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-1 ␤ 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.

Chronic inflammation is a key feature of vascular disease states such as atherosclerosis. Multiple clinical studies have shown that a class of medications termed statins lower cardiovascular morbidity and mortality. Originally developed to lower serum cholesterol, increasing evidence suggests that these medications have potent anti-inflammatory effects that contribute to their beneficial effects in patients. Here, we discuss the clinical and experimental evidence underlying the anti-inflammatory effects of these agents.

Abstract-Krüppel-like factors are members of the zinc finger family of transcription factors that have been implicated as playing key roles in regulating cellular differentiation and tissue development. Studies over the past several years support an important role for this family of factors in vascular biology. This review summarizes the role of Krüppel-like factors in endothelial cell biology.

Activation of macrophages is important in chronic inflammatory disease states such as atherosclerosis. Proinflammatory cytokines such as interferon-␥ (IFN-␥), lipopolysaccharide (LPS), or tumor necrosis factor-␣ can promote macrophage activation. Conversely, anti-inflammatory factors such as transforming growth factor-␤1 (TGF-␤1) 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-␥, LPS, or tumor necrosis factor-␣ and decreased by TGF-␤1 in macrophages. Overexpression of KLF4 in J774a macrophages induced the macrophage activation marker inducible nitric-oxide synthase and inhibited the TGF-␤1 and Smad3 target gene plasminogen activator inhibitor-1 (PAI-1). Conversely, KLF4 knockdown markedly attenuated the ability of IFN-␥, LPS, or IFN-␥ plus LPS to induce the iNOS promoter, whereas it augmented macrophage responsiveness to TGF-␤1 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-␥ and LPS. We further provide evidence that KLF4 interacts with the NF-B family member p65 (RelA) to cooperatively induce the iNOS promoter. In contrast, KLF4 inhibited the TGF-␤1/ 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.Macrophage activation is an integral process in the development of atherosclerosis as well as a number of other chronic inflammatory diseases such as emphysema, inflammatory bowel disease, psoriasis, arthritis, and pancreatitis. Once within the site of inflammation, macrophages elaborate a broad range of cytokines, growth factors, and proteolytic enzymes that may participate in the damage and repair that ensues. Identification of mechanisms that may regulate macrophage activation is, thus, of considerable interest.One of the key events in macrophage response to proinflammatory stimuli is the expression of inducible nitric-oxide synthase (iNOS) 2 and the formation of nitric oxide, an important mediator involved in many host defense actions in macrophages. However, increased amounts of leukocyte-derived nitric oxide can be detrimental by promoting tissue damage in a variety of inflammatory disease states (1, 2). Given the importance of iNOS in a variety of pathophysiological conditions, control of its expression has been the subject of considerable investigation (1). Indeed, several studies have shown that induction or inhibition of iNOS in macrophages by pro-or anti-inflammatory stimuli, respectively, can occur at the level of transcription. For example, proinflammatory stimuli such as LPS or IFN-␥ involve activation of NF-B or interferon-responsive element...

The mechanisms regulating activation of monocytes remain incompletely understood. Herein we provide evidence that Kruppel-like factor 2 (KLF2) inhibits proinflammatory activation of monocytes. In vitro, KLF2 expression in monocytes is reduced by cytokine activation or differentiation. Consistent with this observation, KLF2 expression in circulating monocytes is reduced in patients with chronic inflammatory conditions such as coronary artery disease. Adenoviral overexpression of KLF2 inhibits the LPSmediated induction of proinflammatory factors, cytokines, and chemokines and reduces phagocytosis. Conversely, short interfering RNA-mediated reduction in KLF2 increased inflammatory gene expression. Reconstitution of immunodeficient mice with KLF2-overexpressing monocytes significantly reduced carrageenan-induced acute paw edema formation. Mechanistically, KLF2 inhibits the transcriptional activity of both NF-B and activator protein 1, in part by means of recruitment of transcriptional coactivator p300͞CBP-associated factor. These observations identify KLF2 as a novel negative regulator of monocytic activation.inflammation ͉ transcription ͉ transcriptional factor ͉ NF-B ͉ activator protein 1

Sudden cardiac death exhibits diurnal variation in both acquired and hereditary forms of heart disease 1, 2, but the molecular basis is unknown. A common mechanism that underlies susceptibility to ventricular arrhythmias is abnormalities in the duration (e.g. short or long QT syndromes, heart failure) 3-5 or pattern (e.g. Brugada syndrome) 6 of myocardial repolarization. Here we provide the first molecular evidence that links circadian rhythms to vulnerability in ventricular arrhythmias in mice. Specifically, we show that cardiac ion channel expression and QT interval duration (an index of myocardial repolarization) exhibit endogenous circadian rhythmicity under the control of a novel clock-dependent oscillator, Krüppel-like factor 15 (Klf15). Klf15 transcriptionally controls rhythmic expression of KChIP2, a critical subunit required for generating the transient outward potassium current (Ito). 7 Deficiency or excess of Klf15 causes loss of rhythmic QT variation, abnormal repolarization and enhanced susceptibility to ventricular arrhythmias. In sum, these findings identify circadian transcription of ion channels as a novel mechanism for cardiac arrhythmogenesis.

Abstract-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.

Resistance to the stimulatory effects of insulin on glucose utilization is a key feature of type 2 diabetes, obesity, and the metabolic syndrome. Recent studies suggest that insulin resistance is primarily caused by a defect in glucose transport. GLUT4 is the main insulinresponsive glucose transporter and is expressed predominantly in muscle and adipose tissues. Whereas GLUT4 has been shown to play a critical role in maintaining systemic glucose homeostasis, the mechanisms regulating its expression are incompletely understood. We have cloned the murine homologue of KLF15, a member of the Krü ppel-like family of transcription factors. KLF15 is highly expressed in adipocytes and myocytes in vivo and is induced when 3T3-L1 preadipocytes are differentiated into adipocytes. Overexpression of KLF15 in adipose and muscle cell lines potently induces GLUT4 expression. This effect is specific to KLF15 as overexpression of two other Krü ppel-like factors, KLF2/LKLF and KLF4/GKLF, did not induce GLUT4 expression. Both basal (3.3-fold, p < 0.001) and insulin-stimulated (2.4-fold, p < 0.00001) glucose uptake are increased in KLF15-overexpressing adipocytes. In co-transfection assays, KLF15 and MEF2A, a known activator of GLUT4, synergistically activates the GLUT4 promoter. Promoter deletion and mutational analyses provide evidence that this activity requires an intact KLF15-binding site proximal to the MEF2A site. Finally, co-immunoprecipitation assays show that KLF15 specifically interacts with MEF2A. These studies indicate that KLF15 is an important regulator of GLUT4 in both adipose and muscle tissues.Glucose uptake into cells is regulated by two families of cellular transporters, the sodium-linked glucose transporters (kidney, intestine) and the facilitated glucose transporters (GLUTs). With respect to the latter, GLUT4 is the main effector of insulin-stimulated glucose transport and is located primarily in muscle and adipose tissues (1).Clinical and experimental observations suggest that insulinstimulated glucose transport via GLUT4 is critical in maintaining systemic glucose homeostasis. For example, heterozygous mice deficient in GLUT4 exhibit muscle insulin resistance and develop diabetes (2). Tissue-specific disruption of GLUT4 in adipose tissue and skeletal muscle results in the development of insulin resistance and glucose intolerance (3, 4). In human type 2 diabetic patients, impairment of insulin-stimulated glucose transport is responsible for resistance to insulin-stimulated glycogen synthesis in muscle (5-7).Studies in adipose and muscle tissues reveal that expression of the GLUT4 glucose transporter is controlled at the level of transcription (8, 9). In vitro and in vivo promoter studies support a role for members of the MADS-box family of transcription factors termed MEF2 proteins in the regulation of the GLUT4 promoter. However, these studies suggest that MEF2 binding alone is not sufficient to fully support GLUT4 expression (10 -12).The Krü ppel-like family of transcription factors are important regulators ...