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,1
,
* Institut National de la Santé et de la Recherche Médicale (INSERM), U858, Toulouse, France;
Université de Toulouse, Institut de Médecine Moléculaire de Rangueil, IFR31, Toulouse, France;
Angiogenesis and Thrombosis Department, Sanofi-Aventis Research, Toulouse, France; and
Institut de Pharmacologie et de Biologie Structurale, CNRS UMR 5089, Toulouse, France
1Correspondence: INSERM, U858, Toulouse, Cedex 4, France. E-mail: oxana.koundouzova{at}toulouse.inserm.fr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: apelinergic system • adipocyte • neovascularization • hypoxia • fat grafting • angiogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. Adipose tissue is able to integrate signals from other organs and respond by regulating secretion of adipokines acting locally and distally through autocrine, paracrine, and endocrine interactions. A number of adipocyte-derived factors, including vascular endothelial growth factor (VEGF), nerve growth factor, leptin, plasminogen activator inhibitior type 1, and tumor necrosis factor-
, affect vascular endothelial function to drive angiogenesis (4
5
6)
. Accumulating evidence suggests that hypoxia is the major trigger for the initiation of angiogenesis in most tissues (7
, 8)
. In adipocytes, hypoxia markedly enhances expression and secretion of VEGF and other proangiogenic factors, and it has been suggested that hypoxia might be a modulator of the strong angiogenic process associated with adipose tissue expansion (6
, 9)
. Furthermore, Rupnick and colleagues (10)
have recently speculated that neovascularization may be critical for adipose tissue growth. They showed that systemic treatment with angiogenesis inhibitors resulted in weight reduction and adipose tissue loss in various mouse models of obesity. However, the precise mechanisms and the mediators involved in the crosstalk between adipocytes and endothelial cells remain poorly understood.
We have recently shown that a novel adipocyte-derived factor, apelin, is up-regulated in mouse and human obesity (11
, 12)
. This bioactive peptide is the endogenous ligand of the G protein-coupled receptor, APJ (13)
. The apelinergic system has a widespread pattern of distribution in the central nervous system and periphery (14
, 15)
. Apelin has been shown to lower blood pressure (16
, 17)
and modulate contractility of cardiac tissue and blood vessels (18
, 19)
and food and water intake and pituitary hormone release (20)
and may play a role in osteoblast apoptosis (21)
. More recently apelinergic system has been found to promote embryonic and tumor angiogenesis (22)
. Considering its production in adipocytes, rise in obesity, and potential effects on angiogenesis, apelin would be a good candidate mediator of the interaction of adipocytes and endothelial cells leading to increased angiogenesis during adipose tissue expansion in obesity.
The aim of the present study therefore was to determine whether apelin might play a role in adipose tissue angiogenesis. Therefore, the effect of hypoxia on apelin production in adipocytes and the effect of the apelin/APJ signaling on endothelial cell responses were assessed in vitro. Furthermore, apelin production during adipose tissue hypoxia and the effect of apelin down-regulation were determined in an in vivo model of adipose tissue-induced angiogensis after adipose tissue transplantation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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For hypoxia studies, 3T3F442A adipocytes were cultivated as described previously (11)
up to day 10 of differentiation. Cells were maintained in a hypoxia chamber (In vivo2 400; Ruskinn Inc., Cincinnati, OH, USA) for 8, 24, or 48 h in comparison with normoxia conditions. In this study normoxia was considered as the ambient atmosphere containing 21% O2 and hypoxia, 1% O2.
Proliferation assay
To determine cell proliferation, HUVECs were seeded in 96-well plates (5 wells/condition) and incubated with basic fibroblast growth factor (bFGF; 3 nM; Life Technologies, Inc., Gaithersburg, MD, USA) or apelin-13 (20, 60, 200, or 600 nM; 2, 6, 20, or 60 µM; Bachem, Torrance, CA, USA; H-4566), after serum starvation. The media and factors were replaced twice during experiments. On day 7 after starting proliferation, viable cells were counted using the CellTiter-Glo Luminescent Cell Viability Assay (Promega Corp., Madison, WI, USA).
