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1 From the Wound Healing Research and Glaucoma Units, Department of Pathology; the 2 Department of Molecular Genetics, Moorfields Eye Hospital & Institute of Ophthalmology, London, United Kingdom; and the 3 University of Florida, Gainesville.
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Abstract |
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METHODS. Application of recombinant human TGF-ß1, -ß2, and -ß3 (range 0–10-8 M) was assessed using several assays of HTF function: fibroblast-mediated collagen contraction, proliferation, and migration.
RESULTS. All three isoforms of TGF-ß behaved in a similar manner in vitro. They each stimulated HTF-mediated collagen contraction, proliferation, and migration with a characteristic concentration-dependent response, with peak activities at 10-9, 10-12, and 10-9 M, respectively, that were significantly different from control (P < 0.05). At concentrations above and below peak activities, HTF activity was reduced, demonstrating biphasic effects of TGF-ß.
CONCLUSIONS. TGF-ß1, -ß2, and -ß3 have similar actions in vitro; this is demonstrated by their effects on several HTF-mediated functions. TGF-ß induces a response in HTF that is concentration-dependent, with different functions being maximally stimulated at different concentrations. This biphasic response highlights the significance of the concentration profile of TGF-ß at the wound site. These findings are important in filtration surgery, where constant changes in the local environment occur due to the passage of aqueous and the wound healing process. The varying levels of TGF-ß in the aqueous and subconjunctival tissues may thus significantly modify the conjunctival scarring response.
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Introduction |
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The growth factor transforming growth factor-ß (TGF-ß) is a multifunctional growth factor found throughout the body, intrinsically involved in the processes of scarring.6 7 8 9 In the eye, of the three human isoforms, TGF-ß2 appears to be predominant10 11 and has been identified as important in the pathogenesis of several ocular scarring diseases (e.g., retinal fibrosis and cataract formation12 13 14 ).
TGF-ß2 is also implicated in glaucomatous disease, with elevated levels of this factor being found in the aqueous of glaucoma eyes, compared with normals.15 In addition, the aqueous appears important in the wound healing response after glaucoma filtration surgery, and compared with other growth factors found in aqueous, TGF-ß is the most potent in stimulating in vitro conjunctival fibroblast functions.16 17 However, these and other studies12 18 19 20 have only investigated TGF-ß1 and -ß2; the role of TGF-ß3 in aqueous is relatively unknown.
Moreover, although TGF-ß1 and -ß2 are known potent stimulators of the scarring response in humans,7 8 9 21 22 23 24 the actions of TGF-ß3 in wound healing are unclear, with evidence in some studies that it may behave very differently from the other two human isoforms and inhibit the scarring response in vivo.7 25 The role of TGF-ß3, compared with TGF-ß1 and -ß2, in the processes of conjunctival scarring has still to be delineated.
TGF-ß production in the eye after surgery is locally derived from tissues and inflammatory cells. However, the profile of TGF-ß activity in the aqueous is probably considerably altered by filtration surgery. Furthermore, the passage of aqueous through the filtration wound site will result in a constantly changing environment. The role of different concentrations of the different TGF-ß isoforms on cells in various extracellular environments thus becomes an important consideration.
We have used several in vitro assays to, first, compare the actions of TGF-ß1, -ß2, and -ß3 on different components of the conjunctival scarring response (specifically fibroblast activity), and, second, assess the effect of varying TGF-ß concentrations on cellular function. All the in vitro cell culture techniques in this study were based on human Tenon’s capsule fibroblasts (HTF). This is because HTF are believed to be the key cells involved in the subconjunctival wound healing response (with a number of essential functions, including: proliferation at the wound site, migration to and contraction of the wound, the synthesis of new extracellular matrix components, and, finally, remodeling of this new matrix to produce a scar). Our studies specifically focused on fibroblast proliferation, migration, and collagen contraction.
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Methods |
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Materials
The exogenous TGF-ßs used in these studies were all recombinant
human proteins. TGF-ß2 and -ß3 were generous gifts of Ciba–Geigy
(Basel, Switzerland) and TGF-ß1 was a gift from Gregory
Schultz (University of Florida, Gainesville). Working solutions of
TGF-ß were prepared in DMEM/1% bovine serum albumin (BSA; Sigma,
Dorset, UK). The concentrations of TGF-ß used in the study were as
follows: 0, 10-15 M, 10-14,
10-13, 10-12, 10-11,
10-10, 10-9, and 10-8 M.
