FOXC2 controls Ang-2 expression and modulates angiogenesis, vascular patterning, remodeling, and functions in adipose tissue

Abstract

Adipogenesis is spatiotemporally coupled to angiogenesis throughout adult life, and the interplay between these two processes is communicated by multiple factors. Here we show that in a transgenic mouse model, increased expression of forkhead box C2 (FOXC2) in the adipose tissue affects angiogenesis, vascular patterning, and functions. White and brown adipose tissues contain a considerably high density of microvessels appearing as vascular plexuses, which show redistribution of vascular smooth muscle cells and pericytes. Dysfunction of these primitive vessels is reflected by impairment of skin wound healing. We further provide a mechanistic insight of the vascular phenotype by showing that FOXC2 controls Ang-2 expression by direct activation of its promoter in adipocytes. Remarkably, an Ang-2-specific antagonist almost completely reverses this vascular phenotype. Thus, the FOXC2–Ang-2 signaling system is crucial for controlling adipose vascular function, which is part of an adaptation to increased adipose tissue metabolism.

Obesity is a risk factor for diabetes, dyslipidemia, cardiovascular disease, cancer, and sleep breathing disorders (1–3). Although genetic defects contribute to obesity, high-caloric diet and low physical activity are critical etiological factors responsible for most of today's obesity (4). Unlike most other tissues, adipose tissue constantly experiences expansion and regression throughout adult life. The plasticity of adipose tissue requires continuous remodeling of the vasculature that controls energy expenditure, metabolite exchange, transport of adipokines or hormones, and adipocyte hypertrophy and hyperplasia (5–7). Vascular remodeling and functions are regulated by a number of growth factors and inhibitors. For example, leptin produced by the adipose tissue possesses angiogenic activity, and its expression level is correlated with adipogenesis (8–10). Adiponectin, an adipose tissue-derived hormone, has a reverse correlation with adipose tissue growth and inhibits angiogenesis (11). In addition, known angiogenic factors such as VEGF, angiopoietin (Ang), insulin-like growth factor, hepatocyte growth factor, and FGF produced by adipocytes and nonadipocytes are involved in regulation of adipose angiogenesis (5, 12–21). Indeed, angiogenesis inhibitors could prevent obesity by normalizing metabolisms and without significantly affecting food intake in both leptin-deficient genetic and in high-caloric diet-fed mouse models (22, 23). Interestingly, reduction of fat mass in the angiogenesis inhibitor-treated animals is accompanied by improved insulin sensitivity (22).

Ang is an important group of vascular remodeling factors that control vessel maturation, patterning, and stabilization (24). Although Ang-1 and Ang-2 could bind and activate the same Tie-2 tyrosine kinase receptor on endothelial cells, they seem to display opposing vascular functions (25). Ang-1 and VEGF exert complementary effects during early embryonic development (26). Although VEGF initiates vascular formation, Ang-1 promotes subsequent remodeling, maturation, and stabilization (26). Ang-2 plays a complex role in regulation of vascular remodeling that leads to either vessel sprouting or regression, depending on its expression relation with other angiogenic stimuli. For example, in the presence of VEGF, Ang-2 would potentiate angiogenic sprouting (27). However, in the absence of VEGF, Ang-2 might lead to vascular regression. Despite these known angiogenic factors expressed in adipose tissue, underlying mechanisms of regulation of adipose vascular patterning, remodeling, and functions remain uncharacterized.

