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Regular Articles |
From the Department of Medicine,*
University Clinical
Departments, Royal Liverpool University Hospital, Liverpool; and Wound
Healing Research,
Unit Department of
Pathology, Institute of Ophthalmology, London, United Kingdom
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cell-mediated contraction is a complex process that may involve a number of cellular activities including migration and reorganization of the extracellular matrix. Both of these cell activities are dependent on cell-matrix interactions12 and the matrix of developing PVR membranes is replete with extracellular matrix, including collagen types I and III.8 In general, interactions between these interstitial collagens and cells are thought to involve matrix metalloproteinases (MMPs). RPE cells have been shown to synthesize MMP-1, -2, -3, and -9 in vitro.13,14 Indeed these MMPs have also been implicated in periretinal membranes of eyes with PVR15 and moreover elevated levels of MMP-2 and -9 have been found in the vitreous of PVR eyes.16 Therefore we hypothesized that MMPs may play a critical role in RPE-matrix interactions and specifically, in PVR membrane contraction. To further investigate this concept we examined 1) the expression of MMPs by RPE cells interacting with collagen in an in vitro model of this disease in which both RPE phenotype and collagen type is similar to that of PVR membranes,17 and 2) studied the effects of modulating MMP expression in the in vitro model.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human postmortem eyes up to, but not exceeding, 48 hours postmortem were obtained from the Manchester Royal Hospital Eye bank and complied with the Declaration of Helsinki. Human RPEs (HRPEs) were isolated following the procedure outlined by Edwards18 and modified by Boulton, Marshall, and Mellerio.19 The anterior segment and vitreous were removed after soaking the eyes for 10 minutes in phosphate-buffered saline (PBS) containing fungizone, 100 U of penicillin, and streptomycin. The neural retina was teased away from the RPE and the exposed RPE was washed three times in PBS to remove adherent photoreceptor debris. The posterior eyecup was divided into three segments and areas of the RPE monolayer were isolated with brass cloning rings. The wells formed by the cloning rings were filled with a mixture of 0.25% trypsin, 0.02% ethylenediaminetetraacetic acid, and PBS, and incubated at 37°C for 30 minutes. Dissociated RPE cells were aspirated from Bruch’s membrane and seeded into 25-cm2 tissue-culture flasks coated with 1 ml of newborn calf serum (NCS) each. The RPEs were fed with Ham’s F-10 culture medium (50 ml x10 Ham’s F10, 425 ml distilled water, 5 ml glutamine, 5 ml fungizone, 5 ml penicillin/streptomycin, 8 ml of 7.5% sodium bicarbonate, and 1 ml of 1 mol/L NaOH; all Gibco, Paisley, UK), supplemented with 15% NCS and 3 mg/ml glucose. The cultures were maintained at 37°C in 5% CO2 and air. Primary cultures reached confluence within 2 to 3 weeks and were passaged on. The purity of the cultures was confirmed by immunohistochemical labeling with a wide-spectrum anti-cytokeratin monoclonal antibody (clone K8.13; ICN Biomedicals Ltd., High Wycombe, UK) known to stain the HRPE cell population.20 HRPE cells between the 3rd and 10th passage were used in this study and before collagen gel contraction studies cells were grown in flasks until they were preconfluent. Cells were removed from their flasks by using 0.1% trypsin and 0.04% ethylenediaminetetraacetic acid, then mixed with complete medium and centrifuged for 10 minutes at 800 rpm. After the supernatant was discarded, the cell pellet was resuspended in NCS. Cell numbers and viability were determined by trypan blue exclusion in a hemocytometer.
Preparation of Media
NCS was depleted of exogenous MMPs by following the method described by Azzam and Thompson21 in which gelatin-coated Sepharose beads (Pharmacia) were incubated with NCS at a 1:10 dilution for 2 hours (4°C) on a rotamix. The beads were then removed by passing the mixture through a sterile Millipore filter (0.2 mm) and the resultant NCS was used in subsequent collagen gel formation and in complete tissue culture media (F10 and 15% NCS).
