Members of the Foxo family, Foxo1 (Fkhr), Foxo3 (Fkhrl1), and Foxo4 (Afx), are mammalian homologs of daf-16, which influences life span and energy metabolism in Caenorhabditis elegans. Mammalian FOXO proteins also play important roles in cell cycle arrest, apoptosis, stress resistance, and energy metabolism. In this study, we generated Foxo1-deficient mice to investigate the physiological role of FOXO1. The Foxo1-deficient mice died around embryonic day 11 because of defects in the branchial arches and remarkably impaired vascular development of embryos and yolk sacs. In vitro differentiation of embryonic stem cells demonstrated that endothelial cells derived from wild-type and Foxo1-deficient embryonic stem cells were able to produce comparable numbers of colonies supported by a layer of OP9 stromal cells. Although the morphology of the endothelial cell colonies was identical in both genotypes in the absence of exogenous vascular endothelial growth factor (VEGF), Foxo1-deficient endothelial cells showed a markedly different morphological response compared with wild-type endothelial cells in the presence of exogenous VEGF. These results suggest that Foxo1 is essential to the ability of endothelial cells to respond properly to a high dose of VEGF, thereby playing a critical role in normal vascular development.

No abstract

Abstract-Obese adipose tissue is markedly infiltrated by macrophages, suggesting that they may participate in the inflammatory pathways that are activated in obese adipose tissue. Evidence has suggested that saturated fatty acids released via adipocyte lipolysis serve as a naturally occurring ligand that stimulates Toll-like receptor (TLR)4 signaling, thereby inducing the inflammatory responses in macrophages in obese adipose tissue. Through a combination of cDNA microarray analyses of saturated fatty acid-stimulated macrophages in vitro and obese adipose tissue in vivo, here we identified activating transcription factor (ATF)3, a member of the ATF/cAMP response element-binding protein family of basic leucine zipper-type transcription factors, as a target gene of saturated fatty acids/TLR4 signaling in macrophages in obese adipose tissue. Importantly, ATF3, when induced by saturated fatty acids, can transcriptionally repress tumor necrosis factor-␣ production in macrophages in vitro. Chromatin immunoprecipitation assay revealed that ATF3 is recruited to the region containing the activator protein-1 site of the endogenous tumor necrosis factor-␣ promoter. Furthermore, transgenic overexpression of ATF3 specifically in macrophages results in the marked attenuation of proinflammatory M1 macrophage activation in the adipose tissue from genetically obese KKA y mice fed high-fat diet. This study provides evidence that ATF3, which is induced in obese adipose tissue, acts as a transcriptional repressor of saturated fatty acids/TLR4 signaling, thereby revealing the negative feedback mechanism that attenuates obesity-induced macrophage activation. Our data also suggest that activation of ATF3 in macrophages offers a novel therapeutic strategy to prevent or treat obesity-induced adipose tissue inflammation. (Circ Res. 2009;105:25-32.) Key Words: adipocytes Ⅲ ATF3 Ⅲ fatty acids Ⅲ inflammation Ⅲ macrophages Ⅲ TLR4 K nown as the metabolic syndrome, the cluster of wellestablished risk factors for cardiovascular disease (visceral fat obesity, impaired glucose metabolism, atherogenic dyslipidemia, and blood pressure elevation), is an increasing health problem worldwide. [1][2][3] The pathophysiology underlying the metabolic syndrome is not fully understood and visceral fat obesity appears to be an important component. 4 There is considerable evidence that obesity is a state of chronic low-grade inflammation, which may play a critical role in the pathophysiology of the metabolic syndrome. [1][2][3] Obese adipose tissue is markedly infiltrated by macrophages, suggesting that they may participate in the inflammatory pathways that are activated in obese adipose tissue. 5 Using an in vitro coculture system composed of adipocytes and macrophages, we have provided evidence that a paracrine loop involving saturated fatty acids and tumor necrosis factor (TNF)␣ derived from adipocytes and macrophages, respectively, establishes a vicious cycle that augment the inflammatory change in obese adipose tissue. 6 Recent studies have also poin...

OBJECTIVEWe have provided evidence that saturated fatty acids, which are released from adipocytes via macrophage-induced adipocyte lipolysis, serve as a naturally occurring ligand for the Toll-like receptor (TLR) 4 complex in macrophages, thereby aggravating obesity-induced adipose tissue inflammation. The aim of this study was to identify the molecule(s) activated in adipose tissue macrophages in obesity.RESEARCH DESIGN AND METHODSWe performed a cDNA microarray analysis of coculture of 3T3-L1 adipocytes and RAW264 macrophages. Cultured adipocytes and macrophages and the adipose tissue of obese mice and humans were used to examine mRNA and protein expression.RESULTSWe found that macrophage-inducible C-type lectin (Mincle; also called Clec4e and Clecsf9), a type II transmembrane C-type lectin, is induced selectively in macrophages during the interaction between adipocytes and macrophages. Treatment with palmitate, a major saturated fatty acid released from 3T3-L1 adipocytes, induced Mincle mRNA expression in macrophages at least partly through the TLR4/nuclear factor (NF)-κB pathway. Mincle mRNA expression was increased in parallel with macrophage markers in the adipose tissue of obese mice and humans. The obesity-induced increase in Mincle mRNA expression was markedly attenuated in C3H/HeJ mice with defective TLR4 signaling relative to control C3H/HeN mice. Notably, Mincle mRNA was expressed in bone-marrow cell (BMC)-derived proinflammatory M1 macrophages rather than in BMC-derived anti-inflammatory M2 macrophages in vitro.CONCLUSIONSOur data suggest that Mincle is induced in adipose tissue macrophages in obesity at least partly through the saturated fatty acid/TLR4/NF-κB pathway, thereby suggesting its pathophysiologic role in obesity-induced adipose tissue inflammation.

