Increased Endothelial Mitogen-Activated Protein Kinase Phosphatase-1 Expression Suppresses Proinflammatory Activation at Sites That Are Resistant to Atherosclerosis
Atherosclerosis is a chronic inflammatory disease of arteries. It is triggered by proinflammatory mediators which induce adhesion molecules (eg, vascular cell adhesion molecule [VCAM]-1) in endothelial cells (ECs) by activating p38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein (MAP) kinases by phosphorylation. Blood flow influences atherosclerosis by exerting shear stress (mechanical drag) on the inner surface of arteries, a force that alters endothelial physiology. Regions of the arterial tree exposed to high shear are protected from endothelial activation, inflammation, and atherosclerosis, whereas regions exposed to low or oscillatory shear are susceptible. We examined whether MAP kinase phosphatase (MKP)-1, a negative regulator of p38 and JNK, mediates the antiinflammatory effects of shear stress. We observed that expression of MKP-1 in cultured ECs was elevated by shear stress, whereas the expression of VCAM-1 was reduced. MKP-1 induction was shown to be necessary for the antiinflammatory effects of shear stress because gene silencing of MKP-1 restored VCAM-1 expression in sheared ECs. Immunostaining revealed that MKP-1 is preferentially expressed by ECs in a high-shear, protected region of the mouse aorta and is necessary for suppression of EC activation at this site, because p38 activation and VCAM-1 expression was enhanced by genetic deletion of MKP-1. We conclude that MKP-1 induction is required for the antiinflammatory effects of shear stress. Thus, our findings reveal a novel molecular mechanism contributing to the spatial distribution of vascular inflammation and atherosclerosis.
Early atherosclerotic lesions (fatty streaks) contain monocytes and T lymphocytes that are recruited from the circulation by adhesion to activated vascular endothelial cells (ECs).1 Proinflammatory mediators activate both nuclear factor (NF)-κB and mitogen-activated protein (MAP) kinase signaling pathways, which cooperate to induce proinflammatory proteins such as vascular cell adhesion molecule (VCAM)-1.2 Activation of c-Jun N-terminal kinase (JNK) and p38 MAP kinases by proinflammatory agents requires the activity of upstream MAP kinase kinases (eg, MKK3) and MAP kinase kinase kinases (eg, ASK1).3 Active, phosphorylated forms of JNK and p38 activate transcription factors belonging to the activator protein-1 superfamily (eg, c-Jun, activating transcription factor [ATF]-2) and other proteins through phosphorylation.3 Thus active, phosphorylated forms of JNK and p38 induce VCAM-1 expression by activating ATF-2 and c-jun transcription factors4,5 and also by enhancing VCAM-1 mRNA stability.6 Proinflammatory signaling also induces MAP kinase phosphatase (MKP)-1, a negative regulator which inactivates p38 and JNK by removing phosphate groups.7
Atherosclerosis occurs at distinct sites of the arterial tree located near branches and bends that are exposed to nonuniform hemodynamics, whereas arteries with uniform geometries are relatively protected.8,9 Recent studies revealed that proinflammatory activation of ECs is reduced in protected regions compared to atheroprone sites, thus providing a potential explanation for the differing susceptibilities of these sites to atherosclerosis.10,11 Blood flow influences atherosclerosis by exerting shear stress (mechanical drag) on vascular endothelium, which varies in time, magnitude, and direction according to vascular pulsatility and anatomy. Shear stress alters the physiology of ECs, which respond to it via mechanosensory receptors that convert mechanical forces into numerous biochemical signals.12 Several lines of evidence suggest that the spatial distribution of vascular inflammation and atherosclerosis in the arterial tree is regulated by shear stress. Firstly, the magnitude of shear stress at arterial walls is inversely correlated with their susceptibility to atherosclerosis. For example, analysis of the murine aorta8 or human carotid artery9 by computational fluid dynamics revealed that regions exposed to high mean shear are protected from disease, whereas regions exposed to low or oscillatory shear are not. Secondly, it has been shown that inflammation and atherosclerosis can be induced in murine carotid arteries by the application of a flow-altering device that generates spatial and temporal oscillation of the shear stress field.13 Finally, prolonged high shear suppresses proinflammatory activation in cultured ECs and in arteries perfused ex vivo.9,14–16
Materials and Methods
Reagents and Antibodies
Lipopolysaccharide (LPS) (R&D) and anti–phospho-p38 Tyr180/Thr182 (Cell Signaling Technology), anti–phospho-JNK Tyr183/Thr185 (Cell Signaling Technology), anti-p38 (Cell Signaling Technology), anti-JNK (Cell Signaling Technology), anti–CD31-fluorescein isothiocyanate (FITC) (BD Biosciences Pharmingen), anti–HO-1 (Santa Cruz Biotechnology), anti-tubulin (Sigma Aldrich), and anti–MKP-1 (Santa Cruz) antibodies were obtained commercially. The hybridoma line for anti–VCAM-1 (M/K 2.7 IgG1) was obtained from the American Type Culture Collection (Manassas, Va).
