| BackgroundShear stress induced by blood flow on the vessels is regarded as one of the most etiological factors in many cardio-cerebrovascular diseases diseases such as atherosclerosis, hypertension and stroke. Understanding its molecular mechanisms that lead to the development of atherosclerosis is critical for identifying strategies to limit disease progression before it leads to clinical consequences.Atherosclerotic lesions form preferentially at distinct sites in the arterial tree, especially at or near branch points, bifurcations, and inner curvatures where there is low (<1.5N/m2in humans) or oscillatory blood flow(ie, displaying directional change and boundary layer separation). In contrast, straight regions of the vasculature exhibit uniform laminar shear stress≈1.5N/m2, which is atheroprotective. Shear stress is critically important in regulating vascular physiology and pathobiology of the vessel wall via the modulation of endothelial cell function. However, The relation between shear stress and atherosclerosis is based almost exclusively on clinical observations in humans or experiment in vitro. Cheng C et al developed a perivascular shear stress modifier (referred to as a cast) that can induce changes in shear stress patterns in vivo in a straight vessel and used the model to assess the effect of in vivo alterations of shear stress on the development of atherosclerosis in apolipoprotein E-deficient mice. They found that atherosclerotic lesions developed under conditions of both lowered shear stress and vortices with oscillatory shear stress, whereas no lesions in the increased shear stress region.The c-Jun NH2-terminal kinases (JNKs)'are MAPKs traditionally considered stress-activated protein kinases. This subfamily includes JNK1and JNK2, which are ubiquitously expressed, and JNK3expressed mainly in the heart, brain, and testis(11). JNK is activated by inflammatory cytokines and environmental stresses, including UV irradiation, osmotic stress, redox stress, and mechanical stress. JNK has been shown to be activated in response to onset of laminar shear in vitro and stimulates activation of the activator protein-1transcription factor, resulting in the expression of inflammatory genes such as monocyte chemotactic protein-1and VCAM-1. JNK has also been implicated in atherosclerosis because both feeding mice the JNK inhibitor SP600125and genetic deletion of JNK2decreased atherosclerotic plaque formation in ApoE-/-mice.Meanwhile, pathogenic feature of early atherosclerosis is an inflammatory process in which the endothelium is activated by proinflammatory cytokines. Previous investigators showed that expression of vascular cell adhesion molecule (VCAM)-1and monocyte binding were increased in rabbit carotids exposed chronically to low shear stress compared with carotids exposed to normal shear stress.Because of these links between shear stress, JNK, proinflammatory cytokines and atherosclerosis, we intended to investigate the activation of JNK in the context of fluid shear stress, proatherogenic inflammatory mediators, and atherosclerosis by using the shear stress model in vivo. Objectives1. Shear stress modifier placement around the carotid arteries of apolipoprotein E (apoE)-/-mice to alter the vessel flow and evaluation of shear stress.2. Plaque characteristics induced by different shear stress were analyzed with or without inhibition of JNK activity.3. Furtherly investigating the molecular mechanism of JNK signal pathway in the shear stress induced atherosclerosis.Methods1. Shear Stress Modifier Placement and GroupingMale ApoE-deficient mice (n=84),8weeks old (25～30g), were obtained from were obtained from Jackson Laboratory (Bar Harbor, Maine). Mice were housed at a constant temperature (24℃) and given a normal diet with free access to water. To induce standardized changes of shear stress in vivo, we used a cast as described above which imposes a fixed geometry on carotid vessel wall and thereby causes a gradual stenosis resulting in different blood flow with cast. On the left side, a straight segment of carotid artery without cast has undisturbed shear stress as control. To validate the shear stress modifier, Micro-ultrasound Imaging Measurement was performed to measure velocity (Vmax) and end-systolic diameters (Ds) before and after operation (3days) calculating changes of shear stress with formula:SS=4μVmax/Ds.One week after surgery,84mice were given a high-fat Western-type diet containing0.25%cholesterol and divided into normal saline (NS) control group (n=42) and JNK inhibitor SP600125(JNKI) group (n=42). The mice of JNK-I group were peritoneal injected with JNK inhibitor SP600125(0.2mg/kg/day for each