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Published as doi: 10.1096/fj.07-097014.
 (The FASEB Journal. 2008;22:2151-2160.)
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Distinguishing fibroblast promigratory and procontractile growth factor environments in 3-D collagen matrices

Hongmei Jiang, Sangmyung Rhee, Chin-Han Ho and Frederick Grinnell1

Department of Cell Biology, University of Texas Southwestern Medical School, Dallas, Texas

1Correspondence: Department of Cell Biology, University of Texas Southwestern Medical School, 5323 Harry Hines Boulevard, Dallas, TX 75390-9039, USA. E-mail: frederick.grinnell{at}utsouthwestern.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Understanding growth factor function during wound repair is necessary for the development of therapeutic interventions to improve healing outcomes. In the current study, we compare the effects of serum and purified growth factors on human fibroblast function in three different collagen matrix models: cell migration in nested matrices, floating matrix contraction, and stressed-released matrix contraction. The results of these studies indicate that platelet-derived growth factor (PDGF) is unique in its capacity to promote cell migration. Serum, lysophosphatidic acid, sphingosine-1-phophate (S1P), and endothelin-1 promote stressed-released matrix contraction but not cell migration. In addition, we found that S1P inhibits fibroblast migration and treatment of serum to remove lipid growth factors or treatment of cells to interfere with S1P2 receptor function increases serum promigratory activity. Our findings suggest that different sets of growth factors generate promigratory and procontractile tissue environments for fibroblasts and that the balance between PDGF and S1P is a key determinant of fibroblast promigratory activity.—Jiang, H., Rhee, S., Ho, C.-H., Grinnell, F. Distinguishing fibroblast promigratory and procontractile growth factor environments in 3-D collagen matrices.


Key Words: cell migration • cell contraction • platelet-derived growth factor • lysophosphatidic acid • sphingosine-1-phosphate • wound repair


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
IN 1979, BELL et al. (1) REPORTED THAT fibroblast contraction of type I collagen matrices resulted in formation of tissue-like structures, which were called "dermal equivalents" in anticipation of their use as part of living skin grafts (2) . Subsequent to Bell’s initial observations, three dimensional collagen and fibrin matrices containing fibroblasts and other cell types have been used to study cellular remodeling of connective tissue-like matrices and to characterize iterative cellular adaptation to the changing biomechanical features of the three-dimensional (3-D) matrix environment (3 , 4) . Mechanical remodeling of collagen and other extracellular matrix (ECM) molecules by fibroblasts and myofibroblasts (5) has been implicated in diverse physiological processes including wound repair (6 7 8) , fibrosis (9 10 11) , and malignant transformation (12 13 14) .

3-D matrices differ from two-dimensional (2-D) planar surfaces in a variety of ways, such as substratum stiffness and porosity and the distribution and density of matrix adhesion sites (3 , 6) . Of particular significance, collagen matrices exhibit viscoelastic properties that resemble connective tissues (15 16 17 18) . These viscoelastic characteristics, i.e., the ability of collagen and other molecules within the tissue matrix to stretch and slip past each other and undergo stable reorganization, permit connective tissue remodeling to occur (19) . Surgical procedures take advantage of connective tissue viscoelastic properties when mechanical stretch is used for tissue expansion (20 , 21) . Unlike connective tissue matrices, ECM molecules adsorbed or covalently linked to conventional planar surfaces (e.g., plastic, glass, and polyacrylamide) tend to stay in register with each other and have little capacity to undergo stable, cell-mediated mechanical and molecular reorganization. Consequently, 3-D matrix models permit analysis of cell-matrix interactions that cannot be studied with ECM-coated materials (22) .

The matrix contraction model introduced by Bell et al. (1) often is referred to as floating matrix contraction. The mechanism of floating matrix contraction involves cell motile activity but not cell contraction (23 24 25) . If, however, fibroblasts develop mechanical stress before the matrices are released and are allowed to float in culture medium, then matrix contraction (stressed-released) depends on cell contraction (26 27 28 29) . Floating and stressed-released matrix contraction show different patterns of growth factor stimulation. Serum, platelet-derived growth factor (PDGF), and lysophosphatidic acid (LPA) promote floating matrix contraction, whereas serum and LPA but not PDGF promote stressed-released matrix contraction (30) . Recently, we observed yet another pattern of growth factor regulation in studies on fibroblast migration in nested collagen matrices (dermal equivalents surrounded by cell-free outer collagen matrices). In nested collagen matrices, fibroblast migration is stimulated by PDGF but not by serum or LPA (31) .