Migration assay
Endothelial cell migration was evaluated in a modified Boyden chamber assay as described previously (23)
. Briefly, HUVECs were detached with trypsin, counted, centrifuged, and resuspended in DMEM serum-free medium. Cells (5x104) were plated on the upper chamber. In the lower chamber of the Boyden apparatus, human recombinant bFGF (6 nM, Life Technologies) or apelin-13 (2, 6, 20, 60 µM) were used as chemoattractants. After incubation at 37°C for 22 h, cells were labeled with calcein AM (Molecular Probes, Eugene, OR, USA) according to the manufacturer’s instructions. Fluorescence of cells that had migrated was measured on a Tecan GENios microplate reader (Tecan, Männedorf, Switzerland). All the experiments were carried out at least 3 times in duplicate.
In vitro angiogenesis assay
Growth factor-reduced Matrigel (BD Biosciences, San Jose, CA, USA) was diluted in collagen (1:6 v/v) and kept on ice. This solution (160 µl) was added to each well of 8-well culture slides precoated with type 1 rat tail collagen and left at 37°C for 1 h. An HUVEC suspension, untreated or treated with bFGF (3 nM) or apelin-13 (60 nM, 200 nM, 600 nM, 2 µM, 6 µM), was seeded into the Matrigel/collagen gel for 24 h at 37°C. Microtubules were quantified by microscopy, as described previously (24)
. Briefly, the culture medium was removed, and cells were rinsed twice with PBS and fixed for 30 min at room temperature in a 4% paraformaldehyde solution. Then the cells were washed twice with PBS and stained with Masson trichrome. The formation of the capillary/tubelike networks were examined using a Nikon Eclipse TE-2000 E microscope (x4) equipped with a DXM1200 digital camera (Nikon, Champigny sur Marne, France). Microcapillary network was manually drawn with a pointing device, and the total length of all the capillary tubes in each well was measured using Morpho Expert Image Analysis software (Biocom, Les Ulis, France). All experiments were performed in triplicate and repeated 3 times.
HUVEC transfection with siRNA duplexes
The Stealth Select RNAi for APJ and Stealth RNAi Negative Control were obtained from Invitrogen (Carlsbad, CA, USA). The siRNA sequence targeting human APJ corresponded to the coding region 184–208 relative to the first nucleotide of the start codon (NM_005161.3). Preliminary transient transfection of HUVECs was carried out to test APJ siRNA efficiency. HUVECs were trypsinized, washed with HBSS, and resuspended (1x106 cells) in HUVEC solution (Amaxa Biosystems, Gaithersburg, MD, USA) containing 1 nM siRNA duplex and were transfected using a Nucleoporator (Amaxa Biosystems) following the manufacturer’s instructions. After transfection, cells were immediately seeded into 12-well plates containing complete media. We evaluated siRNA activity by determining APJ mRNA level 6, 24, and 48 h later. For migration assay, 24 h after transfection HUVECs (5x104) were detached with trypsin, counted, centrifuged, and resuspended in DMEM serum-free medium. This assay provides a simple, convenient method to determine the functional consequences of APJ pathway. Apelin at the dose of 50 µg/ml was used as chemoattractant as described below.
Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR)
In 3T3F442A adipocytes, the total RNAs (1 µg) were reverse transcribed using random hexamers and Superscript II reverse transcriptase (RT; Invitrogen). The same reaction was performed without Superscript II (RT–) to estimate DNA contamination. The range of cycle threshold (Ct) values for Ct gene was 23–26, whereas Ct RT– was 35, meaning that very little (<0.1%) of genomic DNA is present in our samples. Real-time PCR was performed starting with 12.5 ng cDNA and both sense and antisense oligonucleotides in a final volume of 25 µl using the SYBR green TaqMan universal PCR master mix (Applied Biosystems, Warrington, UK). Fluorescence was monitored and analyzed in a GeneAmp 7000 detection system instrument (Applied Biosystems). Analysis of the 18S rRNA was performed in parallel using the rRNA control Taqman assay kit (Applied Biosystems) to normalize gene expression. Oligonucleotide primers were designed using the Primer Express software (Applied Biosystems). All primers used were validated for PCR efficiency.
Human APJ and murine apelin were analyzed by reverse transcription and real-time PCR by using RNA samples from transfected cells or siRNA-injected WAT graft. PCR reactions were carried out using Assays-on-DemandTM Gene Expression Products (PE Applied Biosystems, Weiterstadt, Germany). Reactions were performed as described previously (25)
. The calculations of the initial mRNA copy numbers in each sample were made according to the cycle threshold method (26)
and normalized using TATA binding protein mRNA levels.