Preparation of Cell-Populated Three-Dimensional Collagen Matrices
The formation collagen matrices were prepared with rat tail type I collagen (Sigma, Dorset, UK) at 5 mg/ml in 0.1% acetic acid stock solution. To prepare a collagen matrix at a final concentration of 1.5 mg/ml for each assay, 2.1 ml of concentrated culture medium (15 ml of 10x MEM, 35 ml distilled water, 1.5 ml penicillin/streptomycin, 1.5 ml glutamine, 1.5 ml fungizone, and 3 ml of 7.5% sodium bicarbonate) was added with 3.6 ml of collagen solution at 4°C. To this mixture, 0.9 ml of serum containing the appropriate amount of cells was added. Gel contraction studies were performed in either 6-or 24-well plates in which each well received either 1 ml (6 well) or 0.25 ml (24 well) of this final mixture (Sterilin, Stone, UK) and then was transferred to a humidified 37°C, 5% CO2 incubator where the matrix set within 1 minute. After 10 minutes the matrices were overlaid with either 6 ml (6 well) or 1.5 ml (24 well) of complete medium, detached from the base using a pipette tip and floated.
Cells Seeded on Two-Dimensional Collagen Matrices
Collagen matrix formation followed the above protocol except 0.9 ml of cell-free NCS was used in the matrix formula. After matrix formation, cells were seeded onto the matrix in complete media and allowed to settle for 30 minutes in the incubator. Matrices were then detached, floated, and returned to the 37°C incubator.
MMP Inhibitors and Antibodies
Mouse monoclonal antibodies against either 
2 or ß1 integrins
(Serotec) and mouse monoclonal and rabbit polyclonal antibodies to
MMP-1, -2, -3, and -9 (Biogenesis Ltd., Poole, UK) and were diluted
1:10 (v/v) in complete media. A control solution containing 0.1% w/v
sodium azide and 0.14% v/v mouse or rabbit IgG was diluted 1:10 in
complete media. We also used a broad-spectrum, potent hydroxamic
acid-derived MMP inhibitor developed by Galardy and
associates22,23
known as Galardin. This compound was
dissolved in complete media at logarithmic concentrations ranging from
100 mmol/L to 0.1 nmol/L. Eqimolar hyroxamic acid (100 mmol/L) was
dissolved in complete media as a control solution.
Gel Contraction Experiments
To assess the effect of MMP inhibitors, triplicate wells were used. The overlying media of the gels contained varying concentrations of the inhibitors (see above) or the relevant controls. The gel area was measured at days 1, 3, and 7 after seeding and represented as percentage size ±SEM. Cell morphology within the collagen matrices was followed by phase contrast microscopy throughout the duration of the experiments. To determine whether the effects of the MMP inhibitor Galardin were reversible, gels were incubated with complete media containing 10 mmol/L Galardin for 5 days, which was then removed and replaced with complete media containing 100 mmol/L hyroxamic acid. The gel contraction was measured at days 1, 3, 5, 7, and 10 after seeding.
Morphological Assessment of in Vitro Preparations
A number of different methods were used to study the morphological changes of tissue culture preparations (monolayer cultures and matrices) throughout the duration of the experiments. Living tissue cells were visualized using phase-contrast and time-lapse video microscopy, which allowed experiments to run continuously and to be viewed at numerous time points throughout the experiment. Examination at a higher resolution (at the electron microscopy level) and a more detailed analysis expression using immunohistochemical techniques required fixation of specimens at fixed time points.
Phase-Contrast and Time-Lapse Video Microscopy
Routine observations and photographs of living tissue-culture cells were performed using an inverted phase-contrast microscope (Diaphot, Nikon). In addition to phase-contrast microscopy the movement of cells within collagen matrices was monitored throughout 72 hours using time-lapse video microscopy. Collagen matrices containing cells were prepared as previously described but were floated in a minimal amount of tissue culture media. The matrices were then transferred to a humidified chamber maintained at 37°C and 5% CO2 in air, which was mounted onto an inverted phase-contrast microscope (Nikon). The microscope was connected to a video camera (JVC KS300) and recorder system (JVC-BR906OE) that allowed the experiments to be recorded for up to 72 hours.
Light Microscopy
A number of different methods were used to study the cellular changes visible at the light microscopy level. These methods require different fixation and processing protocols and are presented below individually. All collagen matrices were rinsed in PBS three times, each for 5 minutes, before fixation.