mouse), and the NS group were peritoneal injected with0.2ml NS(containing equal volume of DMSO which was used to dissolve SP600125).2. Serum lipid measurementBlood samples were taken before and after injection to monitor the levels of total cholesterol, high-density lipoprotein cholesterol, triglycerides and low-density lipoprotein cholesterol by use of an automatic biochemistry analyzer (Hitachi, Tokyo, Japan).3. Tissue preparation and histological analysisTo compare the effect of the4different shear stress fields on lesion formation in two groups, fifteen mice were humanely killed at10weeks after surgery. Tissues were harvested from mice with4%polyformaldehyde. Bilateral common carotid arteries were carefully removed and fixed in4%polyformaldehyde overnight. Then we classified the4different shear stress fields of two groups for serially cryosections after being embedded in OCT compound. Serial cryosections (6μm) were stained with hematoxylin (Sigma, St. Louis, MO, USA) and eosin (Merck, Whitehouse Station, NJ, USA); collagen and muscle fibers were stained by MASSON compound staining solution (Sigma). Oil-red0(Sigma) staining was used to identify lipid-rich lesions. Corresponding sections were stained immunofluorescence with antibodies against mouse:VCAM-1(Santa Cruz) and vWF (Abcam), images were acquired by laser scanning confocal microscope (LSM710, ZEISS, Germany).4. Quantitative real-time RT-PCR analysisThree contralateral carotid artery specimens, commensurate ten carotid artery specimens of3changed shear stress regions (LSS, HSS, OSS) were pooled for each treatment group (NS and JNK-I) for RNA isolation with TriZol (Invitrogen, Carlsbad, CA, USA). Purified RNA (1μg) was treated with DNase and reverse transcribed (RevertAid M-MulⅤ Reverse Transcriptase, Fermentas UAB, Mainz, Germany) following the manufacturer's protocol. Real-time PCR involved use of the7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Four technical replicates were run for each gene in each sample. Primers were as follows:VCAM-1(forward)5'-ATGACATGCTTGAGCCAGG-3'and (reverse)5'-GTGTCTCCTTCTTTGACACT-3'; β-actin (forward)5'-TGGACATCCGCAAAGAC-3'and (reverse)5'-GAAAGGGTGTAACGCAACTA-3'. PCR amplification was at95℃for5min,36cycles at95℃for10s, annealing at56℃for30s and elongation at72℃for30s. VCAM-1mRNA expression was normalized to that of β-actin.5. Western Blot AnalysisProteins were extracted from three contralateral carotid artery specimens (USS), commensurate nine carotid artery specimens of different shear stress (LSS, HSS, OSS) for each group (NS and JNK-I). Equal amounts of protein (2mg/ml) were separated on10%SDS-PAGE and transferred to nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). After being blocked with5%non-fat milk, the blots were washed in TBS-T three times for10min and incubated at4℃overnight with an appropriate primary antibody: rabbit anti-actin (1:500dilution), rabbit anti-total-JNK(1:1000dilution), rabbit anti-p-JNK(1:1000dilution), rabbit anti-p-p65(1:500dilution), goat anti-VCAM-1(1:200dilution). Then the blots were washed with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology;1:5000dilution) for2hour at room temperature. After3washes in TBS-T, the membrane was visualized by enhanced chemiluminescence plus reagents (Millipore).6. Statistical AnalysisData were expressed as mean±SD. SPSS for Windows16.0(SPSS Inc., Chicago, IL, USA) was used for statistical analysis. Differences of the plaque formation in different site between two groups were analyzed with the Yates corrected2-sided Fisher exact test. Correlations were determined with Spearman rank correlation test. Other parameters were compared by2-tailed Student t test or ANOVA. P<0.05was considered statistically significant.Results1. Micro-ultrasound Imaging Measurement and Serum lipid measurementDuring the experiments,3mice in the NS group and2mice in the JNK-I group died, other mice were in good health, and SP600125was well tolerated. The values of shear stress after cast implantation were markedly decreased in low regions compared with undisturbed regions (P<0.05). Levels of TC and LDL had no difference between2groups before and after injection (P>0.05). Therefore, SP600125had no significant effect on lipid levels in the circulation system, and diet or lipid levels did not contribute to the observed atherosclerotic lesion differences between2groups.2. Inhibition of JNK Activity Markedly Reduced LSS and OSS-Induced Atherosclerotic Plaque FormationLSS and OSS both induced atherosclerotic plaque formation,15of15mice (100%) in LSS region and9of15mice (62.5%) in OSS region. No atherosclerotic lesions were present in the HSS and USS region at10weeks after cast