Current paradigms suggest that two macrophage subsets, termed M1 and M2, are involved in inflammation and host defense. While the distinct functions of M1 and M2 macrophages have been intensively studied -the former are considered proinflammatory and the latter antiinflammatory -the determinants of their speciation are incompletely understood. Here we report our studies that identify Krüppel-like factor 4 (KLF4) as a critical regulator of macrophage polarization. Macrophage KLF4 expression was robustly induced in M2 macrophages and strongly reduced in M1 macrophages, observations that were recapitulated in human inflammatory paradigms in vivo. Mechanistically, KLF4 was found to cooperate with Stat6 to induce an M2 genetic program and inhibit M1 targets via sequestration of coactivators required for NF-κB activation. KLF4-deficient macrophages demonstrated increased proinflammatory gene expression, enhanced bactericidal activity, and altered metabolism. Furthermore, mice bearing myeloid-specific deletion of KLF4 exhibited delayed wound healing and were predisposed to developing diet-induced obesity, glucose intolerance, and insulin resistance. Collectively, these data identify KLF4 as what we believe to be a novel regulator of macrophage polarization.

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

The vascular endothelium plays a critical role in vascular homeostasis. Inflammatory cytokines and non-laminar blood flow induce endothelial dysfunction and confer a pro-adhesive and pro-thrombotic phenotype. Therefore, identification of factors that mediate the effects of these stimuli on endothelial function is of considerable interest. Kruppel-like factor 4 expression has been documented in endothelial cells, but a function has not been described. In this communication we describe the expression in vitro and in vivo of Kruppel-like factor 4 in human and mouse endothelial cells. Furthermore, we demonstrate that endothelial Kruppel-like factor 4 is induced by pro-inflammatory stimuli and shear stress. Overexpression of Kruppel-like factor 4 induces expression of multiple anti-inflammatory and anti-thrombotic factors including endothelial nitric-oxide synthase and thrombomodulin, whereas knockdown of Kruppellike factor 4 leads to enhancement of tumor necrosis factor ␣-induced vascular cell adhesion molecule-1 and tissue factor expression. The functional importance of Kruppel-like factor 4 is verified by demonstrating that Kruppel-like factor 4 expression markedly decreases inflammatory cell adhesion to the endothelial surface and prolongs clotting time under inflammatory states. Kruppel-like factor 4 differentially regulates the promoter activity of pro-and anti-inflammatory genes in a manner consistent with its anti-inflammatory function. These data implicate Kruppel-like factor 4 as a novel regulator of endothelial activation in response to pro-inflammatory stimuli.

The endothelium regulates vascular homeostasis, and endothelial dysfunction is a proximate event in the pathogenesis of atherothrombosis. Stimulation of the endothelium with proinflammatory cytokines or exposure to hemodynamic-induced disturbed flow leads to a proadhesive and prothrombotic phenotype that promotes atherothrombosis. In contrast, exposure to arterial laminar flow induces a gene program that confers a largely antiadhesive, antithrombotic effect. The molecular basis for this differential effect on endothelial function remains poorly understood. While recent insights implicate Kruppel-like factors (KLFs) as important regulators of vascular homeostasis, the in vivo role of these factors in endothelial biology remains unproven. Here, we show that endothelial KLF4 is an essential determinant of atherogenesis and thrombosis. Using in vivo EC-specific KLF4 overexpression and knockdown murine models, we found that KLF4 induced an antiadhesive, antithrombotic state. Mechanistically, we demonstrated that KLF4 differentially regulated pertinent endothelial targets via competition for the coactivator p300. These observations provide cogent evidence implicating endothelial KLFs as essential in vivo regulators of vascular function in the adult animal. IntroductionThrough the elaboration of numerous biological substances, ECs actively regulate fundamental physiological processes, such as regulation of blood coagulation, homing of immune cells, and barrier function. Studies over the past several decades have also identified key physiologic and pathologic phenotypic modulators of ECs. For example, stimulation of the endothelium with proinflammatory cytokines renders the endothelium dysfunctional, inducing a proadhesive and prothrombotic phenotype. In contrast, laminar flow induces critical genes that confer potent antithrombotic, antiadhesive, and antiinflammatory properties. The significance of fluid shear stress is evidenced by the observation that segments of the arterial tree exposed to laminar flow (e.g., straight regions of the vasculature) are less prone to the development of atherosclerotic lesions than are regions exposed to nonlaminar/disturbed flow (e.g., branch points). These observations have led to the current view that the balance of biochemical and biomechanical stimuli is the central determinant of vascular function under physiologic and pathologic conditions. Given the importance of the endothelium in vessel homeostasis, there is great interest in identifying molecular pathways that mediate the effects of both biochemical and biomechanical stimuli. Prior studies from our group and others have identified 2 members of the Kruppel-like factor (KLF) family of transcription factors, KLF2 and KLF4, as being of particular interest. Both KLF2 and KLF4 are induced by laminar flow and in in vitro stud-

