Mark W. Feinberg

KLF2 Is a Novel Transcriptional Regulator of Endothelial Proinflammatory Activation

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-1β 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.

Kruppel-like Factor 4 Regulates Endothelial Inflammation

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 α-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.

Kruppel-like Factor 4 Is a Mediator of Proinflammatory Signaling in Macrophages

Activation of macrophages is important in chronic inflammatory disease states such as atherosclerosis. Proinflammatory cytokines such as interferon-γ (IFN-γ), lipopolysaccharide (LPS), or tumor necrosis factor-α can promote macrophage activation. Conversely, anti-inflammatory factors such as transforming growth factor-β1 (TGF-β1) 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-γ, LPS, or tumor necrosis factor-α and decreased by TGF-β1 in macrophages. Overexpression of KLF4 in J774a macrophages induced the macrophage activation marker inducible nitric-oxide synthase and inhibited the TGF-β1 and Smad3 target gene plasminogen activator inhibitor-1 (PAI-1). Conversely, KLF4 knockdown markedly attenuated the ability of IFN-γ, LPS, or IFN-γ plus LPS to induce the iNOS promoter, whereas it augmented macrophage responsiveness to TGF-β1 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-γ and LPS. We further provide evidence that KLF4 interacts with the NF-κB family member p65 (RelA) to cooperatively induce the iNOS promoter. In contrast, KLF4 inhibited the TGF-β1/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.

The Kruppel-like factor KLF4 is a critical regulator of monocyte differentiation

Monocyte differentiation involves the participation of lineage‐restricted transcription factors, although the mechanisms by which this process occurs are incompletely defined. Within the hematopoietic system, members of the Kruppel‐like family of factors (KLFs) play essential roles in erythrocyte and T lymphocyte development. Here we show that KLF4/GKLF is expressed in a monocyte‐restricted and stage‐specific pattern during myelopoiesis and functions to promote monocyte differentiation. Overexpression of KLF4 in HL‐60 cells confers the characteristics of mature monocytes. Conversely, KLF4 knockdown blocked phorbol ester‐induced monocyte differentiation. Forced expression of KLF4 in primary common myeloid progenitors (CMPs) or hematopoietic stem cells (HSCs) induced exclusive monocyte differentiation in clonogenic assays, whereas KLF4 deficiency inhibited monocyte but increased granulocyte differentiation. Mechanistic studies demonstrate that KLF4 is a target gene of PU.1. Consistently, KLF4 can rescue PU.1–/– fetal liver cells along the monocytic lineage and can activate the monocytic‐specific CD14 promoter. Thus, KLF4 is a critical regulator in the transcriptional network controlling monocyte differentiation.

Adiponectin Inhibits the Production of CXC Receptor 3 Chemokine Ligands in Macrophages and Reduces T-Lymphocyte Recruitment in Atherogenesis

Obese individuals often have low plasma adiponectin and concomitant chronic inflammation with a predisposition to metabolic and cardiovascular diseases. The present study reports a novel antiinflammatory action of adiponectin in human monocyte-derived macrophages (M&PHgr;) suppressing T-lymphocyte accumulation in atherogenesis. RNA profiling of lipopolysaccharide-stimulated human M&PHgr; identified CXC chemokine ligands (CXCLs), such as IP-10 (interferon [IFN]-inducible protein 10) (CXCL10), I-TAC (IFN-inducible T-cell α chemoattractant) (CXCL11), and Mig (monokine induced by IFN-γ) (CXCL9), T-lymphocyte chemoattractants associated with atherogenesis, among the top 14 transcripts suppressed by adiponectin. Real-time quantitative RT-PCR and ELISA verified that adiponectin inhibited expression of these chemokines at both the mRNA and protein levels in a concentration-dependent manner. Adiponectin reduced the release by lipopolysaccharide-stimulated M&PHgr; of chemoattractant activity for CXC chemokine receptor 3–transfected (receptor for IP-10, Mig, and I-TAC) lymphocytes. Adiponectin decreased lipopolysaccharide-inducible IP-10 promoter activity in promoter-transfected THP-1 M&PHgr; but did not change IP-10 mRNA stability. In lipopolysaccharide-stimulated M&PHgr;, reduction of IFN-β by adiponectin preceded inhibition of IP-10 mRNA expression. Immunoblot and chromatin immunoprecipitation analyses demonstrated that adiponectin attenuated activation of the transcription factor IFN regulatory factor 3, involved in the MyD88-independent pathway of Toll-like receptor 4 signaling, and subsequent IFN regulatory factor 3 binding to IFN-β promoter. In vivo studies further demonstrated that apolipoprotein E/adiponectin double-deficient (apoE −/−APN−/−) mice had increased plasma IP-10 levels, accelerated T-lymphocyte accumulation in atheromata, and augmented atherogenesis compared with apoE single-deficient (apoE−/−APN+/+) mice. This study establishes that low levels of adiponectin associated with obesity, the metabolic syndrome, and diabetes favor T-lymphocyte recruitment and contribute to adaptive immune response during atherogenesis.

