Mesangial cells stimulate differentiation of endothelial cells to form capillary-like networks in a three-dimensional culture system.
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.
Kitahara
Tokuyuki
T
Department of Medicine and Clinical Science, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan. tkita1@jcom.home.ne.jp
Hiromura
Keiju
K
Ikeuchi
Hidekazu
H
Yamashita
Shin
S
Kobayashi
Satsuki
S
Kuroiwa
Takashi
T
Kaneko
Yoriaki
Y
Ueki
Kazue
K
Nojima
Yoshihisa
Y
D000704:Analysis of Variance; D015153:Blotting, Western; D002454:Cell Differentiation; D002478:Cells, Cultured; D018920:Coculture Techniques; D042783:Endothelial Cells / Q000502:physiology*; D004730:Endothelium, Vascular / Q000166:cytology*; D005920:Glomerular Mesangium / Q000166:cytology*; D006801:Humans; D018919:Neovascularization, Physiologic / Q000502:physiology*; D011336:Probability; D014471:Umbilical Veins / Q000166:cytology; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*
D042461:Vascular Endothelial Growth Factor A