Macrophages consist of at least two subgroups, M1 and M2 (refs 1-3). Whereas M1 macrophages are proinflammatory and have a central role in host defence against bacterial and viral infections, M2 macrophages are associated with responses to anti-inflammatory reactions, helminth infection, tissue remodelling, fibrosis and tumour progression. Trib1 is an adaptor protein involved in protein degradation by interacting with COP1 ubiquitin ligase. Genome-wide association studies in humans have implicated TRIB1 in lipid metabolism. Here we show that Trib1 is critical for the differentiation of F4/80(+)MR(+) tissue-resident macrophages--that share characteristics with M2 macrophages (which we term M2-like macrophages)--and eosinophils but not for the differentiation of M1 myeloid cells. Trib1 deficiency results in a severe reduction of M2-like macrophages in various organs, including bone marrow, spleen, lung and adipose tissues. Aberrant expression of C/EBPα in Trib1-deficient bone marrow cells is responsible for the defects in macrophage differentiation. Unexpectedly, mice lacking Trib1 in haematopoietic cells show diminished adipose tissue mass accompanied by evidence of increased lipolysis, even when fed a normal diet. Supplementation of M2-like macrophages rescues the pathophysiology, indicating that a lack of these macrophages is the cause of lipolysis. In response to a high-fat diet, mice lacking Trib1 in haematopoietic cells develop hypertriglyceridaemia and insulin resistance, together with increased proinflammatory cytokine gene induction. Collectively, these results demonstrate that Trib1 is critical for adipose tissue maintenance and suppression of metabolic disorders by controlling the differentiation of tissue-resident M2-like macrophages.

The generation of new blood vessels via angiogenesis is critical for meeting tissue oxygen demands. A role for adult stem cells in this process remains unclear. Here, we identified CD157 (bst1, bone marrow stromal antigen 1) as a marker of tissue-resident vascular endothelial stem cells (VESCs) in large arteries and veins of numerous mouse organs. Single CD157 VESCs form colonies in vitro and generate donor-derived portal vein, sinusoids, and central vein endothelial cells upon transplantation in the liver. In response to injury, VESCs expand and regenerate entire vasculature structures, supporting the existence of an endothelial hierarchy within blood vessels. Genetic lineage tracing revealed that VESCs maintain large vessels and sinusoids in the normal liver for more than a year, and transplantation of VESCs rescued bleeding phenotypes in a mouse model of hemophilia. Our findings show that tissue-resident VESCs display self-renewal capacity and that vascular regeneration potential exists in peripheral blood vessels.

Vasculogenesis, the in-situ assembly of angioblast or endothelial progenitor cells (EPCs), may persist into adult life, contributing to new blood vessel formation. However, EPCs are scattered throughout newly developed blood vessels and cannot be solely responsible for vascularization. Here, we identify an endothelial progenitor/stem-like population located at the inner surface of preexisting blood vessels using the Hoechst method in which stem cell populations are identified as side populations. This population is dormant in the steady state but possesses colony-forming ability, produces large numbers of endothelial cells (ECs) and when transplanted into ischaemic lesions, restores blood flow completely and reconstitutes de-novo long-term surviving blood vessels. Moreover, although surface markers of this population are very similar to conventional ECs, and they reside in the capillary endothelium sub-population, the gene expression profile is completely different. Our results suggest that this heterogeneity of stem-like ECs will lead to the identification of new targets for vascular regeneration therapy.

The efficacy of therapeutic angiogenesis for revascularization in ischemia using genes, proteins, and cells has been established. For further improvement, processes allowing enlargement of the luminal cavity to facilitate efficient blood flow need to be facilitated. Recently, we found that expression of APJ and its specific ligand, apelin, is seen in endothelial cells when angiogenesis is taking place during embryogenesis. Apelin-deficient mice are viable but have narrow intersomitic ves-