Transforming Growth Factor-β1 Inhibits Cytokine-mediated Induction of Human Metalloelastase in Macrophages

Matrix metalloproteinases (MMP) have been identified in vulnerable areas of atherosclerotic plaques and may contribute to plaque instability through extracellular matrix degradation. Human metalloelastase (MMP-12) is a macrophage-specific MMP with broad substrate specificity and is capable of degrading proteins found in the extracellular matrix of atheromas. Despite its potential importance, little is known about the regulation of MMP-12 expression in the context of atherosclerosis. In this study, we report that in human peripheral blood-derived macrophages, MMP-12 mRNA was markedly up-regulated by several pro-atherosclerotic cytokines and growth factors including interleukin-1β, tumor necrosis factor-α, macrophage colony-stimulating factor, vascular endothelial growth factor, and platelet-derived growth factor-BB. In contrast, the pleiotropic anti-inflammatory growth factor transforming growth factor-β1 (TGF-β1) inhibited cytokine-mediated induction of MMP-12 mRNA, protein, and enzymatic activity. Analyses of MMP-12 promoter through transient transfections and electrophoretic mobility shift assays indicated that both its induction by cytokines and its inhibition by TGF-β1 depended on signaling through an AP-1 site at −81 base pairs. Moreover, the inhibitory effect of TGF-β1 on MMP-12 was dependent on Smad3. Taken together, MMP-12 is induced by several factors implicated in atherosclerosis. The inhibition of MMP-12 expression by TGF-β1 suggests that TGF-β1, acting via Smad3, may promote plaque stability.

Systemic Delivery of MicroRNA-181b Inhibits Nuclear Factor-κB Activation, Vascular Inflammation, and Atherosclerosis in Apolipoprotein E–Deficient Mice

Rationale: Activated nuclear factor (NF)-&kgr;B signaling in the vascular endothelium promotes the initiation and progression of atherosclerosis. Targeting endothelial NF-&kgr;B may provide a novel strategy to limit chronic inflammation. Objective: To examine the role of microRNA-181b (miR-181b) in endothelial NF-&kgr;B signaling and effects on atherosclerosis. Methods and Results: MiR-181b expression was reduced in the aortic intima and plasma in apolipoprotein E–deficient mice fed a high-fat diet. Correspondingly, circulating miR-181b in the plasma was markedly reduced in human subjects with coronary artery disease. Systemic delivery of miR-181b resulted in a 2.3-fold overexpression of miR-181b in the aortic intima of apolipoprotein E–deficient mice and suppressed NF-&kgr;B signaling revealed by bioluminescence imaging and reduced target gene expression in the aortic arch in apolipoprotein E–deficient/NF-&kgr;B-luciferase transgenic mice. MiR-181b significantly inhibited atherosclerotic lesion formation, proinflammatory gene expression and the influx of lesional macrophages and CD4+ T cells in the vessel wall. Mechanistically, miR-181b inhibited the expression of the target gene importin-&agr;3, an effect that reduced NF-&kgr;B nuclear translocation specifically in the vascular endothelium of lesions, whereas surprisingly leukocyte NF-&kgr;B signaling was unaffected despite a 7-fold overexpression of miR-181b. Our findings uncover that NF-&kgr;B nuclear translocation in leukocytes does not involve importin-&agr;3, but rather importin-&agr;5, which miR-181b does not target, highlighting that inhibition of NF-&kgr;B signaling in the endothelium is sufficient to mediate miR-181b’s protective effects. Conclusions: Systemic delivery of miR-181b inhibits the activation of NF-&kgr;B and atherosclerosis through cell-specific mechanisms in the vascular endothelium. These findings support the rationale that delivery of miR-181b may provide a novel therapeutic approach to treat chronic inflammatory diseases such as atherosclerosis.

Transforming Growth Factor-β1 Inhibition of Macrophage Activation Is Mediated via Smad3

Activated macrophages are critical cellular participants in inflammatory disease states. Transforming growth factor (TGF)-β1 is a growth factor with pleiotropic effects including inhibition of immune cell activation. Although the pathway of gene activation by TGF-β1 via Smad proteins has recently been elucidated, suppression of gene expression by TGF-β1 remains poorly understood. We found that of Smad1–Smad7, Smad3 alone was able to inhibit expression of markers of macrophage activation (inducible nitric-oxide synthase and matrix metalloproteinase-12) following lipopolysaccharide treatment in gene reporter assays. Transient and constitutive overexpression of a dominant negative Smad3 opposed the inhibitory effect of TGF-β1. Domain swapping experiments suggest that both the Smad MH-1 and MH-2 domains are required for inhibition. Mutation of a critical amino acid residue required for DNA binding in the MH-1 of Smad3 (R74A) resulted in the loss of inhibition. Transient overexpression of p300, an interactor of the Smad MH-2 domain, partially alleviated the inhibition by TGF-β1/Smad3, suggesting that inhibition of gene expression may be due to increased competition for limiting amounts of this coactivator. Our results have implications for the understanding of gene suppression by TGF-β1 and for the regulation of activated macrophages by TGF-β1.

