Integration of flow-dependent endothelial phenotypes by Kruppel-like factor 2 (original) (raw)
Endothelial KLF2 expression is selectively induced by a distinct type of flow in vitro and is dependent on blood flow in vivo. To identify critical transcriptional regulators of endothelial mechano-activated programs, and thus gain mechanistic insights into the molecular basis of the atheroprotected versus the atherosusceptible endothelial phenotypes, we have recreated the shear stress waveform characteristic of the atheroprotected (“atheroprotective waveform”) or atherosusceptible regions (“atheroprone waveform”) of the human carotid artery bifurcation (Figure 1A), using a system recently developed in our laboratory (4). The comprehensive transcriptional activities of endothelial cells exposed to these 2 paradigms of biomechanical stimulation were assessed using genome-wide oligo-microarrays, and further validation of several transcription factors selectively upregulated by the atheroprotective waveform was performed using real-time TaqMan PCR. One of the most robustly upregulated transcription factors arising from this analysis was KLF2 (Figure 1B), a zinc finger–containing transcription factor previously implicated in vascular development and T cell activation (5, 6). Several studies have indicated that the expression of KLF2 in cultured endothelial cells is increased after exposure to laminar shear stress (7, 8), and these findings correlate with the in vivo expression pattern of KLF2 in the developing chick embryo in regions exposed to high shear stress (9). Dekker et al. have demonstrated a relative lack of KLF2 expression in the endothelial lining at the bifurcation in the human aorta, at the outer wall of the bifurcation of the aorta and the iliac arteries, and at the branch of the common carotid artery from the aortic arch (7, 10), regions susceptible to the early formation of lesions of atherosclerosis are prone to develop. To critically assess the dependence of endothelial KLF2 expression on blood flow in vivo_,_ we used previously characterized (11) zebrafish with a silent heart (sih) mutation, which have a noncontractile heart. These mutants are viable for the first week and have functional swim reflexes after hatching. The zebrafish heart first starts to beat at around 24 hours after fertilization, initiating visible circulation of blood soon thereafter. As seen in Figure 1C, WT embryos at 48 hours after fertilization display staining for the endothelial cell marker Flk-1 and KLF2a (a zebrafish homolog of KLF2) along the major trunk vessels. In contrast, while Flk-1 expression in sih is indistinguishable from that in WT zebrafish, the vascular expression of KLF2a is lost. As an internal control, labeling of the anal sphincter by the KLF2a probe was noted to be similar in WT and sih embryos (Figure 1C, arrowheads). These observations demonstrate that endothelial KLF2 expression is dependent on blood flow in vivo and that this mode of regulation may be evolutionarily conserved.
Flow-dependent expression of KLF2 and its regulation by a MEK5/ERK5/MEF2 pathway. (A) Archetypal atheroprotective and atheroprone shear stress waveforms derived from a human carotid artery, as previously described (4). These 2 shear stress waveforms were recreated using a dynamic flow system and applied to cultured HUVECs. (B) HUVECs were cultured under static (no flow), atheroprone, or atheroprotective flow conditions for 24 hours, and KLF2 mRNA expression was measured by RT-PCR (n = 3; mean ± SEM). (C) Whole-mount in situ hybridization of WT or sih mutant embryos at 48 hours, probed for Flk or KLF2a. Inserts show close-ups of the trunk vasculature. Anal sphincter staining is indicated by arrowheads. (D) ChIP of MEF2A and MEF2C with the KLF2 promoter under static conditions and flow. (E) KLF2 mRNA in HUVECs infected with control (GFP) or dominant negative MEF2 (MEF2ASA) adenovirus 24 hours before exposure to the static (no flow) or atheroprotective waveform. (F) Western blot of total immunoprecipitated ERK5 and p-ERK5 from HUVECs under static or flow 24 hours after infection with control (GFP) or MEK5-DN adenovirus. (G) KLF2 mRNA levels from experimental samples represented in F. (H) Western blot of ERK5 immunoprecipitates under conditions described in F from HUVECs infected with GFP or MEK5-CA adenovirus. (I) KLF2 mRNA from samples represented in H. *P < 0.05, **P < 0.01 vs. control; Student’s t test. ctrl, control.
