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B in endothelial cells
* BHF Cardiovascular Medicine Unit, National Heart and Lung Institute, Imperial College London, London, UK; and
Institute for Nutrition Research, University of Oslo, Oslo, Norway
1Correspondence: BHF Cardiovascular Medicine, National Heart and Lung Institute, Imperial College London, Hammersmith Campus, Du Cane Rd., London W12 ONN, UK. E-mail: paul.evans{at}imperial.ac.uk
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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activates NF-
B transcription factors, which potentially exert dual functions by inducing both proinflammatory and cytoprotective transcripts. We assessed whether laminar shear stress protects EC by modulating NF-B function. Human umbilical vein EC (HUVEC) were cultured under shear stress (12 dynes/cm2 for 16 h) using a parallel-plate flow chamber or were maintained in static conditions. Comparative real-time PCR revealed that preshearing significantly alters transcriptional responses to TNF by enhancing the expression of cytoprotective molecules (Bcl-2, MnSOD, GADD45ß, A1) and suppressing proinflammatory transcripts (E-selectin, VCAM-1, IL-8). We demonstrated using assays of nuclear localization, NF-B subunit phosphorylation, DNA-binding, and transcriptional activity that NF-B is activated by TNF in presheared HUVEC. Furthermore, a specific inhibitor revealed that NF-B is essential for the induction of cytoprotective transcripts in presheared EC. Finally, we observed that NF-B can be activated in vascular endothelium exposed to laminar shear stress in NF-B-luciferase reporter mice, thus validating our cell culture experiments. We conclude that shear stress primes EC for enhanced NF-B-dependent cytoprotective responsiveness while attenuating proinflammatory activation. Thus modulation of NF-B function may underlie the atheroprotective effects of laminar shear stress.—Partridge, J., Carlsen, H., Enesa, K., Chaudhury, H., Zakkar, M., Luong, L., Kinderlerer, A., Johns, M., Blomhoff, R., Mason, J. C., Haskard, D. O., Evans, P. C. Laminar shear stress acts as a switch to regulate divergent functions of NF-B in endothelial cells.
Key Words: atherosclerosis • blood flow • proinflammatory activation • cytoprotection
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Early lesions (fatty streaks) contain monocytes and T-lymphocytes which are recruited from the circulation by adhesion to activated vascular endothelial cells (EC) (2)
. Proinflammatory cytokines such as TNF activate NF-B transcription factors which regulate vascular inflammation by inducing adhesion molecules (e.g., E-selectin, VCAM-1, ICAM-1), chemokines (e.g., IL-8) and other proinflammatory molecules in EC (2)
. The most abundant form of NF-B in EC, the p50/RelA(p65) heterodimer, activates several proinflammatory genes including E-selectin (3)
, VCAM-1 (4)
and IL-8 (5)
in response to TNF. This form also governs EC viability through the activation of cytoprotective genes, including Bcl-2, A1, GADD45ß, and manganese superoxide dismutase (MnSOD) (6
7
8)
. TNF activates other forms of NF-B in EC including c-Rel-containing dimers, which can activate IL-8, ICAM-1, and the cytoprotective gene A1 (9
10
11)
. In addition to driving proinflammatory and cytoprotective processes, NF-B also activates antiinflammatory genes, including IBs and A20, which control the resolution of inflammatory responses by feeding back to suppress NF-B (12
13
14)
. Taken together these data reveal that NF-B potentially regulates proinflammatory, antiinflammatory, and cytoprotective processes in EC.
NF-B activity is tightly regulated at multiple levels. In unstimulated cells, NF-B is sequestered in the cytoplasm through binding to inhibitory IB molecules. Proinflammatory signaling leads to phosphorylation, ubiquitination, and degradation of IB, thus releasing NF-B for nuclear translocation (15)
. RelA is phosphorylated at several serine residues in response to proinflammatory stimuli including Ser-276, Ser-468 and Ser-536, modifications that collectively regulate its capacity to bind promoters of target genes and recruit cofactors of transcription (15)
. Interestingly, recent reports have suggested that RelA phosphorylation at Ser-276 or Ser-536 is required for transcriptional activation of some NF-B-dependent promoters but not others (16
, 17)
. Thus, modification of RelA can potentially alter the pattern of gene expression in response to proinflammatory stimuli.
