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Circulation Research. 2007;100:e47-e57
Published online before print February 1, 2007, doi: 10.1161/01.RES.0000259564.92792.93
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(Circulation Research. 2007;100:e47.)
© 2007 American Heart Association, Inc.

UltraRapid Communication

Angiogenic Role of LYVE-1–Positive Macrophages in Adipose Tissue

Chung-Hyun Cho, Young Jun Koh, Jinah Han, Hoon-Ki Sung, Hyuek Jong Lee, Tohru Morisada, Reto A. Schwendener, Rolf A. Brekken, Guson Kang, Yuichi Oike, Tae-Saeng Choi, Toshio Suda, Ook-Joon Yoo, Gou Young Koh

From the National Research Laboratory of Vascular Biology and Department of Biological Sciences (C-H.C., Y.J.K., J.H., H-K. S., H.J.L., O-J.Y., G.Y.K.), Korea Advanced Institute of Science and Technology, Daejeon, Korea; Department of Cell Differentiation (T.M.,Y.O., T.S.), The Sakaguchi Laboratory, Keio University Internal Medicine, Tokyo, Japan; Laboratory of Liposome Research (R.A.S.), Institute of Molecular Cancer Research, University of Zurich, Switzerland; Departments of Surgery and Pharmacology (R.A.B), Hamon Center for Therapeutic Oncology Research, UT-Southwestern Medical Center, Dallas, Tex; Department of Biochemistry and Biophysics (G.K.), University of California, San Francisco; Department of Microbiology (T-S.C), College of Medicine, Dankook University, Chonan, Korea.

Correspondence to Gou Young Koh, Department of Biological Sciences, KAIST, 373–1, Guseong-dong, Daejeon, 305–701, Republic of Korea. E-mail gykoh{at}kaist.ac.kr


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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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+ 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+ 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.


Key Words: adipogenesis • angiogenesis • lymphatic vessel hyaluronan receptor 1 • macrophages • matrix metalloproteinases • monocyte chemoattractant protein-1 • vascular endothelial growth factor receptors


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Adipose 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 stromal-vascular tissue, which contains vascular endothelial cells, macrophages, and poorly characterized stem cells.2–4 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.5–8 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 chemokines and growth factors.9,10 Macrophages, particularly in obese subjects, can constitute up to 40% of the cell population within an adipose tissue depot.11,12 The increased number of macrophages in adipose tissue is a likely source of secreted proinflammatory factors, which are responsible for inducing features of metabolic syndrome.13,14 However, under normal circumstances, macrophages are also widely distributed among adipocytes, and constitute 5% to 10% of the cell population in several adipose tissues depots. Besides their inflammatory role, it is unknown whether macrophages may have other roles in adipose tissue.

Here we demonstrate an angiogenic role for macrophages in the tip region of epididymal adipose tissue (EAT), which may be involved in the outgrowth of adipose tissue. Furthermore, we clarify the main mediators and underlying mechanisms responsible for the recruitment and retention of macrophages in this tissue.


*    Materials and Methods
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 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
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 down arrowDiscussion
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Animals and Treatment
Specific pathogen-free C57BL/6J and CCR2 (–/–) (C57BL/6J genetic background) mice were purchased from Jackson Laboratory (Jackson Labs, Bar Harbor, Maine). GFP+ mice (C57BL/6J genetic background) were a gift from Dr Masaru Okabe (Osaka University, Japan). Mice were bred in our pathogen-free animal facility and 8{approx}9-week-old male mice were used for this study otherwise specifically indicated. Animal care and experimental procedures were performed under approval from the Animal Care Committees of KAIST and Dankook University. For systemic depletion of macrophages, mice were treated with intraperitoneal injections of clodrorate liposome (CDL, 12 or 40 mg/kg) as previously described.15,16 To block endogenous VEGF, mice were treated with intraperitoneal injection of sFlt1-Fc (5 µg/d for 2 weeks, R&D Systems) as previously described.17 As a control, dimeric-Fc protein was applied in the same manner. To block VEGFR2, mice were treated with intraperitoneal injections of SU11248 (60 mg/d for 8 days, Imagene Co, Seoul, Korea) as previously described.18 As a control, vehicle containing 0.1% DMSO was applied in the same manner. To block matrix metalloproteinases (MMPs), mice were treated with intraperitoneal injections of zoledronic acid (ZA, Zometa, 100 µg/kg/d for 2 weeks, Novartis Pharma Stein AG, Stein, Switzerland). As a control, vehicle containing 0.1% DMSO was applied in the same manner. To block SDF-1, mice were treated with intraperitoneal injections of anti-SDF-1 blocking antibody (50 µg thrice per week, R&D systems). As a control, nonspecific anti-rat IgG antibody was applied in the same manner.

Bone Marrow Transplantation
Bone marrow cells (2x106) were harvested from the femurs and tibias of GFP+ mice by flushing with ice-cold Dulbecco’s phosphate buffered saline (DPBS, Sigma-Aldrich). The recipient mice (C57BL/6J), 8 to 10 weeks old, were sublethally irradiated at a dose of 4.5 Gy with a {gamma} irradiator (Gammacell 3000, MDS Nordion Inc, Canada). Bone marrow cells were then injected intravenously into the recipient mice 16 hours after irradiation. The bone marrow transplanted mice were examined at the indicated times.

Isolation of Adipocytes and LYVE-1+ Macrophages, and RT-PCR
Mice were anesthetized by intramuscular injection of a combination of anesthetics (80 mg/kg ketamine and 12 mg/kg xylazine). The tip portion of EATs were harvested and incubated with Hanks’s balanced salts solution (Sigma-Aldrich) containing 0.2% type 2 collagenase (Worthington) for 1 hour at 37°C. After inactivation of collagenase activity with bovine serum, the cell suspension was filtered through a 100 µm nylon filter (BD Bioscience), and centrifuged at 400g for 5 minutes. The floating adipocytes were separated from pelleted SVCs, and both cell types were washed and resuspended with DPBS. LYVE-1+ macrophages in the SVCs were enriched by using rat anti-mouse LYVE-1 monoclonal antibody19 and anti-rat IgG MicroBeads (Miltenyi Biotech) and a Magnetic Cell Sorter (MACS, Miltenyi Biotech) according to the manufacturer’s instructions. Enrichment of LYVE-1+ macrophages was confirmed by FACS analysis. Total RNA was extracted from the adipocytes, enriched LYVE-1+ macrophages, and LYVE-1+ cell-depleted SVCs by using Total RNA Isolation System (Promega) according to manufacturer’s instructions. Each cDNA was made with Reverse Transcription System (Promega), and semi-quantitative PCR was performed with the appropriate primers (see supplementary Table I in the online data supplement available at http://circres.ahajournals.org). For all indicated molecules including MMP9, 30 cycles were used for the PCR, but for MMP-7, MMP-8 and MMP-12, 35 cycles were used.

