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From the Departments of Ophthalmology and Cell Biology/Anatomy, Mount Sinai School of Medicine of New York University, New York, New York.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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METHODS. Cultured corneal myofibroblasts were plated at 200
cells/mm2, and cells were grown in DMEM/F12 containing (1)
10% FBS or (2) 10% FBS with FGF and heparin or (3) 1% FBS or (4) 1%
FBS with TGF-ß. As distinguished from the fibroblast phenotype, the
myofibroblast phenotype was identified by the assembly of 
-smooth
muscle (SM) actin protein into the stress fiber cytoskeleton.
To further characterize growth factor regulation of the two phenotypes,
the phenotypic expression of TGF-ß receptor types I and II,
cadherins, and connexin 43 by immunocytochemistry, Western blot
analysis, and immunoprecipitation and of -SM actin mRNA in Northern
blot analysis were evaluated.
RESULTS. Corneal myofibroblasts replated and grown in the presence of FGF-1 or
FGF-2 (20 ng/ml) plus heparin (5 µg/ml) in 10% FBS medium had
decreased expression of -SM actin protein, TGF-ß receptors, and
cadherins. Thus, FGF–heparin decreased the myofibroblast phenotype and
promoted the fibroblast phenotype. Administration of either 20 ng/ml
FGF or 5 µg/ml heparin alone was not effective. Addition of TGF-ß
further enhanced the expression of -SM actin mRNA and protein and
cell surface expression of TGF-ß receptors in myofibroblast cultures.
CONCLUSIONS. FGF-1 or -2 and heparin promoted the fibroblast phenotype and reversed the myofibroblast phenotype. This finding supports the idea that corneal myofibroblasts and fibroblasts are alternative phenotypes rather than terminally differentiated cell types.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-smooth muscle (SM) actin and
its assembly into stress fibers. Although both fibroblasts and
myofibroblasts contribute to normal wound repair, in a fully healed
wound few if any myofibroblasts are found.1
It is not
known whether during healing all myofibroblasts undergo apoptosis or if
some myofibroblasts revert to a fibroblast or a keratocyte phenotype.
Pathologic conditions are associated with excess numbers of persistent
fibroblasts or myofibroblasts.2
3
4 To characterize each of these three phenotypes, recent studies have used cultures of freshly isolated keratocytes, passaged fibroblasts, and myofibroblasts. For example, freshly isolated keratocytes have the patterns of expression of integrins and metalloproteinases that correspond to that seen in situ.5 6 7 Keratocytes are activated to fibroblasts by addition of serum to the culture medium.8 9 Fibroblasts express novel integrins, cytokine receptors, cell junction molecules, matrix metalloproteases, and matrix components similar to expression patterns seen in situ after wounding.10 11 12 13 14 15 Myofibroblasts are generated by adding TGF-ß to keratocytes or to fibroblasts plated at an intermediate density or by plating fibroblasts at very low density.8 16 17 18 Associated with the myofibroblast phenotype are differential expression patterns of integrins, metalloproteases, and cell junction molecules.6 19 20
There have been no reports that cytokine treatment of corneal myofibroblasts induces them to "revert" to fibroblasts in vivo or in vitro. Therefore, we have evaluated whether treatment of cultures with FGF and heparin can "reverse" their myofibroblast phenotype. FGFs are members of a family whose core amino acid sequences homology allows for a common FGF tertiary structure. These common cores bind to heparin, resulting in dimerized FGF. Signaling through the FGF receptor is induced by dimerization of the FGF receptors and is promoted by dimerized FGF–heparin (reviewed in Ref. 21 ). FGF-1 and -2 have overlapping specificity for isoforms of the FGF receptors (FGFR-1, -2, -3, and -4; Table 1 in Ref. 21 ). The prototypic members of the FGF family, FGF-1 and -2, can regulate differentiation in various cell types.
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-SM actin as the myofibroblast
signature, we have determined several membrane proteins whose
expression is differentially regulated by FGF and TGF-ß in
proliferating corneal stromal cells in culture. These studies extend
our knowledge of the role of these growth factors in regulating
characteristics of the fibroblast and myofibroblast
phenotypes.17
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Corneal fibroblasts were maintained in culture up to eight passages.
