During obesity, chronic inflammation of human white adipose tissue (WAT) is associated with metabolic and vascular alterations. Endothelial cells from visceral WAT (VAT-ECs) exhibit a proinflammatory and senescent phenotype and could alter adipocyte functions. We aimed to determine the contribution of VAT-ECs to adipocyte dysfunction related to inflammation and to rescue these alterations by anti-inflammatory strategies. We developed an original three-dimensional setting allowing maintenance of unilocular adipocyte functions. Coculture experiments demonstrated that VAT-ECs provoked a decrease in the lipolytic activity, adipokine secretion, and insulin sensitivity of adipocytes from obese subjects, as well as an increased production of several inflammatory molecules. Interleukin (IL)-6 and IL-1β were identified as potential actors in these adipocyte alterations. The inflammatory burst was not observed in cocultured cells from lean subjects. Interestingly, pericytes, in functional interactions with ECs, exhibited a proinflammatory phenotype with diminished angiopoietin-1 (Ang-1) secretion in WAT from obese subjects. Using the anti-inflammatory Ang-1, we corrected some deleterious effects of WAT-ECs on adipocytes, improving lipolytic activity and insulin sensitivity and reducing the secretion of proinflammatory molecules. In conclusion, we identified a negative impact of VAT-ECs on adipocyte functions during human obesity. Therapeutic options targeting EC inflammation could prevent adipocyte alterations that contribute to obesity comorbidities.

Vascularization is critical for white adipose tissue (WAT) development and homeostasis by providing oxygen and nutrients to this key organ (1). In close interactions with adipocytes, the capillary network contributes to the export of adipokines as leptin and adiponectin to targeted tissues. Endothelial cells (ECs) and pericytes are the main cell components of blood vessels. These mural cells, through direct interactions with ECs, promote the maturation and stabilization of blood vessels (2).

Obesity is characterized by a chronic and auto-inflammation of hypertrophied WAT. The inflamed WAT is the site of cellular remodeling, including adipocyte hypertrophy and altered immune cell and EC phenotypes. In turn, this remodeling leads to increased secretion of numerous inflammatory mediators, including interleukin (IL)-6, chemokine ligand (CCL)-2, IL-8, and CCL5, some of which are implicated in the development of insulin resistance (3).

The accumulation of visceral fat depots (visceral adispose tissue [VAT]) is associated with metabolic syndrome and adverse obesity comorbidities (4). VAT is more inflammatory and vascularized than subcutaneous depots (subcutaneous adipose tissue [SAT]) (5,6). ECs from VAT (VAT-ECs) play a key role in the inflammatory process, exhibiting a senescent phenotype and expressing inflammatory and angiogenesis-related molecules (5). Though adipocytes have been described to participate in the altered phenotype of ECs during obesity (5,79), the retroactive influence of ECs on adipocyte functions remains poorly described. Most studies have highlighted the requirement for ECs in adipose tissue growth playing a role in the proliferation and differentiation of adipocyte precursors (10,11). Thus, evaluating the impact of ECs on the metabolism of unilocular adipocytes is of particular importance in pathophysiological situations such as obesity.

Here, we focus on direct interactions between human adipocytes and ECs isolated from VAT of obese subjects by coculturing these cells in a three-dimensional (3D) setting. This is the first report showing an impact of VAT-ECs on adipocyte functions, identifying IL-6 and IL-1β as key factors in these alterations. Then, we propose an experimental strategy to reverse these effects by improving EC inflammation with Ang-1 treatment, since this molecule, constitutively produced by pericytes, is known to prevent EC inflammation (12).

Antibodies used in the study are listed in Supplementary Table 1. The human recombinant proteins IL-6 and IL-1β were obtained from Peprotech (Rocky Hill, NJ).

Preparation of Different Cell Types From Human SAT and VAT

Adipocytes, ECs, and pericytes were isolated from WAT of 29 lean (SAT) and 87 obese subjects (SAT and VAT, omental depot) whose clinical parameters are described in Supplementary Table 2. SAT from lean subjects were obtained after elective surgery. All clinical investigations were performed according to the Declaration of Helsinki and approved by the ethics committee of Hôtel-Dieu Hospital (Paris, France). In an accepted protocol related to the pathophysiology of low-grade inflammation in obesity (Assistance Publique/Hôpitaux de Paris, Clinical Research Contract), obese subjects (mean BMI >30 kg/m2) were candidates for gastric surgery programs as previously described (6).

WAT biopsies were dissociated by collagenase treatment isolating unilocular adipocytes from the stromavascular fraction (SVF). The adipocytes were then washed three times in 10% sucrose solution. The contamination of adipocyte preparation by preadipocytes was <5% as assessed by the expression of Pref-1/Dlk1, a preadipocyte marker. WAT-ECs were isolated from the SVF of the same WAT biopsies as previously described (13) using immunoselection cocktails (EasySep Do-It-Yourself Selection kit; Stemcell Technologies, Grenoble, France) for the endothelial marker CD31 (M0823 DAKO). The endothelial phenotype of CD31-positive cells was verified by immunofluorescence analysis of an endothelial-specific marker, von Willebrand Factor (vWF) (Supplementary Fig. 1A), and tube formation assay on Matrigel-GF reduced (BD Biosciences, Franklin Lakes, NJ) (Supplementary Fig. 1B). The purity of the WAT-ECs was ~90% with an absence of macrophage contamination, as verified by measuring CD14 expression. To isolate pericytes, the CD31-negative cell fraction was incubated with NG2-positive selection cocktail (ab83508; Abcam). The phenotype of WAT pericytes was verified by comparing the expression of CD146 and RGS5 mRNA, two pericyte markers (14,15), among other cell types (WAT-ECs, preadipocytes, and skin fibroblasts [Supplementary Fig. 2A and B]) and by immunofluorescence analysis of the expression of pericyte markers chondroitin sulfate proteoglycan (NG)-2 and α smooth muscle actin (αSMA). NG2+ cells were distinguished from CD31+ cells in the WAT SVF (Supplementary Fig. 2C). The proportion of pericytes (CD31NG2+) to ECs (CD31+) in SAT of lean and from SAT/VAT of obese subjects was evaluated by counting the corresponding cells.

Cocultures of Adipocytes and ECs From Human WAT in a 3D Setting

Adipocytes and ECs both isolated from VAT of obese subjects or SAT of lean subjects were cocultured in the 3D setting. The difficulty getting access to VAT from lean subjects prevented us from performing coculture experiments. After sonication for 30 min to decrease viscosity, the hydrogel (Puramatrix; BD Biosciences) was diluted in 20% sucrose solution according to the manufacturer’s recommendations. Adipocytes were embedded in this gel at a concentration of 1 × 104 cells/100 µL gel preparation into 96-well plates containing 150 µL endothelial cell basal medium (ECBM) (PromoCell, Heidelberg, Germany), 1% BSA, 1% antibiotics, and human insulin (50 nmol/L). The CD31+ cells isolated from the same individual were then incorporated into 96-well plates containing 3D hydrogel/adipocytes in the ratio of 2 × 104 cells for 1 × 104 adipocytes, which is representative of the proportion of ECs in the WAT vasculature (16). (See detailed protocol in Supplementary Fig. 3.) The culture medium was changed every other day and kept at −80°C for experimental measurements. Additional details can be found in the international patent (PCT/IB2011/052241) (17). The VAT-ECs were prone to migrating into the hydrogel in response to VAT adipocytes and then interacting directly. ECs also maintained their inflammatory profile in the 3D setting (Supplementary Fig. 4A–C).

Immunofluorescence Analysis and Confocal Microscopy

A portion of VAT biopsies and VAT adipocytes/ECs/pericytes was fixed in 4% paraformaldehyde and processed for immunofluorescence analysis. Samples were incubated with the appropriate primary antibody and then the corresponding anti-IgG. The samples were examined with an OlympusBX41 fluorescence microscope (Olympus, Lake Success, NY) or Zeiss 710 confocal laser-scanning microscope (Carl Zeiss, Thornwood, NY).

Senescence of SAT and VAT-ECs

ECs isolated from SAT of lean and SAT/VAT of obese subjects were assessed for senescence-associated β-galactosidase activity (SA-β-gal) according to the company’s instructions (Sigma, St. Louis, MO) and as previously described (18). Five phase-contrast images were recorded on a digital camera (Olympus, Tokyo, Japan). The number of SA-β-gal–positive cells was normalized to the total cell number counterstained with hematoxylin-eosin.

Lipolysis Assay

The adipocytes/hydrogel were incubated in Krebs-Ringer bicarbonate buffer supplemented with 3% BSA for 4 h at 37°C. The release of glycerol (R-Biopharm, Marshall, MI) and nonesterified fatty acids (Randox Laboratories, Antrim, U.K.) was evaluated after stimulation with forskolin (10 µmol/L), dibutyryl-cAMP (DcAMP) (0.5 mmol/L), and isoproterenol (1 µmol/L) (Sigma).