Endothelial Cells and Exposure to Flow
Human umbilical vein endothelial cells (HUVECs) were collected using collagenase and cultured as described previously.16 Confluent HUVEC cultures were exposed to steady, unidirectional shear stress (12 dyn/cm2) for 24 or 48 hours using a parallel-plate flow chamber (Cytodyne) as described previously.16
Silencing of MKP-1
RNA interference was carried out using a specific small interfering (si)RNA that is known to silence MKP-1 in ECs.17 MKP-1–specific double-stranded siRNA oligonucleotides with 3′-dTdT (5′-AAGCUGGACGAGGCCUUUGAGUU-3′; Dharmacon, Chicago, Ill) or nontargeting scrambled controls (Silencer Negative control no. 1 siRNA; Ambion, Foster City, Calif) were synthesized. Cell cultures that were 80% to 90% confluent were transfected with siRNA (5 μmol/L final concentration) by microporation (Digital Bio Technology, Seoul, Korea) following the instructions of the manufacturer and then incubated in growth medium without antibiotics for 48 hours before analysis.
Comparative Real-Time PCR
Transcript levels were quantified by comparative real-time PCR using gene-specific primers for MKP-1 (sense, 5′-CAGCTGCTGCAGTTTGAGTC-3′; antisense, 5′-AGGTAGCTCAGCGCACTGTT-3′), MKP-2 (sense, 5′-TGCAAGGTAGCATGATGAGC -3′; antisense, 5′- TGAGCCTTGGCAACATAGTG-3′), MKP-3 (sense, 5′-GGGCAAGAACTGTGGTGTCT-3′; antisense, 5′-AGCAGCTGACCCATGAAGTT-3′), heme oxygenase-1 (sense, 5′-CGAGAAGGCTTTAAGCTGGT-3′; antisense, 5′-TAGACCGGGTTCTCCTTGTT-3′), VCAM-1 (sense, GGTGGGACACAAATAAGGGTTTTGG; antisense, CTTGCAATTCTTTTACAGCCTGCC), and β-actin (sense, 5′-CTGGAACGGTGAAGGTGACA-3′; antisense, 5′-AAGGGACTTCCTGTAACAATGCA-3′). Total RNA was extracted and reverse transcribed as described previously.16 Real-time PCR was carried out using the iCycler system and SYBR green master mix (Sigma) according to the instructions of the manufacturer. Reactions were incubated at 95°C for 3 minutes before thermal cycling at 95°C for 10 seconds and 56°C for 45 seconds. Reactions were performed in triplicate. Relative gene expression was calculated by comparing the number of thermal cycles that were necessary to generate threshold amounts of product (CT) as described previously.16 CT was calculated for the genes of interest and for the housekeeping gene β-actin. For each cDNA sample, the CT for β-actin was subtracted from the CT for each gene of interest to give the parameter ΔCT, thus normalizing the initial amount of RNA used. The amount of each target was calculated as 2−ΔΔCT, where ΔΔCT is the difference between the ΔCT of the 2 cDNA samples to be compared.
Detection of MKP-1 Protein in Cultured Cells
MKP-1 expression in cultured cells was measured by immunostaining of methanol-fixed cells using rabbit anti–MKP-1 antibodies and Alexa fluor 488–conjugated secondary antibodies followed by laser-scanning confocal microscopy (LSM 510 META; Zeiss). Nuclei were identified using a DNA-binding probe with far-red emission (Draq5; Biostatus). Image analysis was performed using Zeiss LSM 510 META software to calculate average fluorescence values after subtracting background fluorescence values from cells stained with secondary antibody alone. Alternatively, levels of MKP-1 were measured in cell lysates prepared using the Nuclear Extraction Kit (Active Motif) by Western blotting using anti–MKP-1 antibodies, horse radish peroxidase-conjugated secondary antibodies, and chemiluminescent detection.