Blood flow exerts shear stress (mechanical drag) on vascular endothelium. Regions of the arterial tree with uniform geometry are exposed to unidirectional, undisturbed laminar flow (LF), which exerts high shear stress, whereas arches and branches are exposed to disturbed flow (DF), which exerts low shear. Atherosclerotic lesions occur predominantly at sites of low shear, whereas regions of the vasculature exposed to high shear are protected (18
, 19)
. Flow regulates atherogenesis by altering endothelial cells (EC), which detect shear stress via a trimolecular complex expressed at their surface (20)
. This and other mechanosensory receptors, convert mechanical forces into numerous biochemical signals. Prolonged high shear exerts several effects on EC that could potentially suppress atherogenesis, including inhibition of the cell cycle (21)
, suppression of prothrombotic tissue factor activity (22)
, and promotion of viability (23)
. Several lines of evidence suggest that chronic high shear also suppresses inflammation. Firstly, EC adhesion molecules are expressed at reduced levels at atheroprotected sites of the vasculature exposed to high shear (24
, 25)
. In addition, prolonged high shear can suppress the induction of adhesion molecules by proinflammatory cytokines in perfused aortae (26)
or in cultured EC (18
, 27
28
29)
.
The molecular mechanism underlying the antiinflammatory and cytoprotective effects of shear stress on EC is uncertain. Here we assessed the effects of shear stress on the capacity of TNF to activate NF-B, a central regulator of proinflammatory, antiinflammatory, and cytoprotective processes. Our study revealed that laminar shear stress alters the function of NF-B by inhibiting its capacity to induce proinflammatory molecules and simultaneously enhancing the induction of NF-B-dependent cytoprotective transcripts. Thus, alteration of NF-B function may contribute to the mechanism by which shear stress protects arteries from atherosclerosis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, human IL-1 (R&D, Minneapolis, MN, USA), anti-RelA antibodies (sc372; Santa Cruz Biotechnology, Santa Cruz, CA, USA), antiphosphoRelA antibodies (Ser-276, Ser-468 or Ser-536; Cell Signaling Technology, Danvers, MA, USA), anti-IB (Cell Signaling Technology, Danvers, MA, USA), anti-tubulin antibodies (Sigma Aldrich, St. Louis, USA), and anti-lamin B antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were obtained commercially. Other reagents were purchased from Sigma Aldrich (St. Louis, MO, USA) unless otherwise stated. Adenoviruses containing an NF-B-luciferase reporter gene (Ad-NF-B-luc) or cDNA encoding IB super-repressor (IBSR) were provided by Prof. Brian Foxwell, Imperial College London (30)
.
Cells and exposure to laminar flow
Human umbilical vein EC (HUVEC) were collected using collagenase as described previously (31)
and were used before passage six. Cells were grown on glass slides coated with 25 µg/ml fibronectin using growth medium [M199, 20% fetal calf serum, 100 U/ml penicillin G, 100 µg/ml streptomycin, 30 µg/ml endothelial cell growth supplement, and 100U/ml heparin (CP Pharmaceuticals, Wrexham, UK)] until confluent. Growth medium was replaced with basal medium [M199, 20% fetal calf serum, 100 U/ml penicillin G, 100 µg/ml streptomycin] before HUVEC were used in experiments. Confluent HUVEC cultures were exposed to high shear (12 dynes/cm2) unidirectional LF for 16–24 h using a parallel-plate flow chamber (Cytodyne, LA Jolla, CA, USA) as described previously (32)
.