implantation. The incidence of plaque in OSS region was considerably less than the LSS region (P<0.05). In JNK-I group, incidences of plaque were2of15mice (13.3%) in LSS and OSS region and was markedly decreased when differently compared with NS group (P<0.05).In the LSS and OSS regions of NS group, atherosclerotic lesion formation was obvious, and measurements of intima-media ratio (I/M Ratio, Figure IB) showed that plaque size was significantly larger than in the undisturbed region (P<0.05). The lesions had a strikingly different morphology, atherosclerotic lesion areas in LSS region was much more extensive than those in OSS region (1.21VS.0.42; P<0.05). However in the HSS region showed no plaque, the appearance of the vessel was very similar to that of the USS region (P>0.05). In contrast, JNK-I group appeared none atherosclerotic lesion formation in the three changed shear stress and the undisturbed shear stress regions (P>0.05), and the I/M Ratio were significantly lower in OSS and LSS regions than NS group (0.02,0.07VS.0.42,1.21; P<0.05).3. Inhibition of JNK Activity Stabilized Carotid Artery Vessel Composition In Different Shear Stress RegionsWe further performed Oil-red0to identify lipid deposition, MASSON compound solution to stain collagen and muscle fibers. In NS group, HSS regions showed also no plaque formation and less lipid deposited when compared with USS regions (P>0.05), while lipids were abundantly present in lesions of both LSS and OSS regions(P<0.05). The lipid content was significantly higher in the LSS than OSS regions (45.53%VS.13.48%, P<0.05). Obviously different from NS group, there was almost no lesions developed in JNK-I group and less lipid deposition in all regions (P<0.05).LSS and OSS regions with lesions in NS group contained similar thin layers of collagen in the cap of the lesion (18.19%VS.19.41%, P>0.05) and more smooth muscle cells(28.98%VS.30.36%, P>0.05). Whereas in JNK-I group, all regions contained more collagen (17.62%VS.24.41%, P>0.05) and fewer vascular smooth muscle cells (26.17%VS.21.24%, P>0.05) when compared with the same region in NS group.4. Inhibition of JNK Activity Obviously Reduced LSS and OSS-Induced Expression of Proatherogenic Inflammatory MediatorIn NS group, VCAM-1was up-regulated by12.5-fold in the LSS region (P<0.05), meanwhile4.3-fold in the OSS region (P<0.05) when compared with USS regions. In contrast, expression in HSS region was20%of USS (P>0.05). While JNK inhibition significantly decreased OSS, LSS and HSS induced VCAM-1mRNA levels as compared with NS group (12.6%,4.4%and10.67% expression of different regions in NS group; P<0.05). VCAM-1protein in NS group was up-regulated in LSS and OSS regions (9.56and7.13-folds) compared with USS regions, as shown by Western Blot analysis. Immunofluorescence analysis showed that VCAM-1expression was up-regulated in LSS and OSS regions (5.20and4.73-folds, P<0.05) compared with USS regions. VCAM-1leves in LSS were higher than OSS(1.53VS.1.14and0.78VS.0.41; P<0.05). Inhibition of JNK significantly reduced VCAM-1protein levels in LSS, OSS and HSS regions as compared with NS group (50.33%,39.47%,51.85%; P<0.05) and Immunofluorescence analysis (56.41%and48.05%in LSS and OSS regions; P<0.05), with no changes in USS region (P>0.05).The ratio of p-JNK/t-JNK protein expressions were all reduced in OSS, LSS and HSS of JNKI group (26.79%,41.27%and36.84%of different regions in NS group; P<0.05). Furthermore, the expression of p-p65which was regarded as an inflammation transcription factor were markedly down-regulated in the JNKI group, p-p65/t-NF κB were especially decreased in OSS and LSS regions(64.71%and30.26%of those regions in NS group; P<0.05).Conclusions1. In the present study, we applied shear stress modifier on carotid vessel and caused different blood flow identified by Micro-ultrasound Imaging Measurement in apoE-/-mice.2. In vivo study demonstrated that Lowered shear stress and oscillatory shear stress were both essential conditions in plaque formation, while high shear stress and normal shear stress were atheroprotective force. Inhibition of JNK markedly reduced LSS and OSS induced plaque formation in apoE-/-mice.3. Moreover, the expression of JNK and p-p65were relevant to the expression of proatherogenic inflammatory mediators (VCAM-1). JNK and its downstream target (NFκB) may take part in LSS and OSS induced adhesion molecule (VCAM-1) expression in promoting plaque formation. BackgroundThe pathogenic feature of early atherosclerosis is an inflammatory process in which the endothelium is activated by proinflammatory cytokines. Previous investigators showed that the expression of VCAM-1and monocyte binding were increased in