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1 From the Ocular Surface and Tear Center, Department of Ophthalmology, Bascom Palmer Eye Institute; 2 Singapore National Eye Center, Singapore; and 3 Department of Cell Biology and Anatomy, University of Miami School of Medicine, Miami, Florida.
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Abstract |
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 Top Abstract IntroductionMethodsResultsDiscussionReferences |
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METHODS. EGF, TGF-
, PDGF-BB, IL-1ß, bFGF, TGF-ß1, TNF-, or IL-6 were
added at 10 ng/ml to early passaged primary pterygium body fibroblasts
(PBF) or normal human conjunctival fibroblasts (HJF) in a serum-free
medium. Expression of transcripts and proteins of MMP-1, MMP-2, MMP-3,
MMP-9, TIMP-1, TIMP-2, and uPA was determined by Northern
hybridization, ELISA, and Western blotting, respectively. Gelatin and
casein zymographies were performed in their serum-free conditioned
media with or without enzyme inhibitors to determine the activity of
MMP-2 and -3, respectively.
RESULTS. IL-1ß and TNF- dramatically increased the mRNA and protein
expression of MMP-1 and MMP-3 in cultured PBF when compared to normal
HJF and to their nonstimulated counterparts cultured in a serum-free
medium. EGF and TGF- also upregulated MMP-3 in PBF when compared to
HJF. The transcript levels of MMP-2 were high but stable for the two
cell types regardless of the cytokine treatment. Both TIMP-1 and TIMP-2
expressions were not influenced by the cell type or the cytokine
treatment. MMP-9 was not expressed in either of these two types of
fibroblasts. Both IL-1ß and TNF- induced a significant decrease in
uPA expression in PBF, whereas bFGF induced a slight increase in both
HJF and PBF.
CONCLUSIONS. Chronic inflammatory stimulation by IL-1ß and TNF-, which
potentially can be derived from the ocular surface and tears, may be
responsible for increased expression of MMPs in cultured PBF. These
data have clinical implications on progression of pterygium and
recurrence associated with incomplete excision of primary PBF under the
influence of ocular surface inflammation. Suppression of intraoperative
and postoperative inflammation may be a new strategy to prevent
pterygium recurrence.
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Ocular dryness and environmental insults may contribute to the
progression of pterygium and its recurrence. These stimuli, enhanced by
UV irradiation, can trigger the secretion of such proinflammatory
cytokines as interleukin (IL)-1, IL-6, IL-8, and tumor necrosis factor
(TNF) from the corneal8
and conjunctival9
epithelium. In addition, conjunctival epithelial cells can secrete
TNF-, IL-6, IL-8, and GM-CSF after inflammatory
stimulation.8
Certain proinflammatory cytokines, which
originate from the ocular surface epithelium or the tear fluid,
specifically TNF- and IL-1ß, can stimulate proliferation of
cultured Tenon’s capsule fibroblasts.10
Furthermore,
several potent fibroangiogenic growth factors such as basic fibroblast
growth factor (bFGF), platelet-derived growth factor (PDGF)-BB, and
transforming growth factor (TGF)-ß1 have been localized by
immunohistochemistry in different cell types of the pterygium
specimen.11
Tissue remodeling is a key process involved in normal development, wound healing, and pathologic conditions such as tumor invasion, metastasis, and angiogenesis. The degradation of the extracellular matrix that is associated with these processes is mediated by several families of extracellular proteinases. These families include the zinc-dependent matrix metalloproteinases (MMPs), and the serine proteases, such as the plasminogen–urokinase plasminogen activator (uPA) system. MMPs are a family of enzymes that act to modify or degrade the extracellular matrix (ECM).12 13 14 These enzymes are synthesized and secreted by a variety of cell types including fibroblasts. At least 17 members of the MMP family have been identified.15 MMPs are normally coexpressed with a family of tissue inhibitors of metalloproteinases (TIMPs), which inhibit active forms of MMPs. At least four inhibitors, have been characterized and are also produced by many cell types including fibroblasts.12 13 The MMPs and TIMPs have important roles in the process of fibrovascular proliferation. For example, MMPs have been clearly implicated in angiogenesis, whereas their inhibitors are shown to inhibit the angiogenic responses.16 Overexpression of MMP-1 in the skin has led to acanthosis and increased basal-cell proliferation, suggesting that such overexpression may be coupled with a phenotype capable of increased proliferation.17 Besides MMPs and TIMPs, the other proteolytic cascade leading to tissue degradation and remodeling involves uPA, a serine protease. Overexpression of uPA is correlated with the invasiveness of human cancer cells.18
We hypothesized that certain environmental stimuli known to be associated with pterygium, induce secretion of proinflammatory cytokines by the ocular surface epithelium, inflammatory cells in the tear fluid, or both. These cytokines in turn activate pterygium body fibroblasts (PBF), resulting in a phenotype capable of expressing various proteinases associated with ECM remodeling, angiogenesis, and fibroblast proliferation. These traits are important for pterygium progression as a primary lesion and for its recurrence after incomplete surgical removal. Hence, the purpose of this study was to investigate the effects of ocular surface–derived cytokines and growth factors on the expression pattern of proteinases associated with matrix degradation in cultured primary PBF compared with normal conjunctival fibroblasts.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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, PDGF-BB, IL-1ß,
bFGF, TGF-ß, TNF-, and IL-6 were from R & D systems (Minneapolis,
MN). BCA protein assay kit was from Pierce (Rockford, IL).