FOXC2 is a member of the forkhead/winged helix transcription factor family, playing an important role in regulation of metabolism, arterial specification, and vascular sprouting (28). Based on patient studies involving measurements of FOXC2 mRNA levels in white adipose tissue (WAT), identification of a single nucleotide polymorphism in the FOXC2 promoter region and detection of a frame-shift mutation in FOXC2 has led us and others to suggest that FOXC2 plays a role in human conditions such as obesity, dyslipidemia, and type 2 diabetes (29–32). Human FOXC2 mutations are associated not only with obesity and type 2 diabetes but also with defects in lymphangiogenesis (lymphoedema-distichiasis syndrome) (33). Previous work showed that specific expression of FOXC2 in adipocytes led to conversion of WAT to a “brown”-like adipose tissue phenotype in transgenic mice (FOXC2-TM) (34). Adipocytes from such mice exhibit a 4-fold increase in oxygen consumption (34). Thus, this is an optimal model to study how vascularization is regulated in response not only to tissue remodeling (more brown-like adipocytes) but also to an increased metabolic rate. Our present results show that FOXC2 directly controls the promoter activity of Ang-2, which alters vascular patterning, remodeling, maturation, and functions.

Results

Vascular Phenotypes of WAT and Brown Adipose Tissue (BAT).

Necropsy analysis of FOXC2-TM mice revealed that both axillary and inguinal WAT appeared as a reddish or brownish tissue, which was similar to the color of BAT (34) (Fig. 1A). Immunohistochemical analysis of the epididymal and inguinal WAT with an anti-CD31 antibody showed that a high density of “plexus-like” vascularity existed in FOXC2-TM. These microvessels appeared as “honeycomb-like” vascular networks, which encapsulated adipocytes (Fig. 1 B and C). In contrast, microvascular networks of WATs derived from WT mice were highly organized and appeared in a relatively low density. Similar to WAT, interscapular BAT of FOXC2-TM appeared as an exceptionally high density of vascular networks, which virtually consisted of only vascular plexuses (Fig. 1D). Significantly fewer vascular plexuses were detected in WATs or BAT of WT mice (Fig. 1F). It should be emphasized that the vascular phenotype of WAT in FOXC2-TM resembled the phenotypes found in BAT in WT mice, suggesting a possible transition from WAT toward BAT (Fig. 1 C and D). Quantification demonstrated 2- to 3-fold increases of vascular density of both WAT and BAT in FOXC2-TM compared with those of controls (Fig. 1E). These findings demonstrate that overexpression of FOXC2 in the adipose tissue leads to alterations of both vessel numbers and vascular structures.

Fig. 1.

Patterning, remodeling, and maturation of adipose vasculature in FOXC2-TM. (A) Necropsy analysis of subcutaneous adipose tissue of FOXC2-TM and WT mice. Arrows point to reddish WAT of FOXC2-TM. (B–D) Epididymal (B) and inguinal (C) WATs and interscapular BAT (D) of both FOXC2-TM and WT mice were stained with an anti-CD31 antibody. Vascular patterns and structures were revealed by 3D projection by using confocal laser scanning microscope analysis of the whole-mount tissues. Arrows point to disorganized vascular plexuses. (Bar: 50 μm.) (E and F) Total vessel areas (E) and numbers of disorganized vascular plexuses (F) were quantified, and the data are presented as mean (±SD). epiWAT, epididymal WAT; ingWAT, inguinal WAT; intBAT, interscapular BAT.

Vascular Phenotype of Dermal and Subcutaneous Tissues of FOXC2-TM.

We examined blood vessels in the dermal and subcutaneous tissues by immunohistological analysis using an anti-CD31-specific antibody. A high microvessel density was detected in the dermal and subcutaneous tissues of FOXC2-TM compared with those of the WT mice [supporting information (SI) Fig. S1 A and B]. The structure of these vessels appeared to be a dense network with irregular and tangled vascularity. In some areas, blood vessels tended to form ragged vascular plexuses due to fusion of multiple capillaries/microvessels. Strikingly, the average intercapillary distance in the subcutaneous tissue of FOXC2-TM was significantly shorter than that of the adipose tissue of WT mice. Quantification showed that the total area of the CD31-positive structures and the number of vascular plexuses were significantly higher in both dermal and subcutaneous tissues of FOXC2-TM than those of WT mice (Fig. S1 C and D).

Alteration of Vascular Remodeling and Maturation.

