Abstract-Here we report the discovery of a characteristic dense vascular network (DVN) in the tip portion of epididymal adipose tissue in adult mice. The DVN is formed by angiogenesis rather than by vasculogenesis, and has functional blood circulation. This DVN and its subsequent branching may provide a new functional route for adipogenesis. The recruitment, infiltration, and accumulation of bone marrow-derived LYVE-1 ϩ macrophages in the tip region are crucial for the formation of the DVN. Matrix metalloproteinases (MMPs) and the VEGF-VEGFR2 system are responsible not only for the formation of the DVN, but also for the recruitment and infiltration of LYVE-1 ϩ macrophages into the epididymal adipose tissue tip region. SDF-1, but not the MCP-1-CCR2 system, is a critical factor in recruitment and ongoing retention of macrophages in this area. We also demonstrate that the tip region of epididymal adipose tissue is highly hypoxic, and thus provides a microenvironment conducive to the high expression and enhanced activities of VEGF, VEGFR2, MMPs, and SDF-1 in autocrine and paracrine manners, to create an ideal niche for the recruitment, retention, and angiogenic action of macrophages. These findings shed light on the complex interplay between macrophage infiltration, angiogenesis, and adipogenesis in the tip region of adult epididymal adipose tissue, and provide novel insight into the regulation of alternative outgrowth of adipose tissue. (Circ Res. 2007;100:e47-e57.) Key Words: adipogenesis Ⅲ angiogenesis Ⅲ lymphatic vessel hyaluronan receptor 1 Ⅲ macrophages Ⅲ matrix metalloproteinases Ⅲ monocyte chemoattractant protein-1 Ⅲ vascular endothelial growth factor receptors A dipose tissue is a unique organ that has reversible growth depending on the balance of fat metabolism. 1 It is mainly composed of adipocytes supported by stromalvascular tissue, which contains vascular endothelial cells, macrophages, and poorly characterized stem cells. [2][3][4] Developmental growth of adipocytes through adipogenesis (defined as development of adipoblasts into differentiated adipocytes) is accompanied by the growth of vasculature in adipose tissue. 2,3 Recent studies using pharmacological agents or cell implantation have proposed that the growth of adipose tissue is angiogenesis-dependent. [5][6][7][8] However, little is known about the nature of how angiogenesis governs the growth of adipose tissue and, inversely, how the growth of adipose tissue affects the growth of vasculature.Macrophages are released from the bone marrow as immature monocytes and circulate in the blood before extravasation into their target tissues, where they differentiate into resident macrophages. Thus, macrophages are found in every tissue of the body and, depending on the local microenvironment, acquire specialized functions including phagocytosis, antigen presentation, tissue remodeling, and the secretion of a wide range of growth factors and cytokines. 9 The distribution and accumulation of macrophages in certain tissue are mediated by several CC chemok...

Two vascular growth factor families, VEGF and the angiopoietins, play critical and coordinate roles in tumor progression and metastasis. A single inhibitor targeting both VEGF and angiopoietins is not available. Here, we developed a chimeric decoy receptor, namely double anti-angiogenic protein (DAAP), which can simultaneously bind VEGF-A and angiopoietins, blocking their actions. Compared to VEGF-Trap or Tie2-Fc, which block either VEGF-A or angiopoietins alone, we believe DAAP is a highly effective molecule for regressing tumor angiogenesis and metastasis in implanted and spontaneous solid tumors; it can also effectively reduce ascites formation and vascular leakage in an ovarian carcinoma model. Thus, simultaneous blockade of VEGF-A and angiopoietins with DAAP is an effective therapeutic strategy for blocking tumor angiogenesis, metastasis, and vascular leakage.