Adipocyte Secretory Function

Conditioned media obtained from different experimental conditions of adipocytes/WAT-ECs cocultured for 3 days in the 3D setting were analyzed using human cytokine/chemokine Panel I 39 plex and human from Millipore according to the manufacturer’s instructions. Multianalyte profiling was performed on the Luminex-200 system and Xmap Platform (Luminex, Austin, TX). Acquired fluorescence data were analyzed by Xponent software, version 3, using standard curves obtained with serial dilutions of standard cytokine mixtures (as previously described [18]). The detection threshold was fixed as >2 pg/mL. Heat maps were created using MEV MultiExperiment Viewer software (version 4.8; MeV, Boston, MA). Colorimetric ELISA kits were used to determine the concentrations of adipokines (leptin and adiponectin; Duoset, R&D Systems, Minneapolis, MN) and inflammatory factors (IL-6, G-CSF, and CCL2, Duoset, R&D Systems, and IL-8 and CCL5, Peprotech) in the medium from 3D adipocyte/EC cocultures according to the provider’s instructions.

Adipocyte Insulin Sensitivity

The adipocyte/hydrogel was insulin deprived overnight. Next, the cells were stimulated with 10 nmol/L insulin for 15 min at 37°C. Insulin sensitivity was evaluated by the level of Ser473 phosphorylation of Akt (pS473 Akt) normalized to total Akt.

Western Blot Analysis

Cell extracts were prepared as previously described (19). Protein samples were separated by SDS-PAGE and blotted on nitrocellulose transfer membranes (GE Healthcare, Little Chalfont, U.K.). The membranes were probed overnight at 4°C with the corresponding primary antibodies. Specific signals were detected using the ECL detection solution (GE Healthcare) and immediately exposed to X-ray films. The signals were quantified by densitometry.

Preparation of Conditioned Media From VAT-ECs and Experiments on Adipocytes

VAT-ECs (8 × 104 cells) from obese subjects were cultured in 1 mL ECBM with 1% BSA for 7 days at 37°C (medium was changed twice). After washing, VAT-ECs were placed in 800 µL ECBM with 1% BSA for 24 h at 37°C, after which conditioned medium was collected and stored at −80°C until needed. VAT adipocytes cultured in hydrogel were treated for 5 days with conditioned medium prepared from VAT-ECs. After washing, adipocytes were placed in fresh medium for 48 h at 37°C, which was collected and stored at −80°C.

Lactate Dehydrogenase Cytotoxicity Assay

Cytotoxicity was evaluated by measuring the activity of lactate dehydrogenase (LDH) released from damaged cells according to the manufacturer’s instructions (LDH-Cytotoxicity Assay kit; BioVision Research Products, Mountain View, CA).

Neutralizing Antibodies and Recombinant Proteins Experiments

Adipocytes, ECs, and cocultured cells from VAT of obese subjects were treated with IgG1 (3 µg/mL) or with IL-6 (2.5 µg/mL) and IL-1β (0.5 µg/mL) neutralizing antibodies or with tumor necrosis factor (TNF)-α (0.5 µg/mL) neutralizing antibody during 3 days with every day changes. Recombinant IL-6 (10 ng/mL) and IL-1β (1 ng/mL) were incubated with adipocytes, ECs, and cocultured cells from SAT of lean subjects and cultured in the hydrogel during 3 days with everyday changes.

Ang-1 Treatment of Visceral ECs

Isolated adipocytes (1 × 104 cells) and ECs (2 × 104 cells) from VAT were introduced in separate hydrogels (100 µL in 96-well plates) in control culture medium. ECs were incubated in the presence or absence of Ang-1 (100 ng/mL; R&D Systems) for 24 h at 37°C. Adipocytes and Ang-1–treated ECs were cocultured by associating the two hydrogels in control medium. The metabolic and secretory functions of adipocytes were then studied. (See detailed protocol in Supplementary Fig. 5.)

mRNA Preparation and Real-Time PCR

RNA extraction, reverse transcription, and real-time PCR were performed as previously described (20). The primers that were used are listed in Supplementary Table 3. All values were normalized with regard to 18S expression.

Statistical Analysis

The experiments were performed at least five times using adipocytes and WAT-ECs from different obese and lean subjects. Statistical analyses were performed using GraphPad software (San Diego, CA). Values are expressed as means ± SEM. Comparisons between two conditions were analyzed using the Wilcoxon nonparametric paired test (adipocytes and adipocyte/EC cultures) and the Mann-Whitney nonparametric test (lean vs. obese pericytes). Comparisons among more than two groups were performed using one-way ANOVA followed by post hoc tests. Spearman coefficients were calculated to examine correlations. Differences were considered significant when P < 0.05.

ECs From WAT of Obese Subjects Display a Senescent/Inflammatory Phenotype Compared With Those From Lean Subjects

Senescence level of WAT-ECs was evaluated in lean (SAT) and obese (SAT and VAT) subjects. Although no significant difference among depots were observed in WAT of obese subjects, an increased β-gal activity was detected in VAT-ECs from obese subjects compared with SAT-ECs of lean subjects (Fig. 1A). Moreover, VAT-ECs exhibit an increased secretion of several cytokines and chemokines compared with lean individuals (Fig. 1B). Based on these observations, we attempted to identify the potential impact of ECs on the metabolic and secretory properties of adipocytes in VAT from obese subjects. We cocultured adipocytes and ECs isolated from the VAT of the same individual to reproduce this inflammatory environment. Cocultures experiments with noninflammatory SAT-ECs were also performed as a control.

Figure 1

EC phenotype from human WAT during obesity. A: Senescence assessed by SA-β-gal activity was compared in WAT-ECs between lean (SAT-ECs lean) and obese (SAT/VAT-ECs obese). Representative microphotographs (magnification ×10) and graph quantifications are presented. Scale bar = 50 µm. Data are means ± SEM of 5 separate experiments, each performed with different preparations of WAT-ECs. *P < 0.05 VAT-ECs obese vs. SAT-ECs lean. ns, nonsignificant. B: IL-8, IL-6, CCL2, CCL5, and G-CSF secretions were quantified in WAT-ECs by ELISA tests. Inflammation of WAT-ECs was compared between lean and obese subjects after 3 days of culture in the 3D setting. Data are presented as means ± SEM of five independent experiments. *P < 0.05 VAT-ECs obese vs. SAT-ECs lean. ns, nonsignificant.

Figure 1

EC phenotype from human WAT during obesity. A: Senescence assessed by SA-β-gal activity was compared in WAT-ECs between lean (SAT-ECs lean) and obese (SAT/VAT-ECs obese). Representative microphotographs (magnification ×10) and graph quantifications are presented. Scale bar = 50 µm. Data are means ± SEM of 5 separate experiments, each performed with different preparations of WAT-ECs. *P < 0.05 VAT-ECs obese vs. SAT-ECs lean. ns, nonsignificant. B: IL-8, IL-6, CCL2, CCL5, and G-CSF secretions were quantified in WAT-ECs by ELISA tests. Inflammation of WAT-ECs was compared between lean and obese subjects after 3 days of culture in the 3D setting. Data are presented as means ± SEM of five independent experiments. *P < 0.05 VAT-ECs obese vs. SAT-ECs lean. ns, nonsignificant.

Close modal

To have a longer follow-up of biological activities, we previously developed an original 3D culture of human adipocytes using a self-assembling peptidic hydrogel device (17) (Supplementary Fig. 6 [international patent PCT/IB2011/052241]).

VAT-ECs in the 3D Setting Alter Adipocyte Lipolysis, Adipokine Secretion, and Insulin Sensitivity

We investigated the influence of VAT-ECs from obese subjects on adipocyte secretions in this 3D setting. From the third day of coculture, the VAT-ECs provoked a decreased secretion of the adipokines leptin (−50%, n = 6, P < 0.01) and adiponectin (−20%, n = 6, P < 0.001) (Fig. 2A). Lipolytic activity was also altered with decreased production of glycerol and nonesterified fatty acid using different stimulators: isoproterenol (1 µmol/L) (∼30%, n = 6, P < 0.001), forskolin (10 µmol/L) (∼30%, n = 6, P < 0.001), and DcAMP, the nonhydrolyzable cAMP analog (0.5 mmol/L) (∼20%, n = 5, P < 0.01) (Fig. 2B; Supplementary Fig. 7A). Next, we explored the possibility that ECs alter insulin sensitivity. The ratio of pS473 AKT to total AKT was decreased in adipocytes/ECs cocultures (−41.7%, n = 4, P < 0.05) (Fig. 2C; Supplementary Fig. 7B). We also observed that gene expression of three key markers of ER stress, ATF4, HSPA5, and CHOP, was increased (∼1.5-fold, P < 0.05) after 3 days of coculture (Fig. 2D). However, alterations in adipocyte functions were not associated with the cytotoxicity induced by ECs, as indicated by an unchanged LDH activity in cocultures compared with cells cultured alone (Supplementary Fig. 8).