Atheroprotective flow upregulates endothelial KLF2 via a MEK5/ERK5/MEF2 signaling pathway. Next, we sought to characterize the molecular mechanisms responsible for the flow-mediated upregulation of KLF2. The proximal promoter of KLF2 has been shown to be critical for the upregulation of this gene under laminar shear stress (12). To gain insights into possible mechanisms leading to upregulation of KLF2 by flow, we analyzed the proximal promoter for transcription factor consensus binding sequences and found them to include a myocyte enhancer factor–2 (MEF2) site. To determine whether MEF2 members bind the KLF2 promoter, we performed chromatin immunoprecipitation (ChIP) using antibodies to MEF2A and MEF2C and PCR amplification of the proximal KLF2 promoter. Both MEF2A and MEF2C were observed to specifically bind the KLF2 promoter in intact cells, and no product was amplified using control IgG or no antibody (Figure 1D). This binding was not significantly affected by exposure to flow. We then tested whether MEF2 function is required for the upregulation of KLF2 under this type of flow by infecting human umbilical vein endothelial cells (HUVECs) for 24 hours with control adenovirus or virus expressing a dominant negative MEF2A mutant (MEF2ASA). The MEF2ASA protein blocks the function of all 4 MEF2 family members by preventing transactivation at bound MEF2 sites. MEF2ASA, while having no effect on basal KLF2 expression, dramatically abrogated the upregulation of KLF2 observed under flow (Figure 1E). These results therefore argue that members of the MEF2 family of transcription factors bind the endogenous KLF2 promoter and are a critical component of the transcriptional machinery required for the regulation of KLF2 expression under flow.
The MEF2 family of transcription factors has mostly been shown to be regulated at the level of transactivation by phosphorylation and recruitment of cofactors (13). Among the important upstream activating signals for the MEF2 family are cytosolic calcium and signaling via MAPK cascades. A specific MAPK with particular relevance to both vascular endothelium and MEF2 activation is ERK5, which is robustly activated in endothelial cells exposed to shear stress and has been shown to be a crucial modulator of endothelial apoptosis in vitro and in vivo (14–16). To determine whether ERK5 activation is critical for the MEF2-dependent upregulation of KLF2 under flow, HUVECs were infected with adenovirus expressing GFP or a dominant negative form of MAPK kinase 5 (MEK5-DN), the specific upstream activating kinase for ERK5. Cells under static conditions had no detectable p-ERK5, and a robust p-ERK5 band was found in lysates from cells infected with Ad-GFP and exposed to flow for 24 hours. In contrast, cells infected with the MEK5-DN virus and exposed to flow had no detectable p-ERK5 (Figure 1F). MEK5-DN therefore inhibits the flow-mediated activation of its downstream target ERK5. Importantly, cells expressing MEK5-DN also failed to upregulate KLF2 when exposed to flow (Figure 1G), suggesting that activation of ERK5, the only known target of MEK5, is critical for the flow-mediated upregulation of KLF2. To establish whether activation of MEK5 is also sufficient for KLF2 induction, a constitutively active MEK5 construct (MEK5-CA) was expressed in HUVECs for 48 hours. Compared with GFP-expressing cells, MEK5-CA led, as expected, to the phosphorylation of ERK5 (Figure 1H). This increase in ERK5 activation was accompanied by an upregulation of KLF2 expression, an increase similar in magnitude to that observed in cells exposed to flow (Figure 1I). These results define a mechanism involving a MEK5/ERK5/MEF2 pathway linking flow and increases in KLF2 expression, whereby MEK5 activation is both required and sufficient for the flow-dependent upregulation of KLF2.