Assay of p38 Activity in Cultured Cells
Levels of active, phosphorylated p38 were measured in cell lysates prepared using the Nuclear Extraction Kit (Active Motif) by ELISA (Biosource) and were normalized by measuring total levels of p38.
Male C57BL/6 mice between 2 and 3 months of age were used. The MKP-1 knockout mouse strain (MKP-1−/− [C57BL/6]) used in this study was obtained from Bristol-Myers Squibb. All experiments were performed within guidelines set out by the Federation of European Laboratory Animal Science Associations.
En Face Staining
The expression levels of specific proteins were assessed in ECs at regions of the lesser curvature (high probability [HP] site) and greater curvature (low probability [LP] site) of murine aortae by en face staining as described previously.10,11 Animals were killed by CO2 inhalation. The physiology of vessels in vivo was assessed using aortae that were perfused in situ with PBS (at a pressure of ≈100 mm Hg) and then perfusion-fixed with 2% formalin before harvesting. Alternatively, physiological responses of vessels were assessed ex vivo using aortae that were perfused with PBS before harvesting. In the latter case, aortae were cut longitudinally along the greater curvature to expose the endothelial surface. They were then bathed in cell culture medium for varying times either in the presence or absence of proinflammatory agents and then fixed with 2% formalin. Fixed aortae from in vivo or ex vivo experiments were tested by immunostaining using specific primary antibodies and Alexa fluor 568–conjugated secondary antibodies (red). Stained vessels were then mounted before visualization of endothelial surfaces en face using confocal laser-scanning microscopy (Zeiss LSM 510 META). ECs were identified by costaining using anti-CD31 antibodies conjugated to the fluorophore FITC and nuclei were costained using Draq5 (Biostatus). Isotype-matched monoclonal antibodies raised against irrelevant antigens or preimmune rabbit sera were used as experimental controls for specific staining (online data supplement, Figure I, available at http://circres.ahajournals.org). The expression of particular proteins at each site was assessed by quantification of fluorescence intensity for multiple cells (at least 100 per site) using LSM 510 software (Zeiss).
Differences between samples were analyzed using an unpaired Student t test (*P<0.05, **P<0.01, ***P<0.001).
Expression of MKP-1 in ECs Exposed to High Shear Stress
We tested the hypothesis that high shear is protective by inducing MKPs that act as negative regulators of endothelial activation. We used cultured HUVECs exposed either to laminar shear stress (LSS; 12 dyn/cm2) or to control static conditions. We validated the approach by demonstrating that shear stress can induce heme oxygenase-1 mRNA (Figure 1A, top) and proteins (Figure 1B, top) as reported previously.18 Shear stress also induced MKP-1 transcripts, which were significantly elevated following 24 or 48 hours of shearing (Figure 1A, bottom), but had little or no effect on MKP-2 or MKP-3 (supplemental Figure II). The effects of shear stress on MKP-1 transcripts were paralleled by increases in MKP-1 protein levels in HUVECs, as demonstrated by Western blotting (Figure 1B) or by immunostaining (Figure 1C) using anti–MKP-1 antibodies.
MKP-1 Suppresses p38 Activation and VCAM-1 Expression in Sheared ECs
The function of MKP-1 in sheared ECs was assessed using an MKP-1–specific siRNA that significantly suppressed MKP-1 mRNA and protein in sheared ECs, whereas a nontargeting scrambled control had no effect (Figure 2A, compare 2 and 3). We observed that the application of laminar shear stress to HUVECs for 24 hours simultaneously reduced VCAM-1 expression (Figure 2B, top, compare 1 and 2) and suppressed p38 activation (Figure 2B, bottom, compare 1 and 2). Silencing of MKP-1 enhanced VCAM-1 expression and p38 activation in sheared ECs (Figure 2B, compare 2 and 3), indicating that MKP-1 plays an essential role in the suppression of VCAM-1 expression and p38 activation by flow.
MKP-1 Is Preferentially Expressed at a High-Shear, Protected Region of the Mouse Aorta
We used en face staining with anti–MKP-1 antibodies to examine whether MKP-1 expression in aortic ECs correlated with local hemodynamics in vivo. Computational fluid dynamic models suggest that the greater curvature of the murine aorta which has an low probability of developing atherosclerosis (LP site) is exposed to higher shear stresses than the lesser curvature, which has a high probability of developing atherosclerosis (HP site).8 Analysis of wild-type C57BL/6 mice revealed that MKP-1 was expressed at high levels in ECs in the LP region but only at low levels in the HP region (Figure 3, compare 1 and 3). Staining was not observed in parallel experiments using MKP-1−/− (C57BL/6) animals (Figure 3, compare 1 and 2), indicating that the anti–MKP-1 antibodies were specific for MKP-1. Thus, we conclude that MKP-1 is expressed at elevated levels in ECs at atheroprotected sites exposed to high shear in vivo.