Figure 2

Metabolic and secretory functions of human adipocytes in cocultures with ECs from VAT of obese subjects. A: ELISA experiments of adipokine secretion in cocultures. Black bars represent the percentage of decreased secretions from adipocytes cocultured with VAT-ECs (AD+ECs) compared with adipocytes cultured alone in the 3D setting (control [AD]). B: Lipolytic activity was evaluated by glycerol release from AD or AD+ECs after 3 days in the 3D setting. Glycerol release was measured under basal or stimulated conditions for 4 h. Isoproterenol (Iso) (1 µmol/L), forskolin (FK) (10 µmol/L), and DcAMP (0.5 mmol/L). Black bars represent the percentage of decreased glycerol release from AD+ECs compared with AD in the 3D setting. Data are mean ± SEM of 6 independent experiments. **P < 0.01, ***P < 0.001 AD vs. AD+ECs. ns, nonsignificant. C: The insulin response of AD and AD+ECs cultured 3 days in the 3D setting was evaluated after 10 nmol/L insulin stimulation of pS473 Akt during 15 min. The graph represents quantifications of the immunoblots normalized to total Akt (presented in Supplementary Fig. 7B). Data are means ± SEM of 6 independent experiments. #P < 0.05 basal condition (□) vs. +insulin (■), *P < 0.05 AD vs. AD+ECs. D: mRNA relative expression of ER stress gene markers CHOP, HSPA5, and ATF4 in cocultures. The fold increase of gene expression in AD+ECs compared with AD is shown. Data are represented as mean ± SEM of 7 separate experiments. *P < 0.05 AD vs. AD+ECs.

Figure 2

Metabolic and secretory functions of human adipocytes in cocultures with ECs from VAT of obese subjects. A: ELISA experiments of adipokine secretion in cocultures. Black bars represent the percentage of decreased secretions from adipocytes cocultured with VAT-ECs (AD+ECs) compared with adipocytes cultured alone in the 3D setting (control [AD]). B: Lipolytic activity was evaluated by glycerol release from AD or AD+ECs after 3 days in the 3D setting. Glycerol release was measured under basal or stimulated conditions for 4 h. Isoproterenol (Iso) (1 µmol/L), forskolin (FK) (10 µmol/L), and DcAMP (0.5 mmol/L). Black bars represent the percentage of decreased glycerol release from AD+ECs compared with AD in the 3D setting. Data are mean ± SEM of 6 independent experiments. **P < 0.01, ***P < 0.001 AD vs. AD+ECs. ns, nonsignificant. C: The insulin response of AD and AD+ECs cultured 3 days in the 3D setting was evaluated after 10 nmol/L insulin stimulation of pS473 Akt during 15 min. The graph represents quantifications of the immunoblots normalized to total Akt (presented in Supplementary Fig. 7B). Data are means ± SEM of 6 independent experiments. #P < 0.05 basal condition (□) vs. +insulin (■), *P < 0.05 AD vs. AD+ECs. D: mRNA relative expression of ER stress gene markers CHOP, HSPA5, and ATF4 in cocultures. The fold increase of gene expression in AD+ECs compared with AD is shown. Data are represented as mean ± SEM of 7 separate experiments. *P < 0.05 AD vs. AD+ECs.

Close modal

Inflammation Is Worsened in Human Adipocyte and VAT-EC Cocultures

Using multiplex and ELISA assay, we quantified the secretion of inflammation-related molecules in cocultures. Among the 39 screened molecules, 26 were significantly detected in adipocytes and ECs (>2 pg/mL) (Fig. 3A). CXCL1/2/3, IL-8, IL-6, and G-CSF were highly secreted in adipocytes. The significant changes in the secretion of the inflammatory molecules are presented for each experimental condition in Fig. 3B. For most of the molecules, secretion was enhanced in the presence of adipocytes and ECs in an additive manner. However, we observed a synergistic effect on CXCL1/2/3 (6-fold, P < 0.01), IL-6 (7.6-fold, P < 0.01), IL-8 (3-fold, P < 0.05), G-CSF (3.8-fold, P < 0.01), CCL2 (5.7-fold, P < 0.01), fractalkine (4.3-fold, P < 0.05), and γ-interferon (IFN0γ) (5.6-fold, P < 0.05). Increased release of soluble forms of the adhesion molecules intracellular adhesion molecule-1 (4.4-fold, P < 0.01) and E-selectin (3.3-fold, P < 0.05) by cocultured cells was also observed (Fig. 3B). However, the cell origin of these inflammatory mediators could not be identified in our coculture experiments exploring paracrine interaction, but the observation strongly suggests a direct effect of VAT-ECs on adipocyte function. In fact, cocultures associating adipocytes with an increased number of VAT-ECs exhibited a strong dose-dependent effect on adipocyte functions (lipolytic activity and adipokine secretions) and the secretion of inflammatory mediators (IL-6 and IL-8) (Supplementary Fig. 8).

Figure 3

Adipocyte and VAT-EC release of inflammatory and angiogenic-related molecules. Inflammatory response of adipocytes and VAT-ECs from obese subjects in the 3D cocultures was evaluated by comparing the secretion of inflammatory molecules between adipocytes (AD), cocultured adipocytes/VAT-ECs (AD+ECs), and VAT-ECs (ECs). A: Heat map representation of cytokine and chemokine secretions measured by multiplex assay. Graded shades from green to red represent the secretion levels (pg/mL). Cytokines and chemokines were classified from high to low secretion. ADs were used as a control. B: Significant changes in the secretion of inflammatory molecules in 3D cocultures (AD+ECs) compared with adipocytes, and ECs are represented in the graphs. Results are expressed as the fold variation between AD, AD+ECs, or ECs and to take into account human interindividual variations. Data are presented as means ± SEM of 6 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 AD+ECs vs. AD and ECs vs. AD. VEGF, vascular endothelial growth factor.

Figure 3

Adipocyte and VAT-EC release of inflammatory and angiogenic-related molecules. Inflammatory response of adipocytes and VAT-ECs from obese subjects in the 3D cocultures was evaluated by comparing the secretion of inflammatory molecules between adipocytes (AD), cocultured adipocytes/VAT-ECs (AD+ECs), and VAT-ECs (ECs). A: Heat map representation of cytokine and chemokine secretions measured by multiplex assay. Graded shades from green to red represent the secretion levels (pg/mL). Cytokines and chemokines were classified from high to low secretion. ADs were used as a control. B: Significant changes in the secretion of inflammatory molecules in 3D cocultures (AD+ECs) compared with adipocytes, and ECs are represented in the graphs. Results are expressed as the fold variation between AD, AD+ECs, or ECs and to take into account human interindividual variations. Data are presented as means ± SEM of 6 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 AD+ECs vs. AD and ECs vs. AD. VEGF, vascular endothelial growth factor.

Close modal

We emphasized the benefits of our 3D experimental design for direct coculture of adipocytes and VAT-ECs. The conditioned media prepared from VAT-ECs had no effects on adipocytes similar to those of direct cocultures. The inflammatory response of adipocytes to conditioned VAT-EC media remained lower than the response in direct cocultures and failed to induce alterations in adipokine secretion (Supplementary Fig. 9). Taken together, these findings support a putative role of labile factors or direct cell contact in adipocyte dysfunction.

To identify the potential factors involved in the inflammatory response and adipocyte alterations, we tested the effects of neutralizing antibodies of two candidates, IL-6 and IL-1β, in adipocytes and VAT-ECs cocultured or alone in the 3D setting. After 3 days of culture, the presence of the neutralizing IL-6/IL-1β antibodies significantly rescued some deleterious effects induced by cocultures as lipolytic activity (n = 5, P < 0.01) (Fig. 4A) and leptin secretion (n = 7, P < 0.01) (Fig. 4B). Insulin-stimulated Akt phosphorylation was improved by this treatment (n = 5, P < 0.05), while the presence of neutralizing TNF-α antibody failed to rescue the insulin response (Fig. 4C; Supplementary Fig. 10A). In the presence of the neutralizing IL-6/IL-1β antibodies, the inflammatory response of cocultured cells was reduced with decreased levels of IL-6 level (−84.8%, P < 0.001), G-CSF (−79%), and chemokines (CXCL1/2/3, −76%, and IL-8, −67.8%) (n = 7, P < 0.001) (Fig. 4D). However, benefits of IL-6 and IL-1β neutralizing antibody treatments on inflammation and adipocyte functions could be attributed to an effect on both adipocytes and VAT-ECs. Indeed, these treatments 1) tended to increase leptin production by adipocytes and 2) decreased G-CSF secretion by either adipocytes or VAT-ECs cultured alone in the 3D setting (−32% and −49%, respectively; P < 0.001) (Supplementary Fig. 10B–D). Importantly, no significant effects were observed with a treatment of neutralizing IL-6 antibody alone (data not shown), suggesting the importance of a combined action of the two cytokines in WAT inflammation and dysfunctions. Taken together, these results underlined important effects of these inflammatory molecules overproduced in cocultures on inflammation and adipocyte dysfunctions.