Within 16–18 hr of plating cells at intermediate density
(104 cells/ml or 200 cells/mm2), we induced the
myofibroblast phenotype by adding 0.25–1.0 ng/ml TGF-ß1
in DMEM-F12 with 1% FBS (human platelet TGF-ß1;
Collaborative Biomedical Products, Becton Dickinson Labware, Bedford,
MA or recombinant human TGF-ß1; R&D Systems, Inc.,
Minneapolis, MN). Media plus specific growth factors were replaced
every 2 days. At 3 days, the majority of cells expressed -SM actin
microfilaments, and at 4 to 5 days the cultures were confluent and were
100% myofibroblasts. The proportion of myofibroblasts was quantified
by fluorescence microscopic colocalization of Hoechst-stained nuclei
and immunodetected -SM actin microfilaments (see
Immunocytochemistry, below; Fig. 1
).
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-SM
actin to a minimum (Fig. 2)
. In contrast, we produced a totally
myofibroblast culture when we added TGF-ß, 0.25 ng/ml, in DMEM/F12
medium, 1% FBS. Therefore, two different FBS concentrations served as
controls for specific growth factor addition: cultures were grown in
DMEM/F12 medium, 10% FBS, to compare with FGF–heparin, or in DMEM/F12
medium, 1% FBS, to compare with TGF-ß. Phenotype reversal was
evaluated by Western blot analysis and immunocytochemistry, at
intervals between 3 and 5 days after initial treatment with growth
factors. The cultures were confluent at 5 days.
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-SM Actin mRNA-SM actin protein and its
incorporation into microfilaments, which we detect after 3 to 5 days
(see above). We lysed the cells in TriZol Reagent, following the
manufacturer’s instructions (Molecular Research Center, Inc.,
Cincinnati, OH). Samples were separated on a 1% agarose-formaldehyde
gel and transferred to a Nytran nylon membrane (Schleicher & Schuell,
Keene, NH). The membrane was hybridized with a
32P-labeled cDNA probe corresponding to a unique
111 nucleotide sequence of the 3' untranslated region of rabbit -SM
actin (nucleotides 1114–1335)23
at 37°C for 16 to 18
hours. To normalize the RNA loading in each lane, the same filter was
hybridized in NorthernMax Hybridization Buffer (Ambion, Austin, TX)
with a 32P-labeled plasmid cDNA insert of the
internal standard, 18S ribosomal RNA (DECA templates; Ambion), and
washed using solutions provided by Ambion (NorthernMax Wash Buffer
System).24
25
In each detection, the blot was exposed to
Biomax MS film (Eastman Kodak, Rochester, NY) and developed in an
X-Omat (Eastman Kodak). The autoradiograms were scanned with a BioRad
1650 densitometer (Hercules, CA) and quantified using Image
Quant software (Molecular Dynamics, Sunnyvale, CA).
Western Blot Analysis
At 4 to 5 days after passage, when the cultures were confluent,
cells in 100-mm plates were scraped and suspended in PBS with protease
inhibitors (Boehringer Mannheim, Indianapolis, IN), centrifuged
at 1000 rpm, and then lysed in a small volume of Nonidet P-40 (NP-40)
lysis buffer (0.5% NP-40, 150 mM NaCl, 10 mM tris-acetate buffer, pH
8.0) containing protease inhibitors.14
Protein
concentration of lysates was determined using Micro BCA kit (Pierce,
Rockford, IL). Twenty-microgram samples were electrophoresed in 10%
SDS-PAGE. Proteins were separated by SDS-PAGE on 8% gels and
transferred from gels to nitrocellulose for Western blot analysis
(Schleicher & Schuell). We verified that equal amounts of protein were
loaded in each lane by staining the Western blot with Ponceau-S
(Sigma). Immunoblotting was performed with monoclonal anti–-SM
actin (Sigma), monoclonal anti–pan-cadherin (Sigma), or
affinity-purified polyclonal anti–cadherin-11 (Karen Knudsen,
Lankenau Medical Center, Wynnewood, PA), anti–TGF-ß receptor
I (anti–TGF-ßRII) and anti-TGF-ßRII (V-22 and C-16, respectively;
Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and polyclonal
anti–connexin 43 (Cx43; Elliot Hertzberg, Albert Einstein College of
Medicine, Bronx, NY). Primary antibody was followed by the appropriate
secondary antibodies conjugated to horseradish peroxidase (HRP; Jackson
Laboratories, West Grove, PA) and detected by enhanced
chemiluminescence (ECL; Pierce).