KLF2 acts as a transcriptional integrator of multiple endothelial functions. Previous work in our laboratories demonstrated that KLF2 overexpression increases the level of expression of eNOS and inhibits the IL-1β–dependent induction of the proinflammatory adhesion molecules VCAM-1 and E-selectin and the TNF-α–dependent induction of tissue factor (TF) in cultured human endothelial cells (8, 17). These observations, together with the selective upregulation of endothelial KLF2 by a waveform derived from an atheroprotected region of the human carotid and the pattern of expression of KLF2 in the human vasculature, suggested to us that KLF2 might play a critical role in a global regulation of the transcriptional programs that lead to the acquisition of flow-mediated endothelial phenotypes. To test this hypothesis, we sought first to define the global transcriptional targets of KLF2 using genome-wide transcriptional profiling on endothelial cells overexpressing GFP or KLF2-GFP in the presence or absence of IL-1β (10 U/ml, 4 hours, a well-characterized proinflammatory stimulus). Figure 2A shows a self-organizing map of regulated genes clustered according to similar patterns of expression across the 3 experimental conditions (KLF2, IL-1β, KLF2 plus IL-1β) relative to control (GFP). These global patterns revealed 4 distinct clusters of similarly regulated genes. Cluster I represents genes that are downregulated by KLF2 but are not modulated by IL-1β (e.g., angiopoietin-2 [_Ang-2_], endothelial lipase). Cluster II represents genes that are upregulated by KLF2 and not regulated by IL-1β (e.g., NFATc3, eNOS). Cluster III includes genes whose upregulation by IL-1β is antagonized by KLF2 (e.g., IL-6, RANTES). Finally, cluster IV contains genes that are synergistically upregulated by KLF2 and IL-1β (e.g., ELAFIN, prostaglandin E synthase–1). A web-based searchable database of all the microarray data can be found at http://vessels.bwh.harvard.edu/Parmar1\. These observations coupled with functional categorization of regulated genes (Figure 2B) suggest that KLF2 serves as a global transcriptional regulator of multiple endothelial functions, including blood vessel development, vascular tone, thrombosis/hemostasis, and inflammation.
Elucidation of KLF2-regulated global gene expression programs. (A) Visualization of whole-genome expression patterns controlled by KLF2 and/or IL-1β in HUVECs. HUVECs were infected with either Ad-GFP or Ad-KLF2 and incubated for 24 hours before RNA was isolated and analyzed using whole genome microarrays. Self-organizing map software was used to cluster similarly regulated genes and colorize them based on intensity of expression relative to GFP-expressing (control) cells. (B) Selected KLF2-responsive genes grouped by clusters shown in A, with major associated biological functions listed. Dev, development.
To further assess the role of KLF2 in the regulation of pathophysiologically relevant genes belonging to these 4 major functional categories, we characterized the gene expression and functional changes identified through the microarray analysis. As shown in Figure 3, A and C, KLF2 overexpression robustly increased Tie2 mRNA and protein levels, while suppressing Ang-2 at the level of mRNA and secreted protein (Figure 3, A and B). Furthermore, KLF2 potently upregulated the mRNA expression of elastin (Figure 3A) and NFATc3 (Figure 2B), 2 genes critical for elastogenesis and formation of the arterial wall (18), respectively. These observed effects of KLF2 on genes implicated in the Ang-1/Tie2 axis, elastogenesis, and vessel wall formation may explain the lack of assembly of the vascular tunica media and vessel wall instability observed in the KLF2-knockout mice (19).
KLF2 regulates genetic programs controlling blood vessel development, vascular tone, and thrombosis/hemostasis. (A) HUVECs under the same conditions as in Figure 2A were analyzed by quantitative TaqMan RT-PCR (n = 3) of the indicated genes; Ad-GFP was performed as the control. (B) Ang-2 protein levels in supernatants were measured by ELISA under the same conditions (n = 3) as in A, but with an 8-hour incubation with IL-1β. Ang-2 levels shown are after subtraction of the amount in the media before conditioning. (C) Western blot of Tie2 and GAPDH (loading control) on whole-cell lysate of HUVECs infected with Ad-GFP or Ad-KLF2 for 24 hours. (D) Quantitative TaqMan RT-PCR was performed to assess the levels of expression of eNOS, ASS, CNP, and ET-1. (E) CNP protein levels in supernatant measured by ELISA. Supernatant was collected from HUVECs infected with the indicated adenoviruses for 24 hours with or without IL-1β. nd, not detectable. (F) Effect of KLF2 and/or IL-1β on the expression of major genes involved in thrombosis. Quantitative TaqMan RT-PCR of indicated genes was performed as described in A. EDG-1, endothelial differentiation gene 1; PAI-1, plasminogen activatory inhibitor 1. (G) FACS analysis of surface TM (TM/CD141) expression on HUVECs infected with Ad-GFP or Ad-KLF2 for 24 hours. (H) FACS analysis of cell-surface TF expression on HUVECs infected with the indicated virus followed by incubation with media or IL-1β (10 U/ml) for 6 hours. Bar graphs represent mean ± SEM (n = 3). *P < 0.05, **P < 0.01 vs. control; Student’s t test.