Whole Mount Preparations
Whole collagen matrices were fixed in 10% neutral-buffered formalin overnight. Matrices were either then stained for histological examination with hematoxylin or by a fluorescent immunohistochemical method.
Indirect Immunofluorescence of Whole Collagen Matrices
The matrices were fixed in 10% neutral-buffered formalin as described earlier and were initially rinsed by immersion in Eppendorfs, which contained PBS (10 minutes). Nonspecific binding was blocked by exposure to 5% normal goat serum (Sigma) in PBS (v/v) for 120 minutes at room temperature on a rotamix. Serum was removed and replaced with the primary antibody to cytokeratins (clones CK8.13 and CK18; Sigma) at the relevant dilution for 24 hours at +4°C on a rotamix. Extensive washing of matrices was performed by immersing the matrices in rotating 7-ml tubes containing PBS (four 30-minute washes). The matrix was then returned to a new Eppendorf and the goat anti-mouse fluorescein isothiocyanate conjugate (1:40; Sigma) secondary antibody was added via a Millipore filter and left overnight on a rotamix at +4°C. The specimens were further washed (as earlier described) three times on the rotamix in PBS (30 minutes each) and mounted onto a glass slide under a glass coverslip, using fluorostab mounting medium (Euro-Path Ltd.). The specimens were then viewed at an excitation/emission wavelength of 490/525 nm. Photographs were taken on both black and white (Tmax 400) and color slide film (Agfa chrome RS1000).
Scanning Electron Microscopy (SEM)
Collagen matrices were initially rinsed in PBS (3 x 5 minutes) and subsequently fixed in 2.5% glutaraldehyde (EM grade vacuum-distilled Fisons Polaron, Loughborough, UK) in Sorensen’s phosphate buffer, pH 7.4, for 1 hour. After washing in Sorensen’s phosphate buffer (three changes of buffer) for 15 minutes, the specimens were postfixed for 1 hour in 1% (w/v) osmium tetroxide in the same buffer, followed by a further 15-minute wash in buffer alone. The specimens were then washed in distilled water, with one change at 30 minutes, followed by three changes at 10 minutes. The specimens were then dehydrated through a series of graded alcohols, each for 15 minutes (25%, 50%, 70%, 80%, 90%, and three times at 100%; AnaLar grade, BDH).
The specimens were then critical point-dried (Polaron, Hemel Hampstead,
UK), mounted on metal stubs with double-sided adhesive tape, and gold
coated (
12 to 16 nm thickness) using a Polaron sputter coater. The
three-dimensional matrices were then examined in a Hitachi S-520
scanning electron microscope, using secondary electron detection.
Preparation and Collection of Media for Zymography and Enzyme-Linked Immunosorbent Assay (ELISA)
Collagen matrices were prepared as above containing 1 x 105 cells/ml in 6-well plates. Media overlying the collagen lattices was removed at days 1, 3, and 7 after seeding and stored at -70°C until required. Samples were then lyophilized and reconstituted in 2 ml of PBS before analysis by gelatin zymography or ELISA.
Gelatin Zymography
Samples were denatured with an equal volume (15 ml) of dissociating buffer (125 mmol/L Tris-HCl, pH 6.8, 20% (v/v) glycerol, 4% (w/v) sodium dodecyl sulfate, 0.005% (v/v) bromophenol blue; Novex, R&D Systems Ltd.) for 10 minutes at room temperature. The samples were then run on a 10% (v/v) Tris-glycine polyacrylamide gel (Novex, R&D Systems Ltd.) containing 0.1% (w/v) gelatin for 90 minutes with constant 125 V voltage and 40 mA current, within running buffer (25 mmol/L Tris base, 192 mmol/L glycine, 0.1% (w/v) sodium dodecyl sulfate, pH 8.3; Novex, R&D Systems Ltd.). Prestained molecular weight markers (Mr 7200 to 208,000; Bio-Rad) were also ran with the samples. The gels were then placed in renaturing buffer with gentle agitation for 30 minutes. The renaturing buffer was removed and replaced with developing buffer (2.5% (v/v) Triton X-100; Novex, R&D Systems Ltd.) for 30 minutes, which was then replaced with fresh developing buffer and incubated overnight at 37°C. The gel was then stained with 0.5% (w/v) Coomassie blue (Bio-Rad) in 45% (v/v) methanol/45% (v/v) distilled water/5% (v/v) glacial acetic acid for 2 hours. The gel was destained in 45% (v/v) methanol/45% (v/v) distilled water/5% (v/v) glacial acetic acid to visualize the clear bands of protease activity against the blue background.