Fibroblast-Mediated Collagen Contraction Assays
One milliliter free-floating collagen type-1 (Sigma) lattices were
prepared using the method previously described.27
Both
three-dimensional (3D) and two-dimensional (2D) models were
investigated. For 3D lattices, 250 µl aliquots of a collagen/cell
suspension mixture (250,000 cells/ml) were pipetted into single 24-well
plates and allowed to polymerize. For 2D models, collagen lattices were
prepared without any cells, after which HTF (in serum-free DMEM) were
placed on the surfaces of the lattices (20,000 cells/48-well plate).
Each lattice underwent treatment with 500 µl/well of different
concentrations of TGF-ß. They were incubated up to 14 days at 37°C
in 5% humidified CO2 in air and medium, and
TGF-ß was replaced every 3 days.
Measurements of the collagen lattice area were performed from digitized images (model VQV-100; Casio, Tokyo, Japan), which were analyzed using ImageTool software (UTHSCSA software, University of Health Sciences San Antonio, Texas). The results from the sextuplicate wells were expressed as mean percentage reduction in surface area (compared with original surface area, in mm2).
Fibroblast Proliferation Assays
The effects of different concentrations of all three TGF-ß
isoforms on fibroblast proliferation were investigated using several
methods: a manual count technique; by demonstrating cell DNA activity
using bromodeoxyuridine (BrdU) and Ki67 immunocytochemistry; and,
finally, using the colorimetric WST-1 assay.
Fibroblast monolayers (from 96-well plate; initial density, 2000 cells/well) were treated with different concentrations of TGF-ß. On days 0, 3, 7, 14, and 30, monolayers were fixed with 100% methanol, stained with hematoxylin for 2 minutes, washed gently, and air-dried. Cells were manually counted in 10 randomly selected high-power fields (magnification, x400). The results for each experiment were expressed as a mean percentage compared with control (with 95% confidence intervals [CIs]).
Fibroblast monolayer and 3D lattices were assessed for BrdU uptake and Ki67 immunostaining. On days 1, 3, and 7 after treatment, HTF were pulsed with 10 mM BrdU (Amersham, Aylesbury, UK) in DMEM/1% BSA for either 4 or 12 hours, after which monolayers were gently washed in phosphate-buffered saline (PBS), fixed in 95% ethanol/5% acetic acid for 30 minutes at room temperature, air-dried, and frozen at -20°C. Three-dimensional collagen lattices were washed in PBS and fixed in 10% formal saline. Bromodeoxyuridine incorporation was demonstrated by immunocytochemistry using a monoclonal mouse anti-BrdU antibody (Amersham) and immunoprecipitation with diaminobenzamine (DAB). For Ki67 assessment, monolayers were fixed in 70% methanol (Fisons) at -20°C for 30 minutes, and 3D lattices were fixed in 10% formal saline, embedded in paraffin wax, and sectioned before being rehydrated in PBS. Specimens were then microwaved to unmask antigenic sites. Immunocytochemistry was performed using a biotin–streptavidin peroxidase technique with a monoclonal mouse anti-Ki67 antibody (Dako, Sussex, UK) and immunoprecipitation with DAB.
Fibroblast proliferation in the monolayer and 2D models was also assessed using the WST-1 (Boehringer–Mannheim GmBH, Mannheim, Germany) assay. On days 0, 3, 6, 9, 14, and 30, 10 µl of WST-1 was added to the 200 µl of medium in each 96-well plate. A standard calibration curve was constructed at each of these time points, by assessing the absorbance of known cell densities set up in triplicate, and determining the logarithmic equation (R2 calculated for each experimental run). The results were expressed as a percentage of mean absorbance compared with control (with 95% CI).
Fibroblast Migration Assays
Cell migration consists of several components, including
chemotaxis,28
chemokinesis,29
and
haptotaxis.30
For the purposes of the present study, both
chemotaxis and chemokinesis were assessed with HTF, using a Transwell
assay system with tissue culture inserts (Costar; High Wycombe, UK) for
24-well plates.
