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Blood, 15 May 2007, Vol. 109, No. 10, pp. 4249-4257. Prepublished online as a Blood First Edition Paper on January 23, 2007; DOI 10.1182/blood-2006-07-036020.
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY Prolonged shear stress and KLF2 suppress constitutive proinflammatory transcription through inhibition of ATF21 Department of Medical Biochemistry, Academic Medical Center, University of Amsterdam, The Netherlands; 2 Department of Pathology, Cardiovascular Research Institute Maastricht (CARIM), University of Maastricht, The Netherlands; 3 Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug Research, Gorlaeus Laboratories, Leiden, The Netherlands
Absence of shear stress due to disturbed blood flow at arterial bifurcations and curvatures leads to endothelial dysfunction and proinflammatory gene expression, ultimately resulting in atherogenesis. KLF2 has recently been implicated as a transcription factor involved in mediating the anti-inflammatory effects of flow. We investigated the effect of shear on basal and TNF-  –induced genomewide expression profiles of human umbilical vein endothelial cells (HUVECs). Cluster analysis confirmed that shear stress induces expression of protective genes including KLF2, eNOS, and thrombomodulin, whereas basal expression of TNF-–responsive genes was moderately decreased. Promoter analysis of these genes showed enrichment of binding sites for ATF transcription factors, whereas TNF-–induced gene expression was mostly NF- B dependent. Furthermore, human endothelial cells overlying atherosclerotic plaques had increased amounts of phosphorylated nuclear ATF2 compared with endothelium at unaffected sites. In HUVECs, a dramatic reduction of nuclear binding activity of ATF2 was observed under shear and appeared to be KLF2 dependent. Reduction of ATF2 with siRNA potently suppressed basal proinflammatory gene expression under no-flow conditions. In conclusion, we demonstrate that shear stress and KLF2 inhibit nuclear activity of ATF2, providing a potential mechanism by which endothelial cells exposed to laminar flow are protected from basal proinflammatory, atherogenic gene expression.
Atherosclerosis is a vascular disease with a clear focal nature, which has been shown to correlate with shear stress levels on the endothelium, resulting from specific blood flow patterns.1 Absence of shear stress due to oscillatory blood flow at arterial bifurcations and curvatures leads to endothelial dysfunction as characterized by a diminished barrier function and proinflammatory gene expression. These conditions facilitate the entry of lipids and inflammatory cells in the vascular wall, ultimately leading to the formation of an atherosclerotic plaque. On the other hand, endothelial cells exposed to high levels of shear stress maintain an atheroprotective gene expression profile and have a differentiated, quiescent phenotype.2 Transcription factors, being the integrators of various mechanical and biologic stimuli, play a pivotal role in the regulation of gene expression and determine the resulting biologic effect. A possible candidate that could be critical in the protection from atherogenesis is the shear-inducible transcription factor Krüppel-like factor 2 (KLF2). It has become clear in recent publications that KLF2 plays a significant role in maintaining an atheroprotective, quiescent endothelial phenotype.3–5 Protective genes like endothelial nitric oxide synthase (NOS3) and thrombomodulin (TM) are induced by KLF2, whereas expression of the proatherogenic monocyte chemoattractant protein 1 (CCL2) and endothelin (EDN1) is reduced
The inflammatory component in atherosclerotic pathology suggests that inflammatory gene expression is activated in the atherosclerotic vascular wall, which is mediated by transcription factors associated with inflammation. Inflammatory gene expression has been detected at vascular sites prone to atherosclerotic plaque formation.6 Furthermore, the transcription factor nuclear factor–
Given the absence of translocated, nuclear NF-
