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Genomic analysis of smooth muscle cells in 3-dimensional collagen matrix¹

SONG LI², JIANMIN LAO, BENJAMIN P. C. CHEN, YI-SHUAN LI, YIHUA ZHAO, JULIA CHU^*, KUANG-DEN CHEN, TSUI-CHUN TSOU, KONAN PECK and SHU CHIEN³

Department of Bioengineering and The Whitaker Institute of Biomedical Engineering University of California, San Diego, La Jolla, California, USA;
^* Department of Bioengineering, University of California, Berkeley, California, USA; and
Institute of Biomedical Sciences, Taipei, Taiwan, ROC

³Correspondence: Department of Bioengineering and, The Whitaker Institute of Biomedical Engineering, SERF room 228, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0427, USA. E-mail: shuchien{at}ucsd.edu

SPECIFIC AIMS

The interaction of vascular smooth muscle cells (SMCs) with extracellular matrix (ECM) is important for vascular remodeling. The objectives of this study were to profile the genomic remodeling of SMCs in 3-dimensional (3D) collagen matrix and on collagen matrix surface (2D) and to elucidate the signaling events leading to the differential expression profile.

PRINCIPAL FINDINGS

1. SMCs had fewer actin stress fibers, fewer focal adhesions, and a lower proliferation rate in 3D matrix
Human aortic SMCs (HASMCs) were cultured on either 2D or 3D collagen matrix (1 mg/ml) for 24 h and stained for actin and vinculin. Confocal microscopy showed that HASMCs on 2D matrix spread out and had prominent stress fibers, lamellipodia and more focal adhesions (FAs). In contrast, HASMCs in 3D matrix did not spread as much, had fewer actin stress fibers and FAs, and usually had multiple filopodia that followed the collagen fibrils.

To assess proliferation, HASMCs were detached from the matrices, stained with propidium iodide, and subjected to flow cytometry analysis for DNA content. HASMCs in 3D matrix had fewer cells in S phase (5.6±0.6%, P<0.05) than on 2D matrix (9.4±0.8%) and more cells in G0/G₁ phase, suggesting a lower SMC proliferation rate in 3D matrix.

2. Matrix geometry regulates gene expression related to SMC phenotypes and functions
To compare the gene expression of SMCs on 2D and 3D collagen matrices, HASMCs on 2D or 3D collagen matrix were lysed, and the extracted mRNAs were hybridized with DNA microarrays containing 9600 human ESTs. The 3D/2D ratios for each gene from three independent paired experiments were determined, and paired Student’s t test was used to assess the significance of the difference of the mean ratio from unity. Statistical analysis of DNA microarray experiments showed that the 3D/2D ratio of 99 genes was significantly different from 1 (P<0.05) and had a magnitude greater than 2 or lower than 0.5. Of these, 77 genes had 3D/2D ratios greater than 2, and 22 genes less than 0.5. The 3D/2D ratios of some representative genes (with or without significant change) are plotted in Fig. 1 .

View larger version (42K): [in this window] [in a new window]   Figure 1. Analysis of gene expression in SMCs on 2D and 3D collagen matrix with DNA microarray. RNA samples of SMCs from 24 h 2D and 3D cultures were used for microarray experiments. The ratios (3D/2D) of selected genes from the microarray experiments are presented. Bars represent mean ± SE from three independent experiments. *Statistical significance (P<0.05) compared with unity.

Several genes involved in cell cycle regulation were modulated by the matrix geometry. Compared to 2D matrix, the expression of CDKI p21 was higher and that of M phase phosphoprotein 6 was lower in 3D matrix. The results on p21 were confirmed with RT-PCR and real-time RT-PCR. The protein expression of p21 increased, suggesting that the higher level of p21 may be a factor responsible for the decreased cell proliferation in 3D culture. The expression of cyclin A1 was also significantly lower in 3D matrix (P<0.05), though the difference was < twofold (ratio 0.70±0.09). Consistent with these results, the expression of TGFß1, an inhibitor of SMC proliferation, was higher in 3D matrix (1.80±0.34, P<0.05) (Fig. 1) . There was no significant difference in the gene expression of p53 and Bcl2 between 3D and 2D matrices (Fig. 1) .

3D matrix yielded higher levels of gene expression for matrix proteins collagen I and fibrinogen, indicating that SMCs were more synthetic in 3D matrix. Some other genes involved in matrix remodeling such as lysyl oxidase and MMP-1 did not change with matrix geometry (Fig. 1) . There was no significant change in integrin ß1 and ß3 subunits (Fig. 1) . None of the cytoskeleton marker proteins had a > twofold statistically significant change. The microarray results showed that the expression of SM -actin in 3D matrix was lower (0.66±0.03, P<0.05) (Fig. 1) and that the level of ß-actin did not change. The results on collagen I and -actin were confirmed by RT-PCR and real-time RT-PCR. Protein expression of -actin in 3D culture decreased slightly and the collagen synthesis (normalized with DNA content) increased twofold in 3D culture. These results indicate that SMCs had increased matrix synthesis and were less contractile in 3D matrix.

The expression of signaling molecules such as FAK and protein kinase A did not change (Fig. 1) . Small GTPase Rho regulates the formation of actin stress fibers and SMC contraction. RhoC expression was lower in 3D matrix (Fig. 1) . This is consistent with the lower -actin in 3D matrix, and may reflect an autoregulatory mechanism due to the lesser cell spreading and stress fiber formation in 3D matrix. There was no significant difference in HSP70, suggesting the cells in 3D matrix were not stressed differently from those on 2D matrix.