The chemotaxis assay was performed as previously described.31 Cultured HTF in serum-free DMEM were placed in the inner chamber (10,000 cell/well), and serum-free DMEM/1% BSA medium containing TGF-ß1, -ß2, and -ß3 at different concentrations was added to the outer chamber. Cells were left to migrate over a period of 16 hours at 37°C in 5% CO2 in air. Similarly, a checkerboard analysis of migratory activity was set up as previously described,31 32 with different concentrations of TGF-ß in the inner and outer chambers.
For each well, the total number of cells that had migrated to the undersurface of the membrane was calculated. Sets of triplicate membranes were counted. Results were expressed as the mean number of migrated cells (±95% CI) compared with the negative control with only serum-free medium in both chambers (±95% CI).
Statistical Analysis
For each assay, quantitative data were statistically analyzed at
individual time points using a one-way ANOVA (SPSS for Windows; SPSS
Inc., Cary, NC). The observed significance levels from multiple
comparisons were adjusted using the Bonferroni test, with
P < 0.05 indicating significance.
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Results |
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Comparison of TGF-ß concentrations showed significant differences in contraction on days 7, 10, and 14 in 3D models, and on days 3, 7, 10, and 14 in 2D models (P < 0.05). Significant differences were also found between concentrations of 10-12 to 10-8 M in 3D lattices and 10-13 to 10-8 M in 2D lattices, with respect to their stimulation of contraction throughout the course of the study (P < 0.05).
Effect of TGF-ß on HTF Proliferation
TGF-ß stimulated HTF proliferation in monolayer and 2D lattice
models. All isoforms behaved similarly, inducing proliferation in a
biphasic, concentration-dependent manner, with greatest activity at
10-12 M.
In monolayer assays (Fig. 2) , TGF-ß1, -ß2, and -ß3 displayed similar concentration-dependent activities. All three TGF-ß isoforms at concentrations of 10-13 to 10-10 M stimulated proliferation more than control after day 7; and on day 30, all isoforms at all concentrations significantly stimulated HTF proliferation compared with control. For each concentration of TGF-ß, significant differences (P < 0.05) were found between proliferation on days 3, 7, 14, and 30 after treatment. Significant differences were also found between concentrations of 10-14 to 10-9 M with respect to their stimulation of monolayer HTF proliferation throughout the course of the study (P < 0.05).
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Effect of TGF-ß on HTF Migration
TGF-ß stimulated HTF chemotaxis. All isoforms showed similar
concentration-dependent activities in stimulating HTF chemotactic
migratory activity. Maximal stimulation of HTF migration occurred at a
concentration of 10-9 M (Fig. 4)
. All isoforms and concentrations, apart from TGF-ß2
10-12 M, were found to significantly stimulate
migratory activity compared with control.
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Discussion |
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TGF-ß stimulation of fibroblast-mediated collagen contraction
occurred with a biphasic response with peak activity at
10-9 M. These results were similar both for 2D
and 3D lattices. However, previous studies have mainly assessed
TGF-ß1, and there is no work comparing the effects of the three
TGF-ß isoforms on wound contraction. In addition, assays have used 3D
collagen lattices only, and there is but one study, recently reported,
investigating ocular cell-mediated collagen contraction33
and showing a dose-related stimulatory response to 4 x
10-13 to 4 x 10-10
M TGF-ß1. Similar results have been obtained with BHK-21 (infant
hamster kidney), 3T3-L1 (mouse embryo) fibroblasts, and human foreskin
fibroblasts.34
35
In an experiment comparing TGF-ß2 to
the TGF-ß1 isoform in relation to their ability to contract rabbit
dermal fibroblast–populated collagen gels, Pena et al.36
showed equal effects with 2 x 10-10 M of
each isoform. The mechanism by which TGF-ß may stimulate collagen
contraction may via its effect in altering the expression of the
integrin family of cell adhesion receptors, specifically the
2ß1
receptor37
38
and 1,2,3,5 and
ß1 subunits.39