Cell culture and shear stress experiments HUVECs were isolated and cultured in medium-199 (M199; Invitrogen, Carlsbad, CA) supplemented with 20% (vol/vol) fetal bovine serum (FBS), 50 µg/mL heparin (Sigma, St Louis, MO), 6 to 25 µg/mL endothelial-cell growth supplement (ECGS; Sigma), and 100 U/mL penicillin/streptomycin (Invitrogen) as described.10 The 24-hour shear stress experiments were performed in a parallel platetype flow chamber with pulsatile flow (12 ± 7 dynes/cm2) as described,4,14 using a CellMax Quad positive-displacement pump (Cellco, Germantown, MD). Long-term shear stress exposure (6 days) was as described.4,14 In brief, HUVECs were seeded in fibronectin-coated artificial capillary cartridges (Polypropylene 70, Cellco; DIV-BBB cartridge, Flocel, Cleveland, OH) in medium containing 10 µg/mL ECGS. Cells were allowed to adhere and reach confluency overnight, with medium flowing through the extracapillary space using the CellMax Quad pump system to provide oxygen and nutrients. Next, flow was guided through the capillaries and gradually increased to correspond to a pulsatile shear stress of 19 ± 12 dynes/cm2, which was maintained over the next 6 days with intermediary medium changes. For static controls, HUVECs from the same isolate were seeded in fibronectin-coated cell-culture flasks and grown to confluency. After indicated treatments, either total RNA was obtained using Trizol reagent (Invitrogen) or nuclear extracts were made. Inflammatory cytokine stimulation during shear stress experiments
TNF- Real-time RT-PCR cDNA from 0.5 to 1 µg total RNA was synthesized according to the manufacturer's protocol (Invitrogen) and diluted 10x for gene-specific analysis with real-time reverse transcriptase–polymerase chain reaction (RT-PCR). All RT-PCR reactions were performed in a 15 µL reaction on an iCycler thermal cycler system (Bio-Rad Laboratories, Veenendaal, Netherlands). Measured mRNA level were expressed as normalized ratios compared with ribosomal phosphoprotein P0 expression levels. Gene-specific primers were designed using Beacon Designer 3 software (Premier Biosoft International, Palo Alto, CA), and optimal melting temperature was obtained using a temperature gradient reaction. Microarray probe synthesis and hybridization A human oligonucleotide library containing 18.659 gene-specific 65-mer sequences was purchased from Sigma/Compugen (Sigma, Saint Louis, MO; Compugen, San Jose, CA) and spotted on glass slides by the Microarray Department of the University of Amsterdam. Microarrays and coverslips were pretreated for 1 hour at 40°C in a buffer containing 25% formamide (vol/vol), 5x SSC, 0.2% (wt/vol) SDS, and 0.1% (wt/vol) BSA. All microarray experiments were performed using a common reference RNA composed of a pool of RNA from HUVECs, the monocytic cell line THP-1, and wholemount human carotid and aortic arteries. Up to 1 µg total RNA from samples or common reference was amplified in a single round using the T7-based Ambion MessageAmp kit (Ambion, Huntingdon, United Kingdom), with 50% of rUTP ribonucleotides replaced by aminoallyl-rUTP (Sigma). Aminoallyl-modified amplified RNA (aRNA) was labeled with either Cy3 (common reference) or Cy5 (samples) monoreactive dyes (GE Healthcare, Uppsala, Sweden). Next, labeled probes were fragmented followed by purification using the RNeasy mini kit (Qiagen, Hilden, Germany). RNA concentration as well as dye incorporation was measured using the Nanodrop Spectrophotometer (Nanodrop Technologies, Wilmington, DE). Equivalent amounts of labeled aRNA were applied to pretreated oligonucleotide microarrays in duplicate and hybridized for 16 hours at 40°C. After hybridization, slides were washed and subsequently scanned using an Agilent-II Scanner (Agilent Technologies, Palo Alto, CA). Feature extraction was done using Arrayvision 8.0 software (GE Healthcare Europe, Diegem, Belgium), and background subtracted intensities were subsequently normalized using locally weighted linear regression smoothing (LOESS) in the R software environment (LIMMA package; Bioconductor software15). Normalized data were imported into Rosetta Resolver (Rosetta Biosoftware, Seattle, WA). Microarray data analysis Re-ratio–based experiment definitions were constructed in Rosetta Resolver, followed by initial analysis consisting of marker gene verification and hierarchical clustering. For promoter analysis, data were exported from Rosetta Resolver and analyzed using whole genome rVista software.16 These calculations used a database of all transcription factor binding sites (TFBSs) conserved in the human to mouse whole genome alignment of May 2004. Locus link identifications of the genes from each group (Table S1, available on the Blood website; see the Supplemental Materials link at the top of the online article) were used as input. Calculated were the TFBSs overrepresented in 500 base pair upstream regions of these genes using all upstream regions of human genes in release 3 of the Reference Sequence (RefSeq) database (http://www.ncbi.nlm.nih.gov/RefSeq/) as outgroup (Table S2). Immunohistochemistry Human vascular tissue specimens were collected from organ donors after obtaining informed consent with approval of the Academic Medical Center (AMC) Medical Ethical Committee, and procedures conformed to the Declaration of Helsinki. Paraffin sections were deparaffinized and dried, followed by antigen retrieval by boiling the slides for 10 minutes in a 10-mM citrate buffer at pH 6.0. Primary antibody incubation with Thr71-phosphorylated ATF2 antibody was performed overnight at 4°C. Next, biotinylated secondary antibodies were used for 1 hour at room temperature, followed by incubation with streptavidin-biotin complexes conjugated to horseradish peroxidase (Dako, Glostrup, Denmark). Peroxidase substrate coloring with the VECTOR NovaRED substrate kit (Vector Laboratories, Burlingame, CA) was allowed to proceed for 10 minutes. Sections were examined using a Zeiss Axiophot microscope equipped with 2.5x/0.075 numeric aperture (NA), 5x/0.15 NA, 10x/0.3 NA, 20x/0.5 NA, 40x/0.65 NA, and 63x/0.8 NA Plan Neofluar objective lenses (Zeiss, Oberkochen, Germany) and photographed using a Sony DXC-950P digital camera (Sony, Tokyo, Japan) operated with Leica QWin software (Leica Imaging Systems, Cambridge, United Kingdom). Images were processed using Adobe Photoshop CS2 9.0 software (Adobe Systems, San Jose, CA). Overview images of entire vessels were obtained by scanning the slides on an Epson EU-35 flatbed scanner (Seiko Epson, Nagano, Japan) with a resolution of 6400 dpi and importing the images into Adobe Photoshop CS2 9.0 (Adobe Systems, San Jose, CA) using Epson TWAIN Pro software (Seiko Epson). Lentiviral KLF2 overexpression and knockdown Stable lentiviral KLF2 overexpression or knockdown experiments were performed by transducing HUVECs with lentiviral vectors expressing KLF2 cDNA or specific short hairpin RNA sequences directed against KLF2 and firefly luciferase (FLUC), as described.4,5 Nuclear extract preparation and transcription factor enzyme-linked immunosorbent assay (ELISA)
Nuclear proteins were prepared with the nuclear extract kit in accordance with the manufacturer's protocol (Active Motif Europe, Rixensart, Belgium). Transcription factor activity was determined with TransAM MAPK family kit (Active Motif Europe). In brief, 2 to 20 µg nuclear extract was added to each microtiter plate well into which an oligonucleotide with an ATF2 or NF- Immunofluorescence For immunofluorescence, mock- and KLF2-transduced HUVECs were grown on gelatin-coated glass coverslips and fixed with 4% (vol/vol) formaldehyde. Primary antibody incubation using Thr71-phosphorylated ATF2 antibody (Cell Signaling Technology, Danvers, MA) was performed overnight at 4°C. Alexa 488–labeled secondary antibodies were used for 1 hour at room temperature, followed by Hoechst nuclear staining, mounting, and fluorescence microscopy analysis. Photomicrographs were acquired using a Zeiss Axioplan 2 microscope equipped with 10x/0.3 NA, 20x/0.5 NA, 63x/1.4 NA, and 100x/1.5 NA Plan Neofluar objective lenses (Zeiss) and a Coolsnap HQ digital camera (Roper Scientific, Ottobrunn, Germany). Images were processed with Adobe Photoshop C52 9.0 software. RNA interference with duplex siRNA and Western blotting
HUVECs from 3 different isolates were grown to 80% confluency according to cell-culture methods described in "Cell culture and shear stress experiments." Cells were then changed to 1 mL per well of Optimem reduced serum medium (Invitrogen) and transfected with 325 pmol nonspecific (5'-CAGUCGCGUUUGCGACUGG-3' synthesized small interfering RNA [siRNA]; Ambion) or ATF2 siRNA (Silencer predesigned siRNA no. 16704; Ambion) using the Oligofectamine reagent (Invitrogen) according to manufacturer's protocol. After 4 hours, 2 mL M199 supplemented with 20% (vol/vol) FBS, 50 µg/mL heparin, 12.5 µg/mL ECGS, and 100 U/mL penicillin/streptomycin were added to the Optimem medium in each well. After 24 hours, medium was changed to 2 mL of full M199 containing 12.5 µg/mL ECGS, and RNA or protein was harvested after 48 hours. Where applicable, cells were stimulated with 10 ng/mL of TNF- Statistical analysis Experimental data are shown as mean of normalized ratios ± standard error of the mean (SEM) for the indicated number of experiments. The paired or unpaired Student t test was used to calculate statistical significance of expression ratios or optical densities versus controls. P values of less than .05 were considered statistically significant.