Altered patterning of adipose vasculatures prompted us to study vascular remodeling and maturation in both WAT and BAT of FOXC2-TM. In WT mice, vascular smooth muscle cells (VSMCs) were all associated with large arterial blood vessels, and no α-smooth muscle actin (α-SMA)-positive signals were found in microvessels in either WAT or BAT (Fig. 2A). Interestingly, the total number of α-SMA-positive arterial vessels was significantly decreased in both WAT and BAT of FOXC2-TM as compared with WT mice (Fig. 2B). Surprisingly, a considerable number of α-SMA-positive VSMCs were associated with microvessels, including the dense vascular plexuses, suggesting redistribution of VSMCs (Fig. 2A). There was a similar trend for redistribution of NG2-positive pericytes in microvasculatures of WAT and BAT of FOXC2-TM. In WT mice, NG2-positive pericytes were mainly associated with arterial vessels and broadly distributed in the intervascular spaces but remained less associated with microvessels (Fig. 2A). However, virtually all NG2-positive pericytes were found to be associated with primitive vascular networks in both WAT and BAT of FOXC2-TM. In addition to microvessels, large arterial vessels are also partially coated with NG2-positive pericytes (Fig. 2A). Quantification demonstrated that the total area of α-SMA-positive structures in both WAT and BAT was significantly increased in FOXC2-TM (Fig. 2C). In contrast, total numbers of NG2-positive pericytes were significantly decreased in WAT and BAT of FOXC2-TM, but nearly all pericytes remained associated with microvessels. Taken together, these findings demonstrate that FOXC2 affects adipose vessel maturation by regulating distribution of both VSMCs and pericytes. Vascular remodeling is probably a prerequisite for blood vessels to coordinate with the high metabolic rate in FOXC2-expressing adipose tissue.

Fig. 2.

Association of smooth muscle cells and pericytes. (A) Inguinal WAT and interscapular BAT were triple-stained with an anti-CD31, an anti-α-SMA, and an anti-NG2 antibody. Blood vessels (red), VSMCs (green), and pericytes (blue) were revealed by confocal laser scanning microscopy of the frozen tissues. Arrows point to triple-positive signals. (Bar: 50 μm.) (B–E) The total number of α-SMA-positive large arterial vessels (B), the total α-SMA-positive area (C), the total CD31-positive area (D), and the total NG2-positive area were quantified as per optical field (×20), and the data of 9 or 10 random fields are presented as mean (±SD). intBAT, interscapular BAT; ingWAT, inguinal WAT.

Up-Regulation of Angiogenic Factors and Their Signaling Components.

To study the underlying mechanisms of the angiogenic phenotype switched by FOXC2, we compared gene expression profiles of the abdominal WAT of FOXC2-TM and WT mice by using an Affymetrix microarray gene chip and PCR analyses. Sequences of primers used for quantitative real-time PCR assays can be found in Table S1. Interestingly, among up-regulated gene products, the expression levels of several potent angiogenic factors were significantly increased (Table S2). These angiogenic gene products include members of the VEGF, PDGF, Ang, TGF-β, TNF, ephrin, insulin-like growth factor, and endothelin families. Interestingly, several of the significantly up-regulated angiogenic factors such as VEGF-C, Ang-2, and PDGF-AA have been reported to induce angiogenesis and are involved in regulation of vascular maturation, remodeling, and stabilization (35–37). It should be emphasized that Ang-2 was one of the most up-regulated gene products in the adipose tissue of FOXC2-TM. Thus, these data support the active angiogenic phenotype in FOXC2-TM.

Additionally, levels of several receptor signaling molecules expressed in endothelial cells or VSMCs/pericytes, which are directly or indirectly involved in the angiogenesis and vascular remodeling signaling systems, were also increased (Table S3). Importantly, tyrosine kinase receptors, including the PDGF receptor-β (PDGFR-β) and FGF receptor type 2 (FGFR-2), were significantly up-regulated. In addition to tyrosine kinase receptors, the levels of a couple of GTP-coupled signaling components were also significantly increased. These data provide a possible molecular basis for the observed angiogenic phenotypes.

