sole member of the CX3C subfamily of chemokines which exists in both membrane-bound and soluble forms [8,37]. It has been shown to be associated with a variety of inflammatory diseases [38,39]. In a recent study, Nie and Chen [40] demonstrated increased expression of FKN and its receptor CX3CR1 in the aortic-arch atherosclerotic plaques in a rabbit model of atherosclerosis. Inhibition of expression of FKN and CX3CR1 was shown to alleviate the severity of atherosclerosis [12,38,41]. These findings indicate that FKN/ CX3CR1 pair is closely involved in the pathogenesis of atherosclerosis.
We used EA.hy926 cells as an in-vitro model and examined the effect of shear stress (range, 0-19.64 dyne/cm 2 ) on FKN expression. FKN mRNA expression and protein secretion significantly increased at a shear stress level of 4.58 dyne/cm 2 . FKN protein expression in the perfusate was consistent with FKN gene expression level. FKN expression began to increase within 30 min of loading and peaked at 1 h at shear stress level of 4.58 dyne/cm 2 . In parallel with the increase in FKN expression, endothelial cells released FKN into the perfusate. In a recent study, expression of ICAM-1, an important mediator of adhesion responses, was shown to be regulated by low shear stress in a time-dependent manner. In the current study, expression of FKN, an adhesion molecule like ICAM-1, showed a similar response to low shear stress. Our results show that low shear stress may induce inflammatory response during the development of atherosclerosis. Subsequently, we investigated the mechanism by which low shear stress induced expression of FKN.
Studies have shown that low shear stress increased expression of inflammatory cytokines through activation of JNK and NF-kB. We investigated the mediatory role of the MAPK pathway in low shear-stress (4.58 dyne/cm 2 )induced FKN upregulation using relative signal pathway inhibitors and the results obtained by qRT-PCR analysis. Low shear stress promoted ERK1/2, p38, and JNK phosphorylation in a time-dependent manner, and this activation was inhibited by their corresponding inhibitors. This indicates that low shear stress may stimulate MAPK signaling pathways in endothelial cells. Of   pathways in mediating the increase in the expression of FKN induced by low shear stress, and that ERK1/2, p38, and JNK pathways were associated with endothelial dysfunction in cells exposed to shear stress.
It could be inferred that the low shear-stress-induced FKN expression promoted atherosclerosis through the above-mentioned mechanism, at sites subjected to 4.58 dyne/cm 2 of shear stress (e.g., at vascular bifurcation). However, as FKN expression was reduced under high shear stress, it may contribute to antiatherosclerotic processes. Indeed, it is conceivable that MAPK inhibitors may be used as potential antiatherosclerotic therapeutic targets. However, cellular signaling pathways typically involve a cascade of subpathways such as NF-kB and Ca 2þ signaling pathways, which increases the complexity of their interlinkages; it is very difficult to exclude factors other than the MAPK pathways. To further characterize low shear stress-induced FKN expression, other molecular mechanisms such as those involving the cytoskeleton, integrin, and ion channels should also be investigated.
In this study, exposure to low shear stress (of the order of 4.58 dyne/cm 2 ) induced a time-dependent increase in FKN synthesis and secretion by endothelial cells. Low shear stress appeared to activate ERK1/2-related, p38related, and JNK-related MAPK signaling pathways, which in turn induced upregulation of FKN expression. Inhibitors of ERK1/2, p38, and JNK pathways downregulated FKN expression induced by low shear stress. Our findings serve to advance the theoretical basis for prevention and treatment of atherosclerosis.