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Cardiovascular Research 2006 72(2):231-240; doi:10.1016/j.cardiores.2006.07.008
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Copyright © 2006, European Society of Cardiology

Shear stress sustains atheroprotective endothelial KLF2 expression more potently than statins through mRNA stabilization

Johannes V. van Thienena, Joost O. Fledderusa, Rob J. Dekkera, Jakub Rohlenaa, Gerben A. van IJzendoorna, Neeltje A. Kootstrab, Hans Pannekoeka and Anton J.G. Horrevoetsa,*

aDepartment of Medical Biochemistry, Academic Medical Center, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands
bDepartment of Clinical Viro-Immunology, Sanquin Research, Amsterdam, The Netherlands, and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, The Netherlands

* Corresponding author. Room K1-114, Department of Biochemistry, Academic Medical Center, Meibergdreef 15, 1105 AZ, Amsterdam, The Netherlands. Tel.: +31 0 20 5665129; fax: +31 0 20 6915519. Email address: a.j.horrevoets{at}amc.uva.nl

Received 21 April 2006; revised 21 June 2006; accepted 10 July 2006


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
Objective: The transcription factor KLF2 is considered an important mediator of the anti-inflammatory and anti-thrombotic properties of the endothelium. KLF2 is absent from low-shear, atherosclerosis-prone sites of the vascular tree but is induced by HMG-CoA reductase inhibitors (statins) in vitro. We studied KLF2-dependent induction of important determinants of the atheroprotective status of the endothelium to determine whether pharmacological intervention, e.g. by statins, can potentially replace shear stress.

Methods: Shear stress and statin effects in combination with TNF-{alpha} were determined in human umbilical vein endothelial cells by quantitative measurements of the steady-state levels and stability of mRNA for KLF2 and its downstream target genes thrombomodulin (TM) and endothelial nitric oxide synthase (eNOS).

Results: We demonstrate that prolonged shear stress has a potential that is superior to that of statins to induce the KLF2-dependent expression of eNOS and TM, especially in the presence of the pro-inflammatory cytokine tumor necrosis factor-{alpha} (TNF-{alpha}). These effects can be attributed to the sustained stabilization of KLF2 mRNA by shear, leading to an increased KLF2 protein expression and concomitant strong induction of KLF2 downstream targets. The stabilization of KLF2 mRNA is demonstrated to be dependent on signaling involving phosphoinositide 3-kinase (PI3K).

Conclusion: The stabilization of KLF2 steady-state levels, as induced by prolonged shear stress but not by statins, may be essential for sustaining the quiescent, atheroprotective status of the vascular endothelium under inflammatory conditions.

KEYWORDS Statins; Endothelial function; Gene expression; Hemodynamics; Atherosclerosis

This article is referred to in the Editorial by T. Thum and J. Bauersachs (pages 193–195) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
The vascular endothelium is the primary mediator of vessel wall homeostasis. Endothelial dysfunction is currently considered to be at the base of many diseases involving the vasculature, of which atheroslerosis is one of the most prominent examples. This complex multifactorial disease takes decades to develop into a clinically manifest entity and is considered a chronic inflammatory process, occurring in the vessel wall of large- and medium-sized muscular arteries [1]. Although many of the known risk factors for atherosclerosis are systemic in nature, atherogenesis initiates specifically at sites in the arterial tree where blood flow patterns are disturbed, due to the presence of branches or bifurcations [2,3]. The endothelial cell dysfunction resulting from exposure to turbulent flow is in clear contrast to the strong anti-inflammatory and anti-thrombotic properties displayed by cells exposed to high shear stress, essential characteristics that enable these endothelial cells to protect the underlying vessel wall from atherogenesis [4]. Studying the regulation of genes involved in endothelial cell (dys-) function in relation to shear stress is therefore pivotal for our understanding of the pathogenesis of vascular disease.