MKP-1 Suppresses the Activities of p38 and JNK in Atheroprotected Regions of the Aorta
We examined whether MKP-1 regulates MAP kinase activation in protected regions of the aorta by performing en face staining using antibodies that recognize active, phosphorylated p38. In untreated wild-type mice, phosphorylated p38 was detected in the HP region but was not identified in the LP region (Figure 4, compare 1 and 3). LPS treatment elevated phosphorylated p38 levels by ≈2-fold in the HP region (Figure 4, compare 3 and 7) but did not generate detectable levels of phospho-p38 in the LP site (Figure 4, compare 5 and 7). The suppression of phosphorylated p38 in the LP region was not caused by reduced protein expression of p38, which was similar in HP and LP sites (supplemental Figure III). We used MKP-1−/− mice to examine whether MKP-1 is necessary for suppression of p38 activation in LP regions. In contrast to wild-type animals, p38 was phosphorylated constitutively and could be activated further by LPS in LP regions of MKP-1−/− animals (Figure 4, compare 1 and 2 and compare 5 and 6). Thus, we conclude that MKP-1 is essential for the inhibition of p38 activation at the LP site. We observed only modest differences in the levels of active, phosphorylated p38 in ECs at HP sites of wild-type and MKP-1−/− animals (Figure 4, compare 3 and 4 and compare 7 and 8), a finding that is consistent with our observation that MKP-1 is expressed at low levels or is absent from ECs in the HP region of wild-type mice. Interestingly, levels of phosphorylated p38 were ≈2-fold higher in the HP region compared to the LP region of MKP-1−/− mice (Figure 4, compare 6 and 8), suggesting that other negative regulators cooperate with MKP-1 to suppress MAP kinase activities at the LP site. Parallel studies revealed that activation of JNK by phosphorylation occurs constitutively and can be enhanced by LPS treatment at HP sites (supplemental Figure IV, compare 3 and 7) but is suppressed in LP regions of wild-type animals (supplemental Figure IV, compare 1 and 3 and compare 5 and 7). We concluded that MKP-1 is necessary for suppression of JNK activation in the LP site because phosphorylated JNK levels were increased by genetic deletion of MKP-1 (supplemental Figure IV, compare 5 and 6). In contrast to its effects on p38 and JNK activities, genetic deletion of MKP-1 did not alter the activation of the transcription factor NF-κB (supplemental Figure V).
MKP-1 Suppresses VCAM-1 Expression in Atheroprotected Regions of the Aorta
We next assessed whether MKP-1 regulates the spatial localization of VCAM-1 expression in the aorta. En face immunostaining revealed that basal expression of VCAM-1 in untreated wild-type animals was significantly lower in ECs at the LP region compared to the HP region (Figure 5, compare 1 and 3). Injection of LPS induced significantly higher levels of VCAM-1 in the HP region of the mouse aorta compared to the LP region (Figure 5, compare 5 and 7). Thus, our data confirm previous reports that both constitutive and LPS-inducible activation of ECs is suppressed in ECs in the LP region, which is protected from atherosclerosis.10,11 In contrast to wild-type animals, en face staining revealed that VCAM-1 was induced by LPS at similar levels in LP and HP regions of MKP1−/− animals (Figure 5, compare 5 and 6 and compare 7 and 8), indicating that MKP-1 is necessary for suppression of VCAM-1 expression at the LP site. To exclude the effects of MKP-1 genetic deletion on systemic responses to LPS, we isolated aortae from wild-type or MKP-1−/− mice and examined their responses to LPS ex vivo. We observed by en face staining that VCAM-1 was induced by LPS at similar levels in LP and HP sites in aortae of MKP-1−/− mice but was suppressed in LP sites in aortae of wild-type animals (supplemental Figure VI, compare 5 and 7 and compare 6 and 8). Thus, we conclude that it is expression of MKP-1 in the vessel wall that suppresses ECs activation at atheroprotected sites.
