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Keywords:

  • Human CD34+ cells;
  • Stromal derived factor-1;
  • Endothelial differentiation;
  • Chemotaxis;
  • CXCR-4;
  • Matrigel Microvasculature;
  • Progenitor cells

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References

The native CD34+/CD31 cell population present in the stroma-vascular fraction of human adipose tissue (hAT) displays progenitor cell properties since they exhibit adipocyte- and endothelial cell-like phenotypes under appropriate stimuli. To analyze the signals within hAT regulating their phenotypes, the influence of hAT-derived capillary endothelial cells (CECs) was studied on the chemotaxis and differentiation of the hAT-CD34+/CD31 cells. Conditioned medium from hAT-CECs led to a strong chemotaxis of the hAT-CD34+/CD31 cells that was inhibited with pretreatments with pertussis toxin, CXCR-4 antagonist, or neutralizing antibodies. Furthermore, hAT-CECs produced and secreted the CXCR-4 ligand, that is, the stromal derived factor-1 (SDF-1). Finally, hAT-CECs induced the differentiation of hAT-CD34+/CD31 cells toward an endothelial cell (EC) phenotype. Indeed, hAT-CECs and -CD34+/CD31 cell coculture stimulated in a two-dimensional system the expression of the EC CD31 marker by the hAT-progenitor cells and, in a three-dimensional approach, the formation of capillary-like structures via a SDF-1/CXCR-4 dependent pathway. Thus, the migration and differentiation of hAT progenitor cells are modulated by hAT-CEC-derived factors. SDF-1, which is secreted by hAT-derived CECs, and its receptor CXCR-4, expressed by hAT-derived progenitor cells, may promote chemotaxis and differentiation of hAT-derived progenitor cells and thus contribute to the formation of the vascular network during the development of hAT.

Disclosure of potential conflicts of interest is found at the end of this article.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References

The excessive human adipose tissue (hAT) development is associated with adipocyte hypertrophy and hyperplasia [1, 2] as well as an extension of the capillary network [3, [4]5]. The processes involved in the increased number of mature adipocytes as well as the neovascularization within the fat mass are still to be clearly defined. We have described a cell population, present in the native stroma-vascular fraction (SVF) of hAT, characterized as positive for the hematopoietic stem cell marker CD34 and negative for the endothelial cell marker CD31. The CD34+/CD31 cell population was the only cell population of the SVF under adipogenic culture conditions among the capillary endothelial cells (CECs), the macrophages, and the cells negative for the CD34, CD31, and CD14 markers able to express adipocyte-specific markers and metabolic lipolytic and lipogenic activities. Furthermore, under endothelial cell culture conditions, the CD34+/CD31 cells changed their morphology and organization and expressed endothelial cell (EC)-specific markers such as CD31 and von Willebrand factor [5]. In vivo, their injection led to an increase in blood flow and capillary density within the ischemic muscle in the athymic mouse model of hindlimb ischemia, whereas injection with native hAT-derived CD34 cells exerted no effects [5]. Other laboratories have reported similar proangiogenic abilities of the whole adipose tissue (AT) adherent SVF cells [6, 7]. The further characterization of the native CD34+/CD31 cells has shown that they did not express the classic mesenchymal stem cell markers (CD105 and Stro-1) and displayed different features from the adult hematopoietic stem cells since they did not form blood colony in hematopoietic assays [8]. Thus, the native CD34+/CD31 cell population of hAT can be considered a local pool of adipocyte as well as endothelial progenitor cells that might play a role in the formation of adipocytes but also of capillaries accompanying the development of the fat mass. Since the microenvironment, that is, soluble factors such as the stromal derived factor-1 (SDF-1) [9, [10]11], cellular, and extracellular matrix components have been shown to regulate the biology of hematopoietic and mesenchymal stem cells [12, [13]14], the present study was undertaken to characterize the effects of signals arising from hAT microenvironment on the chemotaxis and differentiation of the hAT progenitor cells. Our data delineate the concept that hAT-derived CECs induce the chemotaxis and support the endothelial differentiation potential of the hAT-derived progenitor cells via the SDF-1/CXCR-4 axis within hAT.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References