To detect whether the MMPs were in a proenzyme or activated smaller molecular weight form of the enzyme, samples were also incubated with 2 mmol/L of aminophenylmercuric acetate (APMA; Sigma) for 2 hours at 37°C before zymography. APMA causes autocatalytic cleavage of the inactive proenzyme form to its lower molecular weight active form therefore allowing an assessment of the form of MMPs secreted.
Quantification of MMPs by ELISA
Samples of conditioned media (days 1, 3, and 7) from collagen lattices were quantified using specific ELISA kits (Amersham, UK) to determine the levels of total MMP-1, -2, -3, and -9 and TIMP-1 protein produced. All of the assays were based on a two-site ELISA sandwich format using two antibodies directed against different epitopes of the relevant MMP. The MMP-1 ELISA differed slightly from those for MMP-2, -3, and -9 because these used a horseradish peroxidase-conjugated rabbit-raised polyclonal antibody, whereas the rabbit polyclonal antibody directed to MMP-1 was unconjugated. This procedure therefore required an additional step in the protocol in which a horseradish peroxidase-conjugated donkey and rabbit antibody was added.
All samples of media from the collagen gel experiments were assayed at dilutions 1:1, 1:5, and 1:10 to find readings that fell within those of the standard curves of each assay. Comparisons of MMP quantities within each assay were only made with samples run at the same dilution.
The double-sandwich ELISA method according to manufacturer’s instructions was used for quantifying MMP-1 protein. Briefly MMP-1 standards that were prepared at 0, 6.25, 12.5, 25, 50, and 100 ng/ml and conditioned media were aliquoted into duplicate wells (100 µl/well) coated with monoclonal antibody to MMP-1 and incubated at 25°C for 2 hours. The wells were subsequently washed and emptied four times with wash buffer (350 µl/well; 0.0067 mol/L phosphate buffer, pH 7.5, containing 0.033% Tween 20) before the addition of 100 µl/well of polyclonal antibody (rabbit anti-MMP-1) for 2 hours at room temperature. The wells were then washed four times (350 µl/well) with wash buffer and 100 µl/well of horseradish peroxidase-conjugated donkey anti-rabbit antibody added for 1 hour at room temperature. The wells were washed again as described previously and 100 µl/well of 3,3',5,5'-tetramethylbenzidine/hydrogen peroxide in dimethylformamide (20%, v/v) was added and incubated at 25°C for 30 minutes. The visualization reaction was then stopped with 100 µl/well of 1 mol/L sulfuric acid and the optical densities read on a microplate reader at 450 nm.
MTT Assay
The MTT (3-{4.5-dimethylthiazol-2-yl}-2,5-diphenyltetrazolium bromide; Sigma) assay was used to indirectly assess cellular proliferation as it reflects the cytosolic dehydrogenase activity in cells. This activity has been shown previously to increase in proliferating RPE cells24 and so was used to determine whether the MMP inhibitors had any effect on cellular proliferation in vitro. HRPE cells were harvested as previously described and resuspended in complete media. HRPE cells were aliquoted into 96-well culture plates at seeding densities of 0.625, 1.25, 2.5, 5, and 10 x 104 cells per ml (180 µl per well) in media containing varying concentrations of the inhibitors, antibodies, or controls. Cells were incubated at 37°C for 3 days to allow adequate settlement and growth. The media from each well was carefully removed and replaced with 150 µl of fresh serum-free media. Each well immediately received 20 µl of MTT (5 mg/ml) and was incubated for 4 hours at 37°C.
The media was discarded and replaced with 150 µl of dimethyl sulfoxide and once the MTT crystals dissolved, the wells read at 540 nm on a microtiter plate reader (Dynatech Laboratories). A minimum of six replicates was used in all experiments and results were expressed in terms of percentage survival taking the absorbance of the control wells to be 100% survival.