The vast blood-vessel network of the circulatory system is crucial for maintaining bodily homeostasis, delivering essential molecules and blood cells, and removing waste products. Blood-vessel dysfunction and dysregulation of new blood-vessel formation are related to the onset and progression of many diseases including cancer, ischemic disease, inflammation and immune disorders. Endothelial cells (ECs) are fundamental components of blood vessels and their proliferation is essential for new vessel formation, making them good therapeutic targets for regulating the latter. New blood-vessel formation occurs by vasculogenesis and angiogenesis during development. Induction of ECs termed tip, stalk and phalanx cells by interactions between vascular endothelial growth factor A (VEGF-A) and its receptors (VEGFR1–3) and between Notch and Delta-like Notch ligands (DLLs) is crucial for regulation of angiogenesis. Although the importance of angiogenesis is unequivocal in the adult, vasculogenesis effected by endothelial progenitor cells (EPCs) may also contribute to post-natal vessel formation. However, the definition of these cells is ambiguous and they include several distinct cell types under the simple classification of ‘EPC’. Furthermore, recent evidence indicates that ECs within the intima show clonal expansion in some situations and that they may harbor vascular-resident endothelial stem cells. In this article, we summarize recent knowledge on vascular development and new blood-vessel formation in the adult. We also introduce concepts of EC heterogeneity and EC clonal expansion, referring to our own recent findings.

Immature and unstable tumor vasculature provides an aberrant tumor microenvironment and leads to resistance of tumors to conventional therapy. Hence, normalization of tumor vessels has been reported to improve the effect of immuno-, chemo-and radiation therapy. However, the humoral factors, which can effectively induce maturation of tumor vasculature, have not been elucidated. In this study, we found that the novel peptide apelin and its receptor APJ can induce the morphological and functional maturation of blood vessels in tumors. This apelin-induced tumor vascular maturation enhances the efficacy of cancer dendritic cell-based immunotherapy and significantly suppresses tumor growth by promoting the infiltration of invariant natural killer T cells into the central region of the tumor and thereby robustly inducing apoptosis of tumor cells. Additionally, we showed APJ expression to be enhanced in the tumor endothelium in comparison with normal-state endothelial cells. These findings provide a new target for tumor vascular-specific maturation, which is expected to improve the efficacy of conventional cancer therapies.

Angiogenesis is controlled positively or negatively by extrinsic and intrinsic molecular cues in endothelial cells (ECs); in the tumor microenvironment, the action of positive regulators exceeds that of negative regulators. Thus, overinduction of negative regulators may inhibit tumor angiogenesis. MicroRNAs (miRNAs or miRs) are endogenous short noncoding RNAs regulating gene expression either through translational inhibition or destabilization of target mRNA. Here, we show that miR-125b expression is transiently induced in ECs on stimulation with vascular endothelial growth factor or by ischemia. miR-125b inhibits translation of vascular endothelial (VE)-cadherin mRNA and in vitro tube formation by ECs. Injection of miR-125b into the tumor inhibited VE-cadherin expression by ECs and induced nonfunctional blood vessel formation, resulting in inhibition of tumor growth. It has been suggested that pro-angiogenic signals in ECs also upregulate anti-angiogenic molecules simultaneously via negative feedback. Because miR-125b induction in ECs is transient after pro-angiogenic stimulation, prolonged overexpression of miR-125b could result in blood vessel regression. Thus, miR-125b may be useful in cancer therapy by causing the collapse of the lumen of ECs.

Rationale: Grb2-associated binder (Gab) docking proteins, consisting of Gab1, Gab2, and Gab3, have crucial roles in growth factor-dependent signaling. Various proangiogenic growth factors regulate angiogenesis and endothelial function. However, the roles of Gab proteins in angiogenesis remain elusive.Objective: To elucidate the role of Gab proteins in postnatal angiogenesis. Methods and Results: Endothelium-specific Gab1 knockout (Gab1ECKO) mice were viable and showed no obvious defects in vascular development. Therefore, we analyzed a hindlimb ischemia (HLI) model of control, Gab1ECKO, or conventional Gab2 knockout (Gab2KO) mice. Intriguingly, impaired blood flow recovery and necrosis in the operated limb was observed in all of Gab1ECKO, but not in control or Gab2KO mice. Among several proangiogenic growth factors, hepatocyte growth factor (HGF) induced the most prominent tyrosine phosphorylation of Gab1 and subsequent complex formation of Gab1 with SHP2 (Src homology-2-containing protein tyrosine phosphatase 2) and phosphatidylinositol 3-kinase subunit p85 in human endothelial cells (ECs). Gab1-SHP2 complex was required for HGF-induced migration and proliferation of ECs via extracellular