FOXC2 Transcriptionally Regulates Ang-2 Expression.

To further validate gene expression profiles regulated by FOXC2, quantitative real-time PCR analysis was performed with RNAs extracted from WAT of FOXC2-TM and WT mice. Among all analyzed gene transcripts, Ang-2 was the most up-regulated gene in the adipose tissue of FOXC2-TM, which was consistent with the gene array findings. A nearly 6-fold increase of Ang-2 was detected in WAT of FOXC2-TM as compared with that of WT mice (Fig. 3A). In addition, the level of placental growth factor was also significantly increased. There was a trend for elevated expression levels of EfnB-2, Notch-3, and PDGFR-β. Thus, these results obtained from real-time PCR generally validated the data from Affymetrix array analysis.

Fig. 3.

Detection of gene expression by real-time PCR and analysis of Ang-2 promoter activity. (A) mRNAs extracted from epididymal WAT and mouse embryo fibroblasts (MEFs) were used for quantitative amplification of Ang-2, EfnB2, placental growth factor (PlGF), Notch3, and PDGFR-β by real-time PCR analysis. (B) FOXC2^+/+, FOXC2^+/−, and FOXC2^−/− MEFs were used for correlating Ang-2 and FOXC2 expression levels. (C) The Ang-2 promoter-luciferase reporter construct was used to transfect 3T3 fibroblast-differentiated preadipocytes, and induction of luciferase activity was determined. (Left) The fold increase of luciferase activity after various amounts of FOXC2 DNA was quantified, and the average data of each sample are presented as mean (±SD). (Right) A series of deletion constructs consisting of various regions of the Ang-2 promoter and luciferase reporter were generated and transfected into preadipocytes (see Table S4). delFkh4, deletion of Fkh4.

Sequence analysis of the ligated sequences by using pDRAW (Acaclone Software) revealed five putative forkhead-binding sites by using the forkhead-binding consensus sequence (RYMAAYA; R = A/G; Y = C/T; M = A/C), suggesting that Ang-2 might be a direct target gene for FOXC2. To investigate this possibility, Ang-2 promoter fused with the luciferase reporter was cloned and transfected into 3T3 diffentiated preadipocytes in the presence and absence of FOXC2. Interestingly, Ang promoter activity was increased in a dose-dependent fashion after the addition of FOXC2 (Fig. 3C Left). Nearly an 8-fold increase of luciferase activity was observed at 200 ng of FOXC-2. These findings show that FOXC-2 directly activates Ang-2 promoter activity and controls its expression.

To further delineate the FOXC2-responsive sequences in the Ang-2 promoter, various deletion mutant promoter constructs were used to drive luciferase gene expression. Primers used to make these constructs are listed in Table S4. Among all five Fkh regions in the Ang-2 promoter, deletion of Fkh4 nearly abrogated the promoter activity induced by FOXC2, suggesting that the Fkh4 region was essential for FOXC2-induced transcription activity (Fig. 3C Right). In contrast, deletion of the other four Fkh regions in the Ang-2 promoter did not affect the reporter gene expression in any significant way.

We next isolated preadipocytes from FOXC2^+/+, FOXC2^+/− heterozygous, and FOXC2^−/− knockout mice to quantitatively correlate expression levels of FOXC2 with those of Ang-2. Expectedly, an ideal correlation of expression levels between FOXC2 and Ang-2 existed in FOXC2^+/+ preadipocytes (Fig. 3B). Approximately 50% reduction of expression levels of FOXC2 and Ang-2 was detected in FOXC2^+/− preadipocytes. Interestingly, complete knockout of FOXC2 gene in preadipocytes did not further decrease Ang-2 expression as compared with that of FOXC2^+/− preadipocytes, suggesting that other mechanisms might exist to control the basal expression level of Ang-2 in adipocytes. Taken together, these data demonstrate that FOXC-2 transcriptionally up-regulates Ang-2 expression in adipocytes.