Histological and Morphometric Analysis
Mice were anesthetized by intramuscular injection of a combination of anesthetics (80 mg/kg ketamine and 12 mg/kg xylazine), EAT were fixed by vascular perfusion of 1% paraformaldehyde in PBS, removed, and whole-mounted. For detection of functional blood circulation, 400 µg of fluorescein-conjugated Bauhinia Purpurea lectin (Vector Laboratories, Inc, Burlingame, Calif) was injected intravenously 10 minutes before perfusion-fixation. For detection of hypoxia, Hypoxyprobe-1 (pimonidazole hydrochloride, 60 mg/kg, Chemicon International) was injected 90 minutes before perfusion-fixation. After blocking with 5% goat serum in PBST (0.3% Triton X-100 in PBS) for 1 hour at room temperature, the whole-mounted EAT were incubated overnight at 4°C with one or more of the following primary antibodies: (a) for blood vessels, hamster anti-PECAM-1 antibody, clone 2H8, 1:1000 (Chemicon International, Temecula, Calif); (b) for macrophages and leukocytes, rat monoclonal anti-mouse LYVE-1 antibody, 1:1,000,60 rat anti-mouse F4/80 antibody, clone Cl:A3–1, 1:1,000 (Serotec, Oxford, UK), rat anti-mouse CD11b antibody, clone M1/70, 1:1,000 (BD Pharmingen) and rat anti-mouse CD45, clone 30-F11, 1:1,000 (BD Pharmingen); (c) for adipocytes, guinea pig polyclonal anti-perilipin antibody, 1:1,000 (Research Diagnostics); (d) for apoptotic cells, rabbit polyclonal anti-caspase-3 antibody, 1:500 (Cell Signaling Technology); (e) for hypoxic cells, FITC-conjugated mouse monoclonal anti-Hypoxyprobe-1 antibody, clone 4.3.11.3, 1:500 (Chemicon International); and (f) for VEGFR-2, rabbit polyclonal anti-VEGFR-2 antibody, 1:1000 (TO14). After several washes in PBST, whole-mounted EAT were incubated for 1 hour at room temperature with 1 or more secondary antibodies: (a) Cy3- or Cy5-conjugated anti-hamster IgG antibody, 1:500 (Jackson ImmunoResearch Laboratories); (b) FITC- or Cy5-conjugated anti-rat antibody or anti-rabbit antibody, 1:500 (Jackson ImmunoResearch Laboratories); (c) Cy3-conjugated anti-guinea pig antibody (Jackson ImmunoResearch Laboratories). For control experiments, the primary antibody was omitted or substituted with preimmune serum. Signals were visualized and digital images were obtained using a Zeiss Apotome microscope and a Zeiss LSM 510 confocal microscope equipped with argon and helium-neon lasers (Carl Zeiss). For determining the extent of macrophage infiltration, triple-stained color images with anti-LYVE-1, anti-PECAM-1, and anti-perilipin antibodies were captured with a Zeiss LSM 510 confocal microscope. Using ImageJ software (http://rsb.info.nih.gov/ij), the DVN area was selected as a region-of-interest from PECAM-1/perilipin-stained images, and LYVE-1-stained images were converted to 8-bit gray scale. Area densities of the LYVE-1-stained images were measured from the pixels in the region-of-interest; only pixels over a certain level (>50 intensity value) were taken to exclude background fluorescence. For determining the hypoxic region, anti-Hypoxyprobe-1 antibody-stained images of Hypoxyprobe-1 injected and uninjected EATs were captured from a confocal microscope. After conversion to 8-bit gray scale by ImageJ software, the mean area densities of DVN and body portions of EAT, and section of implanted LLC tumor were measured from the pixels without background fluorescence. To avoid nonspecific fluorescence of anti-Hypoxyprobe-1 antibody, the mean area densities were subtracted from the values of Hypoxyprobe-1-uninjected EAT.

Statistics
Values presented are means ± standard deviation (SD). Significant differences between means were determined by analysis of variance followed by the Student-Newman-Keuls test. Statistical significance was set at P<0.05 or P<0.01.


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
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Presence of a Characteristic Dense Vascular Network in the Tip Region of EAT
To gain insight into the reciprocal interaction between the growth of adipose tissue and the growth of vasculature, we chose to examine whole-mounted EAT of male mice (C57BL/6) during postnatal development. As EAT lacks adjacent tissues, it is relatively easy to visualize blood vessels and adipocytes at the 3-dimensional level by coimmunostaining with antibodies against PECAM-1 (a component of vascular endothelial cells) and perilipin (a membrane protein that surrounds lipid droplets).20 In 2-week-old mice, vascular endothelial cells, which have sprouting and filopodia,21 were readily detected in most of the vasculature of EAT, including both the tip and body regions (Figure 1A). In 8-week-old mice, the sprouting and filopodia were detected mainly in vascular endothelial cells at the EAT tip region, where a characteristic dense vascular network (DVN) was present (Figure 1A). This observation led us to examine both the quantity and the location of DVN in EAT during postnatal development. The DVN was rarely detected in any region of EAT in 2 week-old mice (Figure 1B, 1C, and 1D). However, the number of DVNs exceeding 250 µm in diameter gradually increased from 4 to 8 weeks of age, and then gradually decreased from 10 to 12 weeks of age (Figure 1D). These changes were similar to changes in the whole EAT mass from 2 to 8 weeks (Figure 1D). All DVN were mainly detected in the EAT tip region regardless of age (Figure 1B and 1C). Interestingly, a number of small and variably-sized protrusions of adipose tissues ("adipose bumps") were observed adjacent to the tip (Figure 1B). Furthermore, in an area where a DVN is located, lipid-containing adipocytes were absent, as evidenced by a lack of perilipin-immunostained cells (Figure 1C). From these observations, we sought to address the question of whether there is a relationship between the growth of adipose tissue and the formation of the DVN in the adult EAT tip region. Additionally, we sought the mechanism and reason behind the formation of the DVN in this region.


Figure 1
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  		Figure 1. Presence of a characteristic dense vascular network (DVN) in the tip region of EAT in adult mouse. Vasculature in whole-mounted EAT in mice ranging from 2 to 12 weeks are visualized with PECAM-1 immunostaining (red or reddish brown) (A) vascular endothelial cells having sprouting (white arrowheads) and filopodia (green arrowheads) are detected in most of the vasculature of 2-week-old mice, whereas, in 8 week-old mice, these characteristics are detected mainly in the EAT tip region. B, The DVNs (black arrowheads) are detected mainly in the EAT tip region of 6- and 8-week-old mice. Occasionally, a few variably-sized protrusions of adipose tissue (blue arrows) are observed near the tip region. C, Where a characteristic DVN is present, there are no lipid-containing adipocytes (blue). D, Number of DVN exceeding 250 µm in diameter in the EAT tip region and whole EAT mass (g) at the indicated age. Bars represent means ± SD from 4 to 8 mice. *, P<0.01 vs 2 weeks of age. Scale bar, 100 µm.