Immunocytochemistry
We identified myofibroblasts by the presence of -SM actin in
stress fibers. Cells were fixed with 3% p-formaldehyde
(Fisher Scientific, Fair Lawn, NJ) in PBS, pH 7.4, for 15 minutes at
room temperature or in absolute methanol for 10 to 15 minutes at
-20°C. Nonspecific binding was blocked with 3% normal serum, and
the cells were incubated with cy3 conjugated to mouse monoclonal
antibodies against -SM actin (Sigma). After rinsing with PBS and a
1-minute exposure to Hoechst 33258 (0.06 µg/ml; Sigma), coverslips
were rinsed and mounted with antifade agent.26
Proliferation
Two cytochemical methods were used to evaluate proliferation.
Although we evaluated the myofibroblast phenotype at 3 and 5 days,
cultures are often confluent at that time and are subject to contact
inhibition. Therefore, we assessed proliferation after 2 days of
cytokine treatment to evaluate the impact of the cytokines on
proliferation before contact inhibition as the cells reach confluence.
To detect DNA synthesis as an index of proliferation, bromodeoxyuridine
(BrdU, final concentration 1 µM; Sigma) was added for the last 5
hours of incubation. Cells were fixed with 3%
p-formaldehyde, DNA was denatured with 3 N HCl, and BrdU
labeling was immunodetected with mouse monoclonal anti-BrdU antibody
(Sigma), followed by anti-mouse IgG conjugated to FITC (Jackson
Laboratories). Cells were counted and scored for presence and absence
of BrdU in their nuclei in 5 or 6 randomly selected fields in each
sample (minimum of 243 cells/coverslip were counted). Two independent
proliferation studies detecting BrdU were performed, the results were
averaged, and the mean was determined for each of the two experiments.
As an independent index of proliferation, fixed cells were immunodetected with mouse monoclonal anti-Ki67 (Sigma) followed by anti-mouse IgM-FITC. Ki-67 is a nuclear proliferation factor expressed at all stages of the cell cycle except G0.27 Nuclear staining by Hoechst dye 33258 detected essentially no apoptosis after 2 days in any of the growth conditions.28
Cells were viewed with an epifluorescent Zeiss Axiophot (Thornwood, NY) and photographed on Kodak TMAX 400 film.
Detection of Cell Surface Expression of TGF-ß Receptors
As in the proliferation studies, we detected cell-surface
expression of TGF-ß receptors 2 days after plating. Cell surface
proteins were detected by incubation of cultures with NHS-sulfo biotin,
0.5 mg/ml, for 30 minutes at 0 to 4°C (Pierce). NP-40 lysates with
equalized protein were precleared with normal rabbit serum (Life
Technologies). TGF-ßRI and -RII were immunoprecipitated using
polyclonal receptor specific antibodies (described above; Santa Cruz).
Antibody–antigen complexes were captured with Protein A agarose beads
(Life Technologies) and eluted by boiling in SDS.14
Ten
microliters of each immunoprecipitate was electrophoresed and
transferred to nitrocellulose as described above. Biotinylated cell
surface TGF-ß receptors were detected with streptavidin-HRP (Jackson
Laboratories) followed by ECL.14
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-SM Actin Protein
Expression in Myofibroblast CulturesFigure 1 illustrates the myofibroblastic nature of such a persistent myofibroblast culture (passage 5), fixed and immunodetected 3 days after passaging at intermediate density. Similar to its progenitor culture, the majority of cells are myofibroblasts (Fig. 1A) .
We investigated the ability of FGF to induce fibroblast differentiation
of these persistent myofibroblast cultures, "reversing" the
myofibroblast phenotype. In Western blot analysis the myofibroblastic
nature of the cultures was confirmed by -SM actin expression in
cells grown in 1% or 10% FBS (Fig. 2)
. TGF-ß treatment of the cells increased the immunodetectable -SM
actin protein (Fig. 2)
. In contrast, decreased -SM actin expression
was seen in lysates of cells incubated with 5 µg/ml of heparin plus
FGF-1 or -2 (Fig. 2)
. By immunocytochemistry, we found that the
majority of cells were fibroblasts after 3 days of FGF–heparin
treatment (Fig. 1D)
. Treatment with either 20 ng/ml FGF (Fig. 1B)
or 5
µg/ml heparin alone (Fig. 1C) did not achieve this. On the
basis of the protein and the immunocytochemical data, we used 20 ng/ml
FGF-2 and 5 µg/ml heparin (FGF–heparin) in all subsequent
experiments. FGF-1 with heparin was equally effective in inducing the
fibroblast phenotype.