Isolation of the SVF from hAT

Human AT was obtained from individuals undergoing plastic surgery by lipoaspiration. The study was approved by the ethical committees of Toulouse and Frankfurt am Main hospital universities. Subcutaneous AT was obtained from a total of 38 individuals (mean body mass index: 26.07 ± 1.01 kg/m2). The AT was digested using collagenase (300 U/ml in phosphate-buffered saline [PBS], 2% bovine serum albumin [BSA], pH 7.4) for 30 minutes under constant shaking. Following removal of the floating mature adipocytes, the lower layer containing the SVF was centrifuged (300g, 10 minutes) and the pellet resuspended in erythrocyte lysis buffer (155 mmol/l NH4Cl; 5.7 mmol/l K2HPO4; 0.1 mmol/l EDTA, pH 7.3) for 10 minutes. After successive filtrations through 100-, 70-, and 40-μm sieves, the cells were resuspended in PBS/2% fetal calf serum (FCS).

Isolation of the CD34+/CD31 Progenitor Cells and the CD34+/CD31+ CECs from the SVF of hAT

CD34+ cells were isolated from the SVF using CD34-coupled magnetic microbeads as previously described [5]. Briefly, the SVF was incubated (room temperature, 15 minutes) with the StemCell Technologies (St. Katharinen, Germany, http://www.stemcell.com) positive selection cocktail. Following the addition of magnetic nanoparticles, cells were recovered by successive magnetic sorting steps. The CD34+ cells were suspended in PBS/0.1% BSA. The CD34+ population was depleted from the CD31+ cells using Dynal Biotech (Hamburg, Germany, http://www.dynalbiotech.com) CD31-coupled magnetic microbeads (50 μl/ml). After incubation (4°C, 20 minutes), the cell suspension containing the beads, suspended in 10 ml of PBS/0.1% BSA, was exposed to the magnet for 1 minute. The magnetic bead-free fraction, the CD34+/CD31 cells (hAT-derived progenitor cells), was collected, centrifuged (250g, 10 minutes), and suspended in the culture medium and the CD34+/CD31+ cell fraction (hAT-derived CECs) was removed from the magnet and suspended in endothelial cell culture medium. The CD34+/CD31 cell content of the selected fraction was 92.2% ± 1.2%.

Cell Culture

The hAT-derived CECs were cultured in Endothelial Cell Growth Medium MV (ECGM-MV) (PromoCell, Heidelberg, Germany, http://www.promocell.com) on human fibronectin coated culture dishes. Human AT-derived CECs were used until passage 2 [5, 15]. The human AT-derived progenitor cells were cultured in Endothelial Cell Basal Medium (ECBM, which contains no growth factors) (PromoCell) supplemented with 10% FCS. Progenitor cells were used until passage 1 [5]. Human umbilical vein endothelial cells (HUVECs) were isolated [16] and cultured until passage 8.

Preparation of Conditioned Media and Enzyme-Linked Immunosorbent Assay

Human AT-derived CECs, hAT-derived progenitor cells, hAT-resident macrophages (CD14+/CD31+) that were isolated as previously described [15], and HUVECs were incubated with an equal amount of basal ECBM supplemented with 0.1% BSA for 24 hours. Mature adipocytes (400,000 cells) were included in fibrin gels (1.5 mg fibrinogen/ml ECBM supplemented with 25 units/ml α-thrombin) and cultured in ECBM supplemented with 0.1% BSA for 24 hours. Control gels were prepared without adipocytes. The conditioned media were collected, centrifuged (1,000g, 10 minutes, 4°C), and stored at −70°C until use. Release of SDF-1 was measured in conditioned media by enzyme-linked immunosorbent assay according to the instructions of the manufacturer (RayBiotech Inc., Norcross, GA, http://www.raybiotech.com). Note that the SDF-1 levels were measured in conditioned media from P0, P1, and P2 human AT-derived CECs and no statistical significant differences were found (not shown).