Lung Krüppel-like Factor (LKLF/KLF2) is a zinc-finger transcription factor that we have previously shown to be specifically responsive to prolonged fluid shear stress [5]. We have also shown in ex vivo human vascular tissue that KLF2 is exclusively present in the endothelium and is highly expressed in high-shear regions, correlating the expression of KLF2 to the atheroprotective force of shear stress [5,6]. KLF2 has a distinct function in mediating the anti-coagulant properties of the endothelium [7] and is importantly involved in angiogenesis [8]. Recently, we showed that KLF2 is involved in the control of vascular tone-regulating gene expression in response to prolonged shear stress [6] and that it governs many other processes that ensure proper physiological endothelial function [9]. The induction of KLF2 transcription by shear stress has been studied in great detail in cultured cells and animal models and was shown to be specific for atheroprotective flow patterns [5,6,39]. The molecular signal transduction of shear stress into induction of KLF2 gene transcription depends largely on the activity of the MEK5–ERK5–MEF2 axis [10], although involvement of PI3K-dependent chromatin remodeling and nucleolin has also been suggested [11,38].

KLF2 was shown to exhibit anti-inflammatory potential, not only in endothelium [15], but recently also in monocytes [40]. Recent reports indicate that KLF2 is also a mediator of anti-inflammatory effects of HMG-CoA reductase inhibitors (statins), a class of drugs that, independent of their cholesterol-lowering effects, confers many beneficial properties directly to the endothelium [12]. KLF2 was demonstrated to be particularly important with respect to the statin-induced expression of endothelial Nitric Oxide Synthase (eNOS) and Thrombomodulin (TM), molecules that confer important anti-inflammatory and anti-thrombotic properties to the endothelium [13,14].

In the present study, we directly compared the potentially atheroprotective gene expression regulation downstream of KLF2 with regard to its induction by prolonged shear as well as statin treatment. We found that prolonged shear stress is superior in inducing KLF2-mediated gene expression of eNOS and TM, despite similar transcriptional induction of KLF2 mRNA. Furthermore, we observed that shear-exposed endothelial cells are able to maintain higher levels of eNOS and TM in the presence of a pro-inflammatory stimulus. We now show that the underlying mechanism that may explain this differential response upon prolonged shear or statin treatment is shear-mediated stabilization of KLF2 mRNA, which involves PI3K-dependent signaling.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 
2.1. Cell culture conditions and chemicals
HUVEC were isolated as described [36] and cultured in Medium-199 (Gibco-BRL, Paisley, Scotland), supplemented with 20% (v/v) fetal bovine serum (FBS), 50 µg/ml heparin (Sigma, St. Louis, MO), 6–25 µg/ml Endothelial Cell Growth Supplement (ECGS) (Sigma), 100 U/ml penicillin/streptomycin (Gibco-BRL). The procedures conform with the principles outlined in the Declaration of Helsinki for medical research involving human material. Cells were passaged no more than 3 times before being plated on fibronectin-coated Thermanox coverslips (NUNC, Napierville, IL) in growth medium containing 8.0–12.5 µg/ml ECGS prior to exposure to shear or static conditions. Lovastatin, the PI3Kinase inhibitor LY294002 and the p38 inhibitor SB203580 were purchased from Sigma. Simvastatin was obtained from BUFA B.V. (Uitgeest, the Netherlands). Human recombinant TNF-{alpha} was purchased from R&D Systems (Abingdon, UK).

2.2. Shear stress apparatus and flow conditions
HUVEC were exposed to laminar shear stress either in an artificial capillary system or in a parallel-plate perfusion chamber as described [6]. For prolonged shear stress experiments, HUVEC were seeded into fibronectin-coated artificial capillary cartridges (Polypropylene70, Cat No. 400-025; Cellco) in medium containing 8 µg/mL ECGS. Using the CellMax Quad pump system, flow was gradually increased to correspond to a pulsatile (1 Hz) shear stress of 19±12 dyne/cm2, which was maintained over the next 4–7 days with intermediary medium changes. Alternatively, in the parallel-plate system, steady laminar flow was generated for 24–48 h by dampening the pulsatile flow of a peristaltic pump [Masterflex 7524-05 pump drive with a 7518-10 pump head] (Cole-Parmer, Instrument Company, Chicago, IL) by placing 2 three-way taps with windkessels (~70 mL air), followed by a resistance cannula between the pump and flow cell. This generated a steady flow of 25 dyne/cm2, on top of which an independent 1.2 Hz flow pulse with an amplitude of 5.7 dyne/cm2 could be generated by placing the Celmax Quad pump between the damper assembly and the flow cell.