Abstract. Monocytes/macrophages are key mediators of wound repair, tissue remodeling, and inflammation. However, the molecular mechanisms underlying macrophage recruitment to the site of inflammation is not fully understood. Leptin acts directly on the hypothalamus, thereby regulating food intake and energy expenditure. The leptin receptor, a single transmembrane protein that belongs to the gp130 family of cytokine receptor superfamily, is expressed not only in the hypothalamus but in a variety of peripheral tissues, suggesting the role of leptin as a pro-inflammatory adipocytokine in peripheral tissues. Here, we show that deficiency of leptin signaling reduces renal macrophage infiltration after unilateral ureteral obstruction (UUO). Bone marrow transplantation studies using leptin signaling-deficient db/db mice revealed that leptin signaling in bone marrow cells may not play a major role in the UUO-induced renal macrophage infiltration. Interestingly, central leptin administration reverses the otherwise reduced UUO-induced renal macrophage infiltration in leptin-deficient ob/ob mice. This is effectively abolished by central co-administration of SHU9119, a melanocortin-3 receptor/melanocortin-4 receptor antagonist. This study demonstrates that central leptin administration in ob/ob mice accelerates renal macrophage infiltration through the melanocortin system, thereby suggesting that the central nervous system, which is inherent to integrate information from throughout the organism, is able to control peripheral inflammation. The Adipose tissue is an important endocrine organ that secretes a large number of adipocytokines; pro-inflammatory cytokines such as monocyte chemoattractant protein-1 (McP-1), tumor necrosis factor-α (TNFα) and anti-inflammatory cytokines such as adiponectin [1,2]. Leptin is such an adipocytokine with multiple regulatory potentials; it acts directly on the hypothalamus, where it activates the melanocortin system [3,4], thereby regulating food intake and energy expenditure [5,6]. The leptin receptor (ob-r), a single transmembrane protein that belongs to the gp130 family of cytokine receptor superfamily, has several alternatively spliced isoforms [7], one of which, a biologically active longest isoform or Ob-Rb, is expressed not only in the hypothalamus but in a variety of peripheral tissues including renal parenchymal cells and macrophages [8]. The peripheral actions of leptin include activation of platelet aggregation, modulation of immune functions, and stimulation of vascular endothelial cell proliferation and angiogenesis [9][10][11][12]. These findings, taken together, suggest that leptin acts as a pro-inflammatory adipocytokine in peripheral tissues. Indeed, previous reports have demonstrated that vascular remodeling and neointimal formation are markedly attenuated in leptin-deficient ob/

Definitive hematopoietic progenitor cells have been thought to develop from the vascular endothelium located in the aorta-gonad-mesonephros region of the mouse embryo. However, several recent findings have suggested that most hematopoietic progenitors are derived from non-endothelial precursor cells expressing CD41. We characterized two distinct precursor populations of definitive hematopoietic cell lineages, vascular endothelial (VE)-cadherin + CD41 -CD45 -endothelial cells and CD41 + CD45 -non-endothelial progenitors, both of which are derived from lateral mesoderm. VE-cadherin + endothelial cells obtained from cultures of differentiating embryonic stem cells possessed hematopoietic potential encompassing erythroid, myeloid and B lymphoid lineages, whereas CD41 + progenitors lacked the B lymphopoietic potential. VE-cadherin + endothelial cells in the lower trunk of the embryo proper showed a significant potential for initiating B lymphopoiesis in cultures, while endothelial cells in the yolk sac appeared to have a bias for myeloerythropoietic differentiation. CD41 + progenitors isolated from yolk sac and embryo proper were capable of generating multiple hematopoietic lineages, although mast cell precursors were exclusively enriched in CD41 + progenitors in the yolk sac. These results suggest that hemogenic endothelial cells and CD41 + progenitors possess distinct hematopoietic potential depending on the tissues in which they reside.

Presenilin-1 (PS1) is a gene responsible for the development of early-onset familial Alzheimer's disease. Targeted disruption of the PS1 gene in mice suggested that PS1 might be involved in angiogenesis. We have used an in vitro embryonic stem (ES) cell culture system to prepare endothelial progenitor cells (EPC) lacking PS1 and investigated the roles of PS1 in endothelial cell lineage. With this system, Flk-1+ E-cadherin- EPC were generated from PS1-deficient ES cells, and the EPC lacking PS1 as well as wild-type EPC grew to form VE-cadherin+ endothelial colonies supported by a layer of OP9 stromal cells. Although the endothelial colonies from PS1-deficient EPC showed morphology similar to those from wild-type EPC, the PS1-deficient EPC formed a large number of the colonies compared to wild-type EPC. The enhanced colony-forming ability of PS1-deficient EPC was attenuated by the inductions of wild-type human PS1. To differentiate multiple activities of PS1 for colony-forming ability, we used two types of human PS1 mutants: one (hPS1D257A) with the aspartate to alanine mutation at residue 257 that impairs the proteolytic activity of PS1, and the other (hPS1Deltacat) deleting amino acids 340-371 of the cytosolic loop sequence essential for beta-catenin binding. hPS1D257A showed activity to regulate the colony-forming ability of PS1-deficient EPC, while hPS1Deltacat failed to exhibit this activity. These results suggest that PS1 regulates the growth and differentiation of endothelial progenitor cells through its beta-catenin-binding region and that the defect of PS1 function in endothelial cell lineage could contribute to the induction of vascular pathology.

No abstract