We found that KLF2 overexpression, in addition to upregulating eNOS, also upregulates the expression of argininosuccinate synthetase (ASS) (Figure 3D), a limiting enzyme in eNOS substrate bioavailability (20). KLF2 also upregulates dimethylarginine dimethylaminohydrolase 2 (Supplemental Table 1; supplemental material available online with this article; doi:10.1172/JCI24787DS1), an enzyme that degrades asymmetric dimethylarginine, an endogenous inhibitor of eNOS (21). One of the most strongly upregulated targets of KLF2 is C-type natriuretic peptide (CNP). CNP mRNA and secreted protein levels dramatically increased with KLF2 overexpression (Figure 3, D and E). In contrast to the upregulation of these genes, KLF2 overexpression decreased levels of caveolin-1 mRNA (Supplemental Table 1), a critical negative regulator of eNOS activity (22). Moreover, the expression of endothelin-1 (ET-1), the most potent known endogenous vasoconstrictor, was strongly suppressed by KLF2 in the presence and absence of IL-1β (Figure 3D). These data demonstrate an orchestrated upregulation of endothelium-dependent vasodilatory pathways, in particular the l-arginine/NO pathway, and a downregulation of vasoconstrictive molecules produced by the endothelium.
We next evaluated the KLF2-mediated regulation of genes involved in hemostasis, thrombosis, and inflammation. KLF2 overexpression markedly increased thrombomodulin (TM) mRNA, and surface protein expression (Figure 3, F and G). Inflammatory stimuli have been shown to induce a procoagulant phenotype in endothelial cells (23). KLF2 expression inhibited the IL-1β–mediated increase in endothelial differentiation gene 1 (EDG-1) and plasminogen activatory inhibitor 1 (PAI-1) expression (Figure 3F); the former an important receptor for thrombin-mediated procoagulant signaling (24) and the latter factor an inhibitor of clot dissolution (25). We found that KLF2 overexpression dramatically reduced the IL-1β–mediated increase of cell surface TF, the primary cellular initiator of blood coagulation (Figure 3H) (26). The coordinated regulation of these critical anti- and procoagulant surface proteins suggests that KLF2 expression can confer a robust anticoagulant endothelial phenotype. Interestingly, the selective lack of expression of TF and PAI-1 in regions of the human carotid artery exposed to atheroprotective blood flow (27) is consistent with a role for KLF2 as a suppressor of these genes in vivo. Furthermore, we found that the IL-1β–mediated increase of a large number of proinflammatory genes was muted by KLF2 (Figure 2B, cluster III). Quantitative real-time PCR analysis of numerous endothelial genes associated with inflammation confirmed the global suppression by KLF2 of IL-1β–mediated endothelial activation first unveiled by microarray analysis (Table 1). These genes encode several cytokines and chemokines that mediate inflammatory cell migration into the vessel wall at sites of physiologic homing or pathological inflammation (28). Multiplex ELISA was then used to measure the production of various inflammatory cytokines/chemokines in the supernatants from cultured endothelial cells. KLF2 overexpression suppressed the IL-1β–mediated production of IL-6, IL-8, RANTES, IFN-γ–inducible protein 10 (IP-10), MCP-1, G-CSF, and GM-CSF (Figure 4A). One of the highly KLF2-upregulated genes was prostaglandin D2 synthase (PTGDS), which produces as an end product 15d-PGJ2 (Figure 4, B and C). Interestingly, PTGDS-knockout mice have been recently shown to display accelerated diabetes and atherosclerosis (29). KLF2 overexpression thus globally mutes IL-1β–induced endothelial activation as assessed by cytokine and chemokine production, in addition to promoting an antiinflammatory prostaglandin pathway.