Proliferation, as indicated by incorporation of BrdU, was stimulated by serum, and even more by FGF–heparin in 10% FBS (Table 1) . The lowest incorporation of BrdU was found in cells grown in 1% serum with or without 0.25 ng/ml TGF-ß. Serum and growth factor effects were confirmed in separate coverslips stained for Ki-67 (data not shown).
In some experiments in which the myofibroblast phenotype was induced by 1 ng/ml TGF-ß treatment, we found that replated myofibroblast cultures lacked immunodetectable Ki67 and therefore were in G0. FGF and heparin addition could not reverse the myofibroblast phenotype when the majority of myofibroblasts were in G0, further suggesting that the ability to reverse phenotype is linked to the ability to proliferate.
Serum Factors Influence Growth Factor Induction of Phenotype
As seen in Figure 2
, persistent myofibroblast cultures maintained
their myofibroblast phenotype when grown in the presence of either 1%
or 10% FBS without addition of specific growth factors. However, the
effect of FGF–heparin was dependent on the concentration of serum in
the culture medium and was most effective in promoting the fibroblast
phenotype in 10% FBS. In fact the FGF–heparin–treated cultures in
10% FBS were fibroblastic even if TGF-ß was added. In contrast,
myofibroblast induction by TGF-ß was more effective in low serum (1%
FBS), and in contrast to cultures in 10% FBS, cultures in 1% FBS
remained approximately 50% myofibroblasts even when FGF-2 and heparin
were added. Thus, the myofibroblast phenotype was optimized by TGF-ß
in 1% FBS, and the fibroblast phenotype was optimized by FGF–heparin
in 10% FBS.
TGF-ß Receptor Expression Is Influenced by Serum and Growth
Factors
We confirmed our earlier finding that TGF-ßRI and -RII were
highly expressed in myofibroblasts22
(Fig. 3B
, lanes 1, 3, and 4). In contrast, FGF–heparin treatment decreased both
total and cell surface expression of TGF–ßRI and -RII as well as
decreasing -SM actin (FGF-hep in Figs. 3A
and 3B ).
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-SM actin expression. In contrast, treatment of the replated cells
for 3 days with FGF–heparin induced fibroblastic cultures that
expressed less cadherin than the TGF-ß–treated cultures (Fig. 4
,
lane 2).
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TGF-ß Induced -SM Actin mRNA Expression in Myofibroblasts
We evaluated the impact of replating and TGF-ß or FGF–heparin
on -SM actin mRNA (Fig. 5)
. As we had for protein expression, we also evaluated the impact of the
concentration of FBS, 1% and 10%. Thus, we prepared RNA from
myofibroblasts under various growth conditions: (1) 10% FBS alone or
with (2) FGF-2 and heparin, (3) heparin alone or (4) FGF alone, and (5)
1% FBS alone or with (6) recombinant TGF-ß or (7) human platelet
TGF-ß.
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-SM actin mRNA
(Fig. 5A)
. A 1.3-kb band corresponds to the predicted size for -SM
actin mRNA (arrowhead), based on the size of -SM actin cited in
GenBank (accession no. NM007392). We normalized for loading differences
by detecting with a cDNA probe for 18S ribosomal RNA (Fig. 5B)
.
Myofibroblast cultures, trypsinized and replated, had minimal
detectable -SM actin mRNA except after TGF-ß. Treatment with
TGF-ß increased the expression of the 1.3-kb band 2.3- and 2.9-fold
(Fig. 1C)
. This contrasts with the finding that cells grown with
heparin or FGF or in 1% or 10% FBS continue to express -SM actin
protein (Figs. 1
and 2)
. This suggests that -SM actin protein is
sufficiently stable to maintain the myofibroblast phenotype after
replating in FBS without an increase in -SM actin mRNA synthesis.