Zymogram-ready gels containing gelatin or casein, 4% to 15% Tris-HCl
polyacrylamide gradient ready gel, SDS, and electrophoresis equipment
were from Bio-Rad (Hercules, CA). Human MMP-1 and MMP-3 ELISA kits and
the monoclonal antibodies against human MMP-1, MMP-2, MMP-3, TIMP-1,
and TIMP-2 were from Oncogene Research Products of Calbiochem
(Cambridge, MA). uPA ELISA kit was from Oncogene Science Diagnostics
(Cambridge, MA). Vectastain Elite ABC peroxidase kit was from Vector
Laboratories (Burlingame, CA). Nitrocellulose membranes were from
Scheicher and Schuell (Keene, NH). GeneAmp RNA-PCR kit was from
Perkin-Elmer Cetus (Norwalk, CT). Wizard PCR Preps DNA purification kit
was from Promega (Madison, WI). [-32P]dCTP
was from Du Pont NEN (Boston, MA). XAR-5 and BioMax MS-1 films and
intensifying screens were from Eastman Kodak (Rochester, NY). All other
reagents and chemicals came from Sigma (St. Louis, MO).
Human Conjunctival and Primary Pterygium Fibroblast Cultures
All procedures followed the tenets of the Helsinki declaration and
were approved by the Bascom Palmer Eye Institute Review Board.
Specimens of normal human conjunctiva were obtained from healthy donors
who did not show signs and symptoms of an ocular surface disorder or of
dry eyes. Conjunctival biopsies of 2 x 2 mm were taken from the
superotemporal bulbar conjunctiva during cataract surgery, 2 to 3 mm
from the limbus. Pterygium specimens were obtained after the surgical
removal of primary pterygium. The central portion of the pterygium body
was used for all cultures. All normal conjunctiva and pterygium
specimens came from age-matched donors, whose ages ranged from 45 to 55
years old. These tissue samples were used for explant cultures to
generate normal human conjunctival fibroblasts (HJF) and PBF,
respectively. Each specimen was cut into explants of approximately
1 x 1 mm and placed onto 100-mm tissue culture dishes. Ten
minutes later, each explant was covered with a drop of FBS and placed
overnight in an incubator at 37°C under 95% humidity with 5%
CO2. Ten milliliters of medium (D-FBS) containing
DMEM enriched with 10% FBS, 50 µg/ml gentamicin, and 1.25 µg/ml
amphotericin B was added the next day, and the medium was changed every
2 days thereafter. Fibroblasts were subcultured with 0.05% trypsin and
0.85 mM EDTA in a calcium-free MEM medium at 80% to 90%
confluence with 1:3 to 1:4 split for three passages. Three cell lines
of HJF and three cell lines of PBF were used in this study. Of these,
two cell lines of each were used to study mRNA expression, and one cell
line of each was used for determination of protein expression.
Cytokine Regulation Experiments
Third passage fibroblasts of either HJF or PBF were seeded in
100-mm tissue culture dishes at a density of 106
cells per dish. After 5 days in culture, on confluence, cultures were
switched to a serum-free medium (D-ITS) containing DMEM supplemented
with 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenium, 50
µg/ml gentamicin, and 1.25 µg/ml amphotericin B for 48 hours before
treatment with different cytokines. Some cultures were maintained in
D-FBS. A single concentration of 10 ng/ml was used for all cytokines.