Relationships Between Adipocytes, Vasculature, Functional Circulation, and Macrophage Distribution in the Tip Region of EAT
To define the DVN, the tip region of EAT was examined in more detail with coimmunostaining experiments. This analysis revealed that the DVN is composed of a short segment with a large diameter (Figure 2A), resembling a primitive vascular plexus formed by vasculogenesis during early embryogenesis. To examine distribution of lymphatic vessels in this region, we also performed immunostaining of LYVE-1 (lymph vessel endothelial hyaluronan receptor-1),22 which is a marker of lymphatic endothelial cells in the whole-mounted EAT. However, there was no LYVE-1+ lymphatic vessel. Instead, this region was infiltrated by macrophages having variable shapes and positive staining for LYVE-1 (Figure 2A), suggesting that the formation of the DVN in the tip region could be related to the infiltration of macrophages. Higher magnifications of these images clearly showed frequent vascular sproutings in the outer margins of the DVN and many infiltrated macrophages of varied shapes in the DVN area (supplementary Figure IA in the online data supplement at http://circres.ahajournals.org). In the tip region, our triple coimmunostained images revealed that approximately 90% of these LYVE-1+ cells were CD11b+, which is well-known marker for macrophages, indicating that LYVE-1 staining was an accurate marker for macrophages in this tissue (supplementary Figure IIA). In comparison, approximately 30% to 40% or 40% to 50% of the LYVE-1+ cells were F4/80+ (other macrophage marker) or CD45+ (common leukocyte marker), respectively (supplementary Figure IIB and IIC). Moreover, isolated LYVE-1+ macrophages from the SVCs of EAT displayed active phagocytic activity (supplementary Figure III). These results suggest that the LYVE-1+ cells are different subset of macrophages. In fact, recent report23 has described that LYVE-1 is expressed not only in lymphatic endothelial cells, but also in a subset of macrophages in the wound healing tissues and implanted malignant tumor tissues. Other triple coimmunostained images suggested close interactions between LYVE-1+ macrophage infiltration, formation of the DVN, and adipogenesis in the EAT tip region (Figure 2B). Compared with the lower portion of the tissue, the middle area of the tip region has smaller and nonuniformly-sized lipid-containing adipocytes with less organized blood vessels having wider diameters and an enlarged vascular network (Figure 2B). In the top portion of this newly-formed adipose tissue, the DVN was of characteristic shape without mature lipid-containing adipocytes (Figure 2B). Again, in this DVN, there was a massive infiltration of macrophages, suggesting that macrophages may play a role in the formation of a DVN in the tip portion. Higher magnifications of these images clearly showed such interactions between macrophage infiltration, formation of DVN, and adipogenesis in the EAT tip region (supplementary Figure IB). From these observations, we speculated that longitudinal outgrowth of EAT into the upper part of the abdominal cavity might result from the preceding macrophage-induced formation of the DVN and subsequent adipogenesis in the EAT tip region. For lipid transport from circulation into immature preadipocytes to occur, blood vessels would need to be functional to systemic blood circulation. To examine whether the DVN has blood circulation, fluorescein-labeled lectin was injected intravenously, and the image of fluorescein-lectin was obtained by fluorescence microscopy. Approximately 75% to 85% of the DVN was fluorescein-lectin-positive (Figure 2C and supplementary Figure IV in the online data supplement at http://circres.ahajournals.org), indicating that the DVN in the EAT tip region has blood circulation. Furthermore, red blood cells were often observed in the DVN of the nonperfused adipose tissue (data not shown). Thus, the DVN in the EAT tip region has blood circulation and is able to transport lipids from the blood into immature preadipocytes in order for them to become lipid-accumulating mature adipocytes.


Figure 2
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  		Figure 2. Relationships between adipocytes, vasculature, functional circulation, and macrophage distribution in the tip region of EAT. Whole-mounted EAT from 8-week-old mice are visualized by immunostaining of perilipin (for adipocytes, blue), PECAM-1 (for vasculature, red) and LYVE-1 (for macrophages, green), and merged. A, A characteristic DVN consisting of a short segment and large diameter is present in the tip region, where very few lipid-containing adipocytes and large numbers of macrophages are present. B, Between arrows and arrowheads, blood vessels having large diameters and a large vascular network, lipid-containing adipocytes, and occasional macrophages are present, whereas between arrowheads and bars, a characteristic DVN, very few lipid-containing adipocytes, and large numbers of macrophages are present. C, Fluorescein-labeled lectin was injected intravenously into mice, and the image of fluorescein-lectin (green) in the DVN (red) was obtained. Approximately 75% to 85% (n=4) of the DVN is fluorescein-lectin+, indicating that most DVNs have blood circulation. D, Large numbers of macrophages are localized in the upper portion of and around a characteristic DVN. Scale bar, 100 µm.

Role of LYVE-1+ Macrophages in the Formation of DVN
We then analyzed the regional interaction between the DVN and LYVE-1+ macrophages. Notably, the location of accumulated LYVE-1+ macrophages was always proximal to and around the DVN (Figure 2D and 2E), implying that LYVE-1+ macrophages may affect the formation of the DVN. To examine the role of macrophages in the formation of the DVN, macrophages were depleted by single intraperitoneal treatment of clodrorate liposome (CDL, 12 or 40 mg/kg) as previously described15,16 in the 8{approx}9-week-old mice. This treatment efficiently depletes macrophages in a dose-dependent manner by inducing selective apoptosis of macrophages in all organs, including EAT, for 1 to 2 weeks (Figure 3A). In this situation, the number of the DVN exceeding 250 µm in diameter was dramatically reduced in a dose-dependent manner (Figure 3B). Moreover, vascular sprouting and filopodia were rarely detected in the outer margins of the DVN. However, 3 weeks after this treatment, massive infiltration of macrophages and the characteristic shape of the DVN were similarly observed in the EAT tip region (data not shown), suggesting that circulating macrophages may dynamically replenish this region with fresh macrophages, which then participate to form the DVN.


Figure 3
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  		Figure 3. Angiogenic role and origin of macrophages in the formation of DVN. A and B, 8{approx}9-week-old mice were intraperitoneally treated with 40 mg/kg (A) or the indicated dose (B) of CDL. One week after treatment, LYVE-1+ macrophages are selectively depleted by apoptosis (caspase3, green), but no apoptosis is observed in the vasculature (red) in EAT. Scale bar, 100 µm. Number of DVN exceeding 250 µm in diameter in the EAT tip region. Bars represent means ± SD from 4 mice. *, P<0.01 versus control treated EAT. C and D, BMT with bone marrow cells (green) derived from GFP mice was performed to 8-week-old mice. Three months later, LYVE-1-positive macrophages (blue) and vasculature (red) of EAT were immunostained. More than 85% of LYVE-1+ macrophages are bone marrow-derived cells. Tomographic analysis shows that no PECAM-1+ cells are GFP+ cells. Scale bar, 100 µm. E, FACS analytical profiles of whole SVCs (blue), LYVE-1+ enriched (green) and depleted (red) cells after labeling with FITC-labeled anti-LYVE-1 antibody. x-axis represents fraction of LYVE-1+ cells as M1 (LYVE-1– cells) and M2 (LYVE-1+ cells). F, RT-PCR profiles of various angiogenic factors in LYVE-1+-enriched SVC (a), adipocytes (b) and LYVE-1+-depleted SVC (c) in the dotted line, VEGF-A120.