rabbit carotids chronically exposed to LSS as compared with carotids exposed to normal shear stress. Many studies have revealed that LSS potentiated proinflammatory activation of ECs and regions of the vasculature exposed to LSS might be susceptible to inflammation because of AP-1and NF-κ B pathways and increased expression of adhesion molecules (VCAM-1, ICAM-1)Shear stress and stretch could modulate EC functions by activating mechanosensors, signaling pathways. The JNKs are mitogen-activated protein kinases (MAPKs) traditionally considered stress-activated protein kinases. JNKs are widely activated by inflammatory cytokines and environmental stresses, including osmotic stress and mechanical stress, and are involved in regulation of proinflammatory mediators of ECs. JNK is thought to be among the major regulators of flow-dependent inflammatory gene expression in ECs in atherosclerosis.EC surfaces are equipped with numerous mechanosensors responding to shear stress. PECAM-1(CD31) has recently been shown to form an essential element of a mechanosensory complex that mediates endothelial responses to fluid shear stress. CD31plays a crucial role in the activation of the nuclear factor-κB (NF-κB) and Akt pathways and inflammatory cell accumulation during vascular remodeling. In addition, CD31contributes to atherosclerotic lesion formation in regions of disturbed flow by regulating NFκB mediated gene expression in vivo and in vitro. Cuhlmann et al demonstrated that disturbed blood flow promotes arterial inflammation by NFκB in endothelial cells via JNK-ATF2signaling.Some showed that PECAM-1-knockout mice did not activate NF-κB and downstream inflammatory genes in regions of disturbed flow. However the mechanosensing pathway was discordant, because MAPKs (ERK1/2, p38) and AKT could be phosphorylated by shear stress independently of CD31in vascular ECs. We wondered whether CD31directly transmitted mechanical force to intracellular signaling pathway MAPKs (JNK) and downstream targets. In vitro by use knockdown of CD31, we tested if it played role as one of the major mechanoreceptors in the activation of JNK followed by downstream NF-κB, VCAM-1in HUVECs treated with low shear stress.The in vivo expriments have shown the links between fluid shear stress, JNK, proinflammatory cytokines and atherosclerosis, the accordant results confirmed that low shear stress was highly proatherogenic. And we further investigated the molecule mechanism of JNK in disturbed shear stress induced vessel pathology. To test whether CD31as a sensor and NF-κB were involved in the above signaling pathway, we further validated their actions in low shear stress stimulated HUVECs.Objectives1. To test the time-dependent condition on LSS induced VCAM-1expression in HUVECs. 2. To verify the involvement of JNK in the LSS induced VCAM-1in vitro and the possibly regulating pathway by JNK-NFκB-VCAM1.3. To investigate PECAM1as one of the major mechanoreceptors in the LSS activation of JNK-NFκB-VCAM-1in HUVECs.Methods1. Cell Culture and Flow ExperimentHUVECs purchased from the American Type Cell Collection (USA) were cultured in EBM-2medium (Lonza Walkersville, Walkersville, MD) containing5%fetal bovine serum (FBS). Cells were cultured up to the4th passage for experiments. A parallel-plate flow system was used to impose low shear stress (4dyn/cm2) on HUVECs.2. siRNA Transfection In VitroTo determine the mechanism of low shear stress inducing VCAM-1expression in HUVECs, we transfected cells separately with330pmol of control or human CD31siRNA, human JNK1/2siRNA in3ml of Opti-mem Medium mixed with Lipofectamine2000for6hours and10μmol/L SP600125for15minutes before low shear stress.3. Quantitative real-time RT-PCR analysisLSS stimulated or static HUVECs were pretreated with siRNA or inhibitor. Purified RNA (1μg) was treated with DNase and reverse transcribed following the manufacturer's protocol. Four technical replicates were run for each gene in each sample. Primers were as follows: VCAM-1(forward)5'-ATGACATGCTTGAGCCAGG-3' and (reverse)5'-GTGTCTCCTTCTTTGACACT-3';6-actin (forward)5'-TGGACATCCGCAAAGAC-3' and (reverse)5'-GAAAGGGTGTAACGCAACTA-3'. PCR amplification was at95℃for5min,36cycles at95℃for10s, annealing at56℃for30s and elongation at72℃for30s. VCAM-1mRNA expression was normalized to that of β-actin.4. Immunocytochemistry analysis HUVECs were fixed in4%paraforraaldehyde and permeabilized in PBS containing0.5%Triton X-100. After being blocked with normal serum, cells and cryosections were incubated with rabbit anti-p-JNK, rabbit anti-p-NFκB p65antibody overnight at4℃. Alexa488-conjugated donkey anti-rabbit IgG were used as secondary antibodies. A drop of Prolong Gold antifade