Figure 4

Coculture treatment by IL-6 and IL-1β neutralizing antibodies improves adipocyte functions and inflammation. A: Glycerol release from adipocytes treated with IgG1 (3.0 µg/mL) (control [AD IgG]) and adipocytes cocultured with VAT-ECs treated (AD+ECs abIL-6/IL-1β) or not (AD+ECs IgG) with IL-6 (2.5 µg/mL) and IL-1β (0.5 µg/mL) neutralizing antibodies during 3 days in the 3D setting. Glycerol release was measured after isoproterenol (1 µmol/L) stimulation for 4 h. Results are represented as the fold variation of glycerol release between AD IgG and AD+ECs IgG or AD+ECs abIL-6/IL-1β cocultures. Data are presented as means ± SEM of 5 independent experiments. #P < 0.05 AD IgG vs. AD+ECs IgG, **P < 0.01 AD+ECs IgG vs. AD+ECs abIL-6/IL-1β. B: ELISA experiments of adipokine secretions in cocultures. Shown are the percentages of increased secretions in cocultures treated with IL-6 and IL-1β neutralizing antibodies (AD+ECs abIL-6/IL-1β) compared with cocultures treated with IgG1 (control [AD+ECs IgG]) for 3 days in the 3D setting. **P < 0.01 AD+ECs abIL-6/IL-1β vs. AD+ECs IgG. ns, nonsignificant. C: The insulin response of AD IgG, AD+ECs IgG, AD+ECs abTNF-α, and AD+ECs abIL-6/IL-1β, cultured 3 days in the 3D setting, was evaluated after 10 nmol/L insulin stimulation of pS473 Akt during 15 min. The graph represents quantifications of the immunoblots in insulin-stimulated conditions normalized to total Akt (presented in Supplementary Fig. 10A). Data are mean ± SEM of 5 independent experiments. #P < 0.05 AD IgG vs. AD+ECs IgG, *P < 0.05 AD+ECs IgG vs. AD+ECs abIL-6/IL-1β. D: Multiplex assay of the inflammatory secretome in cocultures. Shown are the percentages of decreased secretions in cocultures treated with IL-6 and IL-1β neutralizing antibodies (AD+ECs abIL-6/IL-1β) compared with cocultures treated with IgG1 (control, AD+ECs IgG) 3 days in the 3D setting. Data are presented as means ± SEM of 7 independent experiments. *P < 0.05, ***P < 0.001 AD+ECs abIL-6/IL-1β vs. AD+ECs IgG.

Figure 4

Coculture treatment by IL-6 and IL-1β neutralizing antibodies improves adipocyte functions and inflammation. A: Glycerol release from adipocytes treated with IgG1 (3.0 µg/mL) (control [AD IgG]) and adipocytes cocultured with VAT-ECs treated (AD+ECs abIL-6/IL-1β) or not (AD+ECs IgG) with IL-6 (2.5 µg/mL) and IL-1β (0.5 µg/mL) neutralizing antibodies during 3 days in the 3D setting. Glycerol release was measured after isoproterenol (1 µmol/L) stimulation for 4 h. Results are represented as the fold variation of glycerol release between AD IgG and AD+ECs IgG or AD+ECs abIL-6/IL-1β cocultures. Data are presented as means ± SEM of 5 independent experiments. #P < 0.05 AD IgG vs. AD+ECs IgG, **P < 0.01 AD+ECs IgG vs. AD+ECs abIL-6/IL-1β. B: ELISA experiments of adipokine secretions in cocultures. Shown are the percentages of increased secretions in cocultures treated with IL-6 and IL-1β neutralizing antibodies (AD+ECs abIL-6/IL-1β) compared with cocultures treated with IgG1 (control [AD+ECs IgG]) for 3 days in the 3D setting. **P < 0.01 AD+ECs abIL-6/IL-1β vs. AD+ECs IgG. ns, nonsignificant. C: The insulin response of AD IgG, AD+ECs IgG, AD+ECs abTNF-α, and AD+ECs abIL-6/IL-1β, cultured 3 days in the 3D setting, was evaluated after 10 nmol/L insulin stimulation of pS473 Akt during 15 min. The graph represents quantifications of the immunoblots in insulin-stimulated conditions normalized to total Akt (presented in Supplementary Fig. 10A). Data are mean ± SEM of 5 independent experiments. #P < 0.05 AD IgG vs. AD+ECs IgG, *P < 0.05 AD+ECs IgG vs. AD+ECs abIL-6/IL-1β. D: Multiplex assay of the inflammatory secretome in cocultures. Shown are the percentages of decreased secretions in cocultures treated with IL-6 and IL-1β neutralizing antibodies (AD+ECs abIL-6/IL-1β) compared with cocultures treated with IgG1 (control, AD+ECs IgG) 3 days in the 3D setting. Data are presented as means ± SEM of 7 independent experiments. *P < 0.05, ***P < 0.001 AD+ECs abIL-6/IL-1β vs. AD+ECs IgG.

Close modal

WAT-ECs From Lean Subjects Do Not Impact the Functions of Cocultured Adipocytes

To establish the importance of the inflammatory environment induced by WAT-ECs on adipocyte dysfunctions, we cocultured adipocytes and ECs isolated from the SAT of lean nondiabetic subjects who did not display an inflammatory/senescent profile. Under these conditions, the secretion of leptin and adiponectin and lipolytic activity was not altered (n = 4) (Fig. 5A and B). Only 10 molecules were significantly detected by the multiplex analysis for which secretion remained much lower than cocultures with cells from VAT (n = 6) (Fig. 5C; Supplementary Figure 11). Although CCL7 and MIP-1α chemokines were induced in SAT cocultures, a synergistic effect of VAT cocultures on CXCL1/2/3, IL-6, IL-8, G-CSF, and CCL2 secretions was not observed in SAT cocultures. Compared with cells from obese subjects, lean cocultures did not exhibit enhanced secretion of soluble adhesion molecules (data not shown). We next confirmed the role of the cytokines IL-6 and IL-1β as key factors in alterations of cocultured adipocytes. Treatment of cocultures by the recombinant cytokines are efficient to induce the inflammatory burst and lipolysis alterations, as observed in cocultured VAT cells (Fig. 6).

Figure 5

Metabolic and secretory functions of human adipocytes cocultured with SAT-ECs from lean subjects. A: Adipokine secretions were measured by ELISA. Comparison of leptin and adiponectin secreted from adipocytes cultured alone (AD) (□) or cocultured with lean SAT-ECs (AD+ECs) (■) for 3 days in the 3D setting. Results are represented as the fold variation between AD and AD+ECs to take into account human interindividual variations. B: Lipolytic activity was evaluated by glycerol release from AD or AD+ ECs in the absence (basal) (□) or presence (1 µmol/L) (■) of isoproterenol (Iso). *P < 0.05 basal vs. Iso. n.s., nonsignificant AD vs. AD+ECs in lean cocultures. C: Heat map representation of cytokine and chemokine secretions measured by multiplex assay. Graded shades from green to red represent the secretion levels (pg/mL). Cytokines and chemokines were classified from high to low secretion. Changes in the secretion of inflammatory molecules in 3D cocultures (AD+ECs) compared with adipocytes (AD) and SAT-ECs are represented in the graphs. Results are expressed as the fold variation between AD, AD+ECs, or ECs. AD were used as a control. Data are presented as means ± SEM of 6 independent experiments. **P < 0.01 AD+ECs vs. AD. VEGF, vascular endothelial growth factor.