Transmigration Assay

Transmigration assays of hAT-derived progenitor cells were performed using 8-μm pore HTS FluoroBlock inserts (Falcon; BD Biosciences, San Diego, http://www.bdbiosciences.com) coated with bovine gelatin (0.15 mg/cm2). The progenitor cells were loaded at 50,000 cells per insert (upper chamber) and conditioned media, SDF-1 in the presence or not of AMD3100 (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) or CXCR-4 neutralizing antibody (clone 12G5; R&D Systems Inc., Minneapolis, http://www.rndsystems.com), were added to the lower chamber. The cells were allowed to migrate into the lower chamber for 20–24 hours. After the assays, the inserts were fixed (4% paraformaldehyde) and the nuclei were stained with 4,6-diamidino-2-phenylindole. For blocking experiments, the progenitor cells were preincubated with the indicated inhibitors at 37°C for 1 hour. The cell transmigration was quantified by counting the number of nuclei using a computer-assisted microscope (Nikon, Düsseldorf, Germany, http://www.nikon.com) at three distinct positions.

RNA Extraction and Real-Time Quantitative Polymerase Chain Reaction Assay

Total RNAs were extracted from human mature adipocytes, hAT-CD34+/CD31 cells, hAT-macrophages, and hAT-CD34+/CD31+ CECs using the RNeasy kit (Qiagen, Hilden, Germany, http://www1.qiagen.com). RNA concentrations were determined using a fluorometric assay (RiboGreen). RNA (0.5 μg) was reverse-transcribed using the SuperScript III RNase H RT system (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) according to the manufacturer's instructions (random hexamers and deoxynucleoside-5′-triphosphates were from Life Technologies, Rockville, MD, http://www.lifetech.com). Reverse transcription was also performed without superscript enzyme on RNA samples to ensure the absence of contaminating genomic DNA. Primers for human SDF-1 (CXCL12), hypoxia inducible factor-1α (HIF-1α), and CXCR-4 were provided by Applied Biosystems (Assay-On-Demand: Hs00171022_m1, Hs00153153_m1, and Hs00607978_m1, respectively; Foster City, CA, http://www.appliedbiosystems.com). All amplification reactions were performed in duplicate from 20 ng cDNA using the Mx4000 Multiplex Quantitative PCR System (Stratagene, La Jolla, CA, http://www.stratagene.com) using the following conditions: 50°C for 2 minutes and 95°C for 10 minutes followed by 40 cycles at 95°C for 15 seconds and 60°C for 1 minute. Results were analyzed with Stratagene MX4000 software, and all values were normalized to the levels of the ribosomal RNAs.

Coculture Between hAT-Derived CECs and hAT-Derived CD34+/CD31 Progenitor Cells

Human AT-derived CECs plated at 60,000 cells per cm2 were cultured in ECGM-MV on human fibronectin-coated 48-well cell culture plates until confluence. Then, hAT-derived progenitor cells were labeled with PHK26 according to the instructions of the manufacturer (Sigma-Aldrich) and 2,000–3,000 cells per well were placed in each well in ECBM/2% SVF. After 10 days of coculture, the cocultured cells were fixed and stained with the indicated antibodies.

Three-Dimensional Coculture Model

Human AT-derived CECs plated at 60,000 cells per cm2 were cultured in ECGM-MV on human fibronectin-coated 48-well cell culture plates. Once confluent, the hAT-CECs were covered with 200 μl of unpolymerized BD Matrigel Matrix Growth Factor Reduced (BD Biosciences) and incubated at 37°C for 30–45 minutes. Human AT-derived progenitor cells were harvested with trypsin and labeled with PHK26 according to the instructions of the manufacturer. The progenitor cells were resuspended in ECBM/0.1% BSA and 2,000–3,000 cells per well were loaded onto the matrigel-coated wells (200 μl of cell suspension per well). After 24 hours of “coculture,” progenitor cell tube formation (red fluorescent network) was assessed with a computer-assisted microscope (Nikon). Microphotographs of the center of each well were taken, and tube formation was quantified by measuring the total tube length using Lucia image software (Nikon, Tokyo, http://www.nikon.com). Tube formation of progenitor cells alone was used as a control. For blocking experiments, the progenitor cells were preincubated with the indicated inhibitors at 37°C for 1 hour.