This was based on preliminary dose–response experiments of cytokine
regulation in ocular surface fibroblasts, using three concentrations of
each cytokine (1, 5, and 10 ng/ml) and on our previous reports on
cytokine regulation in these fibroblasts19
20
as well as
other dose–response data for TNF- showing the 10-ng/ml
concentration to be optimal for MMP-1 and -3 expression in human
cervical smooth muscle cells.21
To study mRNA expression, 10 ng/ml of each of the following human
recombinant cytokines or growth factors were added to the cultures for
4 hours: epidermal growth factor (EGF), TGF-, PDGF-BB, IL-1ß,
bFGF, TGF-ß1, TNF-, or IL-6. In parallel, control cultures with
D-ITS alone or D-FBS alone were compared. Total RNA was then isolated
and subjected to Northern hybridization.
Probe Preparation
Five of human DNA probes, including 185-bp fragment of MMP-1, 480
bp of MMP-2, 155 bp of MMP-3, 551 bp of TIMP-1, and 590 bp of TIMP-2,
were kindly provided by Velidi H. Rao (University of Nebraska Medical
Center, Omaha, NE). Two cDNA probes, 519 bp of uPA and 498 bp of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were purified from
RT-PCR products by electrophoresis through a 1.2% low melting agarose
gel using a Promega Wizard PCR Prep DNA purification kit according to
the manufacturer’s protocol. The primers used for PCR were 487 to 506
(sense) and 982 to 1002 (antisense) for uPA (GenBank accession no.
A18397), and 541 to 561 (sense) and 1018 to 1038 (antisense)
for GAPDH (GenBank accession no. M33197). The
32P-labeled cDNA probes (1 to 2 x
109 cpm/µg DNA) were prepared with
[-32P]dCTP (3000 Ci/mmol) using a random
primers DNA labeling system.
Total RNA Isolation and Northern Hybridization
After 4 hours of incubation with either one of the above
treatments, cells were extracted for total RNA by acid guanidium
thiocyanate–phenol–chloroform extraction with some modifications, as
previously reported.22
Total RNA was quantitated by
measuring the absorption at 260 nm and stored at -80°C before use.
Total RNA at 25 µg/lane was electrophoresed through 1.2% agarose
containing formaldehyde, transferred to nitrocellulose membranes, and
hybridized with 32P-labeled cDNA probes at 2 to
4 x 106 cpm/3 to 8 ng/ml in the
hybridization solution. After visualization of the hybridization
product in the x-ray film, the 32P-label on the
membrane was stripped by washing the membranes at 65°C for 1 hour
twice in 5 mM Tris-HCl, pH 8.0, 0.2 mM EDTA, 0.05% sodium
pyrophosphate, and 0.1x Denhardt’s solution and rehybridized with
other 32P-labeled probes. The relative amount of
each mRNA transcript of interest was determined by scanning its
autoradiogram, analyzing the scanned image with Gel-Pro imaging
software (Media Cybernetics, Silver Spring, MD) and normalizing the
data as a ratio to that of the GAPDH mRNA band.
MMP-1, MMP-3, and uPA ELISA
Third passage HJF and PBF were seeded at a density of 3 x
105 in each plate of 6-well plates. Cells were
cultured for 10 days until confluence in D-FBS, washed three times in
serum-free D-ITS, and then kept in the serum-free D-ITS medium for 24
hours. Cultures were then treated with 10 ng/ml of either TGF-,
IL-1ß, TNF-, or bFGF in serum-free D-ITS and compared to those in
D-ITS or D-FBS alone for 24 hours. Each of these treatments was
performed in triplicate wells. After 24 hours’ incubation with the
different added treatments, the conditioned media were collected,
centrifuged, and stored in -80°C until assayed. Cell lysis solution,
containing 50 mM Tris-HCl, pH 7.6, 300 mM NaCl, and 0.5% Triton X-100,
was added to the wells for 3 hours, and the cellular protein was
collected, centrifuged, and stored in -80°C until assayed. Human
MMP-1 or MMP-3 ELISA in each conditioned medium and human uPA ELISA in
cell lysates were determined in duplicates using their respective
double-sandwiched ELISA kits according to the manufacturer’s protocol.
The total cellular protein content in the cell lysate was determined by the microBCA protein assay reagent kit (Pierce). The protein concentration of MMP-1, MMP-3, or uPA in the culture supernatant was adjusted by its corresponding total cellular protein content for any possible difference in cultured cell numbers. Thus, all ELISA results were expressed as picograms per micrograms of total protein and represented the mean of three triplicates of samples.