Figure 5

Metabolic and secretory functions of human adipocytes cocultured with SAT-ECs from lean subjects. A: Adipokine secretions were measured by ELISA. Comparison of leptin and adiponectin secreted from adipocytes cultured alone (AD) (□) or cocultured with lean SAT-ECs (AD+ECs) (■) for 3 days in the 3D setting. Results are represented as the fold variation between AD and AD+ECs to take into account human interindividual variations. B: Lipolytic activity was evaluated by glycerol release from AD or AD+ ECs in the absence (basal) (□) or presence (1 µmol/L) (■) of isoproterenol (Iso). *P < 0.05 basal vs. Iso. n.s., nonsignificant AD vs. AD+ECs in lean cocultures. C: Heat map representation of cytokine and chemokine secretions measured by multiplex assay. Graded shades from green to red represent the secretion levels (pg/mL). Cytokines and chemokines were classified from high to low secretion. Changes in the secretion of inflammatory molecules in 3D cocultures (AD+ECs) compared with adipocytes (AD) and SAT-ECs are represented in the graphs. Results are expressed as the fold variation between AD, AD+ECs, or ECs. AD were used as a control. Data are presented as means ± SEM of 6 independent experiments. **P < 0.01 AD+ECs vs. AD. VEGF, vascular endothelial growth factor.

Close modal
Figure 6

Effects of recombinant cytokines IL-6 and IL-1β on cocultured cells from SAT of lean subjects. A: Leptin secretion evaluated by ELISA in adipocytes (AD) and cocultures (AD+ECs) treated or not with recombinant IL-6 (10 ng/mL) and IL-1β (1 ng/mL) during 3 days in the 3D setting. Data are presented as means ± SEM of 5 independent experiments. *P < 0.05, **P <0.01, AD vs. AD IL-6/IL-1β or AD+ECs AD IL-6/IL-1β. ns, nonsignificant. B: Glycerol release from AD and AD+ECs treated or not with recombinant IL-6 (10 ng/mL) and IL-1β (1 ng/mL) during 3 days in the 3D setting. Glycerol release was measured after isoproterenol (1 µmol/L) stimulation for 4 h. Results are represented as the fold variation of glycerol release between AD and AD+ECs, AD IL-6/IL-1β, or AD+ECs IL-6/IL-1β. Data are presented as means ± SEM of 5 independent experiments. *P < 0.05 AD vs. AD+ECs AD IL-6/IL-1β. C: Multiplex assay of the inflammatory secretome in cocultures treated (AD+ECs IL-6/IL-1β) or not (AD+ECs) with recombinant IL-6 (10 ng/mL) and IL-1β (1 ng/mL) during 3 days in the 3D setting. Data are presented as means ± SEM of 5 independent experiments. *P < 0.05, **P < 0.01, AD+ECs vs. AD+ECs IL-6/IL-1β. VEGF, vascular endothelial growth factor.

Figure 6

Effects of recombinant cytokines IL-6 and IL-1β on cocultured cells from SAT of lean subjects. A: Leptin secretion evaluated by ELISA in adipocytes (AD) and cocultures (AD+ECs) treated or not with recombinant IL-6 (10 ng/mL) and IL-1β (1 ng/mL) during 3 days in the 3D setting. Data are presented as means ± SEM of 5 independent experiments. *P < 0.05, **P <0.01, AD vs. AD IL-6/IL-1β or AD+ECs AD IL-6/IL-1β. ns, nonsignificant. B: Glycerol release from AD and AD+ECs treated or not with recombinant IL-6 (10 ng/mL) and IL-1β (1 ng/mL) during 3 days in the 3D setting. Glycerol release was measured after isoproterenol (1 µmol/L) stimulation for 4 h. Results are represented as the fold variation of glycerol release between AD and AD+ECs, AD IL-6/IL-1β, or AD+ECs IL-6/IL-1β. Data are presented as means ± SEM of 5 independent experiments. *P < 0.05 AD vs. AD+ECs AD IL-6/IL-1β. C: Multiplex assay of the inflammatory secretome in cocultures treated (AD+ECs IL-6/IL-1β) or not (AD+ECs) with recombinant IL-6 (10 ng/mL) and IL-1β (1 ng/mL) during 3 days in the 3D setting. Data are presented as means ± SEM of 5 independent experiments. *P < 0.05, **P < 0.01, AD+ECs vs. AD+ECs IL-6/IL-1β. VEGF, vascular endothelial growth factor.

Close modal

Considering the importance of the inflammatory profile of VAT-ECs in adipocyte dysfunctions, we proposed to reverse these alterations directly by influencing VAT-ECs. Pericytes are mural cells that promote the maturation and stabilization of blood vessels, in particular through the secretion of Ang-1, exerting prosurvival and anti-inflammatory actions on ECs (12). Therefore, we broadened the topic of this study by analyzing pericyte phenotype in VAT and tried to determine a possible link with EC dysfunction in the context of obesity.

Altered Phenotype of Pericytes From VAT of Obese Subjects

The pericytes, visualized using the specific marker NG2 in WAT from obese subjects (Fig. 7A), were isolated from SAT of lean and from SAT/VAT of obese subjects. The pericyte-to-EC ratio was evaluated in the different conditions. This ratio was strongly reduced in SAT and VAT of obese subjects compared with lean SAT (Fig. 7B). Pericytes from obese WAT displayed an inflammatory profile with increased secretion of G-CSF (∼60-fold), CXCL1/2/3 (∼10-fold), IL-6 (∼30-fold), IL-8 (∼3-fold), and MIP-1α (∼8-fold) compared with lean SAT. No differences were observed between pericytes from SAT and VAT from obese subjects (Fig. 7C). Moreover, Ang-1, secreted by pericytes from lean SAT (37.7 ± 11 pg/mL, n = 4), was not detected in culture media of pericytes from obese pericytes (SAT and VAT).

Figure 7

Pericyte phenotype in WAT from obese subjects. A: Immunofluorescence analysis by confocal microscopy of VAT from obese subjects using antibodies directed against type IV collagen in adipocytes (a) and blood vessels (bv) (green, Cy2-conjugated anti-mouse IgG) and NG2 in pericytes (arrows) (red, Cy3-conjugated anti-rabbit IgG). A representative photomicrograph is presented. Scale bar = 10 µm. B: Comparison of WAT pericyte-to-EC ratio between lean (SAT lean, n = 22) and obese (SAT obese, n = 12, and VAT obese, n = 26) subjects. **P < 0.05, ***P < 0.001, SAT lean vs. SAT/VAT obese. C: Multiplex assay of the inflammatory secretion profile of pericytes isolated from SAT of lean (SAT-PC lean), SAT of obese (SAT-PC obese), or VAT of obese (VAT-PC obese) subjects. Data are presented as means ± SEM of 5 independent experiments. *P < 0.05, **P < 0.01, SAT-PC lean vs. SAT/VAT-PC obese. D: Multiplex assay of the inflammatory secretion profile in culture associating adipocytes and ECs from VAT of obese subjects (AD+ECs) with addition or not of pericytes from the same VAT depot. Pericytes were pretreated (AD+ECs+PC Dex) or not (AD+ECs+PC) with 100 nmol/L Dex) for 1 h. Data are presented as means ± SEM of 5 independent experiments. *P < 0.05 AD+ECs vs. AD+ECs+PC Dex, #P < 0.05, AD+ECs+PC vs. AD+ECs+PC Dex. VEGF, vascular endothelial growth factor.

Figure 7

Pericyte phenotype in WAT from obese subjects. A: Immunofluorescence analysis by confocal microscopy of VAT from obese subjects using antibodies directed against type IV collagen in adipocytes (a) and blood vessels (bv) (green, Cy2-conjugated anti-mouse IgG) and NG2 in pericytes (arrows) (red, Cy3-conjugated anti-rabbit IgG). A representative photomicrograph is presented. Scale bar = 10 µm. B: Comparison of WAT pericyte-to-EC ratio between lean (SAT lean, n = 22) and obese (SAT obese, n = 12, and VAT obese, n = 26) subjects. **P < 0.05, ***P < 0.001, SAT lean vs. SAT/VAT obese. C: Multiplex assay of the inflammatory secretion profile of pericytes isolated from SAT of lean (SAT-PC lean), SAT of obese (SAT-PC obese), or VAT of obese (VAT-PC obese) subjects. Data are presented as means ± SEM of 5 independent experiments. *P < 0.05, **P < 0.01, SAT-PC lean vs. SAT/VAT-PC obese. D: Multiplex assay of the inflammatory secretion profile in culture associating adipocytes and ECs from VAT of obese subjects (AD+ECs) with addition or not of pericytes from the same VAT depot. Pericytes were pretreated (AD+ECs+PC Dex) or not (AD+ECs+PC) with 100 nmol/L Dex) for 1 h. Data are presented as means ± SEM of 5 independent experiments. *P < 0.05 AD+ECs vs. AD+ECs+PC Dex, #P < 0.05, AD+ECs+PC vs. AD+ECs+PC Dex. VEGF, vascular endothelial growth factor.