Immunocytochemistry

Cells were fixed with 4% paraformaldehyde and immunostained with anti-human CXCR-4 monoclonal antibody (Abcam, Cambridge, U.K., http://www.abcam.com) (1:50) or platelet endothelial cell adhesion molecule-1 (CD31) monoclonal antibody (Dako, Glostrup, Denmark, http://www.dako.com) (1:10) followed by staining with fluorescently labeled secondary antibody (1:200) (Invitrogen).

Statistical Analysis

Data are expressed as mean ± SEM from at least three independent experiments. Statistical analysis was performed by Student's t test or one-way analysis of variance followed by a Bonferroni post hoc test when appropriate. Differences were considered significant when p < .05.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References

AT-Derived CECs Induce the Chemotaxis of the CD34+/CD31 Cells

The effect of hAT-CECs on the migration of hAT-derived CD34+/CD31 cells was assessed in modified Boyden chamber assays consisting of two cell chambers separated by a polycarbonate filter (8-μm pores). The lower chamber was filled up with control media or 24 hour-conditioned media from confluent hAT-CECs, mature adipocytes, or confluent HUVECs, and the cell suspension of hAT-derived CD34+/CD31 cells was placed in the upper chamber. Cells that migrated across the filter and attached to the under part of the filter after 24 hours were counted after nuclei staining. As depicted in Figure 1A, conditioned media from mature adipocytes or from HUVECs did not induce any cell migration. Conversely, conditioned media from hAT-CECs led to the robust migration of the CD34+/CD31 cells (5.7 ± 0.7-fold increase, n = 21, p < .001). Human AT-CEC conditioned media failed to induce the migration of hAT-derived CD34+/CD31 cells after protein denaturation of the conditioned media (Fig. 1B). Moreover, the stimulatory effect of conditioned media derived from hAT-CECs was lost when the conditioned media were applied in the upper chamber together with the CD34+/CD31 cells. Taken together, the results showed that secreted proteins from hAT-CECs stimulated the directed chemotactic migration of the hAT-derived CD34+/CD31 (Fig. 1B). In addition, preincubation of hAT-derived progenitor cells with increasing concentrations of pertussis toxin (PTX) (200 and 400 ng/ml) led to a concentration-dependent inhibition of the hAT-CEC-mediated migration (76% ± 9% of inhibition for 400 ng/ml of PTX, p < .001, n = 4) (Fig. 1B).

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Figure Figure 1.. Human adipose tissue (AT)-derived CECs induce the chemotaxis of hAT-derived CD34+/CD31 progenitor cells. (A): Transwell migration of human AT-derived progenitor cells in the presence of control medium (Control) or conditioned media from adipocytes (n = 14), hAT-CECs (n = 21), and HUVECs (n = 3). * p < .001 when compared with control migration. (B): Transwell migration of hAT-derived progenitor cells in response to hAT-CEC conditioned medium in the lower chamber (hAT-CEC conditioned medium), in the upper chamber (upper chamber hAT-CECs) (n = 3), after 1-hour preincubation with increasing concentrations of pertussis toxin (n = 4) or after protein denaturated hAT-CEC-conditioned medium (heated AT-CECs) (n = 4). * p < .01, ** p < .001 when compared with migration induced by hAT-CEC-conditioned medium. Abbreviations: CECs, capillary endothelial cells; hAT, human adipose tissue; HUVECS, human umbilical vein endothelial cells; PTX, pertussis toxin.

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The CXCR-4/SDF-1 Axis Is Involved in the Chemotaxis of the CD34+/CD31 Cells Mediated by AT-Derived CECs

To further identify the nature of the factor(s) involved in such an effect, the CD34+/CD31 cells were preincubated with a CXCR-4 antagonist (AMD3100, 10 μg/ml) or with neutralizing anti-CXCR-4 antibody (10 μg/ml), and the effect of hAT-CEC conditioned media was then assessed. As shown in Figure 2, the hAT-CEC-mediated chemotaxis was suppressed when the progenitor cells were incubated with AMD3100 or with anti-CXCR-4 neutralizing antibody (77% ± 11% and 96% ± 4% of inhibition, respectively, p < .01, n = 6). Moreover, as depicted in Figure 3A, increasing concentrations of recombinant SDF-1α or -1β, ligands for the CXCR-4, induced a concentration-dependent chemotaxis of hAT-derived progenitor cells that was significantly inhibited when the progenitor cells were preincubated with AMD3100 (58% ± 7% of inhibition, p < .01, n = 3) or neutralizing anti-CXCR-4 antibody (72% ± 3% of inhibition, p < .01, n = 3) (Fig. 3B). Of note, the chemotaxis of the CD34+/CD31 cells toward granulocyte monocyte colony stimulating factor (20–100 ng/ml), stem cell factor (20–100 ng/ml), monocyte chemoattractant protein 1 (1–20 ng/ml), and interleukin 8 (20 ng/ml) was also evaluated, and none of the chemokines induced a statistically significant migration (data not shown).