The Kruppel-like factor KLF2 was recently identified as a novel regulator of endothelial pro-inflammatory and pro-thrombotic function. Here it is shown that overexpression of KLF2 potently inhibits vascular permeability factor/vascular endothelial growth factor (VEGF-A)-mediated angiogenesis and tissue edema in the nude ear mouse model of angiogenesis. In vitro, KLF2 expression retards VEGF-mediated calcium flux, proliferation and induction of pro-inflammatory factors in endothelial cells. This effect is due to a potent inhibition of VEGFR2/KDR expression and promoter activity. These observations identify KLF2 as a regulator of VEGFR2/ KDR and provide a foundation for novel approaches to regulate angiogenesis.Angiogenesis, the outgrowth of new vessels from preexisting blood vessels, is an important feature of both normal physiology and pathologic states including chronic inflammatory diseases and tumor development (1-3). The growth factor VEGF-A 1 is a key regulator of physiologic and pathologic angiogenesis (4). Originally identified on the basis of its ability to induce vascular permeability (5), VEGF-A is now recognized as a potent inducer of endothelial proliferation, migration, and survival. Furthermore, VEGF-A also acts as a proinflammatory cytokine and induces the expression of a number of molecules implicated in regulating angiogenesis such specific enzymes (e.g. cyclooxygenase-2 (COX-2)), adhesion molecules (e.g. E-selectin, VCAM-1) (6), and pro-coagulant factors (e.g. tissue factor) (7). The effects of VEGF-A and its family members are mediated by three structurally related receptor tyrosine kinases termed VEGFR1/Flt-1, VEGFR2/KDR/Flk-1, and VEGFR3/Flt4 (8 -13). Among these three receptors, VEGFR2 has emerged as the main receptor mediating VEGF-A effects related to angiogenesis such as endothelial cell proliferation, migration, and proinflammatory activation. In contrast, VEGFR1 is thought to mediate inhibitory and/or decoy effects (14) (15) in vascular endothelial cells. Finally, VEGFR3 is mainly expressed in lymphatics and regulates aspects of lymphatic endothelial cell biology (13). The importance of VEGF-A/VEGFR2 axis is further underscored by the fact that both ligand and receptor levels is increased in pathologic states such as tumor beds (16 -20). It follows that identification of mechanisms that may reduce the expression of either ligand or receptor may serve as the basis for inhibiting angiogenesis in pathologic states.The Sp/Kruppel-like factor (KLF) family of transcription factors is a subclass of the zinc-finger family of transcriptional regulators implicated in the regulation of cellular growth and differentiation (21). To date 20 members have been identified that include 4 Sp factors (Sp1-4) and 16 KLF factors (KLF1-16) (22). Members of this family can bind with varying affinities to the same DNA sequences (termed GC-box or CACCC element) and varying transcriptional activities. Furthermore, members of this family can modulate each other's function through a number of distinct mechanisms s...

Abstract. Antibodies to CD44 have been used to inhibit a variety of processes which include lymphohemopoiesis, lymphocyte migration, and tumor metastasis. Some, but not all, CD44-mediated functions derive from its ability to serve as a receptor for hyaluronan (HA). However, sites on CD44 that interact with either ligands or antibodies are poorly understood. Interspecies rat/mouse CD44 chimeras were used to analyze the specificity of 25 mAbs and to determine that they recognize at least seven epitopes. Amino acid substitutions that resulted in loss of antibody recognition were all located in the region of homology to other cartilage link family proteins. While at least five epitopes were eliminated by single amino acid replacements, multiple residues had to be changed to destroy binding by other antibodies. One antibody was sensitive to changes in any of three separate parts of the molecule and some antibodies to distinct epitopes cross-blocked each other. Certain antibodies had the ability to increase HA binding by lymphocytes but this did not correlate absolutely with antibody specificity and was only partially attributable to CD44 cross-linking. Antibodies that consistently blocked HA recognition were all sensitive to amino acid changes within a short stretch of CD44. Such blocking antibodies interacted with CD44 more strongly than ligand in competition experiments. One large group of antibodies blocked ligand binding, but only with a particular cell line. This detailed analysis adds to our understanding of functional domains within CD44 and requirements for antibodies to influence recognition of one ligand. MONOCLONAL antibodies have provided powerful tools for identifying and characterizing celladhesion molecules. They usually reflect species differences in amino acid sequences between the source of the immunogen and the animal being immunized. Certain antibodies influence adhesive functions and others are informative about ligand-binding states. Particularly well studied in this regard are antibodies to integrins, which appear to undergo conformational changes in response to intracellular or extracellular signals (2,38,48). One antibody that binds only to an "activated" integrin may do so by mimicking the natural ligand (1). Some antibodies dramatically increase the affinity of integrins for ligand, perhaps by stabilizing a particular active conformation (9, 38). Still other reagents block or change the specificity for ligands and there are antibodies which recognize binding site occupancy (10, 36). Finally, antibodies have been described that recognize combinations of particular et and 13 integrin

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Inflammatory mediators like bacterial lipopolysaccharide induce monocytes to express tissue factor (TF), the cell-surface protein that triggers the blood clotting cascade in hemostasis and thrombotic disease. The physiologic ligand for TF is the serine protease, factor VIIa (FVIIa), and the resulting bimolecular enzyme, TF/ FVIIa, can be reversibly inhibited by tissue factor pathway inhibitor (TFPI). Culturing monocytic cells in the presence of both FVIIa and TFPI caused down-regulation of TF expression via reducing its half-life. To exert this effect, FVIIa had to be competent to bind both TF and TFPI, and TFPI had to contain the C-terminal domain required for binding to other cell-surface receptors, including the low density lipoprotein receptor-related protein (LRP). TF down-regulation by FVIIa plus TFPI was abrogated by the 39-kDa receptor-associated protein, which blocks binding of all known ligands to LRP. Furthermore, treatment with FVIIa plus TFPI caused monocyte TF to colocalize with ␣-adaptin, a component of clathrin-coated pits. Thus, in addition to reversibly inhibiting TF/FVIIa catalytic activity, TFPI also mediates the permanent down-regulation of cell-surface TF in monocytic cells via LRP-dependent internalization and degradation. This represents an unusual mechanism for receptor internalization, requiring ligand-dependent bridging of one cell-surface receptor (TF) to a second cell-surface receptor (LRP), the latter being capable of clathrin-mediated internalization.