Close modal

Anti-Inflammatory Strategy Targeting ECs Improves Adipocyte Lipolysis and Inflammation

We observed that pericytes, displaying an altered phenotype during obesity, did not rescue inflammatory profile of ECs when cocultured in the 3D setting (Supplementary Fig. 12A). Addition of pericytes from VAT in adipocytes/ECs cocultures did not further increase the inflammatory response (Fig. 7A) or adipocyte dysfunctions (lipolysis and adipokine secretions [Supplementary Fig. 12D]). However, when pericytes were pretreated with dexamethasone (Dex), their inflammatory profile was reduced (Supplementary Fig. 12B), leading to a global decrease of inflammation in adipocytes/ECs cocultures (Fig. 7D). However, addition of Dex-treated pericytes failed to rescue adipocyte functions in these cocultures (Supplementary Fig. 12D).

Because Ang-1 production was decreased in pericytes during obesity and because its secretion was not rescued by Dex treatment (Supplementary Fig. 12C), we tested the effects of recombinant Ang-1 on VAT-ECs from obese subjects (inflammatory profile) and the consequences on cocultured adipocytes. Briefly, VAT-ECs were incubated in the presence or absence of Ang-1 (100 ng/mL) for 24 h before coculture with unilocular adipocytes. Cells were then cocultured in the 3D setting for 3 days as described in Supplementary Fig. 5.

A 24-h treatment of VAT-ECs with Ang-1 (Ang-1ECs) significantly reduced their secretion of CXCL1/2/3 (−55.8%, P < 0.01) and IL-6 (−37.3%, P < 0.05), whereas it tended to decrease G-CSF and IL-8 secretions (n = 5) (Fig. 8A). Interestingly, when adipocytes were cocultured with Ang-1 ECs, the adipocytes recovered their lipolytic functions (50%, n = 3, P < 0.05) and insulin sensitivity (39%, n = 5, P < 0.05) compared with control cocultures, whereas adipokine secretion remained unchanged (Fig. 8B and C; Supplementary Fig. 13). Moreover, coculturing adipocytes and Ang-1ECs led to reduced secretion of several cytokines and chemokines compared with a control coculture of visceral adipocytes/ECs (n = 5): G-CSF (−42.9%, P < 0.05), IL-6 (−44.1%, P < 0.05), CXCL1/2/3 (−38.6%, P < 0.01), and IL-8 (−47.8%, P < 0.05) (Fig. 8D). Notably, Ang-1 treatment had no direct effect on adipocyte function (data not shown). We did not observe any benefits of Ang-1 when it was added directly to cocultures without WAT-EC pretreatment (data not shown). Taken together, these findings suggest that Ang-1 partially reverses adipocyte dysfunction and inflammation through a specific action on EC inflammation.

Figure 8

Ang-1 pretreatment of VAT-ECs improves inflammation and adipocyte metabolism. A: Multiplex assay of the inflammatory secretome. Shown is percentage decreased in secretions in Ang-1–treated ECs (Ang-1-ECs) compared with control ECs. Data are presented as means ± SEM of 5 independent experiments. *P < 0.05 Ang-1-ECs vs. ECs. B: Effect on glycerol release from adipocytes cultured alone (AD) or adipocytes cocultured with visceral ECs treated (AD+Ang-1-ECs) or not (AD+ECs) with Ang-1 (100 ng/mL, 24 h) after 3 days in hydrogel. Glycerol release was measured under isoproterenol (1 µmol/L)-stimulated conditions. Results are represented as the fold variation of glycerol release between AD and AD+ECs or AD+Ang-1-ECs cocultures. Data are presented as means ± SEM of 5 independent experiments. ##P < 0.01 AD vs. AD+ECs, *P < 0.05 AD+ECs vs. AD+Ang-1-ECs. C: The insulin response of AD, AD+ECs, and AD+Ang-1-ECs, cultured 3 days in the 3D setting, was evaluated after 10 nmol/L insulin stimulation of pS473 Akt during 15 min. The graph represents quantifications of the immunoblots in insulin-stimulated conditions normalized to total Akt (presented in Supplementary Fig. 13C). Data are mean ± SEM of 5 independent experiments. ##P < 0.01 AD vs. AD+ECs, *P < 0.05 AD+ECs vs. AD+Ang-1-ECs. D: Multiplex assay of the inflammatory secretion profile in cocultures. Shown is percentage decrease in secretions in AD+Ang-1-EC cocultures compared with control AD+ECs cocultures. Data are presented as means ± SEM of 5 independent experiments. *P < 0.05 AD+Ang-1-ECs vs. AD+ECs. VEGF, vascular endothelial growth factor.

Figure 8

Ang-1 pretreatment of VAT-ECs improves inflammation and adipocyte metabolism. A: Multiplex assay of the inflammatory secretome. Shown is percentage decreased in secretions in Ang-1–treated ECs (Ang-1-ECs) compared with control ECs. Data are presented as means ± SEM of 5 independent experiments. *P < 0.05 Ang-1-ECs vs. ECs. B: Effect on glycerol release from adipocytes cultured alone (AD) or adipocytes cocultured with visceral ECs treated (AD+Ang-1-ECs) or not (AD+ECs) with Ang-1 (100 ng/mL, 24 h) after 3 days in hydrogel. Glycerol release was measured under isoproterenol (1 µmol/L)-stimulated conditions. Results are represented as the fold variation of glycerol release between AD and AD+ECs or AD+Ang-1-ECs cocultures. Data are presented as means ± SEM of 5 independent experiments. ##P < 0.01 AD vs. AD+ECs, *P < 0.05 AD+ECs vs. AD+Ang-1-ECs. C: The insulin response of AD, AD+ECs, and AD+Ang-1-ECs, cultured 3 days in the 3D setting, was evaluated after 10 nmol/L insulin stimulation of pS473 Akt during 15 min. The graph represents quantifications of the immunoblots in insulin-stimulated conditions normalized to total Akt (presented in Supplementary Fig. 13C). Data are mean ± SEM of 5 independent experiments. ##P < 0.01 AD vs. AD+ECs, *P < 0.05 AD+ECs vs. AD+Ang-1-ECs. D: Multiplex assay of the inflammatory secretion profile in cocultures. Shown is percentage decrease in secretions in AD+Ang-1-EC cocultures compared with control AD+ECs cocultures. Data are presented as means ± SEM of 5 independent experiments. *P < 0.05 AD+Ang-1-ECs vs. AD+ECs. VEGF, vascular endothelial growth factor.

Close modal

We aimed to reproduce the adipocyte/EC dialog participating in the complex intercellular network of the inflamed VAT in a 3D setting. Better knowledge of this dialog could help in the development of new therapeutic strategies targeting inflammatory WAT. We cocultured unilocular adipocytes and ECs isolated from VAT from obese subjects in a 3D hydrogel in which cells cultured alone maintain their functions and their inflammatory phenotype for at least 7 days. Direct cocultures in the 3D setting allow cell-cell contact and the paracrine dialog between cell populations, maintaining labile signals. By following adipocyte functions in the 3D cocultures, we identified an inflammatory cross-talk between adipocytes and VAT-ECs that leads to alterations in lipolytic activity and insulin sensitivity. Adipokine secretion was also impaired in adipocyte-EC cocultures. The decreased secretion of adiponectin and an inflammatory environment are in accordance with most studies exploring EC dysfunctions (8) and insulin resistance (21). In contrast, the influence of inflammation on decreased lipolysis remains a subject of debate. Several studies have described increased basal lipolysis in inflammatory 3T3-L1 adipocytes (3,22), suggesting different mechanisms in unilocular human adipocytes. Interestingly, the effects were observed under β-adrenergic stimulation, which is impaired in obese subjects (23).

In our coculture setting, we observed increased secretion of many relevant inflammatory molecules, particularly IL-6, G-CSF, CXCL1/2/3/8, and IFN-γ, which are proposed to contribute to obesity-related complications, such as type 2 diabetes and atherosclerosis. Overall, these overproduced chemokines and cytokines could aggravate inflammation in VAT by promoting the accumulation and inflammation of immune cells. For example, fractalkine is involved in the recruitment of monocytes and T lymphocytes in atherosclerosis, type 2 diabetes, and obesity (24). Interestingly, adipocytes were recently identified as antigen-presenting cells in response to IFN-γ in high-fat-diet mice with T-lymphocyte activation (25). The adipocyte production of IFN-γ could perpetuate, by a paracrine loop, WAT inflammation. CCL5, which is overproduced by VAT in human obesity, promotes the accumulation and survival of macrophages in WAT (26).

Although being performed with human WAT cells, our 3D setting presents some technical limitations, notably in reproducing the complex inflammatory environment of obese WAT. While we focused on adipocytes and ECs, other cell types could also influence this cross-talk (i.e., preadipocytes, neutrophils, or macrophages). Our team showed that inflammatory macrophages induced an inflammatory and profibrotic phenotype of human preadipocytes (27). More recently, we showed that neutrophils, interacting with VAT-ECs of obese subjects, provoked their inflammation/senescence (18).