Quantitative reverse-transcriptase PCR
Transcript levels were quantified by comparative real-time PCR using gene-specific primers (Table 1
). Total RNA was extracted using an RNAesy kit (Qiagen, Hilden, Germany), and 1 µg was reverse-transcribed using Superscript II (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Real-time PCR was carried out using the iCycler system (Bio-Rad, Hercules, CA, USA) and SYBR green master mix (Bio-Rad) according to the manufacturers’ instructions. Reactions were incubated at 95°C for 3 min before thermal cycling at 95°C for 10 s and 56°C for 45 s. 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 (33)
. CT was calculated for the genes of interest and for the housekeeping gene ß-actin. For each cDNA sample, the CT for ß-actin was subtracted from the CT for each gene of interest to give the parameter DCT, thus normalizing the initial amount of RNA used. The amount of each target was calculated as 2–DDCT, where DDCT is the difference between the DCT of the two cDNA samples to be compared.
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Assays of NF-B intracellular localization
Immunostaining was performed by fixing cells using 4% paraformaldehyde for 15 min prior to the application of anti-RelA antibodies and Alexafluor 568-conjugated secondary antibodies followed by laser-scanning confocal microscopy (LSM 510 META; Zeiss, Oberkochen, Germany). Image analysis was performed using Velocity software (Improvision, Coventry, UK) to calculate the ratio of RelA present in the nucleus compared to the cytoplasm.
The presence of RelA in nuclear lysates prepared using the NucBuster kit (Novagen, San Diego, CA, USA) was assessed by Western blotting using anti-RelA primary antibodies, horseradish-peroxidase-conjugated secondary antibodies (Dako, Glostrup, Denmark) and chemiluminescent detection. Total protein levels in samples were standardized by Western blotting using anti-lamin B antibodies.
Assays of NF-B DNA binding and transcriptional activity
We measured the capacity of nuclear RelA or c-Rel to bind to consensus oligonucleotides by assaying nuclear lysates prepared using the NucBuster kit (Novagen, San Diego, CA, USA) by DNA-binding ELISA (Active Motif, Carlsbad, CA, USA). NF-B transcriptional activity was measured by reporter assay in cells transduced with Ad-NF-B-luc [50 multiplicity of infection (MOI)] as described previously (34)
.
Assay of NF-B transcriptional activation in aortae
The NF-B-luciferase reporter mice used in this study were described previously (35)
. Male mice between 2 and 3 months of age were used. All experiments were performed within guidelines set out by the Federation of European Laboratory Animal Science Associations (FELASA). Aortae harvested from freshly killed mice were dissected from outer layers of fat and then cut longitudinally along the lesser curvature to expose the endothelial surface. All manipulations were performed in culture medium to maintain cell viability. NF-B-dependent luciferase activity at endothelial surfaces was assessed by measuring light production from samples immersed in cell culture medium containing 1.5 mg/ml D-luciferin substrate (Xenogen, Alameda, CA, USA) using an ultrasensitive camera consisting of an image intensifier coupled to a CCD camera (Xenogen). Images of reflected light were obtained before luminescence imaging for reference. Luminescence emitted from the aorta was integrated for 20–30 min starting 2 min after the application of luciferin. Luminesence at the arch or descending aorta was quantified using Image-Pro Plus 4.0 software (Media Cybernetics, Silver Spring, MD, USA).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. TNF induced E-selectin, VCAM-1, and IL-8 transcripts in static cultures of HUVEC but not in presheared cells (Fig. 1
A). Although the induction of ICAM-1 mRNA by TNF was enhanced by preshearing (Fig. 1A
), our data suggest that EC exposed to prolonged high shear are relatively refractory to proinflammatory activation. Preshearing suppressed VCAM-1 expression in response to IL-1 as well as TNF (Fig. 1B
), suggesting that it targets shared components of TNFR and IL-1R signaling pathways. Preshearing also suppressed the induction of IB, IBß, IB
, and A20, which are negative regulators of NF-B activity (Fig. 1A
). In contrast to the effects on E-selectin, VCAM-1, IL-8, IBs, and A20, preshearing either significantly enhanced (Bcl-2, MnSOD, and GADD45ß) or had no effect (A1) on the induction of cytoprotective transcripts by TNF (Fig. 2
). These observations therefore revealed that shear stress primes EC for enhanced expression of cytoprotective molecules in response to TNF, but greatly reduces proinflammatory activation.