KLF2 mediates a global antiinflammatory program in endothelial cells. (A) Protein levels of cytokines in supernatants of HUVECs infected with the indicated virus and then incubated with either normal media or IL-1β (10 U/ml) for 24 hours. Samples were analyzed by multiplex ELISA or cytokine chip. IP-10, IFN-γ–inducible protein 10. (B) Regulation of PTGDS gene expression by KLF2 and/or IL-1β. (C) ELISA of 15d-PGJ2 after extraction from supernatants of cells infected with the indicated virus for 24 hours. (D) ELAFIN secretion measured by ELISA from supernatants under conditions described in A. All data are expressed as mean ± SEM (n = 3). *P < 0.05, **P < 0.01 vs. control; Student’s t test.
Effect of KLF2 and/or IL-1β on inflammatory gene expression by quantitative TaqMan RT-PCR
In contrast to this IL-1β antagonism, KLF2 and IL-1β synergistically upregulated various genes involved in the resolution of inflammation (Figure 2B, cluster IV). IL-11, a cytokine recently found to have a protective effect on endothelium in an allograft rejection model (30), was highly expressed only when both KLF2 was expressed and IL-1β was present (Figure 4A). The upregulation of the elastase inhibitor ELAFIN also displayed a remarkable synergy between KLF2 and IL-1β (Figure 2B, cluster IV) and was found in significant levels only in conditioned media from KLF2-expressing cells exposed to IL-1β (Figure 4D). ELAFIN has been shown to potently suppress smooth muscle hyperplasia in animal models of vascular injury and vein graft degeneration (31). The markedly synergistic mode of regulation of these antiinflammatory targets indicates that KLF2 expression may function to enable the endothelial cell to curtail its responsiveness to inflammatory stimulation and promote physiologic resolution of inflammation.
KLF2 is a critical regulator of the endothelial response to atheroprotective flow. To directly assess the role of KLF2 as regulator of the expression of genes evoked by the waveform derived from an atheroprotected region of the human carotid, we blocked the flow-induced upregulation of KLF2 using small interfering RNA (siRNA) gene silencing. KLF2 was knocked down in cells exposed to flow such that the KLF2 level was approximately equal to that of cells maintained under static control conditions (Figure 5A). Importantly, under these experimental conditions, we did not observe the activation of the double-stranded RNA–triggered IFN-associated antiviral pathways, as determined by induction of the sensitive marker genes 2′-5′-oligoadenylate synthetase or interferon-inducible transmembrane protein 1 (Figure 5B). Blockade of the upregulation of KLF2 in endothelial cells exposed to flow resulted in a significant loss of the regulation of several upregulated genes, including eNOS, ASS, TM, Tie2, CNP, human prostaglandin transporter (hPGT), PTGDS, and nephroblastoma overexpressed gene (NOV) (Figure 5A). The downregulation of IL-8, Ang-2, and ET-1 by flow was also abolished at the mRNA level (Figure 5A). We confirmed these results at the protein level for eNOS and Tie2 (Figure 5C). The flow-mediated increases in cell surface TM expression, secreted levels of CNP, and secreted levels of 15d-PGJ2 were also abolished by blocking the flow-dependent upregulation of KLF2 (Figure 5, D–F).
Endothelial transcriptional programs evoked by atheroprotective flow require KLF2 expression. (A) Effects of suppressing flow-dependent KLF2 upregulation on gene expression. Cells were treated with either scrambled siRNA or siRNA targeting KLF2 for 24 hours and then placed under static or atheroprotective conditions for an additional 24 hours. Shown are RT-PCR data for the indicated genes. hPGT, human prostaglandin transporter; NOV, nephroblastoma overexpressed gene. (B) Monitoring of the interferon response in HUVECs treated with control or KLF2 siRNAs. Gene expression of 2′-5′-oligoadenylate synthetase (OAS2) or interferon-inducible transmembrane protein 1 (IFITM1) was assessed by TaqMan RT-PCR. As a positive control for the interferon response, HUVECs were incubated with long double-stranded RNA (dsRNA) poly I:C for 24 hours (n = 3). (C) Western blot for proteins in the same samples as represented in A. (D) Surface TM FACS on HUVECs under the indicated conditions. (E) ELISA of CNP from supernatants under the conditions described in A. (F) 15-d-PGJ2 levels in supernatants under the indicated conditions. (G) Histogram (blue bars) of genes binned according to fold regulation under flow. Overlaid on the histogram is a graph showing the percentage of genes within each bin that are KLF2 dependent (red diamonds and trend line). Gray shading indicates the portion of the histogram representing the 74 most highly regulated genes under flow (see text). All data are expressed as mean ± SEM (n = 3). *P < 0.05, **P < 0.01 vs. control; Student’s t test.