Adhesion Assay to Collagen Type I
After the effects on matrix contraction, the influence of MMP
inhibitors and integrins on HRPE adhesion to collagen I was assessed
using a semiquantitative colorimetric assay (Cytomatrix; Chemicon).
Briefly, 96-well plates precoated with collagen type I, were washed
with 100 µl of 1%(w/v) bovine serum albumin in PBS for 30 minutes at
37°C to block nonspecific binding sites. Preconfluent HRPE cells were
detached enzymatically (0.1% trypsin and 0.04%
ethylenediaminetetraacetic acid), then mixed with complete medium and
centrifuged for 10 minutes at 800 rpm and resuspended in 1 ml of
complete media and counted using a hemocytometer. Cells were
resuspended at cell concentrations of 1 x
105/ml and 5 x 104/ml
in F10 containing different concentrations of the MMP inhibitors
[Galardin (100 nmol/L to 100 µmol/L) and MMP antibodies (1:10 to
1:100) or antibodies directed against integrin subunits 2 and ß1).
One hundred µl of each sample was added to triplicate wells and
incubated either for 1 or 4 hours at 37°C in a
CO2 incubator. After these incubations the wells
were washed gently four times with 200 µl of PBS before the addition
of 100 µl/well of 0.2% crystal violet in 10% ethanol for 5 minutes
at room temperature. The wells were subsequently washed with 200 µl
of PBS (repeated four times) and the adherent-stained cells lysed with
100 µl/well of 1% sodium dodecyl sulfate (v/v) in PBS added for 5
minutes at room temperature. The absorbance of the stained wells was
read at 540 nmol/L on a microtiter plate reader (Dynatech
Laboratories).
Statistical Evaluations
Statistical analysis of data were performed using the statistics package Unistat (Unistat Ltd., London, UK). Comparisons of data samples from contraction and adhesion assays were by analysis of variance that allowed multiple comparisons between data sets of two or more. Data were presented as mean ±SEM unless otherwise stated and significance was expressed as P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because PVR membranes characteristically involve RPE cells both at membrane surfaces and (as fibroblastic cells) within the avascular collagenous fibrous tissue,7-11 we mimicked this arrangement in vitro by either seeding cells on two-dimensional collagen lattices (corresponding to cells on a membrane) or cells within (three-dimensional) collagen matrices (cells located within the epiretinal membrane). Matrix contraction occurred in both models in a cell number-dependent manner and no contraction occurred in cell-free matrices. Optimal contraction occurred at 4 x 104/ml (two-dimensional) and 4 x 105/ml (three-dimensional) and thus were used in all subsequent experiments unless otherwise stated.
The Production and Distribution of MMPs during RPE-Dependant Collagen Matrix Contraction
Analysis of the matrices and surrounding media for the presence of
MMPs using gelatin zymography revealed both for HRPE seeded in
three-dimensional matrices and HRPE seeded on two-dimensional matrices,
the presence of three major bands at molecular weights of 71, 65, and
56 kd. These bands increased in intensity through 7 days. The molecular
weights of two of these bands corresponded to MMP-2 in both its latent
(71 kd) and active form (65 kd). The 56-kd band corresponds to MMP-1 or
MMP-3 (Figure 1)
. In addition, two faint
minor bands were seen at similar intensities on all days studied. These
two minor bands were at 100 kd and 92 kd and corresponded to MMP-9. A
similar profile for gelanolytic activity also was observed from the
media of HRPE seeded on the surface of collagen matrices.
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. Controls of pure proenzyme standards
for MMP-2 and MMP-9 were also run both with and without APMA
pretreatment. The MMP-2 protein profile showed bands at 71 and 69 kd
that, after activation with APMA, gave an extra band at 65 kd
indicating an active form of the enzyme. The MMP-9 protein showed a
major band at 92 kd that on APMA activation gave a lower molecular
weight at 80 kd corresponding to active MMPs (not shown).