Origin of Macrophages and Angiogenic Role in the Formation of DVN
To clarify the origin of the macrophages in the EAT tip region, 8-week-old mice transplanted with GFP+ bone marrow cells were examined. At 12 weeks after bone marrow transplantation (BMT) with GFP+ cells, approximately 90% to 95% of macrophages in the tip region were GFP+ (Figure 3C), indicating that most of the macrophages were derived from bone marrow through circulation. To evaluate whether bone marrow-derived cells act as endothelial progenitor cells (EPCs) to form, endothelial cells in the DVN, the colocalization of PECAM-1 immunostaining and GFP+ was carefully examined. However, based on our vigorous observations, we found no GFP+ cells in the endothelial cells of any characteristic DVNs (Figure 3D), clearly indicating that the formation of the DVN is induced by angiogenesis through active sprouting, not by vasculogenesis.24,25

Role of MMPs in Macrophage Infiltration and Formation of DVN in the Tip Region of EAT
There is ample evidence that macrophage-derived growth factors and cytokines play a major role in angiogenesis in certain microenvironments.9,10,26 Therefore, we compared the expression patterns of several angiogenic growth factors and cytokines in enriched LYVE-1+ macrophages, adipocytes, and LYVE-1+ macrophage-depleted stromal-vascular cells (SVCs) (Figure 3E) of the EAT tip region by semi-quantitative RT-PCR. Selective expression of adiponectin in the enriched adipocytes and PECAM-1 in the LYVE-1+ macrophage-depleted SVCs reflected the high efficiency of cell separation and enrichment for each cell type from the EAT (Figure 3E). Interestingly, the enriched LYVE-1+ macrophages more abundantly expressed MMP-9, MMP-12, MMP-7, chemokine receptor-4 (CXCR4) and TNF-{alpha}, whereas the adipocytes more abundantly expressed VEGF-A, VEGF-D, angiopoietin-1 (Ang1) and MMP-8, and the LYVE-1+ macrophage-depleted SVCs more abundantly expressed VEGF-C and MMP-2 (Figure 3F and supplementary Figure V). Angiopoietin-2 (Ang2) was expressed in all 3 cell types, while macrophage chemoattractant protein-1 (MCP-1) was expressed in the enriched LYVE-1+ macrophages and LYVE-1+ macrophage-depleted SVCs, and stromal cell-derived factor-1 (SDF-1) was in the adipocytes and in the enriched LYVE-1+ macrophage-depleted SVCs (Figure 3F and supplementary Figure V). Thus, the RT-PCR analysis strongly suggested that MMPs secreted from the macrophages, adipocytes, and SVCs could play a certain role in the formation of the DVN in the adult EAT tip region. All members of the MMP family contain Zn2+ at the catalytic site.27 ZA is a nitrogen-containing (amino-) bisphosphonate that inhibits proteolytic activities of MMPs possibly through Zn2+ chelation.28,29 ZA has been used for reducing complications of bone metastasis in patients with multiple myeloma and several solid tumors with minimal side effects,30,31 and for targeting MMP-9-expressing macrophages and reducing angiogenesis in experimental mice.32 Therefore, to test the role of MMPs in the formation of the DVN, we inhibited MMP activity in mice by treatment with ZA (100 µg/kg/d for 2 weeks).32 The treatment with ZA exerted significant reductions not only in the formation of the DVN and vascular sprouting and filopodia in the outer margins of the DVN, but also in macrophage infiltration in the tip region (Figure 4A and 4B). These results indicate that the action of MMPs is critically involved in macrophage infiltration and subsequent formation of vascular sprouting, filopodia, and DVN in the EAT tip region. To further clarify the role of MMPs in macrophage infiltration from circulating macrophages into the tip region, mice were treated with intraperitoneal injections of ZA (100 µg/kg/d for 2 weeks) 1 week after macrophage depletion by CDL (40 mg/kg). Two weeks after the treatment with ZA, the treated mice showed not only markedly-reduced macrophage accumulation in the tip region, but also a severely-reduced number of DVN (Figure 4A and 4B), indicating that the MMPs secreted by the macrophages, adipocytes, and SVCs play a critical role in macrophage infiltration into the EAT tip region.


Figure 4
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  		Figure 4. Role of MMPs, VEGF, VEGFR2 and SDF-1 in macrophage infiltration and formation of DVN in the tip region of EAT. 8{approx}9-week-old mice were treated with ZA, sFlt1-Fc (sFlt1), SU11248, anti-SDF-1 blocking antibody (SDF1Ab), or vehicle (control) with or without pretreatment with CDL (40 mg/kg, 3 weeks earlier). A, Two weeks later, macrophages and DVN in the EAT tip region are visualized by immunostaining of LYVE-1 (green) and PECAM-1 (red). Scale bar, 100 µm. B, Numbers of macrophages are quantified by area densities of green signal in the avascular areas of the tip region, and the numbers of DVN exceeding 250 µm in diameter in the tip region are counted. Bars represent means ± SD from 4 to 6 mice. *, P<0.01 vs control.

Role of VEGF-VEGFR System in Macrophage Infiltration and Formation of DVN in the Tip Region of EAT
Because angiogenesis is critically dependent on VEGF action,33,34 and all 3 cell types in EAT express the 2 main forms of VEGF (VEGF164 and VEGF120), we examined the role of VEGF in the formation of the DVN in the EAT tip region by blocking VEGF by intraperitoneal treatment with sFlt1-Fc (5 µg/d for 2 weeks).17 As a control, dimeric-Fc protein was given in the same manner. Two weeks after treatment, mice treated with sFlt1-Fc showed not only a reduced number and size of DVN and attenuated angiogenic sprouting and filopodia in the DVN, but also a moderately reduced number of macrophages in the tip region (Figure 4A and 4B), whereas mice treated with the control dimeric-Fc protein showed no difference from untreated mice. Moreover, variably-shaped protruding bumps, having a small amount of macrophages without lipid-containing adipocytes, were observed in the tip region (Figure 4A and 4B). These data indicate that the action of VEGF in the tip region is important in the formation of the DVN, and that VEGF itself might act on macrophage recruitment and infiltration, possibly through the VEGF-VEGFR1 system.35,36 To test whether VEGF affects the recruitment and infiltration of bone marrow-derived macrophages into the tip region, the mice were treated intraperitoneally with sFlt1-Fc (5 µg/d for 2 weeks) 1 week after macrophages depletion by CDL. Two weeks after treatment with sFlt1-Fc, the treated mice showed not only a markedly-reduced number of macrophages in the tip region, but also severely inhibited sprouting and filopodia in the outer margins of the DVN (Figure 4A), indicating that VEGF itself in the tip region could act on macrophage recruitment and infiltration. The role of VEGFR2 is critical in angiogenesis and angiogenic vasculature expresses VEGFR2 abundantly.24,25,33,34 Therefore, levels of VEGFR2 in the vasculature of EAT were examined. Indeed, VEGFR2 was more highly expressed in the vasculatures of the DVN than in the mature vasculature in the body region of EAT (supplementary Figure VI). To examine role of VEGFR2 in the formation of the DVN, VEGFR2 was blocked by intraperitoneal treatment with SU11248 (60 mg/d for 8 days) as previously described.18 This treatment markedly inhibited the formation of the DVN and, to a lesser extent, macrophage infiltration, but the effects were less than those in response to VEGF neutralization with sFlt1-Fc (Figure 4B). In addition, lipid-containing adipocytes were not detected in the protruding bump. These results indicate that VEGFR2 plays a key role in the formation of the DVN, and that VEGFR2-dependent formation of the DVN could facilitate subsequent adipogenesis in the adult EAT tip region.

The Tip Region of EAT Is Highly Hypoxic
Hypoxia is well known to induce angiogenesis, linking vascular oxygen supply to metabolic demand.37 Massive infiltrations of macrophages have been found in hypoxic areas, including avascular areas of tumors.9,10,26 Furthermore, considering that the expression of VEGF and VEGFR2 are mainly dependent on hypoxia and that the actions of the MMPs, VEGF, and VEGFR2 are enhanced in hypoxia, we have reasoned to determine the extent of hypoxia in EAT using immuno-detection of Hypoxyprobe-1. Consistent with previous predictions,38,39 mature adipocytes in the body region of the adipose tissue were highly hypoxic (Figure 5A and 5K), showing a strong Hypoxyprobe-1 signal in the cytoplasm compared with other cells and tissues of other organs, including the lung (Figure 5C and 5K). Noticeably, the tip region of EAT was more hypoxic compared with the body region (Figure 5D-5K). This difference could be because of insufficient blood supply through the immature blood vessels to this locale. In fact, the extent of hypoxia in the tip region of EAT was higher than that of hypoxic areas in solid cancer tissues (Figure 5J and 5K). Thus, the extreme hypoxia in this locale may contribute to high expression levels of VEGF and VEGFR2 and may enhance the actions of VEGF/VEGFR2/MMPs in the formation of the DVN.


