Mesangial cells stimulate differentiation of endothelial cells to form capillary-like networks in a three-dimensional culture system

Tokuyuki Kitahara, Keiju Hiromura, Hidekazu Ikeuchi, Shin Yamashita, Satsuki Kobayashi, Takashi Kuroiwa, Yoriaki Kaneko, Kazue Ueki and Yoshihisa Nojima

Department of Medicine and Clinical Science, Gunma University Graduate School of Medicine, Maebashi, Japan

Correspondence and offprint requests to: Tokuyuki Kitahara, MD, Department of Medicine and Clinical Science, Gunma University Graduate School of Medicine, 3-39-15 Showa, Maebashi, Gunma 371-8511, Japan. Email: tkita1{at}jcom.home.ne.jp



   Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. There are conflicting results regarding the role of periendothelial mural cells in angiogenesis. In the current study, we investigated the role of mesangial cells (MCs) in endothelial vascularization by using a three-dimensional co-culture system in basement-membrane reconstruct gel (Matrigel).

Methods. Human umbilical vein endothelial cells (ECs) and human MCs were used. In the contact co-culture system, ECs and MCs were mixed and then plated together onto Matrigel. In the non-contact co-culture system, MCs were cultured within an intercup chamber, which prevented direct physical contact with the ECs on the Matrigel but allowed both cell types to share culture medium. To visualize ECs and MCs, the cells were labelled with two different fluorescent dyes prior to the co-culture. A capillary-like network formation was observed under a fluorescent microscope and confocal microscope, and the length of the network formation was quantified by the image analyzer.

Results. ECs barely formed capillary-like networks when cultured alone in growth factor-free medium. However, ECs cultured with MCs in a contact condition remarkably formed capillary-like networks (9.10±0.96 vs 0.20±0.07 mm/mm2 at 6 h, contact vs ECs alone, P<0.001). Direct contact between ECs and MCs was clearly demonstrated by confocal microscopy. Differentiation into branching capillary-like structures was also observed in the non-contact co-culture system (3.02±1.21 mm/mm2 at 6 h, P<0.001 vs ECs alone), but less prominently than in the contact co-culture condition. Vascular endothelial growth factor (VEGF) was secreted from MCs, as determined by enzyme-linked immunosorbent assay and immunofluorescent study. Adding neutralizing antibodies against VEGF into the co-culture system partially inhibited capillary network formation.

Conclusions. Our data indicate that MCs help ECs differentiate toward vascularization, in which the direct cell–cell contact between ECs and MCs plays an important role. VEGF is a mediator in this process.

Keywords: angiogenesis; co-culture; mesangial cells; three-dimensional; vasculogenesis; VEGF



   Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Previous studies have reported conflicting results regarding the role of periendothelial mural cells such as pericytes, smooth muscle cells and fibroblasts in angiogenesis [1–4]. Some reported that these mural cells suppressed angiogenesis, whereas others showed stimulatory effects. For example, pericytes negatively regulated endothelial cell (EC) proliferation in vitro and in vivo [2]. On the other hand, pericytes were reported to stimulate angiogenesis through secretion of growth factors such as fibroblast growth factor (FGF) [3], vascular endothelial growth factor (VEGF) and placenta growth factor [4]. In addition, pericytes appear to promote EC survival [5] and affect EC behaviour such as sprouting [6]. Although the origin of mesangial cells (MCs) has not been defined precisely, they are sometimes referred to as microvascular pericytes, which are contractile cells similar to the smooth muscle cells that encircle microvessels in many different tissues [7]. In the repair process of glomerular injury, such as in an experimental model of Thy-1.1 nephritis, interaction between ECs and MCs is considered to be important in the capillary growth [8]. In the development of the embryonal kidney, mice deficient for platelet-derived growth factor (PDGF)-B lack microvascular pericytes and MCs, and develop numerous capillary microaneurysms in the glomeruli, which rupture at late gestation, suggesting that MCs contribute to the formation and mechanical stability of capillary walls [9].

In the present study, we hypothesized that MCs have an ability to promote vascularization. To address our hypothesis, we attempted to develop a three-dimensional co-culture system in vitro and to analyse the cellular interaction between MCs and ECs. This system allowed us to visualize and manipulate developing vasculatures in an ongoing process. Using this three-dimensional co-culture system, we demonstrated that MCs promote EC differentiation into a capillary-like microvasculature.



   Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Cells
ECs were isolated from the human umbilical cord vein by collagenase treatment as described previously [10]. The cells were cultured on type I collagen-coated dishes in RPMI 1640 (Sigma, St Louis, MO) supplemented with 5% fetal bovine serum (FBS; Sigma), 50 µg/ml EC growth supplement (Harbor Bio-Products, Norwood, MA) and 20 U/ml heparin (Sigma). The ECs were studied between the third and fourth passages. Human MCs at the third passage were purchased from Clonetics (San Diego, CA) and cultured in RPMI 1640 supplemented with insulin–transferrin–selenium (Gibco-BRL, Gaithersburg, MD) and 10% FBS. The MCs were studied between the fourth and sixth passages. All cells were incubated at 37°C in 98% humidified air containing 5% CO2.

Fluorescence labelling of living cells
To visualize and discriminate each cell type, cells were labelled with two different fluorescent membrane dyes, PKH2 and PKH26 (Zynaxis, Malvern, PA), with excitation peaks at 490 and 551 nm and emission peaks at 504 and 567 nm, respectively. MCs were harvested by treating with trypsin (Gibco-BRL) for 5 min at room temperature and washing twice in RPMI 1640. Three million cells were resuspended in 1 ml of diluent A according to the manufacturer's instructions. A 7 µl aliquot of PKH2 dye solution (1.0 mM in ethanol) was diluted with 1 ml of diluent A. The cell suspension in diluent A was then added to the diluted dye solution to obtain a final dye concentration of 3.5 µM. After incubating for 10 min at room temperature, 2 ml of FBS and 4 ml of RPMI 1640 with 5% FBS were added to stop the staining process. The labelled MCs were washed three times with RPMI 1640 and resuspended in serum-free media to a cell density of 1.0 x 105 cells/ml. The ECs were washed twice in RPMI 1640 and then labelled with 4 µM PKH26 in diluent C, similar to the MC labelling procedure with PKH2.

Three-dimensional co-culture system
The MCs were cultured in serum-free medium for 48 h prior to the experiments. The ECs were more sensitive to serum-free conditions in a regular plate, and they were therefore kept serum free for 12 h prior to the experiments. The fluorescent-labelled cells were washed three times with RPMI 1640 before co-culturing in basement membrane reconstruct gel (Matrigel). Matrigel is a soluble basement membrane extract of the Engelbreth–Holm–Swarm mouse sarcoma that commonly contains some growth factors [11]. In the current study, we used growth factor-reduced Matrigel (Becton Dickinson, Mountain View, CA) that was depleted of a variety of growth factors (basic FGF, epidermal growth factor, insulin-like growth factor-1, PDGF and nerve growth factor) except for transforming growth factor-ß (TGF-ß), in order to minimize the effect of growth factors in the gel and to determine the effect of MCs on EC capillary formation. The ECs and MCs were adjusted to 1.0 x 105 cells/well, except in the titration study. In the direct contact co-culture system, MCs and ECs were mixed and then plated onto 300 µl of Matrigel with 400 µl of serum-free medium in 24-well plates. In the non-contact co-culture system, ECs were cultured in Matrigel, whereas MCs were cultured within intercup chambers (Kurabo, Japan) in order to prevent physical contact. Cells were observed under a fluorescent microscope (Nikon, Japan) and a confocal laser scanning microscope (Bio-Rad, Richmond, CA). The nuclei were stained with Hoechst 33342 (bisbenzamide at 10 µg/ml, Sigma) for 10 min just before taking the pictures.

In some experiments, the effect of anti-VEGF antibody on EC capillary formation was examined. Goat anti-human VEGF165,121 IgG (R&D Systems, Inc., Minneapolis, MN) or control goat IgG (Sigma) was added to the co-culture system at 100 ng/ml. This anti-VEGF antibody neutralizes the bioactivity of recombinant human VEGF (rhVEGF) in a dose-dependent manner with 100% neutralization at 10 ng/ml [1].

Quantification of EC network formation
Photomicrographs were taken at 0, 3, 6 and 9 h after co-culture and the total length of the PKH26-stained tube-like structures was measured by an image analyser as previously described [12]. The tube structures were defined as ≥3 cells which are connected lengthwise. Photomicrographs were taken in random in order to avoid bias of measurements. Four fields for every three wells were measured, and the total length per field was calculated.