Animals
All experiments were performed in accordance with the recommendations of the French Accreditation of the Laboratory Animal Care and were approved by the local Centre National de la Recherche Scientifique ethics committee. In the current study we used 7-wk-old male C57BL6/J mice purchased from Charles River Laboratories (l’Arbresle, France). Donor and recipient mice were age-matched to minimize rejection. The recipients were anesthetized with an anesthesia cocktail composed of 5 mg/kg xylazine and 50 mg/kg ketamine. Donor mice were killed by cervical dislocation. Implantation was performed with aseptic techniques. After shaving the hair on the dorsal side, 1 subcutaneous air pocket in the inguinal region was prepared, and whole pieces of epididymal WAT (EWAT; 600 mg) from 2 donors were inserted into the air pocket of the recipient through a 1 cm midline incision. Incisions were closed with sterile surgical staples. All animals were housed individually for the duration of the experiment. At various times after implantation, grafted WATs were photographed, half of each EWAT was cut into small pieces and stored at –80°C for RNA isolation, or used for evaluation of EWAT graft vascularization. Normal EWAT was employed as a control.
For histological examination, EWAT was fixed in Zn-formalin for 24 h and then embedded in paraffin for subsequent trichrome staining of 10 µm thickness sections.
In vivo delivery of siRNA/apelin
In this research we make use of siRNA to silence apelin within adipose tissue. First, in vitro studies are conducted for developing siRNA carrier/delivery technique. Those are then followed by in vivo studies to optimize the technique for siRNA delivery. The siRNA duplex targeting murine apelin was commercially obtained from Ambion, Inc. (Austin, TX, USA; ID 184196). For local delivery, siRNA (10 µg/10 µl per graft) was diluted in PBS and delivered locally. The single injection was given by a 32-gauge Hamilton syringe (Hamilton Co., Reno, NV, USA) just before implantation.
Quantification of angiogenesis by hemoglobin (Hb) measurement
The extent of the vascularization of the EWAT grafts was assessed by the amount of Hb detected in the tissue using the Drabkin method (27)
. Each implant was homogenized (TR-10; Tekmar, Mason, OH, USA) in 2.0 ml of Drabkin reagent (Labtest, São Paulo, Brazil) and centrifuged at 10,000 g for 15 min. The supernatants were filtered through a 0.22 µm filter (Millipore Co., Bedford, MA, USA). Hb in the samples was determined spectrophotometrically by measuring absorbance at 540 nm using an ELISA plate reader and compared against a standard curve of Hb. The content of Hb in the granulation tissue was expressed as µg Hb per g wet tissue.
Tissue hypoxia
Pimonidazole hydrochloride (Hypoxyprobe-1; Chemicon International Inc., Temucula, CA, USA) was injected intraperitoneally into mice (200 mg/kg). One hour after injection, adipose tissue was dissected, fixed in formalin, and embedded in paraffin. Sections were cut at 10 µm thickness, and tissue hypoxia was detected by Hypoxyprobe-1 and horseradish peroxidase-conjugated F(ab')2 fragment of anti-mouse immunoglobulin G (IgG) antibody. Brown color was generated using diaminobenzidine as substrate. Control experiments were performed by injecting saline in place of Hypoxyprobe-1, and WAT sections were labeled exactly as described above.
Statistical analysis
Data are expressed as means ± SE. Statistical analyses were performed by 1-way ANOVA followed by the Bonferroni t test. Values of P < 0.05 were considered statistically significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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shows that vs. time-matched normoxic control cells, hypoxia increased both apelin and VEGF mRNA expression in a time-dependent manner. Exposure of 3T3F442A to hypoxia caused a rapid increase in apelin mRNA with a peak induction (17-fold) at 8 h. The apelin mRNA remained elevated above the control (normoxia) level for the entire exposure to hypoxia (48 h). At the same time, VEGF mRNA expression significantly increased by 4-fold at 8 h and reached a peak value (7-fold) at 48 h of hypoxia.
