Sucharita SenBanerjee

The vascular endothelium is a critical regulator of vascular function. Diverse stimuli such as proinflammatory cytokines and hemodynamic forces modulate endothelial phenotype and thereby impact on the development of vascular disease states. Therefore, identification of the regulatory factors that mediate the effects of these stimuli on endothelial function is of considerable interest. Transcriptional profiling studies identified the Kruppel-like factor (KLF)2 as being inhibited by the inflammatory cytokine interleukin-1beta and induced by laminar shear stress in cultured human umbilical vein endothelial cells. Overexpression of KLF2 in umbilical vein endothelial cells robustly induced endothelial nitric oxide synthase expression and total enzymatic activity. In addition, KLF2 overexpression potently inhibited the induction of vascular cell adhesion molecule-1 and endothelial adhesion molecule E-selectin in response to various proinflammatory cytokines. Consistent with these observations, in vitro flow assays demonstrate that T cell attachment and rolling are markedly attenuated in endothelial monolayers transduced with KLF2. Finally, our studies implicate recruitment by KLF2 of the transcriptional coactivator cyclic AMP response element-binding protein (CBP/p300) as a unifying mechanism for these various effects. These data implicate KLF2 as a novel regulator of endothelial activation in response to proinflammatory stimuli.

The vascular endothelium maintains blood fluidity by inhibiting blood coagulation, inhibiting platelet aggregation, and promoting fibrinolysis. Endothelial cells lose these nonthrombogenic properties on exposure to proinflammatory stimuli. We recently identified the Kruppel-like factor KLF2 as a novel regulator of endothelial proinflammatory activation. Here it is found that KLF2 differentially regulates key factors involved in maintaining an antithrombotic endothelial surface. Overexpression of KLF2 strongly induced thrombomodulin (TM) and endothelial nitric oxide synthase (eNOS) expression and reduced plasminogen activator inhibitor-1 (PAI-1) expression. Furthermore, overexpression of KLF2 inhibited the cytokine-mediated induction of tissue factor (TF). In contrast, siRNA mediated knockdown of KLF2 reduced antithrombotic gene expression while inducing the expression of pro-coagulant factors. The functional importance of KLF2 was verified by in vitro clotting assays. By comparison to control infected cells, KLF2 overexpression increased blood clotting time as well as flow rates under basal and inflammatory conditions. In contrast, siRNA-mediated knockdown of KLF2 reduced blood clotting time and flow rates. These observations identify KLF2 as a novel transcriptional regulator of endothelial thrombotic function. The full text of this article is available online at http://circres.ahajournals.org.

BACKGROUND: Although 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are known to modulate endothelial function, the transcriptional mechanisms underlying these effects are incompletely understood. We hypothesized that Lung-Kruppel-like factor (LKLF/KLF2), a novel and potent regulator of endothelial gene expression, may mediate the downstream effects of statins. Here we report that statin-induced expression of endothelial NO synthase (eNOS) and thrombomodulin is KLF2 dependent. METHODS AND RESULTS: KLF2 mRNA was induced by treatment with multiple statins in a concentration-dependent manner. Multiple lines of evidence suggest that this induction is dependent on inhibition of the Rho pathway and requires de novo transcription. Furthermore, promoter deletion and mutational analyses suggest that mevastatin induced KLF2 promoter activity through a single myocyte enhancer factor binding site. Finally, small-interfering RNA-mediated knockdown of KLF2 strongly attenuated the ability of mevastatin to increase eNOS and thrombomodulin accumulation in endothelial cells. CONCLUSIONS: Taken together, these observations indicate that statin-dependent induction of eNOS and thrombomodulin requires KLF2 and thereby provides a novel molecular target for modulating endothelial function in vascular disease.

Activation of macrophages is important in chronic inflammatory disease states such as atherosclerosis. Proinflammatory cytokines such as interferon-gamma (IFN-gamma), lipopolysaccharide (LPS), or tumor necrosis factor-alpha can promote macrophage activation. Conversely, anti-inflammatory factors such as transforming growth factor-beta1 (TGF-beta1) can decrease proinflammatory activation. The molecular mediators regulating the balance of these opposing effectors remain incompletely understood. Herein, we identify Kruppel-like factor 4 (KLF4) as being markedly induced in response to IFN-gamma, LPS, or tumor necrosis factor-alpha and decreased by TGF-beta1 in macrophages. Overexpression of KLF4 in J774a macrophages induced the macrophage activation marker inducible nitric-oxide synthase and inhibited the TGF-beta1 and Smad3 target gene plasminogen activator inhibitor-1 (PAI-1). Conversely, KLF4 knockdown markedly attenuated the ability of IFN-gamma, LPS, or IFN-gamma plus LPS to induce the iNOS promoter, whereas it augmented macrophage responsiveness to TGF-beta1 and Smad3 signaling. The KLF4 induction of the iNOS promoter is mediated by two KLF DNA-binding sites at -95 and -212 bp, and mutation of these sites diminished induction by IFN-gamma and LPS. We further provide evidence that KLF4 interacts with the NF-kappaB family member p65 (RelA) to cooperatively induce the iNOS promoter. In contrast, KLF4 inhibited the TGF-beta1/Smad3 induction of the PAI-1 promoter independent of KLF4 DNA binding through a novel antagonistic competition with Smad3 for the C terminus of the coactivator p300/CBP. These findings support an important role for KLF4 as a regulator of key signaling pathways that control macrophage activation.