Understanding the timing and functions of physiological growth factors in the wound environment is necessary to develop therapeutic strategies to improve healing outcomes (32 33 34 35) . During wound repair, fibroblasts are believed to migrate into the wound region, develop mechanical stress, and undergo contraction (7 , 9 10 11) . The possibility that fibroblast migration and contraction might be regulated by different growth factors was already implicit in the original work that identified PDGF and serum lysophospholipids as activators of the small G proteins Rac and Rho, respectively (36 , 37) . The current study was carried out to investigate the above possibility in more detail, using 3-D matrix assays to compare serum, PDGF, LPA, and several other growth factors that have been shown under diverse conditions and with different cell types to promote matrix contraction, namely, transforming growth factor-β (TGF-β; ref. 38 ), sphingosine-1-phosphate (S1P; ref. 39 ), epidermal growth factor (EGF; ref. 40 ), endothelin-1 (endo-1; ref. 41 ), and basic fibroblast growth factor (bFGF; ref. 42 ).

The results indicate that PDGF is unique in its capacity to promote human fibroblast migration. Serum, LPA, S1P, and endo-1 promote stressed-released matrix contraction but not cell migration. In addition, we found that S1P inhibits fibroblast migration, and treatment of serum to remove lipid growth factors or treatment of cells to interfere with S1P2 receptor function increases serum promigratory activity. Our findings are consistent with the idea that different sets of growth factors generate promigratory and procontractile tissue environments for fibroblasts and that the balance between PDGF and S1P is a key determinant of fibroblast promigratory activity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Type I collagen (3 mg/ml, Vitrogen) was purchased from Cohesion (Palo Alto, CA, USA). Dulbecco modified Eagle medium (DMEM), TRIzol reagent, Superscript II, Opti-MEM I, oligofectamine, Platinum Pfx DNA polymerase, and 0.25% trypsin/EDTA solution were purchased from Invitrogen (Gaithersburg, MD, USA). Fetal bovine serum (FBS) and adult human serum (HS) were purchased from Gemini (Woodland, CA, USA). PDGF (BB isotype) was purchased from Upstate Biotechnology (Lake Placid, NY, USA). Fatty acid-free bovine serum albumin (BSA), LPA, S1P, EGF, endo-1, bFGF, and activated charcoal were obtained from Sigma (St. Louis, MO, USA). TGF-β, Rho kinase inhibitor Y27632, and S1P2 receptor inhibitor JTE-013 were obtained from Calbiochem-Novabiochem (La Jolla, CA, USA). Fluoromount G was obtained from Southern Biotechnology Associates (Birmingham, AL, USA).

S1P and other lipids were removed from serum by charcoal stripping (43) . Ten milliliter serum samples were incubated overnight at 4°C with 1 g of activated charcoal. Samples were centrifuged at 2000 g for 10 min, and the supernatants were filtered using a 0.22 mm cellular acetate filter.

Cell culture and collagen matrix measurements
Use of human foreskin fibroblasts was approved by the University Institutional Review Board (Exemption 4). Early passage human foreskin fibroblasts (hTERT-immortalized; ref. 44 ) were cultured in DMEM supplemented with 10% FBS at 37°C in a 5% CO2 humidified incubator. All experiments were carried out two or more times.

Methods for preparing collagen matrix cultures and measuring floating collagen matrix contraction, stressed-released matrix contraction, and cell migration in nested collagen matrices have been described previously (29 , 31) . Briefly, to measure floating matrix contraction, collagen matrices (200 µl, 1.5 mg/ml collagen, 2x105 cells/matrix) were polymerized for 1 h, then released from the culture surface, and incubated for 4 h floating in basal medium (DMEM+5 mg/ml BSA) and growth factors as shown. To measure stressed-released matrix contraction, collagen matrices as above were cultured overnight in DMEM/10% FBS, rinsed, and then released from the culture surface and incubated for 1 h floating in basal medium and growth factors as shown. At the end of the incubations, samples were fixed with 3% paraformaldehyde in phosphate buffered saline, and the extent of matrix contraction was measured as decrease in matrix diameter.