Shear stress inhibits basal but not TNF- –induced expression of inflammatory genes
The artificial capillary system14 was used to obtain gene expression profiles of endothelial cells stimulated by inflammatory cytokines, comparing their inflammatory response under prolonged, unidirectional pulsatile laminar flow to static conditions. We studied the effects of these conditions using genomewide expression profiling. Figure 1A shows a hierarchical clustering of a selection of genes modulated more than 2-fold by shear, TNF-
To gain insight into the coordinate regulation of genes within a specific group, a search for transcription factor binding elements in upstream regions of constituent genes was performed using whole genome rVISTA. This software package evaluates which TFBSs, conserved between pairs of species, are statistically significantly overrepresented in upstream regions in a group of genes.18 Using the human-mouse alignment of May 2004 and setting a 500 bp upstream search region, we found overrepresentation of distinct TFBSs in each of the 3 main groups (Table S2). Importantly, the TNF- –responsive group B is enriched in binding sites for NF-B and the AP-1 family of transcription factors (indicated in Table S2 as AP1FJ). Group B also shows enrichment of an ATF4 binding site. Furthermore, ATF3 and ATF4 binding sites are overrepresented in group A, which is composed of genes down-regulated by shear stress but not modulated by TNF-. The latter is also evident from a lack of enrichment of NF-B binding sites in group A. Thus, it seems that group A and B share ATF binding site enrichment as well as down-regulation of unstimulated basal expression by shear stress. In group C, composed predominantly of shear induced genes, enrichment for AP-1 and NRF2 binding sites is detected. Evidently, there seems to be a clear distinction in identity of different members of the AP-1/ATF families in the different clusters. Detailed inspection showed this to be based on subtle sequence differences, because homodimers or heterodimers of the ATF family bind the cAMP response element (CRE) 5'-TGAC'GTCA-3', which differs by 1 nucleotide from the consensus AP-1 binding site 5'-TGAC'GTCA-3'.19,20 One of the most studied members of the ATF transcription factor family, ATF2, is crucial for cytokine-induced expression of E selectin in endothelial cells.21,22 Furthermore, the ATF2 transcription factor has been described to be constitutively expressed,19 including in HUVECs,23 which makes it a prime target through which down-regulation of unstimulated basal expression of group A and B genes by shear stress could be mediated. In contrast to this, key members of the AP-1 family, c-Jun and c-Fos, are known to be inducible transcription factors at the expression level in response to growth factors, cytokines, and stress.24 ATF3 and ATF4 also are inducible transcription factors, acting mostly through increased expression in response to endoplasmic reticulum (ER) stress,25 with ATF3 having almost no detectable levels in unstimulated endothelial cells.23 We validated this in our microarray expression profiles from unstimulated endothelial cells. Signal intensity levels for ATF3 and ATF4 are around or below reliable detection levels, whereas the ATF2 signal is well above this threshold. The constitutive expression of ATF2 in unstimulated endothelial cells and its key role in proinflammatory signaling prompted us to further investigate its role in the atheroprotective effect of shear stress. Human lesional endothelial cells are positive for phosphorylated ATF2 To our knowledge, ATF2 has not been described in the context of shear stress and/or atherosclerosis; therefore, its potential physiological relevance was first assessed. The presence of active, phosphorylated ATF2 in endothelial cells from healthy or early atherosclerotic lesions was probed by immunohistochemistry (Figure 2). After determining the presence of a continuous layer of endothelial cells by CD31 staining in human iliac and carotid arteries, a clear and consistent positive signal for phosphorylated ATF2 could be seen in endothelial cells overlying early atherosclerotic lesions. In contrast, endothelium overlying morphologically healthy vessel wall is completely devoid of phosphorylated ATF2, although a strong positive signal for phosphorylated ATF2 was present in the media, presumably in smooth muscle cells.