The transcriptional factors c-fos and c-jun regulate the expression of many genes involved in cell proliferation and differentiation. The expressions of both c-fos and c-jun were higher in 3D matrix (Fig. 1) , whereas other transcriptional factors (e.g., CREB binding protein) showed no difference between 3D and 2D matrices (Fig. 1) . The elevated levels of c-fos and c-jun expression may contribute to the changes in expression of some other genes in 3D matrix.

3. Three-dimensional matrix decreased the tyrosine phosphorylation of FAK
Since SMCs in 3D and on 2D matrices had different numbers of stress fibers and FAs, we postulated that cell adhesion-mediated signaling could be modulated by matrix geometry. We found that the tyrosine phosphorylation of focal adhesion kinase (FAK) in 3D matrix was significantly lower (Fig. 2 A), implying that FAK could be an upstream modulator of gene expression in 3D matrix via its change in phosphorylation.

View larger version (21K): [in this window] [in a new window]   Figure 2. The role of FAK in the expression of p21 and collagen I in 3D and 2D matrix. A) Equal amounts of protein lysates of SMCs from 24 h 2D and 3D cultures were immunoprecipitated with a polyclonal antibody against FAK. The precipitated proteins were subjected to IB with PY20 antibody (top panel). The membrane was striped and reprobed with the antibody against FAK (bottom panel). Bar graph on the top shows the relative degree of tyrosine phosphorylation of FAK (p-FAK) after normalization with the amount of FAK (bottom panel); data are presented with the 2D results taken as a reference (=1). B) Effects of FAK overexpression on the mRNA expression of p21 and collagen. HASMCs were transfected with GFP-FAK or GFP (as control). Two days later, the cells were cultured on 2D or in 3D collagen matrix an additional 24 h. The RNA samples of SMCs from 2D and 3D culture were used for real-time RT-PCR to analyze the relative amount of p21 and collagen I. A, B) Bars represent mean ± SE from three independent experiments. *P < 0.05 for the difference between 2D and 3D samples.

4. Overexpression of FAK blocked induction of p21 and collagen synthesis by 3D matrix and enhanced SMC proliferation in 3D culture
To determine whether the decreased phosphorylation of FAK was involved in 3D matrix-induced gene expression, plasmids encoding green fluorescence proteins (GFP) or GFP-FAK were transfected into HASMCs and cultured on 2D or in 3D matrix. The expression of two representative genes, p21 and collagen, as determined by real-time RT-PCR is shown in Fig. 2B . FAK overexpression abolished the increases of p21 and collagen I expression in 3D matrix, suggesting that dephosphorylation of FAK was responsible for the higher expression levels of p21 and collagen I in 3D matrix. Overexpression of FAK also increased DNA content and inhibited collagen I synthesis in 3D culture.

CONCLUSIONS

Results of DNA microarray studies not only provide quantitative information on genomic programming of SMCs in 3D matrix, but also allow the deduction of information on relevant cellular functions. Our microarray data help to define the molecular mechanisms involved in the phenotypic modulation by matrix geometry, as the results suggest that SMCs in 3D matrix are more synthetic, less proliferative, and less contractile. We also identify some unknown genes regulated by the matrix geometry. This information can direct our attention to previously unknown or ignored genes for further analysis of their potential functions. Such investigations can lead to new experimental analysis to unravel the complex regulatory mechanisms in physiological and pathophysiological conditions.

Our data suggest that decreased cell spreading and the consequent dephosphorylation of FAK lead to the up-regulation of p21, and this may be responsible for the lower proliferation rate in 3D matrix. Our findings also suggest that FAK may function as a molecular switch between the cell proliferation and matrix synthesis phenotypes of SMCs. The decrease of FAK activity in 3D matrix decreases cell proliferation and increases collagen synthesis (Fig. 3 ).

View larger version (29K): [in this window] [in a new window]   Figure 3. Schematic diagram showing the hypothesis that compared to 2D matrix, 3D matrix decreases cell-matrix adhesion and FAK phosphorylation. Dephosphorylation of FAK then increases the expression of p21 and collagen I to result in a decrease in proliferation and an increase in matrix synthesis. Cells were taken from actual photomicrographs and molecules were drawn in accordance with the hypothesis.

Our results indicate that the expression of -actin in 3D matrix is only 66% of that on 2D matrix. It was reported that 3D collagen matrix could retard the transition from contractile to synthetic phenotype compared with 2D culture. The discrepancy between these reports and our study could be due to the different experimental systems. For example, other studies used rabbit SMCs and in some cases compared 3D culture in suspension with those on culture dish by histology examination, whereas we grew subcultured human SMCs in adherent collagen gels and compared gene expression in 3D matrix vs. that on 2D matrix. It remains to be determined whether rabbit and human SMCs respond differently to 3D culture in collagen matrix. Some differences of SMC behavior in different species have been demonstrated.

To maintain SMCs in the contractile state, other matrix components (e.g., laminin and elastin) and mechanical forces may be necessary. It has been shown that mechanical forces such as stretch and shear stress can increase the contractile markers of SMCs on 2D matrix, enhance the mechanical property of vessel wall, and mediate release of growth factors from SMCs. It will be interesting to determine whether mechanical forces can enhance the contractile phenotype of SMCs in 3D matrix. Our study provides a basis for manipulating the phenotype of SMCs through modulation of the extracellular matrix.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0256fje; to cite this article, use FASEB J. (November 15, 2002) 10.1096/fj.02-0256fje

² Present address: Department of Bioengineering, University of California, Berkeley, CA, USA.

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