To measure cell migration using nested collagen matrices, floating matrices were incubated 4 h in DMEM/10% FBS, after which the cell-containing contracted matrices were re-embedded in 200 µl of collagen matrices (1.5 mg/ml collagen) and incubated for an additional 24 h in basal medium and growth factors as shown. At the end of the incubations, samples were fixed as above and then stained with propidium iodine (Molecular Probes, Eugene, OR, USA) to detect cell nuclei. Cell migration index was calculated by counting the average number of cells that had migrated out of dermal equivalents in five x10 microscopic fields selected arbitrarily. Each field included the border of the dermal equivalent (detected by dark field microscopy) and the furthest moving cells (detected by nuclear staining with propidium iodide).

To measure cell migration by scrape wounding, fibroblasts were incubated overnight on collagen-coated coverslips in DMEM/BSA and 0.1% FBS. The samples were scrape wounded with a pipette tip and then incubated an additional 24 h in basal medium and growth factors as shown. At the end of the incubations, samples were fixed and then stained with propidium iodine and with phalloidin to detect actin.

Actin staining, immunofluorescence microscopy, and immunoblotting
Immunostaining of matrices was carried out as described previously (31) . For actin staining, we used Alexa Fluor 488- or 594-conjugated phalloidin (Molecular Probes). Images were collected at 22° with a Nikon Elipse 400 fluorescent microscope using x10/0.45, x20/0.75, and x40/0.75 Nikon Plan Apo infinity corrected objectives (Nikon, Tokyo, Japan), Photometrics SenSys camera (Photometrics, Tucson, AZ, USA), and MetaVue acquisition software (Molecular Dynamics, Downingtown, PA, USA). Subsequent image processing was carried out using Adobe Photoshop 5.5 or 7.0 (Adobe Systems, San Jose, CA, USA).

Dendritic cell index was determined using the MetaVue Integrated Morphometry Analysis function. For each condition, region measurements were made after outlining 20 cells. Dendritic index was calculated as perimeter2/4{pi}xarea. The dendritic index for a round cell is 1.0.

S1P receptor gene silencing and reverse transcriptase-polymerase chain reaction (RT-PCR)
Small interfering RNA (siRNA) silencing of gene expression in human fibroblasts was accomplished as described previously (25) . Primer pairs were designed by and obtained from Dharmacon (Chicago, IL, USA): S1P1 siRNA: 5'-GCUCAAGACCGUAAUUAUCUU-3' and 5'-GAUAAUUACGGUCUUGAGCUU-3'; and S1P2 siRNA: 5'-UUGCCAAGGUCAAGCUGUAUU-3' and 5'-UACAGCUUGACCUUGGCAAUU-3'. To obtain high-efficiency transfection, fibroblast cultures (60–70% confluent) were rinsed with antibiotic-free DMEM and treated with trypsin-EDTA for 1 min to elicit cell rounding but not detachment. Subsequently, antibiotic-free DMEM/10% FBS was added to quench the trypsin. After cells were rinsed with antibiotic-free DMEM, they were incubated with 1 ml Opti-MEM I and oligofectamine containing 300 nM siRNA. After 24 h, 1 ml of DMEM/20% FBS was added to the transfection medium and incubations were continued for an additional 36 h, at which time cells were subcultured. Mock-transfected cells were treated with the transfection reagent but no siRNA.