2.3. Real-time RT-PCR and RACE-PAT
Real-time RT-PCR was performed using the MY-IQ single color real-time PCR detection system (BioRad) on total RNA isolated, using the Absolutely RNA RT-PCR miniprep kit (Stratagene, La Jolla, CA) as described [6]. Gene-specific primers for human KLF2, eNOS, TM, and the housekeeping control ribosomal phosphoprotein P0 were designed using Primer3 (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi), resulting in the following primer sequences: KLF2 Fw: 5'-gcaagacctacaccaagagttcg-3' Rv: 5'-catgtgccgtttcatgtgc-3', eNOS Fw: 5'-tggctttcccttccagttc-3' Rv: 5'-agaggcgttttgctccttc-3', TM Fw: 5'-ttgtggaattgggagcttgg-3' Rv 5'-tctcatgaactggatggggtg-3', P0 5'-tgcacaatggcagcatctac-3' Rv 5'-atccgtctccacagacaagg-3'. After normalization to the P0 control, the mRNA levels were expressed as ratios of the control cultures.

Rapid Amplification of cDNA Ends-PolyA Test (RACE-PAT) was performed as described [20]. In brief, 2 µg total RNA was hybridized with 0.5 µmol/L of the oligo (dT)12 primer 5'-gcgagctccgcggccgcgt(12)-3' and then incubated at 50 °C for 60 min with 1 µl Superscript III reverse transcriptase (200 U/µl, Invitrogen). RT-generated heterologous pools of cDNA were amplified by polymerase chain reaction (PCR) using the KLF2-specific Fw-primer 5'-cacctggcgctgcacat-3' and the RT oligo(dT)12 Rv-primer. PCR was performed using 1 µl cDNA and Taq DNA polymerase (Invitrogen). The settings for the thermal cycler were 94 °C for 3 min; 35 cycles of 94 °C for 45 s, 55 °C for 30 s, 72 °C for 1 min 30 s; and termination at 72 °C for 10 min. The RACE-PAT products were analyzed by 2% agarose gel electrophoresis.

2.4. Immunofluorescence and p-Akt measurement.
Fluorescence immunomicroscopy was performed as described [6,9] using either antisera against two separate synthetic peptides of human KLF2, raised in rabbits by the Eurogentec Double-X program (Eurogentec, Seraing, Belgium) or a commercial anti-eNOS antibody (BD Transduction Laboratories, San Diego CA), and images were obtained using the MRC 1024 (BioRad). Linear color corrections to the photomicrographs were made using Adobe Photoshop version 5.0 (Adobe Systems Inc., San Jose, CA). Levels of phospho-Akt were determined using the colorimetric assay of the FACE-Akt Elisa kit (ActiveMotif, Rixensart, Belgium) according to the manufacturer's instructions. In short, HUVEC exposed to shear- or static conditions were fixed, washed, blocked and incubated with the provided phospho-Akt primary antibody overnight at 4 °C. After washing and incubation with the secondary antibody for 1 h at room temperature, the colorimetric assay was performed and the absorbance at 450 nm was determined. The values were subsequently corrected for cell number using the absorbance values at 595 nm resulting from the optional Christal Violet cell staining.

2.5. RNA interference
A stable knockdown of the KLF2 mRNA was achieved using a Lentiviral expression vector carrying an siRNA expression cassette directed against KLF2, as described previously [6]. An otherwise identical vector containing an siRNA cassette directed against the sequence (5'-GATATGGGCTGAATACAAA-3') of firefly Luciferase was used as a control [37]. First passage primary HUVEC cultures were transduced with the Lentiviral KLF2 siRNA at 50% confluency for 24 h and cultured for an additional 2 days before seeding on Thermanox coverslips (Nunc, Rochester, NY) or into the artificial capillaries, followed by a 4-day flow exposure as described. Effective KLF2 silencing was confirmed by real-time RT-PCR.