We have demonstrated that MKP-1 protects arteries from exhibiting a proinflammatory state by suppressing the activities of p38 and JNK MAP kinases in the vascular endothelium. Our findings complement previous studies of the murine aorta demonstrating that ECs at atheroprotected regions express relatively low levels of proinflammatory molecules, eg, Toll-like receptors19 and bone morphogenetic protein 4,20 and contain activated KLF2, which suppresses NF-κB.11,21,22 Thus, the findings presented herein and those from previous studies show that protected regions of the arterial tree resist proinflammatory activation via multiple mechanisms that suppress both MAP kinase and NF-κB signaling pathways. Interestingly, we observed that VCAM-1 expression at the atheroprotected site was restored fully by genetic deletion of MKP-1. We suggest, therefore, that although suppression of Toll-like receptor and bone morphogenetic protein 4 expression and inhibition of NF-κB activation is likely to play a role in the inhibition of EC activation at atheroprotected regions, these mechanisms are not sufficient to protect ECs in the absence of MKP-1.
We provide evidence that shear stress is an important factor in determining the expression pattern of MKP-1 in aortic ECs because, firstly, MKP-1 was induced by unidirectional shear stress in cultured ECs and, secondly, we observed endothelial expression of MKP-1 in the greater curvature of the murine aorta (protected site), which is exposed to high, unidirectional shear stress but not in the lesser curvature (susceptible site), which is exposed to relatively low and oscillatory shear.8 Recent studies have shown that shear stresses in protected regions of the aorta are ≈20 times higher in the mouse compared to humans.23 We assessed MKP-1 expressed in cultured ECs following the application of 12 dyn/cm2 shear stress, a value that approximates flow conditions in the human aorta. However, it will be interesting in future studies to assess the effects of higher shear stresses that approximate flow conditions in the murine aorta and complex waveforms on the expression of MKP-1 in ECs.
Several signaling pathways have been shown to upregulate MKP-1 in ECs, and further work is now required to identify the precise molecular mechanisms responsible for induction of MKP-1 in response to shear stress. The vascular endothelial growth factor receptor pathway is an interesting candidate because it is activated by shear12 and is known to induce MKP-1 transcription.17 It has also been demonstrated that shear stress suppresses proinflammatory signaling by promoting a reducing state via the induction of reduced forms of glutathione and thioredoxin24 and via activation of Nrf2,25 a transcription factor that induces numerous antioxidants in ECs. Given the exquisite sensitivity of MKP-1 to cellular redox,26 it is plausible that shear stress enhances the catalytic activity of MKP-1, as well as its expression levels in ECs by promoting a reducing environment.
Our finding that MKP-1 can be induced by shear stress is consistent with previous studies that revealed that shear stress can suppress the activation of JNK, p38, and downstream transcription factors and inhibit the induction of VCAM-1 and other adhesion proteins in cultured ECs and in arteries perfused ex vivo.9,14–16 Previous reports have revealed that several intermediaries in the JNK/p38 MAP kinase signaling pathway are negatively regulated by shear stress. For example, the recruitment of TRAF2 (tumor necrosis factor [TNF] receptor–associated factor 2) ubiquitin ligase to the TNF receptor signaling complex, an event that is required for JNK activation in response to TNFα, is reduced in ECs exposed to shear stress.14 In addition, shear stress reduces the activity of ASK1,27 a MAP kinase kinase kinase that functions as a positive regulator of JNK and p38, by enhancing the expression of the reduced form of thioredoxin that acts as a negative regulator of ASK1. Shear stress also induces SHP-2 (Src homology 2–containing protein tyrosine phosphatase-2), a phosphatase that suppresses JNK and p38 activation by targeting MKK3 for dephosphorylation.28 Finally, shear stress also reduces the activation of JNK by TNFα by suppressing the generation of a processed form of protein kinase Cζ that acts as a positive regulator of the JNK activation pathway.29 Our findings reveal that the suppression of MAP kinase activity by shear stress also involves the induction of MKP-1. Thus, although shear stress modulates JNK and p38 activation by targeting multiple signaling intermediaries, the induction of MKP-1 appears to be essential for full protection.
In summary, our findings suggest that high shear stress protects arteries from inflammation by inducing persistent endothelial expression of MKP-1, which suppresses the activities of p38 and JNK. MKP-1 is expressed in atherosclerotic lesions,30 and further work is now required to assess its potential role in protecting arteries from atherosclerosis.
Sources of Funding
This study was funded by the British Heart Foundation.
Original received April 27, 2008; resubmission received July 23, 2008; accepted August 13, 2008.
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