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Figure Figure 2.. The stromal derived factor-1/CXCR-4 axis is involved in the hAT-derived progenitor cell chemotaxis toward hAT-CEC conditioned media. Transwell migration of hAT-derived progenitor cells in the presence of hAT-derived CEC-conditioned media after 1 hour of preincubation with 10 μg/ml of the CXCR-4 antagonist AMD3100 (+AMD3100) (n = 6) or with 10 μg/ml of an anti-CXCR-4 neutralizing antibody (+Anti-CXCR-4) (n = 6). * p < .01 when compared with migration induced by hAT-CEC-conditioned media. Abbreviations: CECs, capillary endothelial cells; hAT, human adipose tissue.

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Figure Figure 3.. Functionality of the CXCR-4/SDF-1 axis in human adipose tissue. (A): Human adipose tissue (AT)-derived progenitor cells were exposed to increasing concentrations of SDF-1α or SDF-1β (10–100 ng/ml), and transwell migration was evaluated after 24 hours (n = 3–8); * p < .01 and ** p < .001 compared with control. (B): Human AT-derived progenitor cells were preincubated (1 hour) with 10 μg/ml CXCR-4 antagonist AMD3100 (n = 3) or with 10 μg/ml anti-CXCR-4 neutralizing antibody (n = 3). Transwell migration toward 100 ng/ml SDF-1α was then evaluated after 24 hours. $ p < .001 compared with control, * p < .01 and ** p < .001 compared with SDF-1α induced migration. Abbreviation: SDF-1, stromal derived factor-1.

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CXCR-4 Is Expressed on CD34+/CD31 Cells and SDF-1 Is Produced by hAT-Derived CECs

The expression of CXCR-4 in hAT-derived CD34+/CD31 cells was then investigated by real-time polymerase chain reaction (PCR) analysis. The results showed that transcripts for CXCR-4 were present in hAT-derived CD34+/CD31 cells (Fig. 4B). Moreover, as presented in Figure 4A, the CXCR-4 protein expression was detected by immunocytochemistry on hAT-CD34+/CD31 cells. Additionally, there was a positive correlation between the level of transcripts encoding for CXCR-4 in hAT progenitor cells and the one of HIF-1α in the corresponding hAT-CECs (Fig. 4B, p = .04, n = 14). In parallel, SDF-1 mRNA expression was assessed in hAT-derived CECs and compared with the one of HUVECs and other cells from the SVF such as hAT-macrophages. As depicted in Figure 4C, the SDF-1 mRNA was mainly expressed by hAT-derived CECs and was below Ct values of 40 in HUVECs. Consistent with the mRNA expression, SDF-1 release was significantly higher in hAT-derived CECs as compared with other cell fractions present in AT-derived SVF such as macrophages (412.2 ± 92.9 pg/ml vs. 34.4 ± 18.1 pg/ml, respectively, p < .001, n = 3–9) (Fig. 4D). The production of SDF-1 by HUVECs was 42 times lower than the one of hAT-CECs (9.84 ± 3.81 pg/ml, n = 4).