2.6. Statistical analysis
Expression data are given as mean±SEM for the indicated number of experiments. The unpaired Student's t-test was used to calculate the statistical significance of the expression percentage versus control cultures. P-values less than 0.05 were considered statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Appendix A. Supplementary data
 References
 
3.1. Induction of KLF2, eNOS and TM by statins and prolonged shear
To compare the transcriptional effects of prolonged shear stress to the effects of statin treatment, HUVEC were exposed to prolonged pulsatile flow at arterial levels and compared to HUVEC treated for 24 and 48 h with Lovastatin (10 µM) (Fig. 1). For this experiment, this specific concentration and duration of exposure to statins was chosen because it resulted in the most pronounced increase in mRNA levels of KLF2 and its downstream targets eNOS and TM (see online supplemental Fig. 1). Lovastatin was chosen, as in our hands it produced the most pronounced atheroprotective effects on HUVEC when compared to other statins, like Simvastatin (see online supplemental Fig. 2). This confirms earlier reports that showed moderate differences in responses by HUVEC to various statins [13,14]. As can be seen (Fig. 1A and B), both prolonged shear stress and statin treatment were able to induce KLF2 mRNA levels to a similar extent. However, a significant difference in the magnitude of induction of the KLF2-target genes eNOS and TM was observed. Shear stress (48 h) was able to induce eNOS and TM mRNA approximately 8 to 12-fold, whereas treatment with Lovastatin (48 h) resulted in an increase of only ~2 to 3-fold for these genes (Fig. 1C–F). These high inductions were maintained in shear-exposed HUVEC for up to 7 days (Fig. 1D,F).


Figure 1
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  		Fig. 1 Transcriptional effects of prolonged shear stress and statin treatment in HUVEC. Depicted are mRNA levels of control and stimulated HUVEC cultures as determined by real-time RT-PCR. KLF2 (A–B), eNOS (C–D) and TM (E–F) expression levels upon exposure to prolonged pulsatile shear stress and treatment with Lovastatin are compared. Data are represented as means±SE (N=3). *P<0.05; **P<0.01.

 
3.2. Induction of eNOS and TM by statins and shear is directly dependent on KLF2
We next performed stable siRNA-mediated knockdown of KLF2 by using our previously described Lentiviral system [6] to demonstrate the direct involvement of KLF2 in the induction of eNOS and TM by both statins and shear in our HUVEC model. Lovastatin-induced KLF2 mRNA levels were significantly reduced by the KLF2 siRNA as compared to mRNA levels in cells transduced with control siRNA (Fig. 2A). The inductions of eNOS and TM mRNA by statins were completely inhibited by knocking down KLF2 mRNA (Fig. 2A), which could also be observed at the protein level (see online supplemental Fig. 3). Efficient KLF2 knockdown in HUVEC exposed to prolonged shear also resulted in a significant inhibition of the shear-induction of eNOS and TM (Fig. 2B).


Figure 2
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  		Fig. 2 Real-time RT-PCR analysis of KLF2 and its transcriptional targets eNOS and TM in HUVEC cultures exposed to shear or statin treatment combined with siRNA-mediated KLF2 knockdown. HUVEC cultures were (A) treated with Lovastatin (10 µM) for 24 h or (B) exposed to prolonged shear (4 days). Both treatments were performed in the presence of lentivirus-mediated expression of either KLF2 siRNA or control siRNA. Expression levels of control cultures and stimulated cultures are compared. Data are represented as means±SE (N=3 to 5). *P<0.05; **P<0.01.