Ang-2 Is the Essential Mediator for the FOXC2-Induced Angiogenic Phenotype.

Transcriptional regulation of Ang-2 expression by FOXC2 in adipocytes suggested that Ang-2 might be responsible for switching on the angiogenic phenotype. To explore this possibility, a known Ang-2-specific inhibitor, L1-10 (38), was used to reverse the FOXC-2-induced angiogenic phenotype in FOXC2-TM. Interestingly, administration of L1-10 to FOXC2-TM at a dose of 4 mg/kg, a dose known to block Ang-2 function in vivo, virtually completely reversed the FOXC-2-induced angiogenic phenotype. Morphologically, the reddish appearance of axillary and inguinal adipose tissue in FOXC2-TM was converted into a relatively pale color in the L1-10-treated group as compared with that of the buffer-treated group (Fig. 4A). Immunohistological analysis showed that the primitive vascular plexus-shaped vessels in inguinal WAT were normalized to well structured vascular networks, which were indistinguishable from those in WT adipose tissues (Fig. 4 C and E). In addition to structural changes, the unusually high density of microvessels in adipose tissues in FOXC2-TM was also normalized to the level of WT adipose tissues (Fig. 4 B and C). Similarly, redistribution of α-SMA-positive VSMCs was reversed to the relatively large arterial vessel, as seen in those in both WAT and BAT of WT mice (Fig. 4D). Quantification showed that blockage of Ang-2 led to decreased total number of VSMC-coated vascular area and increased large arterial vessel association (Fig. 4 F and G). These data provide compelling evidence that Ang-2 is responsible for the FOXC-2-induced vascular maturation and patterning in the adipose tissue.

Fig. 4.

Reversal of vascular phenotype by blocking Ang-2. Ang-2 inhibitor L1-10 was administrated into 3-week-old FOXC2-TM and WT mice for 4 weeks. (A) Axillary, inguinal, and other subcutaneous adipose tissues were exposed after euthanizing mice. Relatively pale WATs were found in the L1-10-treated FOXC2-TM animals. (B) Quantification of CD31-positive blood vessels. (C) Whole-mount tissues of inguinal WAT (ingWAT) and interscapular BAT (intBAT) were stained with an anti-CD31 antibody. (D) Frozen sections of inguinal WAT and interscapular BAT were used for double staining of endothelial cells (red) and VSMCs (green, revealed by presence of α-SMA). White arrows point to double-positive large arterioles, and arrowheads point to microvessel double-positive signals (yellow). (Bar: 50 μm.) (E–G) Quantification of vascular plexuses (E), α-SMA⁺ large arterioles (F), and the total area of α-SMA⁺ area (G). Data are presented as mean (±SD) of 7–10 randomized fields.

Impairment of Vascular Function.

The vascular phenotype in dermal and subcutaneous tissues of FOXC2-TM suggested possible alterations of vascular functions. To explore this possibility, we performed full skin wound-healing experiments. At day 11 after the creation of the wound, all WT mice exhibited complete healing of the wound beds (n = 10). In contrast, a significantly delayed wound healing was observed in FOXC2-TM. Notably, significantly larger diameter wounds already became obvious at day 4 after the creation of the wound and significant differences remained throughout the entire experiments (Fig. 5 B–D). Approximately 3 days of delayed wound healing was observed in FOXC2-TM, which showed complete healing at day 14 (Fig. 5 C and D). Immunohistochemical analysis showed that a significantly higher number of CD31-positive vessels were present in wound tissue of FOXC2-TM than in that of WT mice, suggesting that impairment of wound healing was not due to defects of neovascularization but abnormality of vascular function (Fig. 5 A and E). These results demonstrate that the vascular adaptation seen in FOXC2-TM results in remodeling of existing vessels and formation of premature new vessels that lead to delayed wound healing.