The stromal vascular fraction (SVF) in adipose tissue contains a pool of various stem and progenitor cells, but the existence of hematopoietic stem and progenitor cells (HSPCs) in the SVF has not been seriously considered. We detected the presence of HSPCs in the SVF by phenotypically probing with Lin ؊ Sca-1 ؉ c-kit ؉ (LSK) and functionally confirming the presence using colony-forming cell assay and assessing the long-term multilineage reconstitution ability after SVF transplantation. The LSK population in the SVF was 0.004% plus or minus 0.001%, and 5 ؋ 10 5 freshly isolated SVF cells gave rise to 13 plus or minus 4 multilineage colonies. In addition, 0.15% plus or minus 0.03% of SVF cells was home to bone marrow (BM), especially near vascular and endosteal regions, 24 hours after blood transplantation. SVF transplantation was capable of generating a longterm (> 16 weeks), but variable extent (2.1%-32.1%) multilineage reconstitution in primary recipients, which was subsequently transferred to the secondary recipients by BM transplantation. All HSPCs within the SVF originated from the BM. Furthermore, the granulocyte-colonystimulating factor (G-CSF) mobilization of HSPCs from BM markedly elevated the number of phenotypic and functional HSPCs in the SVF, which induced a high efficiency long-term reconstitution in multilineage hematopoiesis in vivo. Our results provide compelling evidence that adipose tissue is a novel extramedullary tissue possessing phenotypic and functional HSPCs. (Blood. 2010;115:957-964) IntroductionHematopoietic stem and progenitor cells (HSPCs) in the complicated hierarchy of the hematopoietic system maintain themselves and generate many kinds of blood cells throughout life. To maintain their proper functioning, HSPCs reside in a special microenvironment called a niche. [1][2][3] Bone marrow (BM) is a dominant organ possessing a niche for HSPCs during adulthood. It is well known that a small fraction of HSPCs constantly circulate between the BM and peripheral blood without any stimulation. 4,5 The exiting and re-entering of HSPCs is mediated by interactions between several surface molecules, such as selectins and integrins. 1 In principle, on the way from peripheral blood back to the BM, HSPCs can settle down if the proper environment is provided. Supporting this concept, several extramedullary tissues were identified as HSPC-containing organs, including the liver, 6 spleen, 7 muscle, 8,9 and thoracic duct. 10 Among these organs, the liver and spleen are well-known locations for extramedullary hematopoiesis. 1 In both tissues, HSPCs reside close to sinusoidal endothelial cells, which may provide a preferential microenvironment. 6,11 The mechanism underlying HSPC maintenance in these extramedullary organs remains unclear, though the HSPCs still conserve their function properly. [6][7][8][9][10] Adipose tissues contain many different types of adult stem cells that can be used for therapeutic purposes, especially for tissue regeneration. 12 Adipose tissue is composed of lipid-filled mature ad...

Objective-Tremendous efforts have been made to establish effective therapeutic neovascularization using adipose tissue-derived stromal vascular fraction (SVF), but the efficiency is low, and underlying mechanisms and their interaction with the host in a new microenvironment are poorly understood. Methods and Results-Here we demonstrate that direct implantation of SVF derived from donor adipose tissue can create a profound vascular network through the disassembly and reassembly of blood endothelial cells at the site of implantation. This neovasculature successfully established connection with recipient blood vessels to form a functionally perfused circuit. Addition of vascular growth factors to the SVF implant improved the efficiency of functional neovasculature formation. In contrast, spheroid culture of SVF before implantation reduced the capacity of vasculature formation, possibly because of cellular alteration. Implanting SVF into the mouse ischemic hindlimb induced the robust formation of a local neovascular network and salvaged the limb. Moreover, the coimplantation of SVF prevented fat absorption in the subcutaneous adipose tissue graft model. Conclusion-Freshly isolated SVF can effectively induce new vessel formation through the dynamic reassembly of blood endothelial cells and could be applied to achieve therapeutic neovascularization for relieving ischemia and preventing fat absorption in an autologous manner.

Highly metastatic and chemotherapy-resistant properties of malignant melanomas stand as challenging barriers to successful treatment; yet, the mechanisms responsible for their aggressive characteristics are not fully defined. We show that a distinct population expressing CD133 (Prominin-1), which is highly enriched after administration of a chemotherapeutic drug, dacarbazine, has enhanced metastatic potential in vivo. CD133 coupled with dacarbazine efficiently inhibited both tumor growth and metastasis; dacarbazine alone could not attenuate tumor metastasis. The current study demonstrates a previously unidentified role of the lymphatic microenvironment in facilitating metastasis of chemoresistant melanoma cells via a specific chemotactic axis, SDF-1/CXCR4. Our findings suggest that targeting the SDF-1/CXCR4 axis in addition to dacarbazine treatment could therapeutically block chemoresistant CD133 þ cell metastasis toward a lymphatic metastatic niche.