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Apelin-induced angiogenic responses on HUVECs
The formation of microvessels involves the coordinated proliferation, migration, and morphogenetic organization of endothelial cells into capillary tubes. We further examined the effects of apelin on HUVEC proliferation, migration, and angiogenesis. The cellular responses of HUVECs to apelin treatment were compared to those of bFGF. As shown in Fig. 2A
, the treatment of HUVECs with apelin significantly increased cell proliferation in a dose-dependent manner up to100 µg/ml of apelin (Fig. 1A
). In addition, apelin at high doses also induced a significant increase in endothelial cell migration (Fig. 2B
) and Matrigel capillary/tubelike structure formation (Fig. 2C
).
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To determine the functional consequences of the APJ pathway, we assessed the specific silencing effect of siRNA/APJ on the endothelial cell migration. To address this question, HUVECs were first transfected with either negative control siRNA or APJ/siRNA. Twenty-four and 48 h following siRNA transfection, RNAs were prepared to examine APJ expression using real-time PCR analysis. In cells transfected with negative control siRNA, APJ expression was not diminished. By contrast, APJ mRNA was significantly reduced after 24 and 48 h of APJ siRNA transfection in HUVECs (Fig. 3A
). As shown in Fig. 3B
, siRNA-mediated down-regulation of APJ in HUVECs specifically and significantly reduced cell migration induced by apelin as compared with the control nonsilencing siRNA.
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Apelin-dependent angiogenesis in adipose tissue
Although angiogenesis appears to be linked to the activity of adipose tissue-derived proangiogenic factors, very little is known about angiogenesis in adipose tissue, primarily because of the absence of reliable in vivo model. To address this question, we have set up a murine model allowing reliable and quantitative assessment of local adipose tissue-related angiogenesis that involves EWAT implantation. The surgical implantation of EWAT (600 mg) into mice was well tolerated by all animals. No sign of infection or rejection was observed in the implant compartment during the 14 day period of the experiment. As shown in Fig. 4A
, the formation of new blood vessels in EWAT grafts was already observed on day 2. In contrast to control (EWAT on day 0), the grafted EWAT shows a well-developed vasculature on day 14. The development of a vascular supply within adipose tissue was associated with a concomitant and significant elevation in Hb content (Fig. 4B
) and VEGF-R2 level (Fig. 4C
) in grafted EWAT on days 5 and 14. In this active phase of adipose tissue-derived angiogenesis, we found high apelin mRNA expression in EWAT grafts (Fig. 4D
). In addition, the functional neovascularization of adipose tissue was also confirmed by histological analysis of EWAT grafts (Fig. 5A
). Light microscopic examination of WAT sections revealed an enhanced collagen deposition (blue) and a numerous blood vessels (red) in EWAT grafts on days 5 and 14 compared with control (EWAT on day 0).
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As shown by our in vitro experiments, hypoxia induced a time-dependent increase in apelin of differentiated 3T3F442A. We next studied the tissue hypoxia during neovascularization of grafted EWAT on days 0, 2, 5, and 14 using pimonidazole hydrochloride, which forms protein adducts in tissue that experience an oxygen level of 
1% O2. Pimonidazole staining revealed that peripheral regions of EWAT experienced a discrepancy between oxygen supply and demand as early as 2 days after transplantation (Fig. 5B
), which was sustained throughout the early phase of angiogenesis in the grafted EWAT (day 5). In contrast, no hypoxia was observed in grafted EWAT on day 14.
To elucidate the role of apelinergic system in WAT-derived angiogenesis, we down-regulated apelin using siRNA-mediated gene silencing in WAT grafts. In the murine model of adipose tissue derived angiogenesis, we found that local intragraft administration of stable complex apelin-targeting siRNA (50µM, 40 µl), but not of control siRNA, led to the significant decrease in Hb content (36% vs. control) of WAT grafts in the active phase of adipose tissue neovascularization (Fig. 6A, B
). As shown in Fig. 6C
, this effect was correlated with a down-regulation (52% vs. control) of apelin mRNA expression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Angiogenesis is a multistep process that involves several interrelated reactions, such as endothelial cell migration, proliferation, and capillary tube formation. These processes are tightly regulated by actions of proangiogenic factors such as VEGF, bFGF, and other adipocyte-produced proteins (28
, 29)
. We have previously shown that both human and mouse adipocytes secrete apelin, a newly identified adipokine, associated with obesity-associated metabolic abnormalities (11)
. In the vascular system apelin and APJ are known to be expressed in endothelium and smooth muscle cells, but the biological activity of apelinergic system in the human endothelial cell functions remain to be explored. Kasai et al. (30)
have previously reported that apelin stimulates proliferation and migration of RF/6A cells, a rhesus macaque choroid-retina-derived endothelial cells. In our study we demonstrate that apelin is able to induce a proangiogenic response in human endothelial cells. This response includes apelin-dependant stimulation of HUVEC cell migration, proliferation, and matrigel tube formation. Moreover, by using the siRNA approach we have demonstrated that knocking down APJ inhibited apelin-induced HUVEC migration, suggesting a role of the APJ pathway in the functional activity of apelin in the human endothelial cells. In line with our observation, Kalin et al. (22)
recently reported that apelin-APJ signaling is essential for embryonic and tumor angiogenesis. However, the mechanisms by which the apelinergic system promotes physiological and pathological angiogenic processes remain to be clarified.