Western Blot Analysis
To identify MMP and TIMP proteins present in each
fibroblast-conditioned medium, Western blot analysis was performed
using their specific antibodies. Conditioned media from different
fibroblast cultures were adjusted to a final volume of 25 µl to
represent the same quantity of cellular protein (8.3 µg) and
electrophoresed under reducing condition at 4°C in a 4% to 15%
gradient polyacrylamide gel. After electrophoretic transfer to a
nitrocellulose membrane at 4°C, the membrane was immersed with 0.1%
(v/v) Tween 20 in Tris-buffered saline (100 mM Tris, 0.9% NaCl, pH
7.5; TTBS) for 30 minutes with agitation. The primary antibody (i.e., 1
µg/ml of mouse monoclonal antibody against human MMP-1, MMP-2, MMP-3,
TIMP-1, or TIMP-2) in TTBS containing 1% horse serum was placed on
each membrane and incubated at room temperature for 60 minutes with
agitation. After being washed with three to four changes of TTBS over
15 minutes, each membrane was transferred to a 1:200 diluted solution
of biotinylated second antibody (goat anti-mouse IgG; Vectastain Elite
ABC kit) in TTBS containing 1% horse serum and incubated for 30
minutes. After three to four washes with the same solution, they were
incubated with 1:50 diluted Vectastain Elite ABC reagent conjugated
with peroxidase for 30 minutes and processed for color development in
0.5 µg/ml diaminobenzidine in 50 mM Tris-HCl, pH 7.2 containing
0.05% H2O2 for 10 to 20
minutes.
Zymography of Metalloproteinase Activity
To determine gelatinolytic and caseinolytic activities of the
various fibroblast cultures, zymography was performed using a method
similar to that previously described.23
Each conditioned
medium (40 µl), after being adjusted to represent the same quantity
of cellular protein (13 µg) or cell number (8000 cells), was treated
with sample buffer without boiling or reduction. SDS-PAGE was performed
using a 10% polyacrylamide gel containing 0.1% gelatin or a 12% gel
containing 0.1% casein. The gels were soaked in 2.5% Triton X-100 for
30 minutes at room temperature to remove the SDS and incubated in a
reaction buffer (50 mM Tris-HCl, pH 7.5, 200 mM NaCl, 5 mM
CaCl2, and 0.02% Brij-35) at 37°C overnight to
allow proteinase digestion of its substrate. Gels were rinsed again in
distilled water, stained with 0.5% Coomassie brilliant blue R-250 in
40% methanol and 10% acetic acid for 1 hour, and destained with 40%
methanol and 10% acetic acid. Proteolytic activities appeared as clear
bands of lysis against a dark background of stained gelatin or casein.
To verify that the detected gelatinolytic and caseinolytic activities
were specifically derived from metalloproteinases, the gels were
treated with Triton X-100 solution and
Tris/NaCl/CaCl2 reaction buffer containing 5 mM
phenylmethylsulfonyl fluoride with or without 10 mM EDTA in the
parallel experiments.
Statistical Analysis
Student’s t-test was used for analyzing the ELISA
results. The ELISA data are expressed as means ± SD, and the
differences were considered statistically significant at
P < 0.05.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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and PDGF-BB (each 12-fold) and to a lesser
extent by IL-1ß (7-fold), TNF- (4-fold), EGF (3-fold), and bFGF
(2-fold). In cultured PBF, such MMP-1 expression was most markedly
upregulated by the proinflammatory cytokines IL-1ß and TNF-
(34-fold each, compared to D-ITS) and was also markedly upregulated by
EGF and TGF- (31-fold each) and by PDGF-BB (26-fold). A lesser
increase was noted by bFGF (7-fold) and TGF-ß1 (4-fold). For both HJF
and PBF, IL-6 had no effect on MMP-1 expression.
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but was not affected
by other cytokines (Fig. 1
, lane 3). In contrast, the expression of
MMP-3 transcript by PBF was most dramatically increased by IL-1ß
(12-fold) and to a lesser extent by TGF- (11-fold), EGF (7-fold),
TNF- (5-fold), PDGF-BB (3-fold), and bFGF (2-fold). The 3.1-kb MMP-2 transcript was uniformly expressed by both HJF and PBF without notable variation among different cytokine treatments (Fig. 1 , lane 2). Likewise, there was no difference in the expression of the 0.9-kb TIMP-1 and the 3.5-kb TIMP-2 transcripts between HJF and PBF under these treatments (Fig. 1 , lanes 4 and 5, respectively). The MMP-9 mRNA transcript was not expressed by either HJF or PBF (not shown).
Transcript Expression of uPA in HJF and PBF
The 2.3-kb transcript of uPA expressed by HJF was markedly
upregulated by EGF (2.1-fold), TGF- (2.6-fold), PDGF-BB (2.1-fold),
and bFGF (3.2-fold) but was to a lesser extent by IL-1ß (1.5-fold)
and TGF-ß1 (1.4-fold) (Fig. 2)
. A similar pattern was observed in PBF. The uPA transcript expression
was upregulated by EGF (2.1-fold), TGF- (2.2-fold), PDGF-BB
(2.3-fold), and bFGF (2.5-fold) and to a lesser extent by IL-1ß
(1.3-fold). In contrast, uPA transcript expression was downregulated by
TNF- (0.5-fold) and by TGF-ß1 (0.7-fold).