Our results demonstrate that all three isoforms of TGF-ß produce a similar effect in stimulating HTF proliferation in a concentration-dependent manner, with maximal activity at 10-12 M. This occurs in both monolayer and 2D matrix models, with no proliferation detected in the 3D model. In one of the few studies assessing effects of TGF-ß on ocular cell proliferation, Kay et al.40 recently compared the three TGF-ß isoforms on HTF. They demonstrated that all isoforms behaved similarly, with stimulation of HTF proliferation in a dose-dependent manner, although they studied a much narrower range (4 x 10-12 to 4 x 10-10 M), and hence did not establish a biphasic response. TGF-ß1 and -ß2 have been shown to have a similar dose-dependent effect on human foreskin and dermal fibroblasts,41 although some authors suggest that TGF-ß on its own in serum-free medium has no effect on fibroblast proliferation.42 Compared with a variety of "stressed," attached collagen gels, cell proliferation in a "relaxed," free-floating 3D collagen matrix has been shown not to significantly occur,43 44 45 although this phenomenon is controversial.42 46 TGF-ß–induced proliferation has also been demonstrated in C3H 10T (mouse embryonic fibroblasts),47 NRK-49F (rat kidney fibroblasts), and AKR-2B (mouse embryo-derived) fibroblasts.48
Fibroblast proliferation induced by TGF-ß may occur via its modulation of c-fos, c-myc, and c-sis expression49 50 or its induction of cyclin D and strong downregulation of p27 expression, leading to passage from G1 to S phases of the cell cycle.51 Kay et al.40 have suggested that TGF-ß may have an indirect mitogenic effect on HTF, via its induction of fibroblast growth factor-2 (FGF-2). Recent studies found that TGF-ß effects in NRK fibroblast proliferation are mediated by connective tissue growth factor (CTGF).52 CTGF is known to be a cysteine-rich mitogenic peptide, the secretion and synthesis of which are selectively induced by TGF-ß at the level of gene expression. It is believed to mimic many of the actions of TGF-ß on mesenchymal cells only, it has no action on epithelial cell lines.53 However, CTGF alone is not capable of stimulating anchorage-independent growth of fibroblasts but appears to act as a downstream mediator of the growth promoting effects of TGF-ß.
Our results indicate that all TGF-ß isoforms show similar concentration-dependent activities in stimulating HTF chemotactic migratory activity and that TGF-ß chemotactic activity is greater than chemokinetic activity. Maximal stimulation of HTF migration occurred at a concentration of 10-9 M, with all isoforms showing a biphasic response, and another peak (albeit less significant than 10-9 M) of migratory activity being demonstrated at 10-13 M. A biphasic response to TGF-ß has also been demonstrated in neutrophil chemotaxis, where the authors compared isoform activity and suggested potency in the order of TGF-ß2 > TGF-ß3 > TGF-ß1.54 They postulated that TGF-ß–induced migration occurred via its stimulation of fibronectin production and explained the biphasic response by the fact that at high concentrations TGF-ß causes excessive fibronectin secretion, retarding neutrophil migration.54 TGF-ß also strongly stimulates peripheral monocyte chemotaxis,55 attributed to high affinity receptors for TGF-ß found on their cell surface, making them able to respond to fentomolar concentrations. The effects of TGF-ß on ocular cell migration have been assessed in a number of studies on the cornea, where it appears to be primarily inhibitory,56 57 and is more potent than either epidermal growth factor or FGF.58 Other ocular cell migratory activities that have been investigated include trabecular meshwork cells, where both of TGF-ß1 and -ß2 were assessed, with maximal activity found to be at concentrations of 4 x 10-15 to 4 x 10-13 M.59 In this cell type, however, platelet-derived growth factor was shown to be a more powerful chemoattractant. Like our findings, these authors demonstrated that TGF-ß migratory activity was predominantly chemotactic rather than chemokinetic. Unfortunately, higher concentrations of TGF-ß were not fully investigated in these ocular cell migration studies, with most experiments using a range of TGF-ß concentrations with a maximum of less than 4 x 10-13 M.
One of the most important sites of TGF-ß activity in the eye is the aqueous humor, identified as a factor influencing the wound healing response in filtration surgery.16 This has been supported by the demonstration that compared with other growth factors found in the aqueous, TGF-ß has been shown to be the most potent in stimulating HTF activity.60 TGF-ß2 is the predominantly expressed isoform in the aqueous.12 16 18 However, it is important to note that in all these studies only TGF-ß1 and -ß2 isoforms (and not TGF-ß3) were analyzed. The average concentration of active TGF-ß2 in normal aqueous is between 0.73 and 10.98 x 10-11 M, compared with serum where levels of active TGF-ß in normals lie between 12 x 10-11 and 16 x 10-11 M.