Total RNA was isolated from either mock or siRNA-treated cells using the TRIzol reagent according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1 µg of the RNA in a final volume of 20 µl using 1 µg of random primers and Superscript II reverse transcriptase in the presence of RNase inhibitor (RNase Out, Invitrogen, Gaithersburg, MD, USA) according to the manufacturer’s instructions. To assess mRNA silencing, cDNAs were subjected to PCR analysis with specific sets of primers. For either S1P1 or S1P2 specific amplification, primers were designed as follows: forward S1P1 (nucleotides 501–520): 5'-AACTGACCTCGGTGGTGTTC-3', reverse S1P1 (nucleotides 817–836): 5'-AGTTATTGCTCCCGTTGTGG-3'; and forward S1P2 (nucleotides 90–109): 5'-ACCATGGGCAGCTTGTACTC-3', reverse S1P2 (nucleotide 445–464): 5'-CAGGAGGCTGAAGACAGAGG-3'. PCR was performed with Platinum Pfx DNA Polymerase using Gene Amp PCR system 9700 (Applied Biosystems, Foster City, CA, USA). PCR with GAPDH primers was carried out as an internal control. To ensure that the assay was in the linear range, the cycle number and amounts of RNA were varied.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Growth factor specificity of cell migration and collagen matrix contraction
Figure 1 A compares the ability of diverse growth factors to stimulate floating collagen matrix contraction. The concentrations selected were those shown previously by different laboratories to have agonist activity. PDGF, LPA, and serum (FBS and HS) were the most active in stimulating floating matrix contraction, although all of the growth factors tested appeared to have some agonist effect above basal medium (BSA).


Figure 1
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  		Figure 1. Growth factor and serum stimulation of collagen matrix contraction and fibroblast migration in nested collagen matrices. A) Floating collagen matrix (FMC) was carried out for 4 h in medium containing growth factors and serum at the concentrations indicated. Matrix contraction values shown are the averages ± SD for duplicate samples from 3 separate experiments. Starting matrix diameter = 12 mm. B) Nested collagen matrices were incubated 24 h in medium containing growth factors and serum at the concentrations indicated. Cell migration index values shown are the averages ± SD of duplicate samples from 3 separate experiments. C) Stressed-released collagen matrix contraction (SRMC) was carried out for 1 h in medium containing growth factors and serum as indicated. Matrix contraction values shown are the averages ± SD of duplicate samples. Starting matrix diameter = 12 mm.

Figure 1B compares fibroblast migration in nested collagen matrices using the same growth factors and concentrations as in Fig. 1A . PDGF at 50 ng/ml appeared to be much more promigratory than any of the other growth factors tested including HS and FBS. In HS, the PDGF concentration is ~50 ng/ml (45) . Levels of PDGF in fetal serum are lower (46) . No additional stimulation of contraction occurred for any of the growth factors tested at lower concentrations (up to three orders of magnitude).

Finally, Fig. 1C shows stressed-released matrix contraction in the presence of the different growth factors. In this assay, PDGF had little procontractile activity. Serum, LPA, S1P, and endo-1 were potent agonists even though these growth factors did not stimulate cell migration.

Taken together, the results in Fig. 1 demonstrated that different sets of growth factors act as agonists for floating matrix contraction, stressed-released matrix contraction, and fibroblast migration in nested collagen matrices. PDGF is unique in its ability to promote fibroblast migration in nested matrices but a weak agonist for stressed-released matrix contraction. Serum, LPA, S1P, and endo-1 are potent agonists for stressed-released matrix contraction but not for cell migration. In contrast to stressed-released matrix contraction and cell migration, contraction of floating collagen matrices shows little growth factor specificity.

The results observed in Fig. 1B also suggested that S1P might be an inhibitor of human fibroblast migration in nested collagen matrices because the migration index fell below basal levels. S1P previously has been shown to inhibit fibroblast chemotaxis in transwell migration assays (47 , 48) . Additional experiments were carried out to test the effects of S1P on PDGF-stimulated migration in nested collagen matrices. Figure 2 shows that S1P at 1–10 µM blocked cell migration stimulated by 50 ng/ml of PDGF, whereas adding LPA or FBS over a wide range of concentrations in combination with PDGF had no effect.


Figure 2
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  		Figure 2. S1P inhibits PDGF stimulated cell migration in nested collagen matrices. Nested collagen matrices were incubated 24 h in medium containing growth factors and serum as indicated. Cell migration index values shown are the averages ± SD of duplicate samples.