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, IL-1ß, TNF-, or bFGF.
The amount of MMP-1 in the conditioned medium of nonstimulated HJF (in
D-ITS) was 10.9 ± 0.7 pg/µg protein and was significantly
increased by addition of TGF- (22.1 ± 3.3 pg/µg protein,
P = 0.005), IL-1ß (48.1 ± 10.7 pg/µg protein,
P = 0.004), or bFGF (23.6 ± 6.3 pg/µg protein,
P = 0.026) (Fig. 3A
). However, in PBF, the level of MMP-1 was more dramatically increased
by addition of IL-1ß (229.8 ± 12.8 pg/µg protein or 7-fold
increase, P < 0.001), TNF- (158.6 ± 22.5
pg/µg protein, or 5-fold increase, P = 0.001), or
bFGF (95.8 ± 7.7 pg/µg protein, or 3-fold increase,
P = 0.001) compared with the baseline of D-ITS
(30.8 ± 11.8 pg/µg protein) (Fig. 3B)
. When compared to the
level in HJF, the upregulation by IL-1ß, TNF-, and bFGF in PBF was
more notable.
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(to 315.3 ± 84.6
pg/µg protein, or a 6-fold increase, P = 0.027), and
bFGF (to 123.7 ± 3.1 pg/µg protein, or 2.5-fold increase,
P = 0.006) (Fig. 3D) . The protein levels measured by
ELISA for both MMP-1 and -3 were well correlated with their relative
amounts of transcripts expressed by both HJF and PBF under the
treatment of these cytokines.
ELISA of uPA Protein in Cell Lysates of HJF and PBF
The uPA protein levels in cell lysates of HJF were not changed
significantly by adding any of the cytokines (Fig. 4A
). For PBF, however, a slight nonsignificant increase in uPA protein
secretion was noted by adding bFGF (from 0.39 ± 0.05 pg/µg
protein in D-ITS to 0.47 ± 0.02 pg/µg protein with bFGF,
P = 0.064), whereas a significant downregulation was
noted by TNF- (to 0.18 ± 0.03 pg/µg protein,
P = 0.004) and by IL-1ß (to 0.27 ± 0.02 pg/µg
protein, P = 0.018) (Fig. 4B)
. These data corresponded
well with their respective mRNA data.
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, IL-1ß,
TNF-, or bFGF than that of their corresponding cultures in HJF. Of
note, the amount of MMP-1 protein expressed by PBF after stimulation
with either IL-1ß or TNF- and to a lesser extent with bFGF was
considerably higher than that of nonstimulated PBF (in D-ITS) or than
that of corresponding cultures of HJF. A similar pattern was
demonstrated for MMP-3. The 57-kDa band of MMP-3 expressed by PBF was
more pronounced when stimulated by either IL-1ß or TNF-, and such
levels were higher than their corresponding cultures of HJF. Similar to
our observations for the respective mRNA transcripts in Northern blot
analysis, the protein bands of 72-kDa MMP-2, 28-kDa TIMP-1, and 21-kDa
TIMP-2 did not reveal any notable difference in both HJF and PBF under
these treatments.
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, IL-1ß,
TNF-, or bFGF, to verify the gelatinolytic and caseinolytic
activities of MMP-2 and MMP-3, respectively. As shown in Figure 6
(top 2 lanes), the gelatinolytic activity of the 72-kDa band of MMP-2
was noted with both latent (predominantly) and active forms. This
gelatinolytic activity was completely abolished by incubating the gel
with solutions containing 10 mM EDTA (not shown). HJF produced the
gelatinolytic activity of MMP-2, but PBF produced a higher
gelatinolytic activity of the latent form of MMP-2 in all conditions
and a higher active form under the treatment of IL-1ß or TNF-.
