From the moment the heart first starts to beat, the endothelial lining of the cardiovascular system is constantly exposed to a spectrum of hemodynamic forces that have been postulated to play a role in such diverse processes as blood vessel development, angiogenesis, acute and chronic inflammation, and atherogenesis. Several studies in vitro have demonstrated that endothelial cells are able to differentially sense and transduce distinct biomechanical stimuli and respond to them with changes in gene expression (33
). Moreover, endothelial cells acquire specific functional phenotypes depending on the type of biomechanical stimulation to which they are exposed (4
Dekker et al. first identified KLF2 as a gene regulated by steady laminar shear stress in cultured endothelial cells and, based on its pattern of expression in human arteries, these authors suggested a potential protective role for this transcription factor in atherogenesis (7
). Previous studies from our laboratories demonstrated that overexpression of KLF2 in cultured endothelial cells leads to the upregulation of eNOS and the inhibition of the IL-1β–dependent expression of E-selectin, VCAM-1, and TF (8
). Here we demonstrate the differential upregulation of KLF2 in cultured endothelial cells exposed to a shear stress waveform characteristic of a region of the human carotid artery protected from atherogenesis. Moreover, we document the flow-dependent nature of endothelial KLF2 expression in vivo using the sih
zebrafish mutant, which lacks blood flow. Further analyses have revealed that the mechanisms linking hemodynamic forces and KLF2 expression involve activation of a MEK5/ERK5/MEF2 pathway and that MEK5 activation is both necessary and sufficient for the upregulation of KLF2.
Genome-wide transcriptional profiling experiments using cultured endothelial cells overexpressing KLF2 under control or inflammatory conditions revealed that KLF2 regulates transcriptional pathways involved in multiple endothelial functions, including blood vessel formation, control of vasomotor tone, thrombosis, and inflammation. Thus, the present study identifies a molecular mechanism linking atheroprotective flow to an orchestrated regulation of multiple endothelial transcriptional programs. Our results are consistent with a model in which flow activates MEK5, which in turn phosphorylates ERK5, resulting in activation of the MEF2 family at the KLF2 promoter. Binding of the MEF2 members to the KLF2 promoter is found under static conditions and is not substantially altered upon flow, indicating that this family appears to tonically bind to the KLF2 promoter and act as a switch by receiving upstream signals (e.g., phosphorylation by ERK5). In support of this concept, a recent study showed that MEF2 factors can bind to the KLF2 promoter and induce transcriptional activity. Furthermore, inhibition of MEF2 function by p65 and histone deacetylases (HDAC4 and HDAC5) accounts for the reduction in KLF2 expression observed in endothelial cells treated with TNF-α (38
). Mice lacking KLF2 die during embryogenesis, exhibiting defects in assembly of the vessel wall that lead to massive hemorrhage (39
). Ubiquitous deletion of ERK5 in adult mice causes a specific and marked loss of vascular integrity marked by hemorrhages, vessel dilation, and endothelial apoptosis (14
). The phenotype from ubiquitous deletion is in fact indistinguishable from an endothelium-specific knockout of ERK5, suggesting a prominent role for this kinase in endothelial function and survival. Furthermore, MEF2 activation by serum was demonstrated to be impaired in the ERK5-deficient endothelial cells (14
). Our finding that ERK5 activation is required for flow-mediated KLF2 expression may reveal KLF2 as a major MEF2 transcriptional target responsible for the observed vascular phenotype in ERK5-null mice.
Mice null for MEF2C exhibit defects in vascular development that share features with the KLF2 knockout (40
). Our data demonstrating the importance of MEF2 in KLF2 expression, and the global endothelial functions regulated by this mechano-activated gene, suggest that the vascular phenotypes rendered by inactivation of MEF2 may be due to reduced KLF2 expression. It will be of great interest to test whether any of the components of the mechanotransduction pathway outlined here (MEK5/ERK5/KLF2) and their modulators are genetic modifiers of cardiovascular disease.
Our analysis of the global transcriptional networks downstream of KLF2 offers further insight into possible molecular mechanisms underlying the embryonic cardiovascular phenotype of the KLF2-null mice. The data presented here indicate that increased KLF2 expression increases mRNA levels of Tie2 while potently suppressing Ang-2 levels and that this regulation is observed when endothelial cells are exposed to flow. Because Ang-2 can act as a Tie2 antagonist (41
), KLF2 may transcriptionally orchestrate this pathway to potentiate the Ang-1/Tie2 axis.
Another major endothelial function regulated transcriptionally by KLF2 is the control of vessel tone. In addition to the regulation of eNOS previously described by our laboratories and others (8
), we find that KLF2 concomitantly downregulates caveolin-1, a negative regulator of eNOS activity, and upregulates 2 additional enzymes that promote NO production, namely ASS and dimethylarginine dimethylaminohydrolase. CNP is an endothelial paracrine substance whose function appears to also extend beyond vasodilation to include suppression of smooth muscle proliferation and potentiation of endothelial survival and revascularization (42
). Finally, KLF2 inhibited the expression of the vasoconstrictive factor ET-1. We observed similar regulation of these genes in cells exposed to flow and determined that the flow-mediated upregulation of eNOS, ASS, and CNP as well as the downregulation of ET-1 are KLF2 dependent. In summary, the differential regulation of these antagonistic pathways would be expected to coordinately promote endothelium-dependent vasodilation, the impairment of which has been one of the clinical definitions of endothelial dysfunction.
Two major pathophysiological processes that play a critical role in vascular pathophysiology are inflammation and thrombosis. Our data document a global antiinflammatory and antithrombotic role for KLF2 in endothelial cells. The upregulation of TM expression by shear stress has been previously demonstrated in vivo (43
); here we demonstrate KLF2 as a regulator of TM in a hemodynamic environment modeled after an atheroprotected region of the human carotid. Moreover, we have recently reported that TM is a direct transcriptional target of KLF2 (17
). The global antiinflammatory effects of endothelial KLF2 expression in the context of IL-1β activation are manifested as a coordinated regulation of multiple inflammatory mediators. Interestingly, the IL-1β–mediated upregulation of several proinflammatory cytokines, chemokines, and adhesion molecules is strongly suppressed by the overexpression of KLF2. How KLF2 is able to silence such a large spectrum of the proinflammatory phenotype is still unclear, but this activity is reminiscent of its observed effects on T cell activation, a process that also represents a global phenotypic switch (6
). Mechanistically, our laboratories have shown that KLF2 inhibits NF-κB function, and the subsequent TNF-α–mediated VCAM-1 expression, by competing for the coactivator CBP/p300 (8
). These observations suggest that similar mechanisms may play a role in the KLF2-mediated suppression of at least 29 other IL-1β–upregulated genes containing 1 or more NF-κB binding sites in their promoter regions (promoter analysis can be viewed at http://vessels.bwh.harvard.edu/Parmar1). Notably, the relationship between KLF2 and the proinflammatory stimulus is not a simple antagonism. In our transcriptional profiling analysis, we observed a group of genes that displayed a marked synergistic regulation by KLF2 and IL-1β. Thus, KLF2 not only has the ability to prevent initiation of inflammatory activation in endothelial cells, but can also prime these cells to efficiently promote rapid resolution of the inflammatory process.
Our data indicate that aspects of the atheroprotective flow-mediated endothelial functional phenotype require upregulation of KLF2. Thus, interfering with the flow-mediated expression of KLF2 using RNA interference (RNAi) resulted in an increase in leukocyte adhesion to the endothelial monolayer and the loss of protection against oxidative stress seen in endothelial cells preconditioned with flow. The KLF2-mediated “antiadhesive” endothelial phenotype may involve several signaling pathways observed in the global antiinflammatory effect of KLF2 overexpression experiments. The resistance to oxidative stress conferred by KLF2 can be mediated by numerous pathways. Although eNOS, shown here to be regulated by KLF2 and flow, has been shown to be at least partially responsible for the endothelial oxidant resistance conferred by flow (32
), the relevant mechanisms in this experimental model remain to be elucidated.
We demonstrate that KLF2 is selectively upregulated by a shear stress waveform derived from an atheroprotected region of the human carotid but not by an atheroprone shear stress waveform derived from the carotid sinus. Using RNAi to block flow-mediated KLF2 upregulation, we were able to demonstrate that the expression of 15.3% of all genes regulated by atheroprotective flow are dependent on KLF2 expression, with the most highly regulated genes exhibiting a significantly greater dependence on this transcription factor.
The identification of KLF2-dependent transcriptional programs involved in the regulation of multiple endothelial functions contributing to a distinct endothelial phenotype demonstrates a critical role for this transcription factor in maintaining the functional integrity of normal, healthy endothelium. The requirement for KLF2 upregulation in the atheroprotective flow-mediated phenotype, together with the previous demonstration that endothelial KLF2 is selectively expressed in atheroprotected regions, while relatively absent in atheroprone regions of the human vasculature (7
), strongly suggest that the focal nature of atherosclerosis and its long-established correlation with sites of particular hemodynamic environments may be at least partially explained by the spatial patterns of KLF2 expression. Interestingly, we have recently found that statins, which have been shown to have cholesterol-independent “pleiotropic” effects, are potent inducers of the expression of KLF2 and its downstream targets in endothelial cells (44
). Thus, further understanding of the mechanisms of KLF2 induction by atheroprotective hemodynamic environments will help guide the identification of novel physiological and/or pharmacological molecules that mimic this biomechanical stimulus.