Figure 5
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  		Figure 5. Hypoxia in EAT. 8{approx}9-week-old mice were intravenously treated with Hyproxyprobe-1 (60 mg/kg). Sixty minutes later each organ was harvested, and Hydroxyprobe-1 (green) and blood vessels (red) were immunostained. A, Nonlipid portions of cytoplasm (white arrowheads) in adipocytes contain a moderate amount of Hydroxyprobe-1, (C) whereas almost no Hydroxyprobe-1 is detected in lung tissue. B, Immunostaining of adipocytes without Hydroxyprobe-1 injection as a negative control. D–I, Tip (white arrowheads) and body (white arrows) regions of EAT. J, Implanted Lewis lung carcinoma in mice. White arrows, hypoxic area (HA); white arrowheads, nonhypoxic area (NHA). K, Amounts of Hydroxyprobe-1 in each tissue, each portion of EAT, or each tumor area are quantified by area densities of green signal in the given area (0.05 mm2, tip region of EAT; 0.3 mm2, body region of EAT; 0.18 mm2, tumor section). Bars represent means ± SD from 4 mice.

Role of SDF-1 in Macrophage Infiltration and Formation of DVN in the Tip Region of EAT
Our RT-PCR analysis (Figure 3F) also suggested that it might have a reciprocal interaction for the recruitment and retention of macrophages between CXCR4-expressing macrophages and SDF-1-expressing SVCs. To examine whether SDF-1 is necessary for the ongoing retention of macrophages in the EAT tip region, the function of SDF-1 was blocked by intraperitoneal treatment with an anti-SDF-1 blocking antibody (50 µg/thrice per week). As a control, nonspecific anti-rat IgG antibody was as administered in the same manner. Two weeks after treatment, the mice treated with anti-SDF-1 showed moderately reduced numbers of macrophages and a small number of immature DVN (Figure 4A and 4B), whereas the mice treated with control antibody showed no difference compared with untreated mice. To examine whether SDF-1 is necessary for macrophage mobilization and recruitment to occur in the EAT tip region, mice were treated with an intraperitoneal injections of anti-SDF-1 blocking antibody, as described above, one week after macrophages depletion by CDL. Two weeks after treatment with the anti-SDF-1 blocking antibody, the treated mice showed markedly reduced numbers of macrophages and showed no DVN in the tip region (Figure 4A and 4B). These data indicate that the action of SDF-1 is critically involved not only in macrophage retention, but also in macrophage mobilization and infiltration, and subsequent DVN formation in the EAT tip region.

Role of MCP-1/CCR2 System in Macrophage Infiltration and Formation of DVN in the Tip Region of EAT
These data raise the question of how macrophages are infiltrated into the EAT tip region. We hypothesized that MCP-1 (also known as C-C motif chemokine ligand 2, CCL2) might be involved in macrophage infiltration. MCP-1 is known to be a major chemokine for macrophage infiltration in adipose tissue, particularly during obesity,40,41 and its receptor, CCR2,42 plays a role in macrophage infiltration in adipose tissue.43 Our immunostaining showed that a relatively abundant amount of MCP-1 was detectable not only in the vascular area but also in the avascular area of the EAT tip region (Figure 6A). To examine the role of MCP-1/CCR2 in macrophage infiltration in the EAT tip region, we compared the pattern of macrophage accumulation between CCR2 (–/–) mice and wild-type (WT) littermates. Our data indicated no difference in the pattern of macrophage accumulation or in characteristics of the DVN between CCR2 (–/–) mice and WT littermates (Figure 6B and 6C). To clarify the role of the MCP-1/CCR2 system in the recruitment, infiltration, and ongoing retention of macrophages in EAT, macrophages in both CCR2 (–/–) mice and WT littermates were depleted by CDL, and the pattern of macrophage accumulation was examined. One week after the treatment, all macrophages had completely disappeared from the adipose tissue of both CCR2 (–/–) mice and WT littermates (Figure 6B). Three weeks after the treatment, the accumulation of macrophages and development of DVN in the EAT tip region of CCR2 (–/–) mice were indistinguishable from those of WT littermates (Figure 6B and 6C), whereas fewer infiltrated macrophages were observed in the body region of EAT in CCR2 (–/–) mice compared with WT littermates (Figure 6B). The latter finding is consistent with a previous report.43 These data suggest that the MCP-1/CCR2 system is not a critical factor in macrophage infiltration into the EAT tip region.


Figure 6
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  		Figure 6. Role of MCP-1/CCR2 system in macrophage infiltration and formation of DVN in the tip region of EAT. A, MCP-1 was immunostained (green). B, Macrophages and DVN are visualized by immunostaining of LYVE-1 (blue) and PECAM-1 (red) in the 8{approx}9-week-old wild type (Wt) and CCR2 (–/–) mice during the control period (0 w), and 1 and 3 weeks after CDL (40 mg/kg) treatment. Scale bar, 100 µm. C, The density of macrophages per given density of blood vessels in DVN (Mac/BV in DVN) is expressed as a ratio of the amount of blue signal to the amount of red signal in the EAT tip regionin the control period (0 w) and 3 weeks after CDL (40 mg/kg) treatment in WT and CCR2 (–/–) mice. Bars represent means ± SD from 4 to 6 mice. NS, not significant vs WT.


*    Discussion
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 *Discussion
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Little is known about how angiogenesis governs the growth of adipose tissue. Here we show the angiogenic role of macrophages that accumulate largely in the tip region of adult EAT, which is extremely hypoxic (Figure 7). These macrophages are LYVE-1+, originate from the bone marrow, and induce active angiogenesis through the secretion of MMP-9, MMP-12, and MMP-7, and activation of the VEGF-VEGFR2 system. This macrophage-induced angiogenesis is essential for subsequent adipogenesis, which may prove to be important for longitudinal outgrowth of adipose tissue. SDF-1 and VEGF, but not the MCP-1/CCR2 system, are critical factors for the mobilization, recruitment and retention of macrophages to this region (Figure 7). Thus, we have elucidated the angiogenic role of LYVE-1+ macrophages that accumulate largely in the EAT tip region and show that they could be involved in regulating the outgrowth of adipose tissue. Furthermore, we unveil the main mediators and underlying mechanisms responsible for the recruitment and retention of macrophages in this tissue (Figure 7).