Growth factor specificity of fibroblast migration in 2-D after scrape wounding
To learn if the overall pattern of growth factor stimulation of migration observed in Fig. 1B was unique to cells in collagen matrices, experiments also were carried out using 2-D human fibroblast cultures. Figure 3 shows images of cells 24 h after scrape wounding, double-stained with phalloidin for actin and propidium iodide to identify cell nuclei. It can be seen from the photomicrographs that PDGF was the most promigratory growth factor and S1P appeared to be inhibitory similar to our observations for cell migration in nested collagen matrices.


Figure 3
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  		Figure 3. Growth factor and serum stimulation of fibroblast migration on collagen-coated coverslips. Human fibroblast cultures were scrape wounded and incubated 24 h in medium containing growth factors and serum as indicated using concentrations shown in Fig. 1 . At the end of the incubations, samples were fixed and stained for actin and propidium iodide (PI). Scale bar = 300 µm.

Differential effects of S1P and LPA on fibroblast dendritic extensions
Fibroblasts in collagen matrix form dynamic dendritic cell extensions. PDGF stimulates their protrusion, which depends on Rac and PAK1 activation. LPA causes their retraction, which depends on Rho and Rho kinase/myosin II activation (25 , 29 , 49) . Figure 4 A shows the appearance of fibroblasts in collagen matrices after 6 h. Consistent with previous studies, cells in basal medium form short dendritic extensions that increase in size and complexity in the presence of PDGF but retract in the presence of LPA. Similar to LPA, addition of serum or S1P resulted in retraction of dendritic extensions. Retraction was Rho kinase dependent since addition of the Rho kinase inhibitor Y27632 during the last 4 h of the incubations blocked retraction. Endo-1 affected fibroblast dendritic extensions similarly as LPA (not shown).


Figure 4
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  		Figure 4. S1P, LPA, and FBS stimulate retraction of fibroblast dendritic extensions in collagen matrices. A) Collagen matrices were polymerized 1 h with 2 x 104 cells/matrix. After polymerization, samples were incubated 6 h in basal medium and 50 ng/ml PDGF, 10 µM LPA, 1 µM S1P, or 10% FBS as indicated with 10 µM Y27632 added during the last 4 h where shown. At the end of the incubations, samples were fixed and stained for actin. B) Same as in A, except samples were subjected to morphometric analysis. Dendritic index values shown are the averages ± SD for 20 cells. C) Collagen matrices were polymerized 1 h with 2 x 104 cells/matrix. After polymerization, half the samples were incubated 6 h in basal medium and 1 µM S1P, 10 µM LPA, and 10% FBS as indicated. After 2 h, half the samples were washed and switched to PDGF-containing medium. At the end of the incubations, samples were fixed and stained for actin.

Inhibiting Rho kinase not only blocked retraction but also permitted dendritic extensions to reprotrude. Reprotrusion in the presence of S1P appeared to be less robust than with LPA or serum in the medium. To quantify this effect, morphometric measurements were made to determine dendritic cell index (perimeter2/4{pi}xarea). Figure 4B shows that dendritic index varied markedly, so much so that it was useful to plot the results on a log scale to help appreciate the differences. Fibroblasts incubated in S1P-containing medium had a dendritic index ~1–2 consistent with the round appearance of the cells (dendritic index=1.0 for a round cell), whereas cells in basal medium had a dendritic index ~20 and PDGF-treated cells had a dendritic index of >100. Cells in LPA and serum-containing medium had dendritic indices ~2–4. In the presence of Rho kinase inhibitor, the dendritic index increased to ~40 for LPA and ~80 for FBS (~20-fold increases). With Rho kinase inhibitor and S1P, the dendritic index increased only to ~5 (<5-fold increase), confirming that S1P not only caused retraction of dendritic extensions but also interfered with reprotrusion of dendritic extensions under conditions in which Rho kinase was blocked.

Figure 4C shows that the effects of S1P were completely reversible. When fibroblasts with dendritic extensions that had retracted for 2 h were washed and switched to PDGF-containing medium, then no differences were observed between cells that had been in LPA or serum compared with S1P. Dendritic extensions appeared to reprotrude completely in each case.