Fig. 5.

Wound healing. Full skin wounds were created on the backs of WT and FOXC2 mice. (A) The wound beds were stained with H&E (Left) or double-stained with propidium iodide (PI, red) and an anti-CD31 antibody (green) (Right). Arrows point to CD31-positive vessels. (Bar: 50 μm.) Diameters of wounds were measured every other day (B and D), and the percentages of animals with completely healed wounds were recorded (C). (E) CD31-positive blood vessels were quantified as vessel area per optical field (×10), and data represent mean of 9 or 10 samples. ***, P < 0.001.

Discussion

The plasticity of adipose tissue throughout adult life requires constant vessel growth, regression, and remodeling. In addition to adipose tissue growth, conversion of the WAT into a BAT-like phenotype demands a high metabolic rate by activation of the adrenergic/cAMP/protein kinase A signaling pathway and increasing oxygen consumption, which requires increased blood supply (39). Accumulating evidence shows that adipocytes cross-communicate with neighboring endothelial cells via paracrine signaling pathways, extracellular components, and direct cell–cell interactions (5, 40–42). For instance, the adipocyte-derived hormone leptin induces Ang-2 expression in adipocytes (43). In brown adipocytes, it is well established that an increased oxygen consumption/metabolic rate, induced by cold exposure, is associated with increased synthesis of angiogenic factors such as VEGF, which in turn stimulates vessel growth and remodeling in response to metabolic needs (44–46). However, the molecular identity and genetic control of adipose-derived paracrine factors in regulation of vessel growth, maturation, remodeling, patterning, and function remain poorly characterized. Here we show that in a transgenic mouse model, with elevated metabolism in WAT, FOXC2 transcriptionally switches on an angiogenic phenotype in the adipose tissue by increasing expression of angiogenic factors, including Ang-2.

To reveal the identity of FOXC2-regulated soluble paracrine and endocrine factors produced by adipocytes, Affymetrix gene array analysis showed that Ang-2 is one of a few angiogenic gene products that are up-regulated at high levels. Up-regulation of Ang-2 has been confirmed by quantitative real-time PCR, and its promoter activity could be directly induced by FOXC-2. Among five Fkh regions, Fkh4 is the only essential element in the Ang-2 promoter responsible for FOXC2 regulation. Ang-2 is one of the few vascular factors known for regulation of vascular patterning, remodeling, and maturation (24). Although Ang-2 and Ang-1 bind to the same tyrosine kinase receptor Tie-2, they display opposing activity on blood vessel remodeling and maturation. Although Ang-1 promotes vessel maturation by recruiting VSMCs onto the nascent vasculature, Ang-2 repels mural cell association with blood vessels (25, 27). Without mural cells, the newly formed vasculature remains unstable and experiences either growth or regression depending on the presence of other angiogenic stimuli. The primitive plexus-shaped vascular network observed in the adipose tissue of FOXC2-TM animals is consistent with vascular functions of Ang-2. This FOXC2-Ang-2-induced phenotype resembles the phenotypes in tumors induced by Notch antagonists, which induce primitive, disorganized, and nonproductive vascular networks (47).

Although several other angiogenic factors and receptor signaling molecules are also up-regulated in FOXC-2-expressing adipose tissue, their vascular roles in relation to Ang-2 are unclear. It is known, however, that in the presence of Ang-2, VEGF could further accelerate neovascularization by broadly acting on non-mural-cell-coated endothelium (48). Other angiogenic factors could also exert a similar synergistic effect with Ang-2 on vascularization and remodeling. Although Ang-2 repels pericytes and VSMCs from large vessels, it is unclear why these mural cells are redistributed from large vessels to coat microvessels. This effect probably requires an intimate interplay between Ang and PDGF systems. For example, growing cones of tip endothelial cells are known to produce PDGF-BB, which recruits pericytes and VSMCs onto the newly formed vasculature (49, 50). It is possible that Ang-2 repels mural cells from large vessels and that PDGF-BB redirects them onto microvessels. Because little is known about angiogenesis and vascular remodeling in response to increased metabolism in adipose tissue, our model system might provide a unique opportunity to study the underlying mechanisms by which Ang-2 and other vascular factors cooperatively control vascular maturation, remodeling, and function.