 
3.3. Prolonged shear stress induces resistance to suppression of KLF2 by a pro-inflammatory stimulus
As inflammation is a hallmark of atherogenesis, we next investigated the suppressive effect of the pro-inflammatory cytokine TNF-{alpha} on KLF2 levels [5] in the setting of the present study. Therefore, we determined the influence of TNF-{alpha} treatment (25 ng/ml, 6 h) on KLF2 mRNA levels in control cells and after induction by either prolonged shear or treatment with statins. As depicted (Fig. 3A), KLF2 mRNA levels induced by Lovastatin (10 µM) were strongly suppressed in the presence of TNF-{alpha}, whereas the reduction observed in KLF2 mRNA levels induced by prolonged shear was less pronounced (Fig. 3B). The resulting Lovastatin-induced mRNA levels were no longer significantly elevated compared to control, while shear-induced KLF2 remained significantly elevated above the amount observed in the control cells (P<0.01). We next analyzed whether this resistance to TNF-{alpha} was reflected in the levels of the KLF2 transcriptional targets eNOS and TM (Fig. 3C–F). We observed that combined effects of shear and TNF-{alpha} results in mRNA levels of both genes well above those in control HUVEC. This was in clear contrast to the observations in HUVEC treated with Lovastatin and TNF-{alpha}, where mRNA levels of eNOS and TM were no longer increased and even reduced after 48 h. Using lower concentrations of Lovastatin or TNF-{alpha} did not change these results (see online supplemental Fig. 4).


Figure 3
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  		Fig. 3 Analysis of transcriptional effects of TNF-{alpha} in HUVEC during shear-or statin-exposure. Shown are mRNA levels, obtained by real-time RT-PCR, of HUVEC cultures under control and stimulated conditions, in the presence or absence of TNF-{alpha}. Expression levels of KLF2 (A–B), eNOS (C–D) and TM (E–F) in HUVEC cultures exposed to prolonged shear stress or Lovastatin, followed by addition of TNF-{alpha} (25 ng/ml, 6 h) or vehicle (PBS/0.1%BSA), are compared to expression levels in control cultures. Data are represented as means±SE (N=3 to 6). *P<0.05; **P<0.01.

 
3.4. Shear stress effectively enhances KLF2 protein production
To investigate the underlying cause of the clear distinction between the downstream transcriptional effects of shear- and Lovastatin-induced KLF2, we analyzed induction of KLF2 by shear and Lovastatin at the protein level. To this end, we used rabbit antiserum directed against KLF2 for immune fluorescence, which we have previously employed to demonstrate KLF2-overexpression by Lentiviral transduction and induction of KLF2 by shear stress [6,9]. When comparing HUVEC treated with vehicle only to Lovastatin-treated and shear-exposed cells, we observed that the intensity of the signal was clearly higher in sheared HUVEC than that in Lovastatin-treated cells (Fig. 4A). These findings indicated that an apparent difference in KLF2 protein expression lies at the basis of the increased expression of KLF2 downstream effector genes that is observed under shear. Obtaining a more quantitative determination of varying KLF2 protein levels was not feasible, as there are no antibodies available that detect KLF2 by Western-blot analysis in HUVEC. To further characterize the potential differences between shear- and statin-induced gene regulation in endothelial cells, we also performed staining for F-actin in control, shear-exposed and statin-treated HUVEC cultures (Fig. 4B). We observed marked differences in the distribution of F-actin when comparing these conditions. Whereas in the static control culture the actin fibers showed a classical cortical distribution pattern, the Lovastatin-treated cells displayed an apparent reduction in the amount of F-actin filaments, with a seemingly disorganized arrangement of the fibers that were observed. In the shear-exposed cells, the formation of actin stress fibers was seen, a hallmark phenomenon in endothelial cells exposed to shear stress [16,17].


Figure 4
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  		Fig. 4 Effects of shear-exposure and statin treatment on KLF2 protein expression and cytoskeletal rearrangement in HUVEC. Fluorescence immunochemistry using specific KLF2 antisera or phalloidin-TRITC was performed on HUVEC control cultures and cultures exposed to 24 h of shear stress (25 dyne/cm2) in the parallel-plate system or Lovastatin treatment (24 h, 10 µM), as indicated. (A) KLF2 staining using specific antisera. (B) F-actin staining using phalloidin-TRITC.