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Figure Figure 4.. Expression of SDF-1 and CXCR-4 in hAT. (A): Upper panel: hAT-derived CD34+/CD31 progenitor cells were fixed and stained with antibodies directed against CXCR-4 receptor followed by fluorescein isothiocyanate (FITC)-conjugated secondary antibodies. Lower panel: hAT-CD34+/CD31 cells were stained with FITC-conjugated secondary antibodies. Representative fluorescence microscopy analysis is shown from n = 3. (B): Correlation between mRNA levels of CXCR-4 in hAT-progenitor cells and HIF-1 levels in hAT-CECs quantified by real-time polymerase chain reaction (PCR) experiments (p = .04, n = 14). (C): SDF-1 mRNA expression in hAT-macrophages (n = 3), HUVECs (n = 4), and hAT-CECs (n = 6) quantified by real-time PCR experiments. (D): Enzyme-linked immunosorbent assay of 24-hour conditioned media from hAT-macrophages (n = 3), HUVECs (n = 4), and hAT-CECs (n = 9). * p < .05 versus macrophage SDF-1 secretion. Abbreviations: A.U., arbitrary units; CECs, capillary endothelial cells; hAT, human adipose tissue; HIF-1, hypoxia inducible factor-1; HUVECS, human umbilical vein endothelial cells; SDF-1, stromal derived factor-1.

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AT-Derived CECs Support the Differentiation of AT-Derived Progenitor Cells Toward an Endothelial Cell Phenotype

Coculture experiments of hAT-derived CECs and CD34+/CD31 cells were performed. Human AT-derived CD34+/CD31 cells were labeled with PKH26 and seeded onto confluent hAT-derived CECs (Fig. 5A). After 10 days of coculture, CD31 expression in the formal CD31 negative AT-derived progenitor cells (Fig. 5B) was analyzed. hAT-derived CECs formed a dense CD31 positive network into which PKH26 positive AT-derived progenitor cells incorporated and expressed the CD31 marker (Fig. 5C). To further analyze the effect of the hAT-derived CECs on the differentiation of the CD34+/CD31 cells, a three-dimensional coculture model was developed. PKH26 labeled CD34+/CD31 cells were seeded on the top of a growth factor-reduced matrigel under which confluent hAT-derived CECs were present or not. As shown in Figure 6A, CD34+/CD31 cells alone did neither migrate nor form capillary-like structures. Interestingly, the presence of hAT-derived ECs under the matrigel dramatically promoted the organization of hAT-CD34+/CD31 cells into branched structures and pseudotubes (360% ± 48% of the total length obtained with hAT-CD34+/CD31 cells alone, p < .01, n = 7) (Fig. 6B). Given the chemotactic effect mediated by the CXCR-4/SDF-1 axis, we studied the involvement of this system in the hAT-CEC-dependent tube formation by using a neutralizing anti-CXCR-4 antibody, the CXCR-4 antagonist AMD3100, or pertussis toxin (not shown). As depicted in Figure 6C and 6D, the tube formation induced by hAT-derived CECs was significantly inhibited in a concentration-dependent manner by the inhibitors (24% ± 12% and 67% ± 5% of inhibition with 10 μg/ml and 100 μg/ml AMD3100, respectively; 41% ± 15% and 93% ± 2% of inhibition for 10 μg/ml and 20 μg/ml neutralizing anti-CXCR-4 antibody, respectively; 85% ± 0.5% of inhibition for 200 ng/ml pertussis toxin, n = 4–7).

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Figure Figure 5.. Human AT-derived progenitor cells incorporate into the network formed by hAT-CECs and express platelet endothelial cell adhesion molecule-1 (PECAM-1). (A): Confluent hAT-CECs were fixed and stained with antibodies directed against PECAM-1 followed by fluorescein isothiocyanate (FITC)-conjugated secondary antibodies. (B): Confluent PKH26 hAT-CD34+/CD31− progenitor cells were fixed and stained with antibodies against PECAM-1 followed by FITC-conjugated secondary antibodies. (C): Confluent hAT-CECs were cultured in the presence of PKH26-labeled hAT-derived progenitor cells for 10 days of coculture in endothelial basal medium/2% fetal calf serum. Cells were fixed and stained with antibodies directed against PECAM-1 followed by FITC-conjugated secondary antibodies. Representative fluorescence microscopy analysis is shown from n = 3 independent experiments. Abbreviations: CECs, capillary endothelial cells; DAPI, 4,6-diamidino-2-phenylindole; hAT, human adipose tissue.