Orosomucoid (ORM), also called ␣-1 acid glycoprotein, is an abundant plasma protein that is an immunomodulator induced by stressful conditions such as infections. In this study, we reveal that Orm is induced selectively in the adipose tissue of obese mice to suppress excess inflammation that otherwise disturbs energy homeostasis. Adipose Orm levels were elevated by metabolic signals, including insulin, high glucose, and free fatty acid, as well as by the proinflammatory cytokine tumor necrosis factor-␣, which is found in increased levels in the adipose tissue of morbid obese subjects. In both adipocytes and macrophages, ORM suppressed proinflammatory gene expression and pathways such as NF-B and mitogen-activated protein kinase signalings and reactive oxygen species generation. Concomitantly, ORM relieved hyperglycemia-induced insulin resistance as well as tumor necrosis factor-␣-mediated lipolysis in adipocytes. Accordingly, ORM improved glucose and insulin tolerance in obese and diabetic db/db mice. Taken together, our results suggest that ORM integrates inflammatory and metabolic signals to modulate immune responses to protect adipose tissue from excessive inflammation and thereby from metabolic dysfunction.

Cellular antioxidant enzymes play crucial roles in aerobic organisms by eliminating detrimental oxidants and maintaining the intracellular redox homeostasis. Therefore, the function of antioxidant enzymes is inextricably linked to the redox-dependent activities of multiple proteins and signaling pathways. Here, we report that the VEGFR2 RTK has an oxidation-sensitive cysteine residue whose reduced state is preserved specifically by peroxiredoxin II (PrxII) in vascular endothelial cells. In the absence of PrxII, the cellular H(2)O(2) level is markedly increased and the VEGFR2 becomes inactive, no longer responding to VEGF stimulation. Such VEGFR2 inactivation is due to the formation of intramolecular disulfide linkage between Cys1199 and Cys1206 in the C-terminal tail. Interestingly, the PrxII-mediated VEGFR2 protection is achieved by association of two proteins in the caveolae. Furthermore, PrxII deficiency suppresses tumor angiogenesis in vivo. This study thus demonstrates a physiological function of PrxII as the residential antioxidant safeguard specific to the redox-sensitive VEGFR2.

Differentiation of human pluripotent stem cells (hPSCs) into functional cell types is IntroductionHuman embryonic stem cells (hESCs) derived from an early embryo can self-renew indefinitely and differentiate into a variety of cell types. 1 It has been reported that the "stemness" of hESCs is likely maintained through the harmonious actions of signaling pathway networks. 2 Basic fibroblast growth factor (bFGF) is essential for maintaining the stemness of hESCs by highly activating mitogen-activated protein kinase (MAPK) extracellular signalregulated protein kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling, which plays an important role in the stemness of hESCs. 3 Stemness of hESCs is also supported by bFGF-mediated regulation of transforming growth factor-␤ (TGF-␤) signaling 4 ; activation of the TGF-␤/activin/nodal signaling pathway is required to maintain stemness in cooperation with the FGF signaling pathway, whereas its inhibition results in differentiation of hESCs. 5,6 The effect of Wnt signaling on stemness of hESCs is still controversial. Activation of the Wnt pathway by 6-bromoindirubin-3-oxmie, a specific inhibitor of glycogen synthase-3, sustains the undifferentiated status of hESCs. 7 However, activation of Wnt signaling is insufficient to maintain the undifferentiated status of hESCs, because a canonical Wnt signaling is highly activated during differentiation. 8 The stemness of human induced pluripotent stem cells (hiPSCs), like that of hESCs, seems to be maintained by coordinated networks of signaling molecules, although few differences are observed in the gene expression profile. 9,10 Thus, the stemness and differentiation of hESCs and hiPSCs is regulated by complex networks of signaling pathways. It is likely that the modulation of these signaling pathways can induce differentiation of hESCs into a specialized cell type. In fact, bone morphogenic protein 2/4 (BMP2/4), a member of the TGF␤ superfamily, could differentiate hESCs into trophoblasts, primitive endoderm cells, and mesodermal cells. [11][12][13] However, it has been reported that BMP4 is required for sustaining stemness of mouse ESCs by blocking neural differentiation. 14 hESCs could be differentiated to definitive endoderm cells by activation of activin/nodal signaling and suppression of phosphoinositide 3-kinase (PI3K) signaling. 15 Dual inhibition of SMAD signaling by treatment with Noggin and SB431542 resulted in differentiation of hESCs and hiPSCs into neural cells. 16 Many studies have tried to isolate specialized cell types from spontaneously differentiated cells via formation of hESC-derived embryoid bodies using antibodies against cell-typespecific surface markers. 17,18 However, spontaneous differentiation remains inefficient, in that hPSCs cannot be guided toward a specialized lineage at the initial commitment step.The hPSCs provide a possibility that degenerative or damaged tissues can be replaced with hPSC-derived functional cells. A paucity of number and activity of endothelial progenitor cells is correla...