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Effects of prolonged shear stress on NF-B activity
We examined whether shear stress altered the capacity of NF-B to enter the nucleus and stimulate transcription, either constitutively or in response to TNF. Immunostaining revealed that the intracellular localization of RelA varied considerably between neighboring cells cultured under static conditions, with the majority of cells containing both cytoplasmic and nuclear pools (Fig. 3
A). We observed that shear stress significantly altered the constitutive intracellular localization of RelA, which was identified exclusively in the cytoplasm of HUVEC cultured under laminar flow for 16–24 h (Fig. 3A
). These data were corroborated by Western blotting of nuclear lysates which identified RelA in nuclear fractions of static cultures but not in flow-conditioned EC (Fig. 3B
). Treatment of static or flow-conditioned cultures with TNF led to the accumulation of RelA in the nucleus as revealed by immunostaining (Fig. 3A
) or Western blotting (Fig. 3B
). Nuclear localization of RelA in response to TNF was accompanied by destabilization of IB in both static and presheared cells (Fig. 3C
).
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Given the potential role of RelA modifications in governing patterns of gene expression, we assessed whether phosphorylation of RelA in response to TNF was altered by preshearing. Western blotting of cell lysates revealed that RelA was phosphorylated at Ser-276, Ser-468, and Ser-536 in response to TNF in both static and presheared EC (Fig. 3C
). Thus exposure of EC to shear stress did not suppress subsequent phosphorylation of RelA in response to TNF.
We next examined whether shear stress regulated the capacity of NF-B to bind consensus DNA sequences in vitro. Analysis of nuclear lysates revealed that RelA possessed little or no constitutive DNA-binding activity in either static or presheared HUVEC, but could be activated by TNF in both populations (Fig. 4
). In contrast, we observed little or no activation of c-Rel in response to TNF in either static or presheared EC (Fig. 4)
. Finally, reporter gene assays revealed that NF-B transcriptional activity was strongly induced by TNF in both presheared and static cultures, although it was significantly higher in the latter (Fig. 5
). Thus, although HUVEC cultured under static conditions contained NF-B in the nucleus (Fig. 3)
, this pool of NF-B did not possess the capacity to bind DNA or drive transcription (Figs. 4
and 5)
. Full transcriptional activation of NF-B was observed only when cells were treated with TNF (Fig. 5)
. Preshearing suppressed the nuclear localization of constitutive NF-B (Fig. 3)
but only had modest inhibitory effects on NF-B transcriptional activation in response to TNF (Fig. 5)
.
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NF-B induces cytoprotective transcripts in EC exposed to laminar flow
We assessed whether NF-B was essential for the induction of cytoprotective transcripts by TNF in presheared EC by overexpressing a super-repressor version of IB (IBSR) that is not destabilized in response to TNFR signaling (30)
. Transduction of HUVEC with Ad-IBSR suppressed TNF-induced NF-B RelA nuclear localization and binding to consensus oligonucleotides in vitro (Fig. 6
A), and also reduced the induction of A1, GADD45ß, and MnSOD by TNF in presheared EC (Fig. 6B
). By contrast, cytoprotective transcripts were not altered by an empty adenovirus that served as a control. Thus, our data reveal that NF-B is activated by TNF in flow-conditioned cells and is essential for the induction of cytoprotective molecules. However, NF-B activation in flow-treated cells is not sufficient to drive proinflammatory E-selectin, VCAM-1 or IL-8 (Fig. 1)
.