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Figure Figure 6.. hAT-CECs induce the formation of capillary-like structures by hAT-derived progenitor cells via the stromal derived factor-1/CXCR-4 axis. hAT-CECs were cultured until confluence. Once confluent, hAT-CECs were covered with matrigel and PKH26 labeled hAT-derived progenitor cells were seeded onto the matrigel. After 24 hours, tube length was measured. (A): Representative pictures of hAT-derived progenitor cells alone (phase microscopy and fluorescent microscopy analyses, inset picture). (B): Representative picture of hAT-derived progenitor cells cocultured with hAT-CECs (phase microscopy and fluorescent microscopy analyses, inset picture). (C, D): Representative picture of 1-hour pretreated hAT-progenitor cells with AMD3100 (100 μg/ml) (C) or neutralizing anti-CXCR-4 (20 μg/ml) antibody (D) that were cocultured with hAT-CECs (phase microscopy and fluorescent microscopy analyses, inset picture). (E): Total length of the network formed by PKH26 labeled hAT-derived progenitor cells pretreated or not with increasing concentrations of AMD3100 or a neutralizing CXCR-4 antibody. * p < .05, ** p < .01, *** p < .001 when compared with the network formed by adipose tissue (AT)-derived progenitor cells in the presence of AT-CECs (n = 4–7). Abbreviations: 3D, three-dimensional; CECs, capillary endothelial cells; hAT, human adipose tissue.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References

Although reports show that adipogenesis and angiogenesis are tightly correlated during the growth of fat mass [3, 17, [18], [19]20], little is known about the mechanisms that regulate the extension of its vasculature. In the present study we report that hAT-derived CECs induce the chemotaxis of hAT-derived progenitor cells. Moreover, we demonstrate that the presence of hAT-derived CECs promotes the differentiation and organization of hAT-derived hAT-CD34+/CD31 progenitor cells into capillary-like structures via a SDF-1/CXCR-4-dependent pathway.

In the last years, several reports have contributed to the evidence that the SVF cells are phenotypically heterogeneous and can express under specific culture conditions adipogenic, osteogenic, chondrogenic, and myogenic lineage markers but also endothelial, epithelial, and neural markers [5, 8, 21, [22]23]. We have previously shown that native immunoselected CD34+/CD31 cells present in the hAT-derived SVF display angiogenic and adipogenic potentials. Indeed, this cell population is able to differentiate into adipocyte-like cells [8] and into endothelial-like cells in vitro and in vivo to promote the neovascularization of ischemic muscle in mice [5]. Similar results were also described using adherent hAT-SVF [6, 7]. The mechanisms underlying such proangiogenic effects are still to be defined. Although the hAT-derived progenitor cells were shown to integrate into vessels in vivo [5, 6], they could also contribute to neovascularization through the production of proangiogenic factors [7, 24]. Nevertheless, such a population of progenitor cells may participate locally to the extension of the blood capillary network known to be associated with fat mass development. The present study was undertaken to analyze the potential crosstalk within the hAT between hAT-CD34+/CD31 progenitor cells and CECs. Indeed, the EC barrier has already been shown to interact with progenitor/stem cells [25, [26]27]. Human AT-derived CECs specifically stimulated the migration of hAT-derived CD34+/CD31 progenitor cells through the release of soluble proteins. Human AT-derived CECs induced a chemotactic response since no random motility was observed when the cells were incubated in the same compartment with endothelial cell-derived conditioned media. Moreover, since hAT-derived CD34+/CD31 progenitor cell chemotaxis was inhibited in the presence of pertussis toxin, which specifically prevents activation of Gi proteins coupled to chemokine receptors, the involvement of a chemokine ligand/receptor interaction was strongly suggested. Finally, when CXCR-4 from hAT-CD34+/CD31 progenitor cells was neutralized by antibodies [28] or by the CXCR-4 antagonist (AMD3100) [29], hAT-derived CD34+/CD31 progenitor cells failed to migrate in response to hAT-derived CEC conditioned medium. Since real-time PCR and immunocytochemistry analyses showed the expression of CXCR-4 in native hAT-derived CD34+/CD31 progenitor cells, the present results demonstrate that activation of CXCR-4 of the hAT-derived CD34+/CD31 progenitor cells is involved in the hAT-derived CEC-dependent chemotaxis. In agreement, earlier work reported immunoreactivity to CXCR-4 in hAT [30], and a recent publication showed that CXCR-4 overexpression in hAT-stromal cells regulated cell motility [31]. The sole ligand of CXCR-4, described so far, is the CXCL12 chemokine also known as SDF-1 [32, [33]34]. SDF-1, initially described as a product of bone marrow stromal cells [35], is expressed by several tissues including dendritic cells, endothelial cells and pericytes from normal skin [36], osteoblasts, and ECs from the bone marrow [37, [38]39] and astrocytes and neurons from the brain [40]. SDF-1 exists as two isoforms, α and β, originating from alternative splicing of the CXCL12 gene [41, [42]43]. Human AT-CECs expressed and released more SDF-1 than the other cell types present in hAT. Moreover, SDF-1 was detected in very low amounts in conditioned media from HUVECs that did not stimulate the CD34+/CD31 progenitor cell chemotaxis. Human AT-progenitor cells exhibited a concentration-dependent chemotaxis toward SDF-1α and SDF-1β that was inhibited in the presence of CXCR-4 antagonist or neutralizing antibodies. Taken together, the present findings suggest that SDF-1 released by hAT-CECs plays a major role in the CXCR-4-mediated chemotaxis of hAT-progenitor cells, although further experiments are needed to clearly rule out the involvement of other endothelial-derived factors. The couple SDF-1/CXCR-4 plays a pivotal role in multiple checkpoints of stem/progenitor cell biology in the bone marrow compartment including survival, proliferation, mobilization, and homing [10, 44, 45]. In a two-dimensional coculture system, hAT-derived progenitor cells were shown to incorporate into the network formed by hAT-CECs and to express the EC marker CD31, suggesting that hAT-CECs may, in addition to their effect on the migration, regulate the differentiation of hAT-CD34+/CD31. To better define the role of paracrine factors, three-dimensional coculture experiments were performed. The results clearly showed that hAT-derived progenitor cells migrate and assemble into capillary-like structures only in the presence of hAT-CECs. Moreover, when CXCR-4 either was antagonized or neutralized, hAT-progenitor cells failed to appropriately migrate and assemble into tubular structures on matrigel substrate.