 
3.5. Shear stress stabilizes KLF2 mRNA
We decided to explore the possibility that differential effects on KLF2 mRNA stability could explain the observed differences in shear- or statin-induced KLF2 functional efficacy, based on a number of recent reports of other shear-regulated genes [18,19]. By using Actinomycin D to block initiation of transcription, we studied KLF2 mRNA decay in HUVEC exposed to fluid shear stress, and compared this to mRNA degradation in cells kept under static conditions (Fig. 5A). Before the addition of Actinomycin D, HUVEC were pre-conditioned by exposing them to 25 dyne/cm2 of shear stress for 2 h or were cultured in the absence of flow. In clear contrast to the rapid decay of KLF2 mRNA in cells cultured without flow, mRNA degradation was effectively halted by shear stress, as determined in an elaborate shear time series in which 3 to 6 independent HUVEC isolates were used for each observation. This effect was equally significant (P<0.01) at every individual time point that was analyzed, demonstrating a robust stabilization of a specific sub-fraction of KLF2 mRNA by shear stress, thereby keeping absolute mRNA levels elevated well above levels in HUVEC that were not exposed to flow. The protective effect of shear on KLF2 mRNA stability is established rather rapidly, as we did not find any significant differences in effects between 18 h and 2 h of shear pre-conditioning (data not shown). In marked contrast, analysis of KLF2 mRNA stability in Lovastatin-treated HUVEC revealed no significant difference compared to mRNA stability observed in vehicle-treated cells (Fig. 5B).


Figure 5
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  		Fig. 5 (A) Effects on KLF2 mRNA stability in time upon exposure of HUVEC to shear. After 2 h of pre-conditioning by shear (25 dyne/cm2), transcription was blocked by addition of Actinomycin D (2 µg/ml), after which KLF2 mRNA levels were measured and plotted as a percentage of control cultures taken before pre-shearing. (B) Upon 24 h of Lovastatin (10 µM) or vehicle (EtOH) treatment, Actinomycin D was added and KLF2 expression was determined and depicted relative to levels in control cultures taken at t=0 h. (C) After 2 h of pre-shear (25 dyne/cm2), transcription was blocked by adding Actinomycin D and shear-exposed and static HUVEC were kept in the presence or absence of TNF-{alpha}. KLF2 mRNA levels were depicted as a percentage of control cultures taken before pre-shear. (D) Agarose gel electrophoresis of PCR product obtained from static and shear-exposed HUVEC by RACE-PAT procedure. (E) After 1 h of pre-incubation with the PI3K inhibitor LY294002, KLF2 levels were measured and plotted as a percentage of control cultures at t=0 h. (F) Levels of phospho-Akt in HUVEC exposed to shear and static conditions. Data are represented as means±SE (N=3 to 6). N.S. indicates not significant.

 
3.6. TNF-{alpha} does not influence shear-induced KLF2 mRNA stabilization
We showed (Fig. 3) that prolonged shear stress confers resistance of KLF2 mRNA to the suppressive effect of TNF-{alpha} and thereby enhances the downstream transcriptional effects of KLF2 on eNOS and TM. Therefore, it was of interest to demonstrate whether the observed shear-induced stabilization of KLF2 mRNA may be the cause of this effect. We investigated this by performing experiments in which HUVEC were exposed to shear stress in our parallel-plate system or cultured without flow and kept in the presence or absence of TNF-{alpha}. Again, stability of KLF2 mRNA was studied in this setting by blocking transcription initiation through addition of Actinomycin D, after pre-conditioning the cells in the presence or absence of shear for two hours. As can be seen (Fig. 5C), TNF-{alpha} (25 ng/ml, 4 h.) did not influence KLF2 mRNA stability in HUVEC exposed to shear stress as again, a sub-fraction of the KLF2 mRNA pool displayed resistance to rapid degradation. Furthermore, in shear-exposed HUVEC, KLF2 mRNA levels were again significantly elevated compared to levels in cells kept without flow, confirming the stabilizing effect of shear stress on KLF2 mRNAs.

3.7. Inhibition of PI3K stabilizes KLF2 mRNA
The effect of shear stress on stabilization of KLF2 mRNA prompted us to test several mechanisms or signal transduction pathways known to be involved in shear-stabilization of mRNA. To investigate the possibility that, like for eNOS [20], differential polyadenylation may explain the shear-induced stabilization of KLF2 mRNA, we performed a Rapid Amplification of cDNA Ends-PolyA Test (RACE-PAT). However, experiments exposing HUVEC to increases in shear magnitude did not result in changes in polyadenylation, as the PCR resulted in the expected major product of approximately 600 bp, and further bands that were detected, were not differential when comparing shear and static conditions (Fig. 5D). Next, we used the p38 inhibitor SB203580, to explore a possible role of p38, implicated in the stabilization of e.g. COX-2 [21]. We observed no significant effect of SB203580 on the stability of KLF2 mRNA in HUVEC, thereby making direct involvement of p38 in KLF2 mRNA stabilization unlikely (data not shown).