Examination for capillary tube lumen formation
For assessing capillary tube lumen formation, the gels in which the ECs and MCs were co-cultured for 6 h were fixed in 10% formalin for 4 h at room temperature. They were then embedded in paraffin and serially sectioned for staining with haematoxylin and eosin for a light microscope. The sections were also stained with rabbit anti-von Willebrand factor (vWF) antibody (Sigma), followed by rhodamine isothiocyanate-conjugated anti-rabbit IgG (Sigma) for a fluorescent microscope.

Enzyme immunoassay for VEGF
The VEGF concentration in the culture supernatant was measured using a sandwich enzyme immunoassay kit (Quantikine human VEGF immunoassay kit, R&D Systems, Inc.) according to the manufacturer's instruction.

Immunofluorescence staining of VEGF in aco-culture system
We also attempted to confirm the expression of VEGF by cells in a co-culture system by immunofluorescence. In this experiment, PKH26-labelled MCs and unlabelled ECs were used. At 6 h after co-culture, the medium was discarded and cells were washed briefly with Tris-buffered saline (TBS). Cells were fixed in 2% paraformaldehyde for 10 min at room temperature. After washing for 10 min with TBS, fixed cells were blocked with blocking buffer [1 x TBS, 0.1% Triton X-100, 0.5 M NaCl with 3% bovine serum albumin (BSA)] for 30 min and incubated with primary antibody (anti-human VEGF, Santa Cruz Biotech, Inc., Santa Cruz, CA) diluted in antibody buffer (1x TBS, 0.1% Triton X-100 with 3% BSA) overnight at 4°C. After washing with TBS for 10 min, the fixed cells were then treated with blocking buffer for 10 min. The secondary antibody [fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG; Sigma] was diluted in antibody buffer (1:400) and added to the chamber slides for 1 h at room temperature. Finally, the fixed cells were washed for 30 min with TBS, and their nuclei were counterstained and mounted. For all immunohistochemical studies, the primary antibody was omitted as negative control.

Western blot analysis for VEGF receptor expression in ECs
The ECs were washed twice with ice-cold phosphate-buffered saline and harvested using a cell scraper. The cells were lysed in 50 mM Tris–HCl with 1% SDS and 1% ß-mercaptoethanol. Lysate was then boiled for 1 min for protein denaturation. Glycerol was added to a final concentration of 10%, and bromphenol blue was added before loading onto a 7.5% SDS–polyacrylamide gel. After electrophoresis, the separated proteins were elecrotransferred onto a nitrocellose membrane. The membrane was blocked in 2% non-fat milk in TBS, pH 7.6, with 0.1% Tween 20 (TBS-T) for 1 h at room temperature with shaking. The membrane was then incubated for 1 h with a mouse anti-fetal liver kinase-1 (flk-1) monoclonal antibody (Santa Cruz Biotech), extensively washed in TBS-T, then incubated with horseradish peroxidase-linked antibody (Amersham Biosciences, Freiburg, Germany). Signal was visualized by a chemiluminescent kit (Pierce Biotechnology, Inc., Rockford, IL).

Statistical analysis
The results in this study were expressed as the mean±SD. We compared the data in the time course of capillary formation and the MC ratio for the EC network formation by using a repeated measure ANOVA. Statistical comparisons at each measurement time and at each MC ratio were made by one-way ANOVA. The data on the inhibition of EC network formation by anti-VEGF antibody were compared by an unpaired t-test. All statistical analyses were performed using the StatView 5.0.1 software program (Abacus Concepts, Berkeley, CA). Statistical significance was defined as P<0.05.



   Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
On regular plastic plates, ECs have barely exhibited capillary-like network formation [12]. In the Matrigel that contains soluble basement membrane extract and allows three-dimensional culture, ECs are reported to exhibit capillary-like network formation in certain conditions [12]. Therefore, we attempted to establish an EC and MC co-culture system using Matrigel in order to examine the role of MCs for EC vascularization. When cultured alone in the growth factor-reduced Matrigel, ECs barely formed capillary structures 6 h after plating (Figure 1A). MCs alone elongated their processes slightly, but hardly showed tubule formation (Figure 1B). However, ECs co-cultured with MCs in a contact condition displayed dramatically increased network formation (Figure 1C and D). Within 2–4 h, MCs started to migrate and contact ECs. ECs began to align themselves end-to-end and elongated. At 6 h after plating, the ECs showed abundant networks of branching and anastomosing cords of cells. Most MCs had already contacted ECs by 6 h. Cross- and longitudinal sections of these cords showed a central lumen capillary-like formation (Figure 2). Cells around a lumen were stained with anti-vWF antibody, confirming that these cells were ECs. Under a confocal microscope, MCs elongated their fine processes and contacted ECs as if they supported the capillary-like structure (Figure 3).



