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. 1998 May 4;141(3):805-14.
doi: 10.1083/jcb.141.3.805.

PDGF, TGF-beta, and heterotypic cell-cell interactions mediate endothelial cell-induced recruitment of 10T1/2 cells and their differentiation to a smooth muscle fate

K K Hirschi  1 , S A Rohovsky, P A D'Amore
Affiliations

PDGF, TGF-beta, and heterotypic cell-cell interactions mediate endothelial cell-induced recruitment of 10T1/2 cells and their differentiation to a smooth muscle fate

K K Hirschi et al. J Cell Biol. .

Erratum in

  • J Cell Biol 1998 Jun 1;141(5):1287

Abstract

We aimed to determine if and how endothelial cells (EC) recruit precursors of smooth muscle cells and pericytes and induce their differentiation during vessel formation. Multipotent embryonic 10T1/2 cells were used as presumptive mural cell precursors. In an under-agarose coculture, EC induced migration of 10T1/2 cells via platelet-derived growth factor BB. 10T1/2 cells in coculture with EC changed from polygonal to spindle-shaped, reminiscent of smooth muscle cells in culture. Immunohistochemical and Western blot analyses were used to examine the expression of smooth muscle (SM)-specific markers in 10T1/2 cells cultured in the absence and presence of EC. SM-myosin, SM22alpha, and calponin proteins were undetectable in 10T1/2 cells cultured alone; however, expression of all three SM-specific proteins was significantly induced in 10T1/2 cells cocultured with EC. Treatment of 10T1/2 cells with TGF-beta induced phenotypic changes and changes in SM markers similar to those seen in the cocultures. Neutralization of TGF-beta in the cocultures blocked expression of the SM markers and the shape change. To assess the ability of 10T1/2 cells to contribute to the developing vessel wall in vivo, prelabeled 10T1/2 cells were grown in a collagen matrix and implanted subcutaneously into mice. The fluorescently marked cells became incorporated into the medial layer of developing vessels where they expressed SM markers. These in vitro and in vivo observations shed light on the cell-cell interactions that occur during vessel development, as well as in pathologies in which developmental processes are recapitulated.

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Figures

Figure 8
Figure 8
Incorporation of 10T1/2 cells into developing vessels. 10T1/2 cells were prelabeled with PKH26, a permanent red fluorescent dye, and seeded onto a cross-linked collagen matrix. The matrix was then implanted subcutaneously into the backs of C57 mice and incubated for 10 d. Tissue from the experimental area was excised, fixed, and immunostained for αSM-actin, SM myosin, and calponin. Fluorescent cells in association with the abluminal vessel surface indicated that 10T1/2 cells (arrowhead) had become incorporated into the newly forming vessels. Counterstaining revealed that some of these cells expressed αSM-actin, SM myosin, and calponin. Importantly, mouse skeletal muscle did not stain with any of the three SM-specific antibodies. Bar, 80 μm.
Figure 1
Figure 1
BAE-induced 10T1/2 cell migration. 10T1/2 cells and BAE were cocultured in an under-agarose assay for 48 h, and then fixed and stained with Coomassie blue. (a) 10T1/2 cells (right) migrated directionally toward the BAE (left). (b) Higher magnification of 10T1/2 cells closest to BAE. 10T1/2 cells were elongated, and extended processes toward the BAE. (c) 10T1/2 cells at the back side of the well, furthest from the BAE, migrated less and as a continuous sheet with no processes. Bars: (a) 800 μm; (b and c) 200 μm.
Figure 2
Figure 2
Effects of neutralizing PDGF antibodies on EC-induced 10T1/2 cell migration. 10T1/2 cells were cocultured in the under-agarose assay with either BAE or media (DMEM/2% CS) as a control. In some experiments, neutralizing antibodies to PDGF-B (5 μg/ml) and PDGF-A (5 μg/ml) were incorporated into the agarose before plating cells at a density of 2 × 104 cells/well in 25 μl of media. Cells were incubated for 48 h and then fixed and stained with Coomassie blue. Distance migrated was measured using a Profile Projector™ (Nikon, Inc., Melville, NY). Baseline migration of 10T1/2 cells cultured alone (vs. media) was ∼100 μm. This value was subtracted from all experimental values and the control was set to zero.
Figure 3
Figure 3
Expression of SM-specific markers in BAE-10T1/2 cell coculture. 10T1/2 cells were incubated alone or in coculture in the under-agarose assay for 4 d with BAE that had been prelabeled with Di-I-Ac-LDL. The cultures were then fixed and immunostained for SM-specific markers. BAE exhibited negligible amounts of (a) αSM-actin, (b) SM-myosin, (c) calponin and (d) SM22α. 10T1/2 cells had low but detectable levels of (e) αSM- actin, and lesser amounts of (f) SM-myosin, (g) calponin, and (h) SM22α. 10T1/2 cells cocultured with BAE had significantly higher levels of (i) αSM-actin, (j) SM-myosin, (k) calponin, and (l) SM22α. BAE (arrows) were visualized within each coculture using fluorescent microscopy (m–p). Bar, 100 μm.
Figure 4
Figure 4
Western analyses of SM markers in BAE-10T1/2 cell cocultures. (a) Comparison of SM-specific protein expression in aortic EC and SMC. (b) Comparison of SM-specific protein expression in 10T1/2 cells in solo culture and 10T1/2 cells in coculture with BAE. 10T1/2 cells and BAE were plated alone or simultaneously in coculture (1:1) and incubated for 48 h. Protein was isolated from all cell populations and subjected to Western blot analyses. 10 μg of total protein was loaded into lanes for solo cultures of BAE, 10T1/2, and bovine aortic SMC (BASMC), and 20 μg of total protein was loaded for the cocultures. Blots were probed with antibodies to αSM-actin, SM-myosin, SM22α, or calponin, and then quantified using scanning computer densitometry.
Figure 5
Figure 5
Effects of TGF-β on 10T1/2 cell phenotype. 10T1/2 cells were cultured in 4-well chamber slides in the absence (a and c) or presence (b and d) of 1 ng/ml TGF-β1 for 24 h. The cells were fixed with 4% paraformaldehyde and immunostained for αSM-actin (a and b) and SM-myosin (c and d). Bar, 40 μm.
Figure 6
Figure 6
Immunohistochemical analysis of the effect of anti-TGF-β on αSM-actin and SM-myosin in BAE-10T1/2 cell cocultures. 10T1/2 and BAE cells were cocultured in the under-agarose assay for 4 d in the presence (b, c, e, and f) or absence (a and d) of a neutralizing antibody to TGF-β (10 μg/ml) and then stained for αSM-actin (a and b) or SM-myosin (e and d). BAE prelabeled with fluorescent Di-I-Ac-LDL for identification in the anti-TGF-β-treated cocultures are shown in c and f. Bar, 40 μm.
Figure 7
Figure 7
Western blot analyses of the effect of TGF-β neutralization on expression of αSM-actin and SM-myosin in BAE-10T1/2 cell cocultures. 10T1/2 and BAE cells were cocultured (1:1) for 48 h in the presence or absence of a neutralizing antibody to TGF-β (10 μg/ml). Protein was isolated from all treatment groups and subjected to Western blot analyses for (a) αSM-actin and (b) SM-myosin and quantified using scanning computer densitometry.
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