Figure 7
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  		Figure 7. Model for complex interplay between macrophage infiltration, angiogenesis, and adipogenesis in the tip region of adult EAT. Highly hypoxic unknown cells in the EAT tip region could provide an active microenvironment for the recruitment and retention of macrophages and for the angiogenic effects of macrophages through high-level expression and enhanced activities of MCP-1, VEGF, and SDF-1 in autocrine and paracrine manners. Once bone marrow-derived circulatory macrophages accumulate in this area, the macrophages secrete MMP-9, MMP-12, MMP-7, and other angiogenic factors and cytokines. On this stimulation, quiescent blood vessels become activated, as evidenced by sprouting and VEGFR2 expression. The activated blood vessels undergo angiogenesis to form DVN that has a blood circulation. In turn, triglyceride in blood mobilizes into adipoblasts and preadipocytes, which become lipid-containing mature adipocytes (adipogenesis). This model is supported by the effects of several specific inhibitors at each step. ZA, MMP inhibitor; sFlt1-Fc, VEGF inhibitor; Anti-SDF-1 antibody, SDF-1 inhibitor; CDL, selective macrophages apoptotic agent; SU11248, VEGFR2 inhibitor.

We had initially been interested in examining the distribution of lymphatic vessels in EAT by immunostaining of LYVE-1, which is a marker of lymphatic endothelial cells.22 By chance, we discovered from the coimmunostaining results that most LYVE-1+ cells in EAT were macrophages rather than lymphatic endothelial cells. Indeed, our results reveal that the LYVE-1+ cells are variably-shaped single cells rather than continuous tube-like lymphatic endothelial cells. Moreover, many of the LYVE-1+ cells expressed well-known markers for macrophages: 90% were CD11b+, and {approx}30% to 40% were F4/80+. Importantly, LYVE-1+ cells undergo apoptosis in all organs including EAT by active phagocytosis during 1 to 2 weeks of treatment with clodrorate liposome.15,16 Moreover, isolated LYVE-1+ cells from the EAT display active phagocytic activity. These ample pieces of evidence indicate that the LYVE-1+ cells are different subset of macrophages. In fact, LYVE-1 is structurally related to CD44 and other hyaluronan-binding proteins, and is selectively expressed in lymphatic endothelial cells, a subset of infiltrating macrophages found in tumors and inflamed tissues, and in sinusoidal endothelium in liver and spleen.23,44 Of these LYVE-1+ cells, only lymphatic endothelial cells have the homoeobox domain related transcriptional factor PROX-1,45 distinguishing LYVE-1+ macrophages from lymphatic endothelial cells. The mechanisms of LYVE-1 expression and the functional significance of LYVE-1 beyond hyaluronan transport have not been defined in detail. However, recent reports46,47 showed that LYVE-1 knockout mice displayed an apparently normal phenotype, with no visible alterations in hyaluronan metabolism, lymphatic vessel development and maintenance, dendritic cell trafficking, or tumor growth. In comparison, it is known that the expression and function of LYVE-1 in macrophages is upregulated in hypoxic and inflammatory conditions, and by a variety of growth factors and cytokines.44 These conditions were observed in the microenvironment of the EAT tip region. In turn, LYVE-1+ macrophages, as activated macrophages, could to produce a vast number of factors that promote angiogenesis; as their bone marrow-derived monocytic precursors, they secrete factors that mediate their own migration and infiltration intohypoxic areas.9,10,26 Thus, the large number of infiltrated LYVE-1+ macrophages could actively participate in the formation of DVN in the hypoxic EAT tip region (Figure 7).

MMP-induced degradation of matrix protein plays a critical role in tissue remodeling, including angiogenesis.48 Among MMPs, MMP-9 is of particular interest, because it is thought to be a tumor angiogenic and metastatic factor that signals through the VEGF-VEGFR system.49,50 MMP-9 is also known to play a crucial role not only in macrophage infiltration, but also in macrophage-induced angiogenesis.27 Moreover, evidence indicates that MMP-9 produced by bone marrow-derived cells, including macrophages, contributes to carcinogenesis.51 Furthermore, macrophages are known to express and secrete not only MMP-9, but also MMP-12 and MMP-7,52 and the latter MMPs are also involved in angiogenesis.53,54 Our expression profiles of MMPs in three cell types of the EAT tip region suggest that MMP-9, MMP-12, and MMP-7 from LYVE-1+ macrophages, MMP-8 from adipocytes, and MMP-2 from SVCs could contribute to form the DVN. Therefore, we sought to determine whether the MMPs are essential for macrophage recruitment and infiltration into the locale and for formation of the DVN. Our data obtained from using MMP inhibitor ZA indicate that the MMPs play a critical role in macrophage infiltration and macrophage-induced angiogenesis for the formation of DVN in theEAT tip region (Figure 7).

VEGF is one of the adipokines that are secreted from adipose tissues39,55; such factors could be beneficial in regenerative cell therapy using adipose stromal cells to treat ischemic diseases in a paracrine manner.56 Our study reveals that VEGF plays critical roles not only in DVN formation, but also in macrophage recruitment and infiltration to the EAT tip region in a paracrine manner. Moreover, VEGF, in concert with Ang1, may act to help maintain of vascular integrity in adipose tissue in a paracrine manner. Thus, VEGF in adipose tissues could have multiple functions as one of the adipokines. Our data also indicate that the action of SDF-1 is critically involved not only in the ongoing retention of macrophages, but also in macrophage mobilization and infiltration into the EAT tip region. The importance of chemokine SDF-1 in the recruitment and retention of bone marrow-derived CXCR4 (SDF-1 receptor)-positive circulating progenitor cells into hypoxic ischemic and regenerative tissues for subsequent angiogenesis has been well established.57–61 Likewise, in our study, hypoxia in the EAT tip region could induce expression of SDF-1 and thus spur its role in macrophage retention; bone-marrow-derived macrophage recruitment, mobilization, and infiltration; and subsequent angiogenesis. CXCR4 is the sole SDF-1 receptor and is moderately expressed in some subsets of leukocytes including monocytes/macrophages62; its expression in macrophages is enhanced by hypoxia.63 Likewise, LYVE-1+ macrophages in the EAT tip region express CXCR4, and its expression can be enhanced by hypoxia in EAT. Thus, the recruitment and ongoing-retention of macrophages is the result of a reciprocal interrelationship between CXCR4-expressing LYVE-1+ macrophages and SDF-1-expressing vascular-stromal cells, and this relationship is eventually involved in macrophage-induced angiogenesis and the subsequent angiogenesis-dependent outgrowth of the EAT tip region (Figure 7).

Previous studies indicate that the growth of adipose tissue is angiogenesis-dependent.5–8 However, these observations are obtained from situations of active growth of adipose tissues, such as in obese mice or implantation of preadipocytes. In this study, we have focused on studying the role of locally accumulated LYVE-1+ macrophages and locally active angiogenesis in adipogenesis only at the EAT tip region. It is envisaged that macrophages and blood vessels in the rest of EAT of normal are relatively quiescent and that the process of adipogenesis in this tissue is relatively slow. Our systemic treatments that targeted MMPs, the VEGF-VEGFR2 system, and SDF-1 could mainly have their effects on the tip region of adipose tissues, but not on the main portion of the adipose tissues. This may explain why we did not observe a marked reduction in total EAT fat mass and body weight during the 2 weeks exposure to several different treatments. However, in the future, the role of tip growth in EAT on total growth of EAT remains to be elucidated with long-term treatments with several specific agents that affect the formation of DVN or angiogenesis.

In conclusion, our findings shed light on the close interactions between macrophage infiltration, angiogenesis, and adipogenesis in the tip region of adult EAT.


*    Acknowledgments
 
We thank Jae Hoon Shin and Tae-Youn Kim for technical assistance; Jennifer Macke and Bong Ihn Koh for critical reading of the manuscript.