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Fig. 1. Single culture and contact co-culture on Matrigel. (A) Endothelial cells (ECs) cultured alone for 6 h. ECs were labelled with red fluorescence dye. ECs barely formed capillary structures (x200). (B) Mesangial cells (MCs) cultured alone for 6 h. MCs were labelled with green fluorescence dye. MCs elongated slightly but did not form networks (x200). (C) Tubular-like network formation observed in the co-culture system (x40). (D) MCs and ECs co-cultured in the contact conditions for 2, 4 and 6 h. MCs started to migrate and contacted ECs at 2 h. Marked elongated EC network formation was observed at 6 h (x100).

 


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Fig. 2. Endothelial cell (EC) tube formation. (A) Vertical section of an EC network. Capillary-like tube formation was observed (haematoxylin and eosin, x400). (B) A longitudinal section of an EC network. The section was stained with anti-von Willebrand factor antibody (x200).

 


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Fig. 3. Mesangial cells (MCs) contacted endothelial cells (ECs) with their bodies and fine processes. MCs and ECs were co-cultured in the contact condition for 6 h (x600).

 
We next studied the humoral effect of MCs on EC network formation in a non-contact co-culture system using an intercup chamber that separated MCs from ECs. The endothelial capillary-like structures were also formed in the non-contact co-culture condition but were significantly shorter than in the contact co-culture (Figure 4). To examine the quantitative effect of MCs for vascularization, ECs were co-cultured with different ratios of MCs. By decreasing the ratio of MCs to ECs, the EC network formation was significantly reduced in both co-culture systems (Figure 5).



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Fig. 4. Time course of capillary-like network formation. The lengths of the networks were measured at 3, 6 and 9 h. Endothelial cells (ECs) alone ({blacktriangleup}) hardly formed networks. The network formation in the contact co-culture condition () was significantly accelerated compared with that in the non-contact condition ({bigcirc}). Data are expressed as mean±SD. *P<0.05 vs ECs alone; **P<0.001 vs ECs alone, {dagger}P<0.01 vs non-contact co-culture.

 


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Fig. 5. Effect of the endothelial cell (EC) to mesangial cell (MC) ratio on EC network formation. ECs were co-cultured with different ratios of MCs. The ratios of co-cultured MCs to ECs were 1 (EC:MC = 1:1), 0.5 (1:0.5) and 0.25 (1:0.25). Decreasing the ratio of MCs to ECs reduced EC network formation in both contact (black bars) and non-contact (white bars) conditions. Data are expressed as the mean±SD. The grey bar represents ECs alone. *P<0.05; **P<0.01; ***P<0.001.

 
VEGF is an important growth factor that regulates angiogenesis [1,4,5]. Therefore, we wanted to determine the role of VEGF in EC network formation. We first studied the VEGF production from MCs and ECs by enzyme-linked immunosorbent assay (ELISA) and immunofluorescent study. VEGF was detected in the culture supernatant of MCs (34±2.2 pg/ml, at 6 h), but not of ECs (below the assay limit of detection; <5 pg/ml). By immunofluorescent study, VEGF was expressed primarily by MCs (Figure 6). The expression of VEGF receptor (flk-1) by ECs was confirmed by western blotting (Figure 7).



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Fig. 6. Immunofluorescence staining of vascular endothelial growth factor (VEGF) in co-cultured endothelial cells (ECs) and mesangial cells (MCs). (A) MCs pre-labelled by red fluorescent dye. (B) VEGF expression visualized by anti-VEGF antibody. (C) Hoechst 33342 staining for nuclei. (D) Merged image. (E) Phase contrast image. Network-formed ECs are indicated by arrowheads.

 


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Fig. 7. Vascular endothelial growth factor (VEGF) receptor is expressed in endothelial cells (ECs). Cell lysate from human umbilical vein ECs was subjected to immunoblotting analysis using anti-flk-1 antibody.

 
As shown in Figure 1, ECs cultured alone in growth factor-reduced Matrigel hardly exhibited network formation. To examine the direct effect of VEGF, rhVEGF (1 ng/ml) was added to ECs cultured alone in Matrigel. VEGF stimulated EC network formation (3.5±0.36 mm/mm2 vs 0.21±0.06 mm/mm2, ECs + rhVEGF vs ECs alone, at 6 h).