Treatment of serum with activated charcoal increases serum-stimulated cell migration in nested collagen matrices
PDGF is the major promigratory factor in serum (50 , 51) , and serum frequently has been used as an agonist to study cell migration. Lipid growth factor agonists LPA and S1P are also released from platelets and found in serum (52 53 54) . Since S1P appeared to be an inhibitor of human fibroblast migration, we reasoned that the presence of S1P in serum might be acting as an inhibitor to reduce serum promigratory activity. Incubation of serum with activated charcoal can remove S1P and other lipid agonists (43) , which was the method used to identify serum lysophospholipids as activators of the small G protein Rho (37) .

Human and FBS were treated with activated charcoal. Figure 5 A shows that no differences in the Coomasie blue stained profile of polypeptides could be detected after charcoal treatment. Figure 5B shows that activated charcoal treatment removed the activity from serum responsible for retraction of dendritic extensions, and Fig. 5C shows that charcoal-treated serum had much greater promigratory activity than control serum. These findings were consistent with the idea that lipid agonists in serum (e.g., LPA and S1P) were responsible for retraction of dendritic extensions and inhibited human fibroblast migration. Since LPA does not inhibit cell migration as shown in Fig. 2 , S1P was a likely candidate to explain the migration inhibition effect.


Figure 5
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  		Figure 5. Treatment of serum with activated charcoal removes cell retraction-promoting activity and increases serum-stimulated cell migration. A) Coomasie blue-stained SDS-PAGE profiles of HS and FBS before and after activated charcoal treatment (CT). Samples = 20 µg/lane. B) Collagen matrices were polymerized 1 h with 2 x 104 cells/matrix. After polymerization, samples were incubated for 4 h in basal medium with 10% serum or CT-serum as indicated. At the end of the incubations, samples were fixed and stained for actin. Scale bar = 50 µm. C) Nested collagen matrices were incubated 24 h in medium containing 50 ng/ml PDGF, 10 µM LPA, and 10% HS and FBS, control and activated charcoal treated, as indicated. Cell migration index values shown are the averages ± SD of duplicate samples. Comparison by Student’s t test showed P < 0.0001 for CT-HS vs. HS and CT-FBS vs. FBS.

Blocking S1P2 receptor function increases serum-stimulated cell migration in nested collagen matrices
Inhibition of fibroblast migration by S1P has been linked to S1P2 receptors (47 , 48) , and S1P2 receptor function can be blocked with the receptor antagonist JTE-013 (55 , 56) . Figure 6 A shows that addition of JTE-013 prevented S1P but not LPA-dependent retraction of dendritic extensions. Figure 6B shows that JTE-013 blocked the ability of S1P to inhibit PDGF-stimulated migration and increased almost 3-fold the promigratory effect of serum but had no effect on agonist-stimulated floating collagen matrix contraction (Fig. 6C ).


Figure 6
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  		Figure 6. Blocking S1P2 receptors inhibits S1P-dependent cell retraction-promoting activity and increases cell migration in nested collagen matrices. A) Collagen matrices were polymerized 1 h with 2 x 104 cells/matrix. After polymerization, samples were incubated 4 h in basal medium and 1 µM S1P or 10 µM LPA with 10 µM JTE-013 added as indicated. At the end of the incubations, samples were fixed and stained for actin. Scale bar = 100 µm. B) Nested collagen matrices were incubated 24 h in medium containing 50 ng/ml PDGF, 1 µM S1P, and 10% HS with and without 10 µM JTE-013 as indicated. Cell migration index values shown are the averages ± SD of duplicate samples from 2 separate experiments. Comparison of control vs. JTE-013 samples by Student’s t test showed the following values: BSA, 0.230; PDGF, <0.001; S1P, 0.002; PDGF/S1P, <0.001; 10% HS, <0.001. C) Floating collagen matrix contraction was carried out for 4 h in medium containing growth factors and serum as above. Matrix contraction values shown are the averages ± SD of duplicate samples from 3 separate experiments. Starting matrix diameter = 12 mm.