An important finding by Tripathi et al.15 is the demonstration that aqueous levels of TGF-ß2 are significantly raised in glaucomatous (primary open-angle glaucoma) eyes compared with age-matched controls (1.8 versus 0.71 x 10-11 M active TGF-ß2), in a sample of patients undergoing routine cataract surgery. The authors hypothesized that this difference may account for the excessive extracellular matrix deposition seen in the trabecular meshwork of glaucomatous eyes, leading to decreased aqueous outflow and raised intraocular pressure. Production of TGF-ß in the aqueous is believed to be derived from local tissues. Again, studies have identified TGF-ß2 as the predominant isoform produced by the iris and ciliary body61 and trabecular cells.62 In addition, it has been shown that when secreted, it is mainly in its latent form.
Although the aqueous is a major factor in glaucoma filtration surgery, production of TGF-ß will also be locally derived from the wound site. TGF-ß isoforms can be produced by a variety of cells: TGF-ß1, released predominantly from degranulating platelets; TGF-ß2, locally produced in the aqueous, as discussed above; and TGF-ß3 from inflammatory cells. At least two factors would be expected to alter the amount of TGF-ß secreted and its degree of activation at the filtration wound site. First, surgery will invariably initiate the blood clotting cascade and, second, cause a breakdown in the blood–aqueous barrier. Plasmin and thrombospondin63 64 produced by these events can then activate all three TGF-ß isoforms. Hence, the profile of TGF-ß activity at the wound site may be considerably altered by filtration surgery, complicated by the fact that the passage of aqueous through the filtration wound will result in a constantly changing environment.
The implications of different peak activities of TGF-ß–induced fibroblast functions may be explained physiologically. In a wound environment, the two early functions of fibroblasts are migration and proliferation. TGF-ß is initially released by inflammatory cells and platelets at the wound site. At relatively low concentrations, it can act as a stimulant for fibroblast proliferation and as a weak chemoattractant (range, 10-13 to 10-12 M). At this stage, a provisional matrix is deposited, which attenuates fibroblast proliferation. The concentration of TGF-ß in the wound would then probably be much higher due to the stimulated increase in fibroblast number. Previous authors have suggested that at concentrations of >10-9 M, TGF-ß is a potent stimulant of collagen production.17 Thus, at around 10-9 M, TGF-ß activity is adapted to collagen matrix deposition, with stimulated functions of fibroblast-mediated contraction, secondary to stimulated fibroblast migration and matrix remodeling. Hence, during normal evolution of the scarring process, the key functions of the fibroblast depend on its environment. In particular, the effects of growth factors determine fibroblast activity at any one time. Our in vitro results thus suggest that the biphasic effects of TGF-ß determine the response of fibroblasts and that the role of TGF-ß during HTF-mediated conjunctival wound healing is very much dependent on its concentration at the wound site.
In summary, we have demonstrated that TGF-ß is a potent stimulant of HTF fibroblast activity, suggesting its stimulatory role in the conjunctival scarring response. Because all three isoforms are probably present during the wound healing response after glaucoma filtration surgery, an important finding has been the fact that they behave in a similar manner. Finally, TGF-ß actions are characterized by different concentration-dependent effects and different peak activities for stimulating various fibroblast functions, and its biphasic characteristics have implications for the timing and development of the conjunctival scarring response. Hence, TGF-ß appears to be an important component of conjunctival scarring. Its potent effects make it a possible target agent for modulating the scarring response after glaucoma filtration surgery.
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Footnotes |
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Submitted for publication April 20, 1999; revised August 6, 1999; accepted September 13, 1999.
Commercial relationships policy: N.
Corresponding author: M. Francesca Cordeiro, Wound Healing Research and Glaucoma Units, Department of Pathology, Moorfields Eye Hospital & Institute of Ophthalmology, Bath Street, London EC1V 9EL, United Kingdom. m.cordeiro{at}ucl.ac.uk
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2ß1 (VLA-2) mediates reorganization and contraction of collagen matrices by human cells Cell 67,403-410[Medline][Order article via Infotrieve]
2ß1 integrin expression in osteogenic cells J Biol Chem 270,376-382This article has been cited by other articles:
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