Finally, we determined the role of VEGF on EC network formation in the three-dimensional co-culture system. Blocking VEGF by its neutralizing antibody significantly but partially inhibited EC network formation induced by MCs (84.9% reduction in contact and 84.5% reduction in non-contact co-culture at 6 h) (Figure 8).



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Fig. 8. Inhibition of endothelial cell (EC) network formation by anti-vascular endothelial growth factor (VEGF) neutralizing antibody. EC network formations in the contact co-culture (black bars) and in the non-contact co-culture (white bars) conditions were significantly but partially inhibited by goat anti-VEGF antibody (100 ng/ml). ECs stimulated by recombinant human VEGF alone (1 ng/ml) (grey bars) were almost completely inhibited. Data are expressed as the mean±SD. *P<0.001.

 


   Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In order to investigate the role of MCs for EC vascularization, we developed a convenient MC and EC co-culture system which allowed us to observe MC–EC interactions easily and achieve a rapid quantification of neovascularization. Using this co-culture system, we clearly demonstrated the ability of MCs to induce the differentiation of ECs into capillary-like structures. Previous reports showed that interaction of ECs and MCs is important in the revascularization process during the recovery from glomerular injury and in normal capillary formation and stabilization during the development of glomeruli [8,9]. Though our three-dimensional co-culture system does not completely reproduce the vascularization during recovery from glomerular injury or development of glomeruli, our data indicate that MCs potentially possess an ability to help ECs differentiate toward vascularization.

In the current study, we also showed that the contact interaction of MCs with ECs markedly enhanced vascular formation as compared with the non-contact interaction. Anatomically, MCs are adjacent to ECs in vivo and the contact of MCs with ECs is clearly observed during the revascularization process of glomerular capillaries in Thy-1 glomerulonephritis [8]. There are two possible reasons why contact interaction of MCs with ECs is more effective for vascularization. The first is that local concentrations of angiogenic factors secreted from MCs would be higher in the contact condition. Another reason is that signalling pathways of angiogenic factors in ECs would be enhanced under physical contact with MCs. Signals from adhesion molecules are reported to enhance angiogenesis [13].

Though a contact interaction effectively enhanced EC vascularization, non-contact interaction also increased capillary formation. This result indicates that humoral factors that promote ECs to form networks are secreted by MCs. In the present study, we demonstrated that VEGF is secreted by MCs and acts as a potent molecule for endothelial differentiation and capillary formation. VEGF is reported to be important for the development and repair of renal glomerular capillaries [14–16]. Neutralizing VEGF antibodies interrupted postnatal murine glomerular capillary development [14], and an antagonist of VEGF inhibited glomerular capillary repair in a recovery model of Thy-1 glomerulonephritis [15]. Moreover, in vivo administration of VEGF stimulated growth of embryonic kidney explants [14] and enhanced capillary repair in severe glomerulonephritis [15,16]. Thus, VEGF is an essential molecule for kidney development and repair. In normal human and rat kidneys, VEGF expression is confined to Bowman's capsule, podocytes, distal duct epithelia and collecting duct epithelia [15]. However, VEGF synthesis has also been demonstrated in activated MCs during mesangial proliferative glomerulonephritis [15]. In addition, MCs have been reported to produce VEGF in vitro [17]. We also confirmed VEGF production from MCs in our system. These data suggest that in pathological conditions, not only podocytes but also MCs would be a source of VEGF. Though VEGF would be a crucial factor in our system, blocking VEGF by anti-VEGF neutralizing antibody could not inhibit EC network formation completely in either the contact or non-contact co-cultures. This finding suggests that other angiogenic factors from MCs stimulate endothelial differentiation. It has been reported that activated MCs secrete PDGF [18] and FGF [19], though we have not determined the levels of these factors in our system.

In summary, we demonstrated that MCs regulate endothelial differentiation toward vascularization. The VEGF produced by MCs is one of the important angiogenic factors in this process. Moreover, contact interaction of MCs with ECs significantly enhanced capillary network formation. However, we are lacking in knowledge about how MCs signal the ECs. This three-dimensional co-culture system could be a useful tool allowing the further understanding of the role of MCs in vascularization.

Conflict of interest statement. None declared.



   References
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 Introduction
 Materials and methods
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 Discussion
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Received for publication: 28. 1.04
Accepted in revised form: 6.10.04





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