reagent with DAPI was used to seal coverslips. Images were acquired by laser scanning confocal microscopy (LSM710, Carl Zeiss, Germany) and analyzed by Image Pro Plus6.0.5. Western Blot AnalysisHUVECs for each group were collected and qual amounts of protein (2mg/ml) were separated on10%SDS-PAGE and transferred to nitrocellulose membrane. After being blocked with5%non-fat milk, the blots were washed in TBS-T3times for10min and incubated at4℃overnight with an appropriate primary antibody:rabbit anti-β-actin, rabbit anti-total-JNK (t-JNK) and rabbit anti-p-JNK, rat anti-CD31, goat anti-VCAM-1. Then the blots were washed with TBS-T and incubated with horseradish peroxidase-conjugated secondary antibody for2h at room temperature. After3washes in TBS-T, the membrane was visualized by enhanced chemiluminescence plus reagents.6. Data AnalysisData were expressed as mean±SD. SPSS for Windows16.0(SPSS Inc., Chicago, IL, USA) was used for statistical analysis.Results1. LSS induced Time-dependent Upregulation of VCAM-1in HUVECsWe tested the levels of VCAM-1exposure to LSS (4dyn/cm2) at various times (0,6,12,18,24hr) in HUVECs. The elevation of VCAM-1mRNA was detected within a short time,4.93-fold that of control at6hr (P<0.05). The expression peaked at12hr (17.58-fold) and was sustained to18hr (17.50-fold), then decreased at24hr (P<0.05). The protein detection by western blot analysis showed the same pattern (P<0.05). So we stimulated HUVECs with low shear stress for12hr in the following experiments.2. Activities of JNK and NF-κB Enhanced with Upregulation of VCAM-1Induced by LSSThe activity of JNK (p-JNK/t-JNK) was enhanced4.34-fold (P<0.05) or3.14-fold (P<0.05) by low shear stress as compared with the static control group. Simultaneously, after low shear stress for12hr, the activity of NF-κB (p-p65) was enhanced1.89-fold (P<0.05) or1.86-fold (P<0.05).3. The interference efficiency of siRNA of CD31and JNKBefore LSS treatment, HUVECs were transfected with si-CD31and negative siRNA oligo CD31, a control of si-CD31. In static and low groups, the relative quantitation of CD31with si-CD31pretreatment was significantly downregulated66.50%and69.51%when compared with respective control (0.35±0.04vs.1.00±0.10in static group;0.30+0.02vs.1.54±0.14in low groups; P<0.05).To validate the role of JNK, we used human JNK1/2siRNA (si-JNK) and SP600125to downregulate JNK level. Negative siRNA oligo JNK1/2(si-Neg) and no treatment were considered controls in the low and static groups. As compared to respective control, the inhibition efficiency of si-JNK was significantly downregulated to66.99%and69.39%of their respective control (0.43±0.09vs.1.00±0.12in static group;0.57±0.13vs.1.86±0.11in low groups; P<0.05).4. Downregulation of CD31Depressed the Enhanced of JNK Stimulated by LSSLSS could increase JNK activity compared with static control (2.37,2.74-folds of low control and low si-Neg vs. static control, P<0.05). With si-CD31pretreatment, the activity of JNK was decreased24.68%and31.16%respectively compared with the static control and low control (0.13±0.01 vs.0.54±0.03and0.39±0.03vs.1.27±0.06; P<0.05). The expression of p-JNK by immunofluorescence showed a similar pattern (P<0.05), except that the degree of p-JNK reduction by si-CD31was not statistically significant by the static control group (P>0.05).5. Inhibition of JNK Attenuated the Activity of NF-κB and VCAM-1Immunofluorescence detection of the p-p65expression was significantly increased by low shear stress without interference of JNK (si-JNK or SP600125)(2.67±0.08and2.47±0.02folds in low control and si-Neg groups vs. static control, P<0.05). However, low shear stress stimulation with si-JNK pretreatment or SP600125inhibition showed the activity of p-p65decreased, respectively to39.28%and43.54%as compared with the low control (1.05±0.06and1.16±0.05vs.2.27±0.08; P<0.05). Western blot analysis showed a similar pattern with alteration of p-p65(P<0.05). Meanwhile, VCAM-1expression showed a similar pattern (P<0.05). Changes of p-p65and VCAM-1level were significantly correlated (χ2=0.992, P<0.01).Conclusions1. In vitro experiments, we found LSS induced VCAMl expression in a time-dependent manner, and we chose to stimulate HUVECs with low shear stress for12hr in the following experiments.2. Downregulation of JNK by si-JNK or SP600125inhibition could attenuate NF-κ B activity and VCAM-1expression induced by LSS in HUVECs.3. Knockdown of CD31with siRNA reduced endothelial p-JNK and VCAM-1levels, which indicated JNK might play a critical role in LSS induced VCAMl expression at least in part by CD31dependent sensation and modulating NF-κB activity.
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