Obesity provokes an ER stress in adipocytes (28). Notably, ER stress induces a decreased secretion of leptin and adiponectin, an alteration of insulin signaling and lipolysis, and inflammation in these cells (29). Here, we observed an ER stress in cocultured cells, which could be linked to the adipocyte dysfunctions.

Chronically elevated levels of IL-6, combined with increased IL-1β, favor development of type 2 diabetes (30). As with IL-6 (31), IL-1β secretion is increased in WAT during obesity (32), and both cytokines are known to act in concert in diverse biological process (33). Il-1β is also implicated in a “secondary” inflammatory response, regulating the production of cytokines and chemokines as G-CSF, IL-6, or IL-8 (3436). Interestingly, our study showed that neutralization of IL-6 and IL-1β in cocultures led to a decreased inflammatory response and a rescue of some metabolic and secretory alterations of adipocytes (i.e., G-CSF, CXCLs, and IL-8). Conversely, treatment of SAT cells with recombinant IL-6 and IL-1β provoked roughly the same cell dysfunctions. This strongly suggests that IL-6 and IL-1β are important adipocyte/EC-derived factors provoking cell dysfunctions.

The functional properties and activation state of WAT-ECs play a crucial role in the promotion of both adipocyte alterations and inflammatory cross-talk. WAT-ECs isolated from lean individuals were not able to promote adipocyte alterations or inflammatory responses, highlighting the specificity of the cross-talk of inflammatory WAT cells in the context of obesity. An important purpose of the current study was to reduce WAT-EC inflammation in order to reverse the adipocyte dysfunctions. We chose Ang-1, which is constitutively produced by pericytes and known to prevent EC inflammation (12). Low levels of Ang-1 and a decreased number of pericytes are both hallmarks of type 2 diabetes (37,38). Ang-1 treatment of VAT-ECs tended to decrease the inflammatory profile and reversed, at least in part, the inflammatory response and restored lipolysis and insulin sensitivity in the coculture system. Our results are supported by previous studies showing protective effects of Ang-1 on the vasculature. In in vitro models, Ang-1 has been shown to prevent EC apoptosis and reduce the inflammation of ECs through decreased surface expression of adhesion molecules (39,40). In mice, Ang-1 deficiency exaggerates the wound-healing response to injury, leading to profound damage, such as fibrosis and vascular abnormalities (41). Conversely, Ang-1 treatment of diabetic mice improves vascular density, reduces adipocyte size, and ameliorates metabolic disorders relative to obesity (42).

Several studies have described reduced tissue abundance or altered pericyte phenotype in pathological conditions such as diabetic retinopathy and systemic sclerosis, respectively (43,44). For the first time, to the best of our knowledge, we provide information on the features of pericytes in the WAT from obese subjects. We highlighted a strong decrease of the pericytes-to-EC ratio in obese WAT, suggesting a defect in the vascular protection by pericytes of WAT-ECs. Moreover, these pericytes displayed an altered phenotype with increased expression of several inflammatory molecules (CCL2, CCL5, and TNF-α) and a decreased expression Ang-1 compared with pericytes from lean subjects. Thus, the inflammation-associated senescence of VAT-ECs, which was attributed to adipocyte secretions (5), may also result from pericyte dysfunctions in WAT from obese subjects. Notably, dysregulation of VAT-ECs may also result from disrupted contacts with pericytes (45). However, more information is needed on the direct links between adipocyte, EC, and pericyte dysfunctions in the VAT of obese subjects. The kinetics of these events is not fully understood and cannot be resolved by the present in vitro study. Kinetics studies with high-fat-diet mice models regarding interactions between adipocyte metabolism and EC/pericyte alterations within VAT would be critical to support our conclusions.

Finally, the inflammatory cross-talk between adipocytes and VAT-ECS could position them as important actors in the maintenance of VAT inflammation. Amelioration of VAT-EC dysfunctions by acting on pericyte phenotype or targeting the inflammatory secretions of ECs could prevent the adipocyte alterations responsible for obesity-related complications, such as atherosclerosis and type 2 diabetes.

Acknowledgments. The authors acknowledge patients and the physician Dr. Christine Poitou of the Nutrition Department of Pitié Salpêtrière for patient recruitment. They also acknowledge Christophe Klein from imaging facilities (Cordeliers Research Center). For cellular studies, ethics authorization was obtained from Comité de protection des personnes Pitié Salpêtrière. Human adipose tissues pieces were obtained thanks to a clinical research contract (Assistance Publique/Direction de la Recherche Clinique AOR 02076). The manuscript was edited for language and style by San Francisco Edit.

Funding. This work was supported by a grant from the European Community seventh framework program, Adipokines as Drug to combat Adverse Effects of Excess Adipose tissue project (contract number HEALTH-FP2-2008-201100); Assistance Publique Hopitaux de Paris Clinical Research Contract, Emergence program, University Pierre et Marie Curie (to V.P.); and French National Agency of Research (French government grant, “Investments for the Future,” grant ANR-10-IAHU, ANR AdipoFib).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. V.P. and D.L. conceived and performed the experiments, analyzed data, and wrote the manuscript with the input of all the coauthors. C.R. conceived and performed the experiments and analyzed data. N.V. collected adipose tissue samples of obese patients during bariatric surgery. K.C. wrote the manuscript with the input of all the coauthors. K.C. and D.L. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