Systemic Delivery of MicroRNA-181b Inhibits NF-κB Activation, Vascular Inflammation, and Atherosclerosis in Apoe-/- Mice

Rationale: Activated nuclear factor (NF)-&kgr;B signaling in the vascular endothelium promotes the initiation and progression of atherosclerosis. Targeting endothelial NF-&kgr;B may provide a novel strategy to limit chronic inflammation. Objective: To examine the role of microRNA-181b (miR-181b) in endothelial NF-&kgr;B signaling and effects on atherosclerosis. Methods and Results: MiR-181b expression was reduced in the aortic intima and plasma in apolipoprotein E–deficient mice fed a high-fat diet. Correspondingly, circulating miR-181b in the plasma was markedly reduced in human subjects with coronary artery disease. Systemic delivery of miR-181b resulted in a 2.3-fold overexpression of miR-181b in the aortic intima of apolipoprotein E–deficient mice and suppressed NF-&kgr;B signaling revealed by bioluminescence imaging and reduced target gene expression in the aortic arch in apolipoprotein E–deficient/NF-&kgr;B-luciferase transgenic mice. MiR-181b significantly inhibited atherosclerotic lesion formation, proinflammatory gene expression and the influx of lesional macrophages and CD4+ T cells in the vessel wall. Mechanistically, miR-181b inhibited the expression of the target gene importin-&agr;3, an effect that reduced NF-&kgr;B nuclear translocation specifically in the vascular endothelium of lesions, whereas surprisingly leukocyte NF-&kgr;B signaling was unaffected despite a 7-fold overexpression of miR-181b. Our findings uncover that NF-&kgr;B nuclear translocation in leukocytes does not involve importin-&agr;3, but rather importin-&agr;5, which miR-181b does not target, highlighting that inhibition of NF-&kgr;B signaling in the endothelium is sufficient to mediate miR-181b’s protective effects. Conclusions: Systemic delivery of miR-181b inhibits the activation of NF-&kgr;B and atherosclerosis through cell-specific mechanisms in the vascular endothelium. These findings support the rationale that delivery of miR-181b may provide a novel therapeutic approach to treat chronic inflammatory diseases such as atherosclerosis.

MicroRNA-26a regulates pathological and physiological angiogenesis by targeting BMP/SMAD1 signaling

Rationale: The rapid induction and orchestration of new blood vessels are critical for tissue repair in response to injury, such as myocardial infarction, and for physiological angiogenic responses, such as embryonic development and exercise. Objective: We aimed to identify and characterize microRNAs (miR) that regulate pathological and physiological angiogenesis. Methods and Results: We show that miR-26a regulates pathological and physiological angiogenesis by targeting endothelial cell (EC) bone morphogenic protein/SMAD1 signaling in vitro and in vivo. MiR-26a expression is increased in a model of acute myocardial infarction in mice and in human subjects with acute coronary syndromes. Ectopic expression of miR-26a markedly induced EC cycle arrest and inhibited EC migration, sprouting angiogenesis, and network tube formation in matrigel, whereas blockade of miR-26a had the opposite effects. Mechanistic studies demonstrate that miR-26a inhibits the bone morphogenic protein/SMAD1 signaling pathway in ECs by binding to the SMAD1 3′-untranslated region, an effect that decreased expression of Id1 and increased p21WAF/CIP and p27. In zebrafish, miR-26a overexpression inhibited formation of the caudal vein plexus, a bone morphogenic protein-responsive process, an effect rescued by ectopic SMAD1 expression. In mice, miR-26a overexpression inhibited EC SMAD1 expression and exercise-induced angiogenesis. Furthermore, systemic intravenous administration of an miR-26a inhibitor, locked nucleic acid-anti–miR-26a, increased SMAD1 expression and rapidly induced robust angiogenesis within 2 days, an effect associated with reduced myocardial infarct size and improved heart function. Conclusions: These findings establish miR-26a as a regulator of bone morphogenic protein/SMAD1-mediated EC angiogenic responses, and that manipulating miR-26a expression could provide a new target for rapid angiogenic therapy in ischemic disease states.