One of the most intriguing findings in our study is that a specific Ang-2 inhibitor could reverse the FOXC-2-induced vascular phenotype in adipose tissue. These functional data provide convincing evidence that Ang-2 is the target gene product responsible for the observed vascular phenotype. Several independent studies show that Ang-2 is constitutively up-regulated in expanding adipose tissues (5, 21, 51). In addition to having its direct angiogenic function, Ang-2 is required for vascular patterning, remodeling, and maturation in growing adipose tissue. Ang-2 is a soluble vascular factor that targets both proximal and distal vasculatures (52, 53). Malformation of vascular networks in nonadipose tissues as a response to an “overshoot” in adipose tissue-derived angiogenic factors leads to functional defects. For example, skin wound healing is significantly delayed in FOXC-2-TM.

Taken together, our work provides compelling evidence and molecular mechanisms that FOXC-2 regulates the cross-talk between adipocytes and vascular cells, including endothelial cells and mural cells. Ang-2 as a direct target for FOXC2 is involved in vascular remodeling, patterning, and maturation in adipose tissues. We would like to speculate, based on the findings presented, that via regulation of FOXC2 adipose tissue can adapt its degree of vascularization to meet present metabolic demand. Thus, functional interference with FOXC2 or Ang-2 might provide a therapeutic approach for prevention and treatment of obesity or its related disorders.

Experimental Procedures

Animals.

Animals were anesthetized by an injection of a mixture of Dormicum and Hypnorm (1:1, Roche and VetaPharma) before all procedures and killed by a lethal dose of CO₂ followed by cervical dislocation. All animal studies were reviewed and approved by the Animal Care and Use Committee of the North Stockholm Animal Board. See SI Methods for information about reagents.

Plasmid Construction, Mutagenesis, Cell Culture, and Transfection.

Plasmid construction, mutagenesis, cell culture procedure, and DNA transfection are described in SI Methods.

Whole-Mount Staining and Confocal Analysis.

The whole thicknesses of dorsal middle region skin tissue, inguinal and epididymal WAT, and interscapular BAT were freshly resected from FOXC2-TM and WT mice and were fixed with 4% paraformaldehyde overnight. Tissue samples were transferred into PBS and were digested with proteinase K (20 μg/ml) for 5 min followed by staining overnight at 4°C with a rat anti-mouse CD31 antibody (1:100). After rigorous rinsing, blood vessels were detected with labeled secondary antibodies. After washing, slides were mounted in Vectashield mounting medium (Vector Laboratories) and stored at −20°C in the dark before examination under a confocal microscope (Zeiss confocal LSM510 laser scanning microscope, CLSM). The images were further analyzed with the Adobe PhotoShop CS software program.

Real-time Quantitative RT-PCR.

See SI Methods for details.

Immunohistochemistry.

See SI Methods for details.

Vascular Quantification Analysis.

Vascularization areas were quantified by using an Adobe PhotoShop program. Briefly, 10 random fields (200 × 200 μm per square) of CD31-, NG2-, or α-SMA-positive structures from three to five animals were activated and calculated with a computerized mathematic method.

Ang-2 Blocking Experiments.

Three-week-old female FOXC2-TM mice were randomly divided into two groups (five animals per group), and the same number of WT mice were used as controls in each group. The detailed protocol for Ang-2 blockage experiments is described in the SI Methods.

Statistical Analyses.

Statistical analysis of the in vitro and in vivo results was made by a standard two-tailed Student's t test by using Microsoft Excel 2003. P < 0.05, P < 0.01, and P < 0.001 were deemed as significant, highly significant, and extremely significant, respectively.