As PI3K has been implicated in destabilization of COX-2 mRNA in a single report [22], we tested the effect of its specific inhibitor LY294002, on the stability of KLF2 mRNA. Indeed, inhibition of PI3K by LY294002 resulted in a long-term stabilizing effect on ~50% of KLF2 mRNA (Fig. 5E), very similar to the effect observed for shear stress (Fig. 5A). Previous reports have shown a transient activation of PI3K-dependent Akt phosphorylation [23,41], reason why we exposed HUVEC to 24 h of pulsatile shear and determined the levels of phosphorylated Akt in our experimental setup. As shown (Fig. 5F), levels of phospho-Akt were indeed lowered by approximately 20% in HUVEC exposed to prolonged shear, as compared to HUVEC kept without flow.


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Appendix A. Supplementary data
 References
 
Shear stress is widely regarded as a stimulus that is an absolute prerequisite for proper physiological endothelial function. The observation that high shear protects the vessel wall from atherogenesis has been the subject of myriad investigative efforts into the mechanism by which shear exerts its beneficial influence on endothelial function. The shear-responsive transcription factor KLF2 has recently emerged as an important mediator of many favorable effects on endothelial gene expression [9,10], by virtue of its induction by shear stress [5,6] as well as HMG-CoA reductase inhibitors [13,14]. The expression of TM and eNOS, two major contributors to the anti-thrombotic and anti-inflammatory/anti-adhesive properties of healthy endothelial cells [24,25], has been shown to be dependant on a common mediator, KLF2 [6,7,9,15]. In endothelial cells, the presence of TM is of great importance for maintaining a non-thrombogenic surface at arterial shear-rates [26]. The potential of shear stress to induce the expression of TM was demonstrated previously [27]. The finding that, in addition to shear stress, statins are also able to induce KLF2 transcription [13,14], suggested for the first time that it would be possible to devise novel pharmacological interventions that could mimic the atheroprotective effects of shear stress. We now present direct comparative data that establish a superior potential of prolonged shear to statins to induce the KLF2-dependent expression of eNOS and TM in endothelial cells (Fig. 1). This is highly remarkable as we show that both are direct targets of the transcription factor KLF2 in our model (Fig. 2). We demonstrate that the potent induction of KLF2-targets eNOS and TM by prolonged shear is likely explained by increased stability of KLF2 mRNA (Fig. 5), resulting in an increase in the amount of KLF2 protein (Fig. 4). In the current study, we thus established the effects of the transcription factor KLF2 on the steady-state mRNA levels of eNOS, which was borne out also at the protein level (supplemental Fig. 3). For its full physiological function, however, eNOS vasodilatory and anti-inflammatory actions are also regulated in many convoluted ways at the protein level, e.g. by Akt-driven phosphorylation [23,41], something we did not address in the current study.