The increase in -SM actin mRNA induced by TGF-ß, compared with
cells grown in all other conditions, could occur either by new
synthesis or by stabilization of -SM actin mRNA. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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–SM actin, cadherins, and TGF-ß receptors. The
reverse, the conversion of fibroblasts to myofibroblasts induced by
TGF-ß1, has been well-documented previously. The
resultant myofibroblasts are characterized by increased expression of
SM -actin, TGF-ß receptors and decreased Cx43
expression.16
20
22 The current studies were initiated because we noted that on occasion myofibroblast cultures arose spontaneously. Typical fibroblast cultures have fewer than 25% myofibroblasts. In contrast, we defined "persistent myofibroblast cultures" as cultures passaged and grown under standard culture conditions that were comprised of >60% myofibroblasts at confluence. It is of interest to speculate on the origin of the spontaneous myofibroblast cultures. In the present study we produced persistent myofibroblasts by adding 0.25 to 1 ng/ml TGF-ß to primary cultures. Thus, we reason that the originating keratocytes in the spontaneously persistent myofibroblast cultures may have been exposed to large quantities of TGF-ß before or early in their isolation.
Our current finding that fibroblast phenotype induction by FGF requires heparin is consistent with previous demonstrations that heparin promotes FGF signaling. Most models of FGF–heparin interaction propose that heparin’s impact is on the binding of FGF to its cell surface receptor.30 Heparin causes oligomerization of FGF and binding of oligomerized FGF to its receptors results in receptor dimerization, thus activating receptor tyrosine kinase and initiating the biological responses to the growth factor.31 It is possible that FGF-2, bound to heparan sulfate proteoglycan, is translocated to the nucleus where signaling may occur.32 Although it has been demonstrated recently that the requirement for heparin may be eliminated by raising FGF concentration to 50 ng/ml, this was not true for the corneal myofibroblasts (data not shown).33
We do not know whether the growth factor–induced changes are part of a cassette of genes that is regulated by each growth factor, if the changes are independent effects, or if they occur sequentially. It is of interest that FGF and TGF-ß, which have opposite effects on the phenotype, signal via different pathways: FGF receptors are tyrosine kinases and TGF-ß receptors are serine/threonine kinases. There is recent evidence that these pathways converge on regulation of the Smad proteins, which are downstream of the TGF-ß receptors.34 35 Smads are 45- to 70-kDa proteins with high sequence similarity to the Drosophila "Mad" proteins at their N- and C-terminal ends. Transcriptional activation by TGF-ß requires translocation of Smad proteins into the nucleus.22 36 Because FGF may induce phosphorylation of residues within the region linking inhibitory and stimulatory domains of Smads, signaling through the FGF receptor may prevent TGF-ß–induced Smad translocation.34
A different interplay between TGF-ß and FGF in fibroblast proliferation has been reported, in which proliferation is promoted by TGF-ß via stimulation of FGF secretion.37 We could not evaluate this in our current studies because the influence on proliferation of 1% versus 10% FBS was as great as the specific growth factor. However, in terms of phenotype regulation, our results indicate that TGF-ß and FGF can induce opposite effects in promoting the myofibroblast or fibroblast phenotype. Future studies are needed to evaluate the intersection of serum effects on cell cycle and impact of these and other growth factors on phenotype regulation.38
Our results reinforce the concept that the myofibroblast is not a terminally differentiated cell. We have previously demonstrated that the phenotype in myofibroblast cultures generated by passaging at very low cell density is reversible: if myofibroblasts generated from low-density passage are passaged at high density, the resultant culture is fibroblastic.18 Our current findings demonstrate that FGF-heparin can induce the fibroblast phenotype in previously myofibroblast cultures. Thus, by controlling cell density, growth factor concentration, heparin and serum content, it is possible to push a population of cells to the fibroblastic or myofibroblastic phenotype and foster wound repair and closure while diminishing scar formation. We do not know whether the myofibroblast-to-fibroblast reversion will occur in situ, but these studies provide evidence that it is possible.
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Acknowledgements |
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Footnotes |
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Submitted for publication January 3, 2001; revised May 23, 2001; accepted June 11, 2001.
Commercial relationships policy: N.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. 
1734 solely to indicate this fact.
Corresponding author: Sandra K. Masur, Department of Ophthalmology, Box 1183, Mount Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029-6574. sandra.masur{at}mssm.edu
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-smooth muscle (-SM) actin during corneal stromal wound healing Invest Ophthalmol Vis Sci 36,809-819-smooth muscle actin expression in granulation tissue myofibroblasts and in quiescent and growing cultured fibroblasts J Cell Biol 122,103-111-smooth muscle actin expression and myofibroblast transformation in cultured corneal keratocytes Cornea 15,505-516[Medline][Order article via Infotrieve]
gene expression in cultured corneal fibroblasts by TGF-ß, dexamethasone, and retinoic acid Invest Ophthalmol Vis Sci 40,887-896This article has been cited by other articles:
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