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and to a lesser extent with bFGF. A similar pattern was
observed in PBF. The enhanced caseinolytic activity under the treatment
of IL-1ß or TNF- in PBF also revealed two clear bands,
corresponding to the glycosylated and unglycosylated forms of MMP-3,
respectively. |
Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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This study demonstrates that the expression of both MMP-1 and MMP-3 by
PBF was preferentially promoted at both mRNA and protein levels after
treatment with IL-1ß and TNF-. These two cytokines are potent
proinflammatory mediators, which can be produced by macrophages and
lymphocytes, stimulate the proliferation of certain types of
fibroblasts, and affect the production of MMPs by these cells. The
expression of mRNA correlated well with that of the protein and the
respective gelatinolytic and caseinolytic activities. Such levels were
significantly higher than those expressed by HJF. These data resemble
those previously reported for human synovial
fibroblasts,24
endometrial stromal cells,25
and fibrochondrocytes.26
Besides IL-1ß and TNF-, the
two structurally related growth factors EGF and TGF- also
upregulated MMP-3 in PBF when compared to HJF in our study. EGF is a
ubiquitous fibroblast mitogen that also stimulates the production of
MMP-1 and MMP-3 in several types of fibroblasts.27
28
Similarly, TGF- upregulates MMP-1 and -3 in synergism with IL-1ß
but not alone.29
Both EGF and TGF- can be produced by
ocular surface epithelial cells.22
30
31
The phenotype of MMP-1 and MMP-3 overexpression in fibroblasts is
frequently associated with cellular proliferation. For example, TNF-
and IL-1ß stimulate proliferation of cultured Tenon’s capsule
fibroblasts,10
and TNF- can stimulate the proliferation
of other types of fibroblasts.32
33
PDGF-BB and EGF induce
rheumatoid fibroblasts proliferation while increasing production of
MMP-1 and MMP-3.34
IL-1 and PDGF-BB stimulate synoviocyte
proliferation, whereas IL-1 stimulates collagenase
transcription.35
These data collectively show that a
change in the regulation of the metalloproteinase genes may reflect a
change in cellular proliferation. Further studies will be directed to
correlating the higher expression of MMP-1 and MMP-3 by PBF than HJF
with an intrinsic state of higher cellular proliferation. Furthermore,
such upregulation of MMP-1 and MMP-3 was not accompanied by any change
of expression in TIMP-1 and TIMP-2, which was constitutively expressed
but not differentially affected by any of the cytokines and growth
factors tested. This finding was also reported in rheumatoid synovial
fibroblasts24
and in endometrial
fibroblasts.28
Besides the MMPs system, some cytokines and growth factors also
modulated the uPA system. Expression of uPA is localized at discrete
cell-matrix contact sites and at areas of cell–cell contacts in human
fibroblasts.36
Enhanced expression of uPA is correlated
with local tumor proliferation.37
Unlike the
aforementioned inflammatory cytokines that preferentially upregulated
MMP-1 and MMP-3, TNF- and IL-1ß were found to downregulate uPA
expression at the mRNA and protein levels. On the contrary, bFGF was
the only cytokine that upregulated uPA expression in HJF and PBF (Fig. 2)
. This finding resembles 3T3 fibroblasts in which the expression of
uPA transcript is also induced by bFGF.38
However, we did
not note a significant difference in uPA expression under the influence
of bFGF between HJF and PBF. Interestingly, UV irradiation enhanced the
mRNA expression of uPA in human fetal fibroblasts.39
Further studies are needed to determine whether the basal expression of
uPA in nonstimulated PBF may result from chronic UV exposure.
Recently, our laboratory has discovered that pterygium head fibroblasts
(cultured from the pterygium portion invading onto the cornea)
intrinsically (i.e., without stimulation by any of these
proinflammatory cytokines) overexpress MMP-1 and MMP-3 over TIMP-1 and
TIMP-2.40
Because enhanced expression of MMPs over TIMPs
favors degradation of ECM, we thus speculate that this phenotype
explains how progressive corneal invasion takes place in primary
pterygium. Because the two proinflammatory cytokines IL-1ß and
TNF- can render resting PBF a phenotype similar to that of pterygium
head fibroblasts, we believe that the progression of pterygium in the
primary setting, or the recurrence after excision may be linked to
ocular conditions that are associated with the production of these two
cytokines.
The source of IL-1ß and TNF- can be the conjunctival
epithelium,8
stromal fibroblasts,9
and
inflammatory cells such as macrophages and
lymphocytes.41
42
When produced by these cells, these
cytokines may appear in the tear fluid. Production of IL-1ß and
TNF- is increased by surgical trauma,43
44
lipopolysaccharide,8
and UV irradiation.9
Furthermore, when there is a decrease of tear clearance or turnover,
IL-1ß concentrations increase in the tear fluid.45
Taken
together, these data suggest that chronic inflammatory stimulation of
the ocular surface, by such stimuli as UV irradiation, tear deficiency,
and microtrauma from climatic or occupational environments, can lead
PBF to overexpress MMPs over TIMPs.