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NF-B transcriptional activation in LF and DF regions of murine aorta
We used transgenic NF-B-luciferase reporter mice to assess the spatial distribution of NF-B transcriptional activity in murine aortic endothelium exposed to different forms of blood flow in vivo. Comparisons were made between NF-B activities in the aortic arch, which contains regions exposed to DF, and activities in the descending aorta, which is exposed to high rates of LF. In untreated animals, NF-B-driven luciferase activity was significantly greater in the aortic arch compared to the descending aorta (Fig. 7
). However, TNF treatment elevated NF-B-luciferase activity by similar magnitudes in both regions (Fig. 7)
. Luciferase activities in TNF-treated vessels were abolished by gentle scraping of the endothelial surface, indicating that they were generated by EC rather than by underlying tissues (data not shown). In summary, we observed that NF-B possesses weak constitutive activity in EC at the aortic arch and can be activated strongly by TNF in EC at the arch or descending aorta. Thus, our data reveal that NF-B can be activated in vascular endothelium exposed to high shear stress in vivo, which is fully consistent with our cell culture experiments.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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to induce proinflammatory transcripts is consistent with previous reports (18
, 27
28
29)
. In addition, our novel data reveal that preshearing enhances the induction by TNF of Bcl-2, MnSOD, and GADD45ß, cytoprotective molecules that maintain EC viability in the presence of noxious stimuli such as TNF and oxidative stress (6
7
8)
. We conclude that LF exerts specific modulatory effects on NF-B-driven transcriptional responses to TNF.
The underlying mechanism for simultaneous suppression of proinflammatory mRNA and enhancement of cytoprotective transcripts by shear stress remains uncertain. A previous study revealed that LF inhibited the activity of an E-selectin reporter gene, indicating that shear stress regulates E-selectin at a transcriptional level (29)
. It has been suggested that LF suppresses transcription of proinflammatory genes by inhibiting NF-B activation (29)
but this idea has not been supported by other groups (18
, 26
, 28)
. We observed that NF-B-dependent transcription could be activated by TNF in EC exposed to high rates of LF, where it played an essential role in the expression of A1, MnSOD and GADD45ß. Thus, we conclude that the suppression of proinflammatory transcripts by shear stress does not rely on a complete inhibition of NF-B transcriptional activation. In an attempt to define the level at which shear stress modulates NF-B function, we performed detailed biochemical studies of RelA, an NF-B subunit that is required for transcription of both proinflammatory and cytoprotective transcripts. Our observations suggest that preshearing does not inhibit nuclear localization of NF-B nor its capacity to bind consensus oligonucleotides in vitro in response to TNF. Furthermore, chromatin immunoprecipitation (ChIP) studies performed by our group have revealed that RelA NF-B subunits were recruited to the E-selectin promoter in response to TNF in both static and presheared EC (J. Partridge and P.C. Evans, unpublished). We suggest, therefore, that LF modulates transcriptional responses to TNF by targeting events that occur either downstream from or parallel to the recruitment of RelA to promoters of proinflammatory genes.
Recent studies have revealed that phosphorylation of RelA can potentially alter patterns of gene expression in response to particular stimuli. For example, Anrather et al. have shown that the VCAM-1 induction by TNF relies on phosphorylation of RelA at Ser-276 whereas MnSOD expression was not regulated by this modification (17)
. We reasoned therefore that shear stress may alter transcriptional responses to TNF by modulating RelA phosphorylation. However, our data revealed that RelA is phosphorylated at Ser-276, Ser-468, and Ser-536 in response to TNF in both static and presheared cultures, suggesting that shear stress may not modulate NF-B activation at this level. Further investigations are required to assess the potential effects of shear stress on other post-translational modifications of RelA including acetylation, nitrosylation, poly ADP-ribosylation, and ubiquitination.
A previous study identified several genes that can be induced by c-Rel-containing NF-B dimers in EC treated with TNF. These include IL-8 (9)
, a molecule that was suppressed by preshearing (Fig. 1)
, and also ICAM-1 (9)
, a molecule that was not suppressed by shear stress (Fig. 2)
. In contrast, other studies have suggested that c-Rel is expressed at low levels in HUVEC and displays little or no activity in response to TNF in EC (36)
. The reasons for these discrepancies are unclear. Although we observed that preshearing or TNF had little effect on c-Rel activity, we cannot exclude the possibility that c-Rel-containing NF-B dimers may be important for the activation of some genes, e.g., ICAM-1 in presheared EC exposed to proinflammatory stimuli.
We observed that LF suppressed the induction of IB, IBß, IB, and A20, molecules that collectively feedback to inhibit NF-B activation in response to TNF. However, this did not correlate with altered kinetics of NF-B activation, which were similar in static and flow-conditioned cultures exposed to TNF for 4 h or 8 h (data not shown) suggesting that other negative regulators of NF-B may have compensatory effects in flow-conditioned cells. Thus the significance of suppression of IBs and A20 by LF remains uncertain.
It is plausible that LF may suppress the induction of proinflammatory, but not cytoprotective, transcripts by inducing negative regulators of transcription that specifically target promoters of proinflammatory genes. Candidates include the transcription factor KLF2, which is induced by LF and known to sequester cofactors of NF-B transcription (37)
. However, it is currently unknown whether KLF2 targets specific gene promoters for modulation of transcriptional activity or has broader effects. Alternatively, shear stress may suppress E-selectin, VCAM-1, and IL-8 by targeting MAP kinases which function in concert with NF-B in activated EC. In support of this idea, it has been demonstrated that shear stress inhibits activation of JNK and p38 by TNF (26)
. Furthermore, although MAPKs are essential for the expression of E-selectin (38)
, VCAM-1 (39)
, and IL-8 (40)
by TNF, they do not regulate GADD45ß (41)
. Similarly, recent studies from our group have revealed that pharmacological inhibitors of JNK or p38 block the induction by TNF of E-selectin, VCAM-1, IL-8, and A20 but either have no effect or enhance the induction of A1, GADD45ß and MnSOD (H. Chaudhury and P. C. Evans, unpublished data). Thus uncoupling of NF-B from MAP kinase activation may lead to the simultaneous suppression of proinflammatory molecules and enhancement of cytoprotective transcripts in response to TNF in presheared EC.
We sought to validate the concept that EC exposed to LF in vivo can be activated for NF-B-dependent transcription using a transgenic strain of mice containing an NF-B-luciferase reporter. Reporter gene systems are advantageous in this setting because they are highly sensitive, amenable to quantification, and provide a direct measure of transcriptional activation. Indeed, transgenic reporter mice have been utilized to assess the activity of the eNOS promoter in vascular endothelium in vivo (42)
. Using NF-B luciferase reporter mice, we observed that NF-B could be activated by TNF in EC in the descending aorta, which is exposed to high rates of LF, thus validating our observations with cultured cells. Furthermore, our observations lead us to conclude that NF-B-dependent cytoprotection may contribute to the atheroprotective effects of LF in the arterial tree.
Other groups have used immunostaining and confocal microscopy to define the intracellular localization of NF-B proteins in murine EC (20
, 24)
. Tzima et al. (20)
observed that RelA localizes constitutively to the nucleus of murine aortic EC in atherosusceptible sites exposed to DF. This finding is entirely consistent with our observation that constitutive NF-B activity is significantly elevated in the aortic arch (an atherosusceptible site) compared to the descending aorta (an atheroresistant site) in NF-B reporter mice. These findings are also consistent with our observation that NF-B localized to the nucleus constitutively in EC cultured under static conditions (a surrogate for low shear). However, NF-B was not constitutively active at a transcriptional level in static EC cultures. Thus it is plausible that transcriptional activation of NF-B requires specific conditions, e.g., oscillatory flow, that are found in the aorta but not mimicked in static cultures. Interestingly, Hajra et al. (24)
detected high levels of NF-B proteins in EC at sites of low shear in the aortic arch and suggested that these regions may be primed for enhanced NF-B activation in response to proinflammatory mediators. Although we did not find evidence to support this concept, the resolution of the luminescence detection equipment that we employed may be insufficient to identify highly localized peaks in NF-B activity. Thus we anticipate adapting the imaging system for future studies to define the anatomical location of NF-B activity in the aortic arch more precisely.
In summary, we found that LF modulates the function of NF-B to enhance the induction of cytoprotective transcripts by TNF while attenuating proinflammatory activation. Thus we suggest that modulation of NF-B function may underlie the atheroprotective effects of LF in the arterial tree. Our conclusion that NF-B plays a largely cytoprotective role in EC exposed to LF has obvious implications for the therapeutic targeting of this pathway.
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ACKNOWLEDGMENTS |
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Received for publication January 10, 2007. Accepted for publication May 3, 2007.
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