To obtain an unbiased global picture of the role of KLF2 in the regulation of the flow-mediated transcriptional programs, genome-wide transcriptional profiling was used to characterize gene expression under flow with control siRNA and flow with KLF2 siRNA. These experiments revealed that the expression of 109 genes was dependent on the flow-mediated KLF2 upregulation, representing 15.3% of the total number of genes regulated by flow (713 genes). We next sought to determine whether there was a global relationship between those 109 genes and the entire set of genes regulated by flow. Flow-activated gene expression was plotted as a histogram, in which genes were binned according to fold regulation. Subsequently, the percentage of genes in each bin affected by KLF2 siRNA was overlaid as a scatter plot (Figure 5G). The percentage of flow-regulated genes altered by KLF2 siRNA strongly correlated with their magnitude of regulation by flow (_r_2 = 0.72, as plotted). Of the 74 most highly regulated genes under flow (Figure 5G, gray), 34 of these (46%) depended on KLF2 upregulation. This finding suggests that KLF2 plays a key role in atheroprotective flow-mediated endothelial gene expression.
KLF2 is a mediator of flow-dependent endothelial functional phenotypes. Finally, to determine whether the upregulation of KLF2 and its transcriptional targets is required for the complex functional phenotypes evoked in endothelial cells by atheroprotective flow, we utilized assays that assess the cellular responses to an inflammatory or oxidant stress challenge. First, we examined leukocyte adhesion to endothelial cells challenged with an inflammatory stimulus (IL-1β, 1 U/ml, 6 hours). As shown in Figure 6A, preconditioning with flow significantly decreased the IL-1β–dependent adhesion of human HL-60 cells to endothelial monolayers when compared with static (no flow) controls, and blockade of KLF2 upregulation during the flow preconditioning suppressed this antiadhesive phenotype. Second, we examined the role of KLF2 in the resistance to oxidative stress previously observed in endothelial cells preconditioned with laminar shear stress (32). KLF2 overexpression was capable of inhibiting H2O2-mediated oxidative injury and subsequent cell death as observed by cell morphology (Figure 6B, top row) in cells cultured under static (no flow) conditions. Preconditioning of endothelial cells with flow also induced resistance to the oxidant stress challenge, and this resistance was dependent on the upregulation of KLF2 (Figure 6B, bottom row). Expression of catalase and superoxide dismutase, 2 components of cellular oxidative defense mechanisms, were not found to be regulated by KLF2 (Figure 6C), thus indicating that other flow-dependent, KLF2-mediated antioxidant mechanisms are probably involved. Together, these data demonstrate that KLF2 is necessary and sufficient for 2 important functional phenotypes we observe in endothelial cells exposed to atheroprotective flow and that this transcription factor acts as an integrator of the local humoral and biomechanical milieu.
KLF2 expression is essential for endothelial cellular phenotypes conferred by flow. (A) HL-60 cell adhesion to HUVEC monolayers under the indicated conditions. After preconditioning (static or atheroprotective flow), the cells were treated with 1 U/ml of IL-1β for 6 hours and then incubated with fluorescently labeled HL-60 cells. Shown are representative fields of HUVEC monolayers (blue) and attached monocytes (yellow). The bar graph on the right displays quantification of bound HL-60 cells. (B) Role of KLF2 in flow-mediated resistance to oxidant stress. Cells under static conditions (top row) were infected with either Ad-GFP or Ad-KLF2 for 24 hours, and cells preconditioned with atheroprotective flow (bottom row) were treated with control or KLF2 siRNA. Cells were then incubated with or without 200 μM tert-butyl H2O2 for an additional 4 hours. (C) Total protein from GFP- or KLF2-expressing HUVECs blotted for catalase, manganese superoxide dismutase (MnSOD), or α-tubulin as a loading control. All data are expressed as mean ± SEM (n = 3). *P < 0.05 versus control; Student’s t test.