Taken together, the present results show that hAT-derived CECs, through their production of paracrine factors such as SDF-1, modulate the migration but also the differentiation and organization of the hAT-derived CD34+/CD31 progenitor cells through the activation of CXCR-4. CXCR-4 mRNA levels are known to be upregulated by hypoxia through HIF-1 activation via the hypoxia responsive element in the 5′ region of the CXCR4 gene [46, 47]. In the murine AT, hypoxic areas have been shown to be associated with obesity [48]. Interestingly, the mRNA levels of CXCR-4 from the CD34+/CD31 were found to be positively correlated to HIF-1α transcripts of the CECs. A recent publication has reported that the recruitment of CXCR-4 positive progenitor cells to regenerating foci or tissues was mediated by hypoxic gradients via HIF-1 induced expression of CXCR-4/SDF-1 axis [28]. Moreover, SDF-1 expression in ischemic tissue has been shown to primarily localize in ECs [36]. It is thus tempting to speculate that hypoxia within the hAT may promote the migration of resident hAT-derived CD34+/CD31 progenitor cells at active sites of neovascularization in the fat mass.

All together, the present results show that the migration and differentiation of hAT-derived progenitor cells are modulated by factors originating from hAT-derived CECs. The SDF-1/CXCR-4 axis appears to play a key role. Such an effect may be involved in the recruitment of hAT-derived progenitor cells to foci where SDF-1 is highly expressed such as neovascularization sites to promote the extension of the capillary network that occurs during the growth of the fat mass.

Disclosure of Potential Conflicts of Interest

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References

The authors indicate no potential conflicts of interest.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References

We are grateful to Valérie Wegner and Pauline Decaunes for their excellent technical assistance. We also thank Dr. Marie Sanson for her special help with HUVECs. This work was supported by grants from AVENIR INSERM, Alexander von Humboldt Foundation, and Sofja Kovalevskaja Price (Humboldt Foundation and the German Federal Ministry of Education and Research).

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosure of Potential Conflicts of Interest
  8. Acknowledgements
  9. References