Hypoxia is a major driving force for angiogenesis. Hypoxic conditions induce increased transcription of the mRNA for VEGF, leptin, and other proangiogenic factors in adipocytes (6
, 9)
. It is also known that mature adipocytes are capable of enhanced production and release of the critical angiogenic signals under hypoxic conditions (31)
. In this study, we have examined the adipocyte-specific expression profile of apelin in response to hypoxia. We show that hypoxia up-regulates apelin expression in 3T3F442A adipocytes, suggesting that hypoxia may be a potent regulatory mechanism of activation of the apelinergic system in adipose tissue. Our observations are consistent with a recently reported study (32)
that showed that the apelin gene is regulated by hypoxia in adipocytes via the HIF pathway.
Several lines of evidence suggest a link between blood vessel formation and adipogenesis (33)
. During fetal development, arteriolar differentiation precedes adipocyte development, and differentiation of blood vessel extracellular matrix (ECM) precedes differentiation of adipocyte ECM (34)
. During postnatal development, VEGF expression and resulting angiogenesis may augment adipogenesis in adipose tissue (29)
. Recently Rupnick et al. (10)
have shown that treatment of mice from different obesity models with angiogenic inhibitors decreased body weight, suggesting that adipose tissue accretion can be regulated through the vasculature. In agreement with this concept, using an in vivo model of adipose tissue-derived angiogenesis, we have demonstrated that adipose tissue is one of the active sites of angiogenic processes. In this model system the first appearance of the newly formed blood vessels around the transplanted adipose tissue was observed on days 2 and 5, indicating that angiogenesis in adipose tissue is a dynamic process characterized by the rapid development of the vascular network. Consistent with our in vitro results in adipocytes, we observed tissue hypoxia and up-regulated apelin expression during the early phase (days 2 and 5) of angiogenic processes in adipose tissue. Our data support the idea that local adipose tissue hypoxia may lead to the stimulation of the production and release of proangiogenic factors, the function of which is to sustain adequate blood flow in the expanding adipose tissue. The hypoxia-dependent adipokine dysregulation may be partly responsible for the development of diseases linked to obesity, particularly type II diabetes and the metabolic syndrome.
Using siRNA technology, we provide a strong evidence that apelin signaling pathway plays a critical role in the agiogenic activity of adipose tissue. We show that local intragraft administration of stable complex apelin-targeting siRNA led to the significant inhibition of active angiogenesis in the grafted adipose tissue. Thus, although APJ levels are relatively low in endothelial cells, such data clearly show that the apelin/APJ complex is responsible of the cellular effect observed. These findings, coupled with the recent demonstration of the involvement of apelin/APJ pathways in microvascular proliferations of brain tumors (22)
support the novel notion that the apelinergic system could play an important role in the pathological angiogenesis. Further delineation of the specific upstream molecular pathways through which the apelin/APJ control physiological and pathological angiogenesis is needed.
Taken together, our data provide the first evidence that apelin/APJ signaling pathways play a critical role in the development of the functional vascular network in adipose tissue. In addition, we have shown that adipocyte-derived apelin can be up-regulated by hypoxia. These findings highlight a potential of apelinergic system as a target for developing novel therapeutic strategies to modulate angiogenesis.
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ACKNOWLEDGMENTS |
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Received for publication January 18, 2008. Accepted for publication July 17, 2008.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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  D. D. Briana and A. Malamitsi-Puchner Reviews: Adipocytokines in Normal and Complicated Pregnancies Reproductive Sciences, October 1, 2009; 16(10): 921 - 937. [Abstract] [PDF]
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