The effects of TNF-{alpha} on endothelial gene expression are widely studied because of their relevance to many inflammatory diseases such as atherosclerosis. An inflammatory stimulus like TNF-{alpha} may impart pro-adhesive and pro-thrombotic properties on the endothelium [28]. The expression of both TM and eNOS, two important molecular mediators influencing these effects, has been shown to be efficiently repressed by TNF-{alpha} in static cultures of HUVEC [29,30]. The direct control of the expression of both molecules by KLF2 suggests that the suppressive effects of TNF-{alpha} on eNOS and TM are a direct consequence of the well established KLF2-repression by TNF-{alpha} [5,15,31]. Accordingly, it was also shown that both adenovirus-mediated high overexpression of murine KLF2 and moderate, prolonged Lentiviral overexpression of human KLF2 in endothelial cells, attenuate the inflammatory response elicited upon stimulation with TNF-{alpha} [9,15]. The significance of these findings with regard to physiological protection of the vessel wall seems challenged, however, by the observation that endogenous KLF2 is rapidly downregulated by such cytokines in the absence of flow [5,31]. We now show, however, that even in the presence of TNF-{alpha}, shear-exposed endothelial cells maintain increased mRNA levels of KLF2, eNOS and TM, whereas Lovastatin-treated cells do not show these effects (Fig. 3). Thus, our current findings may resolve this paradox, as we show that prolonged shear stress has the potential to maintain increased expression of TM and eNOS in the presence of inflammation, which may be explained by the inability of TNF-{alpha} to repress the shear-stabilized sub-fraction of KLF2 mRNA. These data provide evidence for the existence of a mechanism by which fluid shear stress increases KLF2 protein levels in HUVEC by influencing the stability of its mRNAs, whereas this mechanism is apparently not operational in Lovastatin-treated HUVEC. One observation related to this issue is a clear difference between the influence of shear and statins on the rearrangement of the actin cytoskeleton (Fig. 4), which has been described to influence gene-expression and mRNA stability [17].

Shear-induced increases in gene expression through stabilization of mRNA have been described for a number of important endothelial genes, including eNOS [18] and COX-2 [19]. We now show that the stabilization of KLF2 involves a novel mechanism, in which the phosphatidyl-inositide-mediated signal transduction pathway and PI3K seems to play a pivotal role, as inhibition of this kinase mimics the observed shear effects (Fig. 5). PI3K is a lipid kinase that is involved in signal transduction controlling a wide array of important cellular functions (e.g. cell survival/apoptosis) in several types of cells [32]. A rapid (15 s) but transient activation of PI3K by onset of shear mediates the endothelial response to turbulent shear, thereby linking PI3K-activation to atherogenesis [33]. In HUVEC exposed to prolonged shear, however, we found levels of phospho-Akt, one of its effector proteins, to be decreased (Fig. 5), which is in full accordance with previous findings showing that shear-induced activation of Akt is transient [23]. Recently, several reports have indicated a possible negative correlation between PI3K activity and increased KLF2 protein expression. First, knock-out mice for the lipid phosphatase PTEN, a negative regulator of PI3K, display a phenotype that is very reminiscent of KLF2 knockout mice [34,35]. Furthermore, impaired PTEN activity in adult animals resulted in increased angiogenesis due to differential regulation of several vascular growth factors [34] analogous to the inhibitory effects of KLF2 on VEGF-mediated angiogenesis in vivo [8] and endothelial cell migration in culture [9]. Second, a recent report shows that increased activity of another inositol-phosphatase, SHIP-1, increases KLF2 expression in T-cells, suggesting a possible direct relation between the PI3K–Akt pathway and the expression of KLF2 [42].

In conclusion, we believe that the observations made in this study are a strong indication that prolonged shear stress is the most important stimulus for increasing the expression of endothelial genes that guard the vessel wall from inflammatory processes and that KLF2 is a pivotal transcription factor for these effects. Induction of an increased stability of KLF2 mRNA is crucial to this robust atheroprotective potency of healthy flow profiles, which is lacked by statins, eventhough both induce similar expression levels of KLF2. This suggests that potential pharmacological interventions could be directed at stabilizing KLF2 mRNA, rather than at inducing its expression. Establishing the molecular mediators of the observed stabilization of an inflammation-resistant KLF2 mRNA pool by shear stress may lead to effective strategies to improve endothelial function in cardiovascular disease.


   Appendix A. Supplementary data
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
References
 
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.cardiores.2006.07.008.


   Acknowledgements
 
This study was supported by the Netherlands Heart Foundation, The Hague (Molecular Cardiology Program grant M93.007); NWO-Genomics, The Hague (grant 050-10-014) and SenterNovem, The Hague (by IOP-genomics grant IGE03012C).


   Notes
 
Time for primary review 28 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Appendix A. Supplementary data
 References
 

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T. T. Tuomisto, H. Lumivuori, E. Kansanen, S.-K. Hakkinen, M. P. Turunen, J. V. van Thienen, A. J. Horrevoets, A.-L. Levonen, and S. Yla-Herttuala
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