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As gelatin zymography only allows semiquantification, samples of
conditioned media from the matrices were also quantified for the levels
of MMPs-1, -2, -3, and -9 and TIMP 1 using specific ELISAs. The
findings demonstrate a time-dependent increase in each of these enzymes
with MMP-2 showing the most significant increase in protein level
detected (Table 1)
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Attempts to inhibit MMP activity during HRPE-mediated collagen
matrix contraction used antibodies and chemical inhibitors (see below).
These experiments were performed to ascertain whether inhibition of
these molecules could have a direct effect on the contractile process.
Because the combined zymography and ELISA results indicated the
presence of MMP-1, -2, -3, and -9 in the matrices, antibodies specific
to each of these enzymes were used to evaluate whether contraction of
the cell-mediated contraction of the collagen matrices was
MMP-dependent. Controls included the use of inhibitory antibodies to
integrins 2 and ß1 subunits, which have been shown to inhibit
RPE-mediated contraction.
The Effect of MMP Inhibition on Contraction of HRPE-Populated Three-Dimensional Matrices
Antibodies directed against MMP-1, -2, -3, and -9 individually had
only a small individual effect on the contraction rate of
HRPE-populated matrix contraction when compared to control matrices.
Although all of the antibodies tested seemed to decrease the rate of
contraction, this reduction was not significant at any time point
tested (analysis of variance, P > 0.05; Figure 3
). Phase-contrast microscopy showed
there was no apparent difference in the settlement and spreading of
treated and control cells within the matrix. Antibodies directed
against the subunits of the 2ß1 integrin significantly reduced
matrix contraction from the fourth day onwards
(P < 0.01).
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) The Effect of MMP Inhibition on Contraction of Matrices Overlain with HRPE (Two-Dimensional)
Antibodies directed alone or in combination against MMP-1, -2, -3,
and -9 had no significant effect on the contraction of collagen
matrices on which HRPE were seeded (two-dimensional). No significant
inhibition of contraction was achieved with any of the antibodies used
at any dilution (Figure 4)
. The
contraction of the matrices tended to be reduced by the fourth day in
the presence of antibodies against MMP-1 (44.3 ± 10.97), MMP-2
(45.67 ± 12.89), MMP-3 (41 ± 8.19), and MMP-9 (41 ±
8.19) compared to controls (32 ± 4.58), but these reductions were
not significant. A cocktail of antibodies directed against
MMP-1, -2, -3, and -9 also did not inhibit contraction, whereas
the antibody directed against the ß1 subunit of the 2ß1
integrin, significantly reduced matrix contraction on the first
day after seeding (P < 0.01).
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As combinations of antibodies inhibited cell-populated contraction, further studies were undertaken to evaluate the role of MMPs in matrix contraction using the broad-spectrum MMP inhibitor, hydroxamic derivative Galardin that is known to inhibit the activity of all MMPs.
No significant inhibition of contraction occurred with HRPE seeded on
collagen matrices when two-dimensional matrices were exposed to
Galardin diluted in normal culture media at any Galardin concentration
(0.1 nmol/L to 100 µmol/L) or time point tested (Figure 5)
. No noticeable morphological
differences between the inhibitor-treated cells and control cells were
observed.
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. At day 7, this inhibition was
obtained with concentrations of Galardin of 100 nmol/L and above for
4 x 105
cells (Figure 6)
. The inhibition of
contraction also seemed to be cell number-dependent as matrices seeded
with fewer cells required a reduced concentration of Galardin to
achieve the same level of inhibition. Thus matrices seeded at a cell
density of 1 x 105
required 10 nmol/L
Galardin to significantly (P < 0.05) reduce
contraction (Figure 7)
.
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Cellular Morphology in Collagen Matrices
During collagen matrix contraction the cells were initially round
within the matrix and most cells progressed from the initial
development of small processes (day 1), through to a stellate and
spindle-shaped appearance by day 7 (Figure 8, A and B)
. No differences in morphology
between any of the MMP antibody-treated groups and control cells could
be distinguished. Whole matrix preparations also showed no differences
in cellular appearance and distribution during contraction. At day 7,
cells within all matrices appeared equally spread and dedifferentiated
and reorganization of surrounding collagen was seen in all matrices.