The above findings have relevance to devising a new strategy of
pterygium surgery. The pivotal concept is that if incompletely excised,
residual PBF can be activated into proliferation and MMPs
overexpression under inflammatory cytokines. Therefore, suppression or
elimination of such ocular inflammation caused by any ocular surface
insults should be desirable in managing pterygium. Hence, the ideal
treatment of pterygium should include a combination of a thorough
removal of the abnormal tissue, with an agent that reduces
inflammation. Without being certain that all such tissues can be
thoroughly removed, pterygium excision must be coupled with agents that
reduce ocular inflammation to the minimum. One such strategy is the
administration of intraoperative and postoperative injections of a
long-acting corticosteroid to the margins of the excised tissue, to
minimize inflammation and further activation of the residual
fibroblasts. Subconjunctival corticosteroids injections have been used
successfully in the treatment of corneal graft
rejection,46
and in nonnecrotizing anterior
scleritis.47
48
Cyclosporin A is another anti-inflammatory
agent, previously shown to inhibit the expression of both
IL-149
and TNF-.50
This drug has been
administered topically for a variety of inflammatory disorders of the
ocular surface,51
or by a subconjunctival injection, as
recently studied in a rabbit model.52
53
Based on our
results, a potent broad-spectrum anti-inflammatory treatment to the
residual conjunctiva after pterygium excision may block the unwanted
effects of ocular surface–derived cytokines on any remaining pterygium
fibroblasts, thereby preventing further activation and proliferation of
these cells.
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Footnotes |
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Submitted for publication December 14, 1999; revised March 1, 2000; and accepted March 8, 2000.
Commercial relationships policy: N.
Corresponding author: Scheffer C. G. Tseng, Bascom Palmer Eye Institute, William L. McKnight Vision Research Center, 1638 NW 10th Avenue, Miami, FL 33136. stseng{at}bpei.med.miami.edu
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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in the pterygium Acta Histochem (Jena) 98,195-201[Medline][Order article via Infotrieve]
, PDGF-BB and IL-1ß Invest Ophthalmol Vis Sci 37,2068-2080 messenger RNA and protein in human corneal epithelial cells Invest Ophthalmol Vis Sci 33,3302-3306 in human tear fluid after excimer laser induced corneal wound Br J Ophthalmol 81,145-149 concentration and fluorescein clearance in ocular rosacea Invest Ophthalmol Vis Sci 40,2506-2512This article has been cited by other articles:
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  L. Zhou, R. W. Beuerman, L. P. K. Ang, C. M. Chan, S. F. Y. Li, F. T. Chew, and D. T. H. Tan Elevation of Human {alpha}-Defensins and S100 Calcium-Binding Proteins A8 and A9 in Tear Fluid of Patients with Pterygium Invest. Ophthalmol. Vis. Sci., May 1, 2009; 50(5): 2077 - 2086. [Abstract] [Full Text] [PDF]
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E. Peiretti, S. Dessi, C. Mulas, C. Abete, C. Norfo, M. Putzolu, and M. Fossarello Modulation of Cholesterol Homeostasis by Antiproliferative Drugs in Human Pterygium Fibroblasts Invest. Ophthalmol. Vis. Sci., August 1, 2007; 48(8): 3450 - 3458. [Abstract] [Full Text] [PDF]
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C.-H. Kuo, D. Miyazaki, N. Nawata, T. Tominaga, A. Yamasaki, Y. Sasaki, and Y. Inoue Prognosis-Determinant Candidate Genes Identified by Whole Genome Scanning in Eyes with Pterygia Invest. Ophthalmol. Vis. Sci., August 1, 2007; 48(8): 3566 - 3575. [Abstract] [Full Text] [PDF]
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 S Kase, N Kitaichi, N Furudate, K Yoshida, and S Ohno Increased expression of mucinous glycoprotein KL-6 in human pterygium. Br. J. Ophthalmol., September 1, 2006; 90(9): 1208 - 1209. [Full Text] [PDF]
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W. Li, H. He, C.-L. Kuo, Y. Gao, T. Kawakita, and S. C. G. Tseng Basement membrane dissolution and reassembly by limbal corneal epithelial cells expanded on amniotic membrane. Invest. Ophthalmol. Vis. Sci., June 1, 2006; 47(6): 2381 - 2389. [Abstract] [Full Text] [PDF]
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Y. Shoshani, J. Pe'er, V. Doviner, J. Frucht-Pery, and A. Solomon Increased Expression of Inflammatory Cytokines and Matrix Metalloproteinases in Pseudophakic Corneal Edema Invest. Ophthalmol. Vis. Sci., June 1, 2005; 46(6): 1940 - 1947. [Abstract] [Full Text] [PDF]
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H.-S. Kim, L. Luo, S. C. Pflugfelder, and D.-Q. Li Doxycycline Inhibits TGF-{beta}1-Induced MMP-9 via Smad and MAPK Pathways in Human Corneal Epithelial Cells Invest. Ophthalmol. Vis. Sci., March 1, 2005; 46(3): 840 - 848. [Abstract] [Full Text] [PDF]
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D.-Q. Li, Z. Chen, X. J. Song, L. Luo, and S. C. Pflugfelder Stimulation of Matrix Metalloproteinases by Hyperosmolarity via a JNK Pathway in Human Corneal Epithelial Cells Invest. Ophthalmol. Vis. Sci., December 1, 2004; 45(12): 4302 - 4311. [Abstract] [Full Text] [PDF]
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T. M. Nolan, N. Di Girolamo, M. T. Coroneo, and D. Wakefield Proliferative Effects of Heparin-Binding Epidermal Growth Factor-like Growth Factor on Pterygium Epithelial Cells and Fibroblasts Invest. Ophthalmol. Vis. Sci., January 1, 2004; 45(1): 110 - 113. [Abstract] [Full Text] [PDF]
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N. Di Girolamo, M. T. Coroneo, and D. Wakefield UVB-Elicited Induction of MMP-1 Expression in Human Ocular Surface Epithelial Cells Is Mediated through the ERK1/2 MAPK-Dependent Pathway Invest. Ophthalmol. Vis. Sci., November 1, 2003; 44(11): 4705 - 4714. [Abstract] [Full Text] [PDF]
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 N. G. Sandler, M. M. Mentink-Kane, A. W. Cheever, and T. A. Wynn Global Gene Expression Profiles During Acute Pathogen-Induced Pulmonary Inflammation Reveal Divergent Roles for Th1 and Th2 Responses in Tissue Repair J. Immunol., October 1, 2003; 171(7): 3655 - 3667. [Abstract] [Full Text] [PDF]
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D. Q. Li, T. Y. Shang, H.-S. Kim, A. Solomon, B. L. Lokeshwar, and S. C. Pflugfelder Regulated Expression of Collagenases MMP-1, -8, and -13 and Stromelysins MMP-3, -10, and -11 by Human Corneal Epithelial Cells Invest. Ophthalmol. Vis. Sci., July 1, 2003; 44(7): 2928 - 2936. [Abstract] [Full Text] [PDF]
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A. Solomon, M. Grueterich, D.-Q. Li, D. Meller, S.-B. Lee, and S. C. G. Tseng Overexpression of Insulin-like Growth Factor-Binding Protein-2 in Pterygium Body Fibroblasts Invest. Ophthalmol. Vis. Sci., February 1, 2003; 44(2): 573 - 580. [Abstract] [Full Text] [PDF]
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 T. M. Nolan, N. DiGirolamo, N. H. Sachdev, T. Hampartzoumian, M. T. Coroneo, and D. Wakefield The Role of Ultraviolet Irradiation and Heparin-Binding Epidermal Growth Factor-Like Growth Factor in the Pathogenesis of Pterygium Am. J. Pathol., February 1, 2003; 162(2): 567 - 574. [Abstract] [Full Text] [PDF]
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A. Leonardi, R. Cortivo, I. Fregona, M. Plebani, A. G. Secchi, and G. Abatangelo Effects of Th2 Cytokines on Expression of Collagen, MMP-1, and TIMP-1 in Conjunctival Fibroblasts Invest. Ophthalmol. Vis. Sci., January 1, 2003; 44(1): 183 - 189. [Abstract] [Full Text] [PDF]
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W. Eichler, U. Friedrichs, A. Thies, C. Tratz, and P. Wiedemann Modulation of Matrix Metalloproteinase and TIMP-1 Expression by Cytokines in Human RPE Cells Invest. Ophthalmol. Vis. Sci., August 1, 2002; 43(8): 2767 - 2773. [Abstract] [Full Text] [PDF]
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N. Di Girolamo, M. T. Coroneo, and D. Wakefield Active Matrilysin (MMP-7) in Human Pterygia: Potential Role in Angiogenesis Invest. Ophthalmol. Vis. Sci., August 1, 2001; 42(9): 1963 - 1968. [Abstract] [Full Text] [PDF]
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 N. Dushku, M. K. John, G. S. Schultz, and T. W. Reid Pterygia Pathogenesis: Corneal Invasion by Matrix Metalloproteinase Expressing Altered Limbal Epithelial Basal Cells Arch Ophthalmol, May 1, 2001; 119(5): 695 - 706. [Abstract] [Full Text] [PDF]
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