In other experiments, fibroblast S1P1 and S1P2 receptors were silenced using siRNA. Commercial antibodies against S1P receptors were not found to be of suitable specificity to analyze the effects of siRNA knockdown; therefore, RT-PCR was carried out. Judging from RT-PCR, human fibroblasts expressed both S1P1 and S1P2 receptors, but the levels of S1P2 appeared higher. Treatment of cells with S1P2 siRNA decreased expression of both S1P1 and S1P2 receptors (Fig. 7 A), prevented S1P inhibition of PDGF-stimulated migration, and increased serum stimulation of migration (Fig. 7B ). Migration stimulated by PDGF alone was unaffected. Treatment with S1P1 siRNA decreased S1P1 receptors (Fig. 7A ), and the effects on cell migration were less pronounced (Fig. 7B ). Control experiments showed that silencing either S1P1 or S1P2 did not change fibroblast activity in floating collagen matrix contraction (Fig. 7C ).


Figure 7
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  		Figure 7. Silencing S1P2 receptors increases cell migration in nested collagen matrices. A) RT-PCR (30 cycles) showing that S1P1 siRNA inhibited expression of S1P1 but not S1P2 and S1P2 siRNA inhibited expression of S1P1 and S1P2 B) Nested collagen matrices were incubated 24 h in medium containing 50 ng/ml PDGF, 1 µM S1P, and 10% HS using mock-, siRNA1-, and siRNA2-treated cells. Cell migration index values shown are the averages ± SD of duplicate samples from two separate experiments. Comparison of mock vs. siRNA samples by Student’s t test showed the following values for S1P1: BSA, 0.013; PDGF, 0.206; S1P, 0.142; PDGF/S1P, 0.011; HS, <0.001; and for S1P2: BSA, 0.040; PDGF, 0.359; S1P, <0.001; PDGF/S1P, <0.001; HS, <0.001. C) Floating collagen matrix contraction was carried out for 4 h in medium containing growth factors, serum, and cells as above. Matrix contraction values shown are the averages ± SD of duplicate samples from two separate experiments. Starting matrix diameter = 12 mm.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The goal of the current study was to compare the effects of serum and purified growth factors on human fibroblast activity in several 3-D collagen matrix models. We found that PDGF is unique in its ability to promote fibroblast migration but is a weak agonist for cell contraction. By contrast, serum, LPA, S1P, and endo-1 are potent agonists for cell contraction but are weak promoters of migration compared with PDGF. In addition, we found that S1P inhibits fibroblast migration in nested collagen matrices, and treatment of serum to remove lipid growth factors or treatment of cells to interfere with S1P2 receptor function increases serum promigratory activity.

The possibility that there might be distinct promigratory/procontractile growth factor environments for fibroblasts already was implicit in the original studies that identified PDGF and serum lysophospholipids as activators of the small G proteins Rac and Rho, respectively (36 , 37) . PDGF is known as a Rac-dependent (57) promigratory factor for fibroblasts (58) and the major promigratory factor for fibroblasts in serum (50 , 51) . PDGF also stimulates protrusion of fibroblast dendritic extensions. Serum, LPA, S1P, and endo-1 are Rho activators, the contractile activity of which can be blocked by inhibition of Rho kinase activity (28 , 39 , 59) . These agonists all caused retraction of fibroblast dendritic extensions.

Previous work using floating collagen matrices confounded the possibility to distinguish promigratory from procontractile growth factor functions in 3-D matrices because, it turns out, floating matrix contraction is stimulated by both promigratory and procontractile agonists. What Bell et al. (1) called floating collagen matrix contraction represents contraction of the matrix, not the cells (23 24 25) . Indeed, the relative lack of growth factor specificity of floating matrix contraction calls into question whether this process has any direct analogy to wound contraction as originally thought. Stressed-released matrix contraction, on the other hand, depends on cell contraction (26 27 28 29) and offers a model of myofibroblast contractile function (3 , 6 , 7) .