Sources of Funding

This work was supported by the Korea Science and Engineering Foundation (KOSEF) through the National Research Lab. Program (2004–02376, GYK) funded by the Ministry of Science and Technology and the Korea Health 21 R&D project (0405-DB01–0104-0006) funded by the Ministry of Health and Welfare.

Disclosures

None.


*    Footnotes
 
Current affiliation of C.-H.C: Department of Physiology, College of Medicine, Chungnam National University, Daejeon, Republic of Korea.

Original received October 10, 2006; revision received November 2, 2006; resubmission received December 18, 2006; revised resubmission received January 16, 2007; accepted January 23, 2007.


*    References
up arrowTop
 up arrowAbstract
 up arrowIntroduction
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 *References
 

  1. Spiegelman BM, Flier JS. Obesity and the regulation of energy balance. Cell. 2001; 104: 531–543.[CrossRef][Medline] [Order article via Infotrieve]
  2. Hausman GJ, Richardson RL. Cellular and vascular development in immature rat adipose tissue. J Lipid Res. 1983; 24: 522–532.[Abstract]
  3. Crandall DL, Hausman GJ, Kral JG. A review of the microcirculation of adipose tissue: anatomic, metabolic, and angiogenic perspectives. Microcirculation. 1997; 4: 211–232.[Medline] [Order article via Infotrieve]
  4. Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, Alfonso ZC, Fraser JK, Benhaim P, Hedrick MH. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell. 2002; 13: 4279–4295.[Abstract/Free Full Text]
  5. Rupnick MA, Panigrahy D, Zhang CY, Zhang CY, Dallabrida SM, Lowell BB, Langer R, Folkman MJ. Adipose tissue mass can be regulated through the vasculature. Proc Natl Acad Sci U S A. 2002; 99: 10730–10735.[Abstract/Free Full Text]
  6. Fukumura D, Ushiyama A, Duda DG, Xu L, Tam J, Krishna V, Chatterjee K, Garkavtsev I, Jain RK. Paracrine regulation of angiogenesis and adipocyte differentiation during in vivo adipogenesis. Circ Res. 2003; 93: e88–e97.[Medline] [Order article via Infotrieve]
  7. Neels JG, Thinnes T, Loskutoff DJ. Angiogenesis in an in vivo model of adipose tissue development. FASEB J. 2004; 18: 983–985.[Abstract/Free Full Text]
  8. Brakenhielm E, Cao R, Gao B, Angelin B, Cannon B, Parini P, Cao Y. Angiogenesis inhibitor, TNP-470, prevents diet-induced and genetic obesity in mice. Circ Res. 2004; 94: 1579–1588.[Abstract/Free Full Text]
  9. Murdoch C, Giannoudis A, Lewis CE. Mechanisms regulating the recruitment of macrophages into hypoxic areas of tumors and other ischemic tissues. Blood. 2004; 104: 2224–2234.[Abstract/Free Full Text]
  10. Pollard JW. Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev Cancer. 2004; 4: 71–78.[CrossRef][Medline] [Order article via Infotrieve]
  11. Weisberg SP, McCann D, Desai M, Rosenbaum M, Leibel RL, Ferrante AW Jr. Obesity is associated with macrophage accumulation in adipose tissue. J Clin Invest. 2003; 112: 1796–1808.[CrossRef][Medline] [Order article via Infotrieve]
  12. Xu H, Barnes GT, Yang Q, Tan G, Yang D, Chou CJ, Sole J, Nichols A, Ross JS, Tartaglia LA, Chen H. Chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance. J Clin Invest. 2003; 112: 1821–1830.[CrossRef][Medline] [Order article via Infotrieve]
  13. Bouloumie A, Curat CA, Sengenes C, Lolmede K, Miranville A, Busse R. Role of macrophage tissue infiltration in metabolic diseases. Curr Opin Clin Nutr Metab Care. 2005; 8: 347–354.[Medline] [Order article via Infotrieve]
  14. Wellen KE, Hotamisligil GS. Obesity-induced inflammatory changes in adipose tissue. J Clin Invest. 2003; 112: 1785–1788.[CrossRef][Medline] [Order article via Infotrieve]
  15. Seiler P, Aichele P, Odermatt B, Hengartner H, Zinkernagel RM, Schwendener RA. Crucial role of marginal zone macrophages and marginal zone metallophils in the clearance of lymphocytic choriomeningitis virus infection. Eur J Immunol. 1997; 27: 2626–2633.[Medline] [Order article via Infotrieve]
  16. Zeisberger SM, Odermatt B, Marty C, Zehnder-Fjallman AH, Ballmer-Hofer K, Schwendener RA. Clodronate-liposome-mediated depletion of tumour-associated macrophages: a new and highly effective antiangiogenic therapy approach. Br J Cancer. 2006; 95: 272–281.[CrossRef][Medline] [Order article via Infotrieve]
  17. Niida S, Kondo T, Hiratsuka S, Hayashi S, Amizuka N, Noda T, Ikeda K, Shibuya M. VEGF receptor 1 signaling is essential for osteoclast development and bone marrow formation in colony-stimulating factor 1-deficient mice. Proc Natl Acad Sci U S A. 2005; 102: 14016–14021.[Abstract/Free Full Text]
  18. Osusky KL, Hallahan DE, Fu A, Ye F, Shyr Y, Geng L. The receptor tyrosine kinase inhibitor SU11248 impedes endothelial cell migration, tubule formation, and blood vessel formation in vivo, but has little effect on existing tumor vessels. Angiogenesis. 2004; 7: 225–233.[CrossRef][Medline] [Order article via Infotrieve]
  19. Morisada T, Oike Y, Yamada Y, Urano T, Akao M, Kubota Y, Maekawa H, Kimura Y, Ohmura M, Miyamoto T, Nozawa S, Koh GY, Alitalo K, Suda T. Angiopoietin-1 promotes LYVE-1-positive lymphatic vessel formation. Blood. 2005; 105: 4649–4656.[Abstract/Free Full Text]
  20. Londos C, Sztalryd C, Tansey JT, Kimmel AR. Role of PAT proteins in lipid metabolism. Biochimie. 2005; 87: 45–49.[Medline] [Order article via Infotrieve]
  21. Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A, Abramsson A, Jeltsch M, Mitchell C, Alitalo K, Shima D, Betsholtz C. VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol. 2003; 161: 1163–1177.[Abstract/Free Full Text]
  22. Banerji S, Ni J, Wang SX, Clasper S, Su J, Tammi R, Jones M, Jackson DG. LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan. J Cell Biol. 1999; 144: 789–801.[Abstract/Free Full Text]
  23. Schledzewski K, Falkowski M, Moldenhauer P, Kzhyshkowska J, Ganss R, Demory A, Falkowska-Hansen B, Kurzen H, Ugurel S, Geginat G, Arnold B, Goerdt S. Lymphatic endothelium-specific hyaluronan receptor LYVE-1 is expressed by stabilin-1+, F4/80+, CD11b+ macrophages in malignant tumours and wound healing tissue in vivo and in bone marrow cultures in vitro: implications for the assessment of lymphangiogenesis. J Pathol. 2006; 209: 67–77.[CrossRef][Medline] [Order article via Infotrieve]