Shear stress suppresses nuclear levels of activated ATF2 via KLF2 Based on the promoter analysis and immunohistochemical data described in the 2 previous headings of "Results," we investigated the role of ATF2 in the modulation of gene expression by shear stress in more detail. For this purpose, we used an enzyme-linked immunosorbent assay (ELISA)–based assay that measures nuclear levels of activated transcription factors that are able to bind to oligonucleotides containing their cognate DNA binding sites. Nuclear extracts from HUVECs exposed to pulsatile flow show a clear reduction in levels of phosphorylated ATF2, most prominently after 5 days of shear (Figure 3A). Stable lentiviral overexpression of KLF2 for 7 days resulted in a suppression of nuclear activated ATF2 to a level similar to that reached by prolonged shear (Figure 3B). The reduced nuclear levels of phosphorylated ATF2 were not due to a change in total ATF2 levels, because neither shear stress nor KLF2 overexpression altered ATF2 mRNA expression compared with static or mock controls, as measured by RT-PCR (Figure S1). Knockdown of KLF2 using lentivirally delivered siRNA abrogates the shear stress–mediated inhibition of ATF2, implicating a direct dependence on KLF2 in this observation (Figure 3C). Even under static conditions, KLF2 siRNA increases ATF2 levels significantly compared with a control siRNA against FLUC. The latter observation correlates with an increase in expression levels of several proinflammatory genes in static cells transduced with KLF2 siRNA compared with mock siRNA (Figure S2).
KLF2 overexpression inhibits TNF- –induced nuclear activation of ATF2
To further investigate the mechanism by which shear stress mediates its effects on ATF2, we measured nuclear activation of ATF2 in cells overexpressing lentiviral KLF2. KLF2 has been shown to be the causal factor in shear-mediated inhibition of several genes involved in inflammation and vascular tone.3–5,13 Figure 4A shows that nuclear ATF2 was potently suppressed by KLF2 compared with mock-transduced cells in the unstimulated control. Furthermore, TNF-
Phosphorylated ATF2 is excluded from the nucleus by KLF2 Because the activated transcription factor assay cannot discern between the degree of nuclear localization and the degree of phosphorylation, immunofluorescence was used to clarify this issue. Fluorescent staining of phosphorylated ATF2 in HUVECs showed a clear and predominant cytoplasmic localization of phosphorylated ATF2 in KLF2-overexpressing cells (Figure 5B), whereas mock transduction resulted in exclusive nuclear staining of phosphorylated ATF2 (Figure 5A). These observations were confirmed by an additional nuclear staining with Hoechst (Figure 5C-D), which led to merged pictures (Figure 5E-F) clearly identifying the major effect of KLF2 on the localization of phosphorylated ATF2. Quantification of the immunofluorescence data from 2 independent experiments show a clear reduction in pATF2-positive nuclei from 85% in mock cells to 25% in KLF2 transduced cells (Figure 5G).
ATF2 knockdown leads to reduction of basal and TNF-induced proinflammatory gene expression levels
Having shown that ATF2 is inhibited by shear stress via KLF2, we next investigated the effect of direct inhibition of ATF2 on proinflammatory gene expression employing duplex siRNA against ATF2. A solid knockdown of ATF2 mRNA and protein levels by about 80% was achieved in HUVECs 48 hours after addition of ATF2 siRNA (Figure 6A-C). The siRNA-mediated inhibition of ATF2 was equally potent in vehicle- and TNF-
There is still no definitive explanation for the cause of the moderate proinflammatory status of endothelium in the absence of flow in vitro or at disturbed flow regions of the vasculature in vivo. In this study we focused on noncytokine-induced gene expression as modulated by prolonged shear stress. The promoters of genes that are down-regulated by prolonged exposure to shear stress show a clear enrichment for ATF binding sites (Figure 1A-D). Our present results show that both shear stress and the shear-induced transcription factor KLF2 inhibit the activity of the constitutively expressed proinflammatory transcription factor ATF2 by inhibiting its nuclear translocation in HUVECs (Figures 3 and 5). Furthermore, constitutive and cytokine-induced expression of the panel of inflammatory genes, several adhesion molecules, and chemokines5 is indeed sensitive to ATF2 activity as shown by siRNA-mediated knockdown (Figure 6C), and their expression is suppressed by both shear stress and KLF2 (Figures 1 and 3). Interestingly, knockdown of residual KLF2 results in a further increase of constitutive expression of these genes under static conditions (Figure S2). Together, these data strongly suggest that blunting of ATF2 activity is one prominent mechanism by which shear stress and KLF2 inhibit the constitutive proinflammatory gene expression observed in the absence of biomechanical stimulation of endothelium. The physiological relevance of these in vitro data is supported by our novel observation that endothelial cells overlying human early atherosclerotic plaques have vastly increased levels of phosphorylated ATF2 compared with healthy endothelium (Figure 2). Interestingly, we consistently observed a much stronger staining for phosphorylated ATF2 in medial smooth muscle cells, located directly underneath the endothelial layer in plaquefree areas, compared with neointimal smooth muscle cells. This finding demonstrates for the first time a differential expression of activated, phosphorylated transcription factor ATF2 between medial and intimal smooth muscle cells, suggesting that it would be one of the molecular mediators of the phenotypic change undergone by smooth muscle cells during plaque formation. Together, these findings warrant further detailed investigation into the role of ATF2 during in vivo atherosclerosis.
The effect of shear stress on basal and TNF-
KLF2 has been shown to control multiple processes that maintain a healthy, functional endothelium and confer protection from initiation of atherosclerosis.3,5,14 For cluster group C, real-time PCR validated that shear stress increased KLF2, NOS3, and TM mRNA expression compared with static conditions and that these genes were inhibited by TNF-
Recruitment of CBP/p300 has recently been implicated in the KLF2-mediated inhibition of cytokine-induced gene expression via NF- In conclusion, our in vitro results show a prominent role for the activated transcription factor ATF2 in basal but also inducible inflammatory transcription in endothelial cells, with endothelial activated ATF2 being present in vivo only at lesional areas of the human vasculature. This proinflammatory status is repressed by both shear stress and KLF2, which are also able to inhibit the nuclear activity of the transcription factor ATF2 in vitro. This strongly suggests that shear stress through KLF2 inhibits proinflammatory gene expression in regions of the vasculature that appear to be protected against the focal initiation of atherosclerosis.
Contribution: J.O.F. designed and performed research, analyzed data, and wrote the manuscript; J.V.v.T. and R.J.D. performed research and contributed vital experimental tools; R.A.B. and O.L.V. performed research; J.R. contributed vital experimental tools; A.-P.J.J.B., M.J.A.P.D., J.K., T.J.C.v.B., and H.P. designed research; and A.J.G.H. designed research, analyzed data, and edited manuscript. Conflict-of-interest disclosure: The authors declare no competing financial interests. Correspondence: A. J. G. Horrevoets, Rm K1-114, Department of Biochemistry, Academic Medical Center, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands; e-mail: a.j.horrevoets{at}amc.uva.nl.
This work was supported by the Netherlands Organisation for Scientific Research (NWO), The Hague (grant 050-10-014); The Netherlands Heart Foundation, The Hague (grant M93.007); and the European Union (European Vascular Genomics Network grant LSHM-CT-2003-503254).
Submitted July 18, 2006; accepted January 11, 2007.
Prepublished online as Blood First Edition Paper, January 23, 2007
DOI: 10.1182/blood-2006-07-036020
An Inside Blood analysis of this article appears at the front of this issue.
The online version of this article contains a data supplement.
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Abstract
Introduction
, significant difference (P < .01) between TNF-