Our observation that S1P inhibits fibroblast migration in nested collagen matrices extends previous work demonstrating that S1P blocks fibroblast chemotaxis in transwell migration assays (47 , 48) . S1P interacts with several heterotrimeric G-protein receptors (53) and affects cells differently depending on their complement of S1P receptors (60 61 62 63) . Inhibitory effects of S1P on fibroblasts have been associated with activation of S1P2 receptors (47 , 48) coupled to Rho stimulation and Rac inhibition. Inhibition of cell migration by S1P cannot simply be attributed to Rho activation and retraction of dendritic extensions since neither LPA nor serum inhibited PDGF-stimulated cell migration even though both agonists activate Rho and retraction of dendritic extensions. Direct measurement of Rho and Rac activity of fibroblasts in collagen matrices is technically difficult given the low cell number and high concentration of collagen with which one has to contend. Nevertheless, our observations on dendritic extensions provide indirect support for the Rho activation/Rac inhibition idea. S1P-treated cells were unable to fully protrude dendritic extensions when Rho kinase activity was blocked, and protrusion is believed to be Rac dependent (25 , 49) .

Inhibition of migration by S1P can explain why serum has much less effect on human fibroblast migration compared with PDGF even though PDGF is the major promigratory factor in serum (50 , 51) . Consistent with this possibility, we found that treatment of serum with activated charcoal, which removes lipid agonists (43) , including those responsible for Rho activation (37) , resulted in an increase in the promigratory activity of serum. Similarly, blocking S1P2 receptor function with JTE-013 prevented S1P inhibition of PDGF-stimulated migration and increased serum promigratory activity.

Previous work showed that deletion of S1P2 receptors from mouse embryo fibroblasts (MEFs) increased serum-stimulated MEF chemotaxis (48) . We found that silencing S1P2 receptors by siRNA blocked S1P inhibition of PDGF promigratory activity and increased serum promigratory activity for the treated cells. In the absence of specific antibodies, receptor silencing was demonstrated by RT-PCR. Based on this method, siRNA silencing of S1P1 receptors also appeared to increase serum promigratory activity, an observation that requires future explanation. Taken together, the findings suggest that S1P-stimulated retraction of dendritic extensions and inhibition fibroblast migration depend on the S1P2 receptor pathway and that the presence of S1P in serum decreases serum promigratory activity for human fibroblasts.

That different sets of growth factors generate promigratory vs. procontractile tissue environments for fibroblasts has important implications with respect to wound repair. It is now well understood that the dynamically changing growth factor environment plays a key role in repair (64 65 66 67) . Until recently (68) , most general reviews of the repair process have tended to ignore the lipid growth factors. These agonists likely are the key activators for fibroblast and myofibroblast contraction. Regulation of fibroblast migration by the balance between PDGF and S1P has additional implications. Conditions that can interfere with appropriate levels or timing of growth factors in the wound environment such as loss of proteinase regulation in chronic wounds (69 , 70) are believed to result in abnormal repair (71 , 72) . In general, it has been assumed that growth factor supplementation should improve wound repair by compensating for insufficient levels of growth factors (32 , 33) . Replenishment of PDGF will be of little consequence, however, if S1P levels in the wound environment are elevated sufficiently so that the environment is unfavorable for fibroblast migration.

In conclusion, our findings support previous work emphasizing the potential importance of the platelet-derived lipid agonists in wound repair (52 , 54 , 68 , 73) . As summarized in Fig. 8 , the lipid agonists LPA and S1P are key factors in determining connective tissue remodeling processes that involve contractile activity of fibroblasts (and myofibroblasts). In addition, the balance between PDGF and S1P is a key determinant of fibroblast promigratory activity in the wound environment. Ironically, most studies on the mechanism of fibroblast migration have been carried out in serum-containing medium using cells in 2-D culture. Pharmacological and other interventions that have been reported to increase cell migration could have resulted from enhancement of the PDGF promigratory pathway or interference with the S1P inhibitory pathway.


Figure 8
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  		Figure 8. Regulation of fibroblast migration and contraction by serum and the tissue environment. Different sets of growth factors generate promigratory vs. procontractile tissue environments for fibroblasts. The balance between PDGF and S1P is a key determinant of fibroblast promigratory activity. See text for other details.


   ACKNOWLEDGMENTS
 
We thank W. Snell and M. Petroll for helpful advice and suggestions. This research was supported by U.S. National Institutes of Health grant GM-31321.

Received for publication September 21, 2007. Accepted for publication January 17, 2008.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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