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. Histological and SEM examination of matrices demonstrated
the presence of holes or tunnels in control matrices that were not
observed in matrices significantly inhibited with Galardin (Figure 8; D
to F
). It was further observed that cells failed to appear on the
matrix surface in Galardin-treated matrices even after 7 days, whereas
control matrices showed the presence of cells (and holes) on the
surface from 24 hours (Figure 8F)
. Time-lapse video microscopy of
contracting matrices also revealed that cells were stationary within
the Galardin treated matrices, whereas control matrices showed
cells acquired a stellate/spindle shape and migrated through the
surrounding matrix (Figure 9, A and B)
.
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To assess whether the effects of Galardin were reversible,
cell-populated matrices were incubated with complete media containing
10 mmol/L Galardin for 5 days (by which time inhibition of contraction
had occurred). The medium was then removed and replaced with complete
media containing 10 mmol/L of hydroxamic acid (control). The matrix
contraction was measured after seeding as before. Whereas matrices
treated with complete media containing 10 mmol/L Galardin for the
10-day duration of the experiment exhibited inhibition of contraction
when compared to untreated controls (P < 0.01).
However, when Galardin was removed from the incubating media at day 5
(exchange experiment) there was an increased rate of contraction
(Figure 10A)
. The difference in
contracted matrix area between exchanged and nonexchanged experiments
reached significance by the tenth day after seeding
(P < 0.01). However, if Galardin was added (at
day 5) to the media of matrices that were already contracting (ie,
incubated with control media for days 1 to 5) then no change (compared
to controls) in the rate of contraction was observed (Figure 10B)
.
Morphological examination of exchange experiment matrices revealed that
after removal, of the Galardin, the cells progressed to stellate and
spindle shapes as in the control untreated matrices.
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Trypan blue exclusion studies showed no cytotoxic effects at any
Galardin concentration studied (not shown). The MTT assay of activity
in HRPE seeded at different cell densities in the presence or absence
of Galardin showed no difference (P > 0.05) in
cytosolic dehydrogenase activity (absorbance) after 4 days
(Figure 11)
. No difference was seen
between any groups of controls or cells grown in media containing
Galardin at concentrations from 0.1 nmol/L to 10 µmol/L. This
confirmed the lack of toxicity observed for HRPE seeded on matrices
(two-dimensional) as well as the reversibility experiments performed
with HRPE seeded within the matrix.
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2 and ß1 (10
µg/ml). The integrin antibodies significantly reduced adhesion to
collagen type I (P > 0.05; Figure 12
). However, no statistical difference
was seen between adhesion of cells seeded in the absence of Galardin
and cells seeded with Galardin at any of the concentrations of the
agent tested (Figure 12)
. Furthermore no difference was seen in cell
morphology or cytoskeletal staining in either the controls or the
inhibitor treated groups (Figure 13)
.
This data supports the two-dimensional model findings, ie, HRPE
adhesion to and subsequent contraction of the underlying collagen
matrix was not significantly affected in the presence of MMP
inhibitors.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The MMP profile in both three-dimensional and two-dimensional matrices
is similar to that the MMP profile observed in PVR membranes by Webster
and colleagues15
that collectively demonstrate the
presence of MMP-1, -2, -3, and -9 in the scars. These observations
prompted us to use the RPE-collagen matrices to further investigate the
role of MMPs in the contraction process, using selective and general
MMP-neutralizing agents. For this part of the study, we used
neutralization of 2ß1
integrins as a control because neutralization of these receptors
previously has been shown to inhibit contraction of two-dimensional
matrices by RPE.27,28
The results of our investigation
revealed fundamental differences between the mechanisms involved in the
contraction of two-dimensional and three-dimensional matrices.
Specific individual antibodies against individual MMPs did not
significantly alter cell-mediated contraction of the collagen matrices
in either the two-dimensional or three-dimensional models, whether
polyclonal or monoclonal antibodies were used. However, a combination
of all of the MMP antibodies did exert an effect, albeit only with
cells seeded within the matrix (three-dimensional). As the combination
of MMP antibodies showed some significant inhibition of cell-populated
matrices, a potent broad-spectrum MMP inhibitor was used to further
investigate the role of MMPs in RPE-populated collagen matrix
contraction. The hydroxamic derivative known as Galardin inhibits the
activity of all MMPs.29,30
Therefore, this compound was
used to determine whether MMP production in the two models is essential
for, rather than incidental to, matrix contraction. The results show
that, whereas the adhesive integrins
2ß1 are required for
both systems, MMPs are only needed for three-dimensional matrix
contraction. Indeed, exposure of RPE seeded within a three-dimensional,
but not on a two-dimensional, collagen matrix to Galardin within normal
culture media inhibits contraction of collagen matrices in a
dose-dependent manner. Moreover, the inhibition of contraction induced
by Galardin in the three-dimensional model was far greater than
observed using the combinations of antibodies directed against MMPs. It
is possible that MMP antibodies may not be as efficient as Galardin at
MMP blockade or that Galardin may block MMPs in addition to those
detected by us in the matrices.
Because Galardin has no effect on RPE adhesion and the anti-contractile effects in three-dimensional matrices are reversed by exchanging the Galardin containing media with normal culture media, the mechanism of action seems to be directly because of inhibition of MMPs rather than Galardin-induced RPE cytotoxicity or interference with RPE-matrix adhesion.
Although, collagen degradation has been shown to be minimal during three-dimensional matrix contraction by fibroblasts,31 only minimal degradation of the total matrix collagen by cells would be expected in such a system. Moreover, in vivo studies on tight-skin mice wounds, which characteristically have delayed wound contraction of full-thickness wounds, have shown a marked decrease in collagenase content compared to control wounds,32 an observation consistent with our finding that MMPs are required for contraction of three-dimensional matrices. However it is not clear why MMPs are important for three-dimensional but not two-dimensional matrix contraction.
It is not clear why MMPs are important for three-dimensional but not
two-dimensional matrix contraction. The morphological and time-lapse
studies of RPE-populated matrices reported here show cells in control
matrices initially form small processes into the surrounding matrix
(day 1). From day 2 they show longer processes penetrating deeper into
the matrix and finally a stellate or a dedifferentiated spindle
morphology is seen by day 7. In contrast, the cells in Galardin-treated
matrices at day 7 exhibit small processes into their surrounding matrix
and not a stellate or spindle shape. Although the precise function of
MMPs in three-dimensional matrix contraction is unclear, it seems
plausible that MMPs permit the penetration of cellular processes into
adjacent matrix and enable reorganization of this matrix. Indeed,
previous ultrastructural studies of RPE-populated matrices have
revealed bundling or clumping of collagen fibers around RPE
cells.17
Moreover, we did not observe cell processes
penetrating the underlying matrix in two-dimensional matrices.
Therefore, we are undertaking ultrastructural studies of MMP
inhibitor-treated matrices, to determine whether the collagen-bundling
process is MMP-dependent. Our findings indicate that adhesive and
collagenolytic cell-matrix interactions are required for
three-dimensional matrix contraction but that MMP activity may not be
necessary for two-dimensional matrix contraction. However adhesive
interactions are still needed for contraction of the underlying
collagen matrix. If collagen reorganization is key to matrix
contraction, it might be expected that agents such as Galardin would
inhibit two-dimensional contraction. However, the finding that
MMP-blockade only inhibits three-dimensional contraction suggests that
contraction is dependent on cell-matrix interactions other than
collagen reorganization. Such mechanism purported to generate
contractile forces is that of cell migration: it is known that
migratory cells impart a force to their substrate as they move and it
is suggested that this force compacts and contracts loose matrices.
Cell migration both in and on a collagen matrix (ie, three-dimensional
and two-dimensional systems) depends on 2ß1 integrins and thus our
finding that blockade of adhesive integrins in both systems is
consistent with the cell migration theory of matrix contraction. In the
two-dimensional system, cells may not require a path to be cleaved to
permit locomotion whereas, in the three-dimensional system RPE may
require such a path and hence MMPs to create such a path. One analogy
that may explain the differences is between a person walking on a path
(two-dimensional) system and a person walking through a jungle: only
the latter would need to clear a pathway whereas both need focal
contact between their feet and the ground to propel themselves forward.
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Footnotes |
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Supported in part by the Foundation for the Prevention of Blindness, Guide Dogs for the Blind Association, Dunhill Medical Trust, International Glaucoma Association and Medical Research Council (grant G9330070).
Accepted for publication July 6, 2001.
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