1.
Christiaens
V
,
Lijnen
HR
.
Angiogenesis and development of adipose tissue
.
Mol Cell Endocrinol
2010
;
318
:
2
9
[PubMed]
2.
Takakura
N
.
Role of intimate interactions between endothelial cells and the surrounding accessory cells in the maturation of blood vessels
.
J Thromb Haemost
2011
;
9
(
Suppl. 1
):
144
150
[PubMed]
3.
Permana
PA
,
Menge
C
,
Reaven
PD
.
Macrophage-secreted factors induce adipocyte inflammation and insulin resistance
.
Biochem Biophys Res Commun
2006
;
341
:
507
514
[PubMed]
4.
Wajchenberg
BL
.
Subcutaneous and visceral adipose tissue: their relation to the metabolic syndrome
.
Endocr Rev
2000
;
21
:
697
738
[PubMed]
5.
Villaret
A
,
Galitzky
J
,
Decaunes
P
, et al
.
Adipose tissue endothelial cells from obese human subjects: differences among depots in angiogenic, metabolic, and inflammatory gene expression and cellular senescence
.
Diabetes
2010
;
59
:
2755
2763
[PubMed]
6.
Cancello
R
,
Tordjman
J
,
Poitou
C
, et al
.
Increased infiltration of macrophages in omental adipose tissue is associated with marked hepatic lesions in morbid human obesity
.
Diabetes
2006
;
55
:
1554
1561
[PubMed]
7.
Kralisch
S
,
Sommer
G
,
Stangl
V
, et al
.
Secretory products from human adipocytes impair endothelial function via nuclear factor kappaB
.
Atherosclerosis
2008
;
196
:
523
531
[PubMed]
8.
Xu
A
,
Wang
Y
,
Lam
KSL
,
Vanhoutte
PM
.
Vascular actions of adipokines molecular mechanisms and therapeutic implications
.
Adv Pharmacol
2010
;
60
:
229
255
[PubMed]
9.
Zhang
H
,
Zhang
C
.
Regulation of microvascular function by adipose tissue in obesity and type 2 diabetes: evidence of an adipose-vascular loop
.
Am J Biomed Sci
2009
;
1
:
133
142
[PubMed]
10.
Aoki
S
,
Toda
S
,
Sakemi
T
,
Sugihara
H
.
Coculture of endothelial cells and mature adipocytes actively promotes immature preadipocyte development in vitro
.
Cell Struct Funct
2003
;
28
:
55
60
[PubMed]
11.
Varzaneh
FE
,
Shillabeer
G
,
Wong
KL
,
Lau
DC
.
Extracellular matrix components secreted by microvascular endothelial cells stimulate preadipocyte differentiation in vitro
.
Metabolism
1994
;
43
:
906
912
[PubMed]
12.
Imhof
BA
,
Aurrand-Lions
M
.
Angiogenesis and inflammation face off
.
Nat Med
2006
;
12
:
171
172
[PubMed]
13.
Curat
CA
,
Miranville
A
,
Sengenès
C
, et al
.
From blood monocytes to adipose tissue-resident macrophages: induction of diapedesis by human mature adipocytes
.
Diabetes
2004
;
53
:
1285
1292
[PubMed]
14.
Cho
H
,
Kozasa
T
,
Bondjers
C
,
Betsholtz
C
,
Kehrl
JH
.
Pericyte-specific expression of Rgs5: implications for PDGF and EDG receptor signaling during vascular maturation
.
FASEB J
2003
;
17
:
440
442
[PubMed]
15.
Zimmerlin
L
,
Donnenberg
VS
,
Pfeifer
ME
, et al
.
Stromal vascular progenitors in adult human adipose tissue
.
Cytometry A
2010
;
77
:
22
30
[PubMed]
16.
Xue
Y
,
Lim
S
,
Bråkenhielm
E
,
Cao
Y
.
Adipose angiogenesis: quantitative methods to study microvessel growth, regression and remodeling in vivo
.
Nat Protoc
2010
;
5
:
912
920
[PubMed]
17.
WIPO Patent WO 2011/148310A1 [Internet]. Available from http://www.sumobrain.com/patents/wipo/method-culturing-adipocytes/wo2011148310a1.pdf. Accessed 29 December 2012
18.
Rouault
C
,
Pellegrinelli
V
,
Schilch
R
, et al
.
Roles of chemokine ligand-2 (CXCL2) and neutrophils in influencing endothelial cell function and inflammation of human adipose tissue
.
Endocrinology
2013
;
154
:
1069
1079
[PubMed]
19.
Dos Santos
EG
,
Dieudonne
MN
,
Pecquery
R
,
Le Moal
V
,
Giudicelli
Y
,
Lacasa
D
.
Rapid nongenomic E2 effects on p42/p44 MAPK, activator protein-1, and cAMP response element binding protein in rat white adipocytes
.
Endocrinology
2002
;
143
:
930
940
[PubMed]
20.
Lacasa
D
,
Taleb
S
,
Keophiphath
M
,
Miranville
A
,
Clement
K
.
Macrophage-secreted factors impair human adipogenesis: involvement of proinflammatory state in preadipocytes
.
Endocrinology
2007
;
148
:
868
877
[PubMed]
21.
Rotter
V
,
Nagaev
I
,
Smith
U
.
Interleukin-6 (IL-6) induces insulin resistance in 3T3-L1 adipocytes and is, like IL-8 and tumor necrosis factor-alpha, overexpressed in human fat cells from insulin-resistant subjects
.
J Biol Chem
2003
;
278
:
45777
45784
[PubMed]
22.
Suganami
T
,
Nishida
J
,
Ogawa
Y
.
A paracrine loop between adipocytes and macrophages aggravates inflammatory changes: role of free fatty acids and tumor necrosis factor alpha
.
Arterioscler Thromb Vasc Biol
2005
;
25
:
2062
2068
[PubMed]
23.
Horowitz
JF
,
Klein
S
.
Whole body and abdominal lipolytic sensitivity to epinephrine is suppressed in upper body obese women
.
Am J Physiol Endocrinol Metab
2000
;
278
:
E1144
E1152
[PubMed]
24.
Shah
R
,
Hinkle
CC
,
Ferguson
JF
, et al
.
Fractalkine is a novel human adipochemokine associated with type 2 diabetes
.
Diabetes
2011
;
60
:
1512
1518
[PubMed]
25.
Deng
T
,
Lyon
CJ
,
Minze
LJ
, et al
.
Class II major histocompatibility complex plays an essential role in obesity-induced adipose inflammation
.
Cell Metab
2013
;
17
:
411
422
[PubMed]
26.
Keophiphath
M
,
Rouault
C
,
Divoux
A
,
Clément
K
,
Lacasa
D
.
CCL5 promotes macrophage recruitment and survival in human adipose tissue
.
Arterioscler Thromb Vasc Biol
2010
;
30
:
39
45
[PubMed]
27.
Keophiphath
M
,
Achard
V
,
Henegar
C
,
Rouault
C
,
Clément
K
,
Lacasa
D
.
Macrophage-secreted factors promote a profibrotic phenotype in human preadipocytes
.
Mol Endocrinol
2009
;
23
:
11
24
[PubMed]
28.
Ozcan
U
,
Cao
Q
,
Yilmaz
E
, et al
.
Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes
.
Science
2004
;
306
:
457
461
[PubMed]
29.
Xu
L
,
Spinas
GA
,
Niessen
M
.
ER stress in adipocytes inhibits insulin signaling, represses lipolysis, and alters the secretion of adipokines without inhibiting glucose transport
.
Horm Metab Res
2010
;
42
:
643
651
[PubMed]
30.
Spranger
J
,
Kroke
A
,
Möhlig
M
, et al
.
Inflammatory cytokines and the risk to develop type 2 diabetes: results of the prospective population-based European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam Study
.
Diabetes
2003
;
52
:
812
817
[PubMed]
31.
Kern
PA
,
Ranganathan
S
,
Li
C
,
Wood
L
,
Ranganathan
G
.
Adipose tissue tumor necrosis factor and interleukin-6 expression in human obesity and insulin resistance
.
Am J Physiol Endocrinol Metab
2001
;
280
:
E745
E751
[PubMed]
32.
Moschen
AR
,
Molnar
C
,
Enrich
B
,
Geiger
S
,
Ebenbichler
CF
,
Tilg
H
.
Adipose and liver expression of interleukin (IL)-1 family members in morbid obesity and effects of weight loss
.
Mol Med
2011
;
17
:
840
845
[PubMed]
33.
Stapp
JM
,
Sjoelund
V
,
Lassiter
HA
,
Feldhoff
RC
,
Feldhoff
PW
.
Recombinant rat IL-1beta and IL-6 synergistically enhance C3 mRNA levels and complement component C3 secretion by H-35 rat hepatoma cells
.
Cytokine
2005
;
30
:
78
85
[PubMed]
34.
Elias
JA
,
Lentz
V
.
IL-1 and tumor necrosis factor synergistically stimulate fibroblast IL-6 production and stabilize IL-6 messenger RNA
.
J Immunol
1990
;
145
:
161
166
[PubMed]
35.
Lai
CF
,
Baumann
H
.
Interleukin-1 beta induces production of granulocyte colony-stimulating factor in human hepatoma cells
.
Blood
1996
;
87
:
4143
4148
[PubMed]
36.
Eskan
MA
,
Benakanakere
MR
,
Rose
BG
, et al
.
Interleukin-1beta modulates proinflammatory cytokine production in human epithelial cells
.
Infect Immun
2008
;
76
:
2080
2089
[PubMed]
37.
Imesch
PD
,
Bindley
CD
,
Wallow
IH
.
Clinicopathologic correlation of intraretinal microvascular abnormalities
.
Retina
1997
;
17
:
321
329
[PubMed]
38.
Singh
H
,
Brindle
NPJ
,
Zammit
VA
.
High glucose and elevated fatty acids suppress signaling by the endothelium protective ligand angiopoietin-1
.
Microvasc Res
2010
;
79
:
121
127
[PubMed]
39.
Kim
I
,
Moon
SO
,
Park
SK
,
Chae
SW
,
Koh
GY
.
Angiopoietin-1 reduces VEGF-stimulated leukocyte adhesion to endothelial cells by reducing ICAM-1, VCAM-1, and E-selectin expression
.
Circ Res
2001
;
89
:
477
479
[PubMed]
40.
Papapetropoulos
A
,
Fulton
D
,
Mahboubi
K
, et al
.
Angiopoietin-1 inhibits endothelial cell apoptosis via the Akt/survivin pathway
.
J Biol Chem
2000
;
275
:
9102
9105
[PubMed]
41.
Jeansson
M
,
Gawlik
A
,
Anderson
G
, et al
.
Angiopoietin-1 is essential in mouse vasculature during development and in response to injury
.
J Clin Invest
2011
;
121
:
2278
2289
[PubMed]
42.
Jung
YJ
,
Choi
HJ
,
Lee
JE
, et al
.
The effects of designed angiopoietin-1 variant on lipid droplet diameter, vascular endothelial cell density and metabolic parameters in diabetic db/db mice
.
Biochem Biophys Res Commun
2012
;
420
:
498
504
[PubMed]
43.
Armulik
A
,
Genové
G
,
Betsholtz
C
.
Pericytes: developmental, physiological, and pathological perspectives, problems, and promises
.
Dev Cell
2011
;
21
:
193
215
[PubMed]
44.
Sims
DE
.
Diversity within pericytes
.
Clin Exp Pharmacol Physiol
2000
;
27
:
842
846
[PubMed]
45.
Hall-Glenn
F
,
De Young
RA
,
Huang
B-L
, et al
.
CCN2/connective tissue growth factor is essential for pericyte adhesion and endothelial basement membrane formation during angiogenesis
.
PLoS ONE
2012
;
7
:
e30562
[PubMed]

Supplementary data