  24. Risau W. Mechanisms of angiogenesis. Nature. 1997; 386: 671–674.[CrossRef][Medline] [Order article via Infotrieve]
  25. Carmeliet P. Angiogenesis in life, disease and medicine. Nature. 2005; 438: 932–936.[CrossRef][Medline] [Order article via Infotrieve]
  26. Murdoch C, Muthana M, Lewis CE. Hypoxia regulates macrophage functions in inflammation. J Immunol. 2005; 175: 6257–6263.[Abstract/Free Full Text]
  27. Birkedal-Hansen H Proteolytic remodeling of extracellular matrix. Curr Opin Cell Biol. 1995; 7: 728–735.[CrossRef][Medline] [Order article via Infotrieve]
  28. Teronen O, Heikkila P, Konttinen YT, Laitinen M, Salo T, Hanemaaijer R, Teronen A, Maisi P, Sorsa T. MMP inhibition and downregulation by biphosphonates. Ann NY Acad Sci. 1999; 878: 453–465.[Abstract/Free Full Text]
  29. Boissier S, Ferreras M, Peyruchaud O, Magnetto S, Ebetino FH, Colombel M, Delmas P, Delaisse JM, Clezardin P. Bisphosphonates inhibit breast and prostate carcinoma cell invasion, an early event in the formation of bone metastases. Cancer Res. 2000; 60: 2949–2954.[Abstract/Free Full Text]
  30. Cohen MH, Dagher R, Griebel DJ, Ibrahim A, Martin A, Scher NS, Sokol GH, Williams GA, Pazdur R. U.S. Food and Drug Administration drug approval summaries: imatinib mesylate, mesna tablets, and zoledronic acid. Oncologist. 2002; 7: 393–400.[Abstract/Free Full Text]
  31. Ibrahim A, Scher N, Williams G, Sridhara R, Li N, Chen G, Leighton J, Booth B, Gobburu JV, Rahman A, Hsieh Y, Wood R, Vause D, Pazdur R. Approval summary for zoledronic acid for treatment of multiple myeloma and cancer bone metastases. Clin Cancer Res. 2003; 9: 2394–2399.[Abstract/Free Full Text]
  32. Giraudo E, Inoue M, Hanahan D. An amino-bisphosphonate targets MMP-9-expressing macrophages and angiogenesis to impair cervical carcinogenesis. J Clin Invest. 2004; 114: 623–633.[CrossRef][Medline] [Order article via Infotrieve]
  33. Coultas L, Chawengsaksophak K, Rossant J. Endothelial cells and VEGF in vascular development. Nature. 2005; 438: 937–945.[CrossRef][Medline] [Order article via Infotrieve]
  34. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med. 2003; 9: 669–676.[CrossRef][Medline] [Order article via Infotrieve]
  35. Barleon B, Sozzani S, Zhou D, Weich HA, Mantovani A, Marme D. Migration of human monocytes in response to vascular endothelial growth factor (VEGF) is mediated via the VEGF receptor flt-1. Blood. 1996; 87: 3336–3343.[Abstract/Free Full Text]
  36. Hiratsuka S, Minowa O, Kuno J, Noda T, Shibuya M. Flt-1 lacking the tyrosine kinase domain is sufficient for normal development and angiogenesis in mice. Proc Natl Acad Sci U S A. 1998; 95: 9349–9354.[Abstract/Free Full Text]
  37. Pugh CW, Ratcliffe PJ. Regulation of angiogenesis by hypoxia: role of the HIF system. Nat Med. 2003; 9: 677–684.[CrossRef][Medline] [Order article via Infotrieve]
  38. Yun Z, Maecker HL, Johnson RS, Giaccia A. Inhibition of PPAR {gamma} 2 gene expression by the HIF-1-regulated gene DEC1/Stra13: a mechanism for regulation of adipogenesis by hypoxia. Dev Cell. 2002; 2: 331–341.[CrossRef][Medline] [Order article via Infotrieve]
  39. Trayhurn P, Wood IS. Adipokines: inflammation and the pleiotropic role of white adipose tissue. British J Nutr. 2004; 92: 347–355.
  40. Sartipy P, Loskutoff DJ. Monocyte chemoattractant protein 1 in obesity and insulin resistance. Proc Natl Acad Sci U S A. 2003; 100: 7265–7270.[Abstract/Free Full Text]
  41. Kanda H, Tateya S, Tamori Y, Kotani K, Hiasa K, Kitazawa R, Kitazawa S, Miyachi H, Maeda S, Egashira K, Kasuga M. MCP-1 contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis in obesity. J Clin Invest. 2006; 116: 1494–1505.[CrossRef][Medline] [Order article via Infotrieve]
  42. Charo IF, Myers SJ, Herman A, Franci C, Connolly AJ, Coughlin SR. Molecular cloning and functional expression of two monocyte chemoattractant protein 1 receptors reveals alternative splicing of the carboxyl-terminal tails. Proc Natl Acad Sci U S A. 1994; 91: 2752–2756.[Abstract/Free Full Text]
  43. Weisberg SP, Hunter D, Huber R, Lemieux J, Slaymaker S, Vaddi K, Charo I, Leibel RL, Ferrante AW Jr. CCR2 modulates inflammatory and metabolic effects of high-fat feeding. J Clin Invest. 2006; 116: 115–124.[CrossRef][Medline] [Order article via Infotrieve]
  44. Jackson DG. Biology of the lymphatic marker LYVE-1 and applications in research into lymphatic trafficking and lymphangiogenesis. APMIS. 2004; 112: 526–538.[CrossRef][Medline] [Order article via Infotrieve]
  45. Oliver G, Alitalo K. The lymphatic vasculature: recent progress and paradigms. Annu Rev Cell Dev Biol. 2005; 21: 457–483.[CrossRef][Medline] [Order article via Infotrieve]
  46. Gale NW, Prevo R, Fematt JE, Ferguson DJ, Dominguez MG, Yancopoulos GD, Thurston G, Jackson DG Normal lymphatic development and function in mice deficient for the lymphatic hyaluronan receptor LYVE-1. Mol Cell Biol. 2006. In press.
  47. Huang SS, Liu I-H, Smith T, Shah MR, Johnson FE, Huang JS. CRSBP-1/LYVE-l-null mice exhibit identifiable morphological and functional alterations of lymphatic capillary vessels. FEBS Lett. 2006; 580: 6259–6268.[CrossRef][Medline] [Order article via Infotrieve]
  48. Stetler-Stevenson WG. Matrix metalloproteinases in angiogenesis: a moving target for therapeutic intervention. J Clin Invest. 1999; 103: 1237–1241.[Medline] [Order article via Infotrieve]
  49. Bergers G, Brekken R, McMahon G, Vu TH, Itoh T, Tamaki K, Tanzawa K, Thorpe P, Itohara S, Werb Z, Hanahan D. Matrix metalloproteinase-9 triggers the angiogenic switch during carcinogenesis. Nat Cell Biol. 2000; 2: 737–744.[CrossRef][Medline] [Order article via Infotrieve]
  50. Hiratsuka S, Nakamura K, Iwai S, Murakami M, Itoh T, Kijima H, Shipley JM, Senior RM, Shibuya M. MMP9 induction by vascular endothelial growth factor receptor-1 is involved in lung-specific metastasis. Cancer Cell. 2002; 2: 289–300.[CrossRef][Medline] [Order article via Infotrieve]
  51. Coussens LM, Tinkle CL, Hanahan D, Werb Z. MMP-9 supplied by bone marrow-derived cells contributes to skin carcinogenesis. Cell. 2000; 103: