|Year : 2015 | Volume
| Issue : 1 | Page : 15-22
The role of matrix metalloproteinases in retinal pigment epithelial cell-induced gel contraction and collapse
Usama Ahmed Shalaby1, Ahmed Mohammed Saeed2, Mohamed Fathy Farid2
1 Department of Ophthalmology, Medical School, University of Aberdeen, Foresterhill, Aberdeen, AB25 2ZD, United Kingdom
2 Department of Ophthalmology, Benha University, Benha, Egypt
|Date of Web Publication||29-Mar-2016|
Ahmed Mohammed Saeed
Department of Ophthalmology, Benha University, Benha, 13621
Source of Support: None, Conflict of Interest: None
Purpose: To determine the role of matrix metalloproteinases (MMP)-1, -2, and -9 in directing the behavior of human retinal pigment epithelial (HRPE) cells during contraction of three-dimensional collagen gels. Materials and Methods: The effect of HRPE cells on collagen gel invasion and contraction were quantified by using phase contrast microscopy. Immunohistochemistry of the gel using monoclonal antibodies against MMP-1, -2, and -9 was performed. The effects of MMP inhibitor, Batimastat (BB-94) on cultured HRPE cells, and gel contraction were observed. Results: HRPE cells mostly proliferated as sheets of cells on the surface of the collagen gel and only minimally invaded the gel. However, some HRPE cells were seen to invade the gel as single cells detaching from the surface sheets. Surface-located sheets of cells exerted a dose-dependent contraction on the gel and generally failed to express MMPs. Single cells invading the gel expressed MMP-2 and -9. No expression of MMP-1 was observed by HRPE cells. BB-94, at a dose of 500 nM and 5 μM, reduced the amount of gel contraction. Conclusions: These data indicate that MMPs are selectively involved in HRPE invasion and collagen gel contraction. Both these processes may be implicated in the pathogenesis of similar conditions in vivo in which contraction of collagen gel is a feature, for example, proliferative vitreoretinopathy.
Keywords: Batimastat, collagen gel, matrix metalloproteinases, proliferative vitreoretinopathy, retinal pigment epithelial cell
|How to cite this article:|
Shalaby UA, Saeed AM, Farid MF. The role of matrix metalloproteinases in retinal pigment epithelial cell-induced gel contraction and collapse. Egypt Retina J 2015;3:15-22
|How to cite this URL:|
Shalaby UA, Saeed AM, Farid MF. The role of matrix metalloproteinases in retinal pigment epithelial cell-induced gel contraction and collapse. Egypt Retina J [serial online] 2015 [cited 2020 May 28];3:15-22. Available from: http://www.egyptretinaj.com/text.asp?2015/3/1/15/179345
| Introduction|| |
Proliferative vitreoretinopathy (PVR) is a pathological process which primarily involves migration of human retinal pigment epithelial cells (HRPE) into the subretinal space on the epiretinal and subretinal surfaces. , HRPE cells, in addition to other mesenchymal and inflammatory cells, particularly macrophages, secrete cytokines, ,, and various enzymes,  which alter the surrounding extracellular matrix (ECM) leading to the formation of periretinal fibrocellular membranes. Upon contraction of these membranes, the retina becomes fixedly detached, and surgical repair is less likely to succeed. ECM degradation by HRPE cells is a critical factor for cellular migration and invasion into the vitreous gel during the process of PVR.  This may be facilitated by cellular secretion of proteolytic enzymes such as matrix metalloproteinases (MMPs). ,,,
MMPs are calcium and zinc-dependent endopeptidases, which play a critical role in the wound healing process by remodeling the fibrillar components of the basement membrane.  In vivo, secretion and subsequent inhibition of these enzymes are largely regulated by interplay between different growth factors/cytokines  and natural inhibitors of MMP tissue inhibitors of metalloproteinase. ,,,, Recently, synthetic MMP inhibitors derived from hydroxamic acid such as Batimastat (BB-94) have been used successfully to antagonize MMP action on tumor cells, and thus prevent metastases. ,
Previous clinical and experimental studies have indirectly shown that MMPs may be involved in the process of PVR. ,,,,,,[19 ] If MMPs do play a role in PVR, a process in which there is an extensive contraction of the vitreous gel, it follows that natural or synthetic MMP inhibitors may modulate the effects of HRPE cells on collagen gel contraction. We have used an in vitro model of PVR ,, to reassess the contribution of HRPE cells to gel contraction, and to study the effect of synthetic MMP inhibitor, BB-94 on gel contraction.
| Materials and Methods|| |
Human retinal pigment epithelial cells
Human donor eyes were obtained from the cornea bank, Netherlands Ophthalmic Research Institute, Amsterdam and handled according to the declaration of Helsinki. HRPE cells were harvested by incubating the eye cups with 0.25% trypsin (ICN Biomedical, Inc.) for 2 h at 37°C. The cells were suspended in Glasgow modified minimum essential medium (GMEM) plus 10% fetal calf serum (FCS) and centrifuged at 1000 rpm for 10 min. The cell pellets were resuspended and cultured in GMEM medium with heat inactivated FCS 10%, nonessential amino acids, L-glutamine, and streptomycin and grown until they approached confluence. ,, The cells were passed 3-4 times before use to form a cell line. Passed cells retained their HRPE characteristics as determined by hexagonal shape in confluent culture [Figure 1]a, the cells took the stellate morphology with extensive stress fibers formation upon contact with the surface of collagen gel [Figure 1]b. HRPE cells immunohistochemical positive staining for cytokeratin- 19 (MO-772, DAKO A/S) and vimentin (DAB technique) [[Figure 1]c and d respectively].
|Figure 1: (a) Confluent human retinal pigment epithelial cells. (b) Spread, stellate morphology with extensive stress fiber formation of the cells cultured on the surface of collagen gel (white arrows) (a and b, ×200). (c). Immunocytochemical staining of retinal pigment epithelial cytoplasm for cytokeratin 19 (alkaline phosphatase nti-alkaline phosphatase technique), and (d) for vimentin (DAB technique)|
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Human monocytic cell line
Human monocytic cell line (THP-1 cells) from European Collection of Cell Culture (HGMP Resource Centre, Hinxton, Cambridge, UK) were grown on 10% FCS and passed several times before use to form a cell line. The cells were collected after centrifuge at 700 rpm for 10 min and used at a concentration of 5 × 10 5 in the collagen gel contraction assay.
Collagen gels formation
Three-dimensional collagen gels were prepared in 24-well plates (Nunclon™) on ice by a modification of the method previously described from rat tail collagen, Type I (Sigma, C-7661) in GMEM + 10% FCS, at a final concentration of 3 mg/ml. ,,, In some experiments, serum was omitted (see Results). Gels were allowed to solidify by incubation at 37°C. NaOH and HEPES were used to bring the pH to 7.1 for cell culture.
Subconfluent HRPE were harvested using trypsin 0.25%, washed twice in GMEM + 10% FCS to inactivate the trypsin, and seeded at various concentrations in a final volume of 300 μl medium onto the surface of gels in 24 well-plates. The degree of gel contraction was determined by the reduction in the surface area of the gel quantified using the digital images and applying an image analysis package (Leica QW, image processing software, Qwin-Lite (Cambridge UK)). The effects of cell number, time course, and serum on the ability of cells to contract and to invade collagen gels were determined. Four replicates were included for each data set, and each experiment repeated at least 3 times. Similar experiments were performed using THP-1 cells. The cells were collected using Roswell Park Memorial Institute medium growth medium and spun at a relatively low speed (700-800 rpm). The cells were seeded at 5 × 10 5 concentration in a final volume of 300 μl medium onto the surface of gels in 24-well-plates. Gels were observed at regular intervals for up to 3 days by phase contrast microscopy.
Immunohistochemistry of collagen gels
At various time intervals, collagen gels were fixed in the 24-well plate with 10% formaldehyde in phosphate buffered saline (PBS) overnight. Gels were then carefully removed from the wells, oriented and embedded in paraffin for sectioning. Sections (5 μm) were mounted on aminopropyl ethyl silane-coated slides. Sections were dewaxed with histoclear 100% for 30 min, then treated with alcohol (100% for 5 min, then 70% for 5 min) followed by microwaving in citrate buffer for two periods of 20 min. The slides were then dehydrated in PBS for 30 min.
Immunohistochemistry was performed as follows: Sections were incubated with the primary monoclonal antibodies against human MMP-1, 2, and 9 (Cat no. MAB901, MAB903, and MAB911, respectively. R and D systems) at doses of 10, 3.5, and 2.5 μg/ml for 1 h at room temperature and then washed 3 times with TRIS-buffered saline (TBS). The alkaline phosphatase-anti alkaline phosphatase method was carried out by incubating the sections with the secondary antibodies (rat anti-mouse; biotin, E-0354, DAKO A/S) for 30 min at room temperature, followed by three washes in TBS and finally the tertiary label (Strep ABC complex/AP; K0391, DAKO A/S) was added and incubated with the slides for another 30 min.  The sections were then incubated with the substrate (naphthol, veronal acetate buffer, levamisole, and fast red TR salt) for 10 min.
Finally, the sections were counter-stained with hematoxylin, washed thoroughly with Scott's tap water and distilled water for 1 min each, and mounted in Apathy's aqueous medium. Experiments were done in triplicate. Negative controls excluded the primary antibodies.
Cell migration/invasion of the gel
Cell migration by HRPE and THP-1 cells into the collagen gels was determined in vitro by phase contrast microscopy of the collagen gels. The degree of collagen invasion by the cells was determined using a micrometer attachment to the microscope and observing the depth from the surface of the gel to which the leading pair of cells migrated (leading front technique). , In this technique, the top layer of the cells is focused, and this position is read on the micrometric fine adjustment of the microscope (Leitz Labovert phase-contrast microscope). The cells are subsequently followed into the gel with the fine adjustment until the two farthest migrated cells are in focus. This position is then read, and the migrated distance of the leading cell front is calculated by subtracting these two readings. Four replicates were included for each data, and each experiment repeated at least 3 times.
In addition, cellular invasion by HRPE cells was assessed by light microscopy of the fixed and stained collagen gel sections. In this assay, cells were considered to have invaded the gel if there was a clear distance (more than one cell size) between the edge of the migrating cell and the surface of the gel. Cells were categorized as having invaded the gel or as remaining on the surface of the gel. The percentage of the cells expressing a specific enzyme (MMP) in each section was counted and correlated with invasion into the gel.
Synthetic matrix metalloproteinases inhibitors (Batimastat)
Effects of synthetic matrix metalloproteinases inhibitors on cell proliferation assay
HRPE cells were passed into fresh flasks, cultured in GMEM/10% FCS for different periods of time, harvested by brief trypsinization (0.25% for 20 min), and counted using a hemocytometer. Cells were examined for their viability by trypan blue exclusion on each of four consecutive days. HRPE cells in the tissue culture reached peak growth (sub-confluence) and viability on the second postincubation day. The effects of different concentrations (5 nM, 50 nM, 500 nM, and 5 μM) of the synthetic MMP inhibitor, BB-94 (British Biotech, Oxford, UK) on cell growth was determined by incorporating the inhibitors into the culture media for the first 48 h.
Effects of synthetic matrix metalloproteinases inhibitors on collagen contraction
The MMP inhibitor (BB-94) was solubilized in a small volume of dimethyl sulfoxide (DMSO) and diluted in PBS. It was then incorporated into the collagen gel assay at three final concentrations of 50 nM, 500 nM, and 5 μM. Three sets of collagen gels were used (a) positive control (cells alone); (b) DMSO control (cells in medium plus DMSO at the same concentration used for inhibitors); (c) test gels (cells grown with the different concentrations of the inhibitor for 48 h), then the cells at a concentration of 5 × 10 5 /well were seeded on to the gels in 300 μl of growth medium containing the inhibitor. Estimation of the amount of gel contraction was done as mentioned before for three postincubation days.
Data and statistical analysis
Control and test samples in the cell proliferation assays and the collagen contraction assays were statistically analyzed using paired Student's t-test. The tested and control materials were performed in triplicate or quadruplicate, and the experiments were repeated at least 3 times. One-tailed distribution was used on comparison with the positive control, and two-tailed distribution was used on comparison with both negative and positive controls. Significance was accepted at 95% confidence intervals.
| Results|| |
Human retinal pigment epithelial cell behavior on collagen gels
HRPE cells cultured on plastic uncoated substrates in GMEM plus 10% FCS, grew as sheets of cells with a characteristic hexagonal and polygonal mosaic [Figure 1]a and b and expressed markers typical of RPE cell (see Methods). On contact with the collagen gels, HRPE cells rapidly adhered, spread, and migrated on the gel surface. HRPE cellular invasion of the gel also occurred but differed significantly when it originated from sheets of cells as compared to individual cells. Invasion, as individual cells were time dependent [Table 1] and reached a maximum by the third postincubation day (41.4 μm). The migrating cells adopted a spindle-shaped morphology in which it was sometimes difficult to identify cell outlines within the gel. HRPE cells invaded the gel to a greater depth in the presence of serum than in its absence [Table 1]. In this way, sheets of HRPE cells embedded themselves into the gel and achieved a form of anchorage to the gel with condensation of the fibrils around the points of adhesions [Figure 1]b. Anchorage points developed at several sites and appeared to be associated with contraction of the gel in the form of external compression by the entire sheet of cells. THP-1 cells migrated as individual cells and showed no tendency to form sheets.
|Table 1: Cell migration into collagen gels (3 mg/ml) was measured by leading front technique (see Methods). Cells were seeded onto the gels in the presence of 10% fetal calf serum at different cell concentrations and observed for up to 72 h. Cells at 50 × 104/ml in the absence of serum were also observed (50)¤ (¤= serum-free). *= P<0.05|
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Contraction of collagen gels by human retinal pigment epithelial cells
Collagen gels failed to undergo contraction in the absence of cells [Figure 2]a. However, when HRPE cells were seeded onto the surface of collagen gels, the gels contracted progressively, especially in the presence of the serum [[Figure 2]b: serum-free cells, c and d: cells with the serum). Quantification of gel contraction indicated that the degree of gel contraction was dependent on the number of cells and the presence of the serum [Figure 3] but even with high doses of cells (5 × 10 5 ) contraction usually never exceeded 50% of the original gel size. This confirms previous experiments in which it was shown that cell types had different capabilities for contraction of vitreous gels in ascending order of contractile potential as follows: Macrophages < glial cells < RPE cells < fibroblasts.  Only the last cell type appeared to have the capability to effectively reduce the gel volume to < 50%. 
|Figure 2: Human retinal pigment epithelial cell-induced collagen gel contraction. A: Collagen gel (3 mg/ml) in tissue culture before adding the cells. B: Mild gel contraction after culture with human retinal pigment epithelial cells in the absence of serum (72 h). C: Moderate gel contraction after incubation with cells and serum (24 h). D: Marked gel contraction after incubation with cells and serum (72 h). E: Mild gel contraction after incubation with cells and serum in the presence of Batimastat (500 nM) (arrows delimit the edge of the gel)|
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|Figure 3: Human retinal pigment epithelial cell-mediated collagen gel contraction (time course). There is a progressive reduction in the gel diameter which commenced from the first postincubation day and reached the maximum contraction on the 3rd and 4th day with the cells grown in 10% fetal calf serum. Cells seeded at a concentration of 5 × 105/well|
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THP-1 cells which are well-known by the production of MMPs spread into the gel but did not affect the gel area at any time point of the experiment; however, several areas of collagen liquefaction (lacuna) have appeared inside the gel.
Expression of matrix metalloproteinases-2 and 9 by human retinal pigment epithelial cells cultured on collagen gels
Immunohistochemistry of collagen gels containing HRPE cells showed differential expression of MMPs. HRPE cells on the surface of the gels [Figure 4]a-d appeared to be embedded in a network of condensed collagen as predicted from the phase contrast images [Figure 1]c and d and failed to express markers for any of the MMPs tested in this study. In contrast, single HRPE cells which had invaded the gel to a depth of several micrometers appeared to reside in clefts in the gel which were made more obvious by fixation during embedding and sectioning. No condensation of collagen fibrils appeared around these cells but instead they strongly expressed markers for MMP-2 and 9 [Figure 4]e-h. The percentage of cells per section expressing MMPs was evaluated after 24 and 72 h. The number of the positive cells increased by days 2 and reached the maximum by the third postincubation day. No expression of MMP-1 by HRPE cells was detected in these experiments.
|Figure 4: Immunohistochemistry of human retinal pigment epithelial cells in collagen gels stained for matrix metalloproteinases-1, -2, and -9. (a) Negative control. (b) Low power view of the surface of a gel showing negative-staining cells embedded near gel surface (matrix metalloproteinases-1). (c and d) Higher magnification of the surface of a gel showing several negatively-staining cells (short arrows) for matrix metalloproteinases-2 and matrix metalloproteinases-9, respectively. Long arrows show folds in the surface of gel caused by contraction of the gel. (e [low] and f [high]) Power views of matrix metalloproteinases-2 positive cells (short arrows) invading the gel in the depths of a lacuna (long arrow). (g and h) Low and high power views of matrix metalloproteinases-9 positive cells in similar position in a further lacuna (long arrow)|
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Effect of matrix metalloproteinases inhibitors on human retinal pigment epithelial cell viability and proliferation
HRPE cells in the tissue culture reached a peak, subconfluent growth on the second postincubation day. In the presence of synthetic MMP inhibitor at 5 and 50 nM doses, there was no significant reduction in the cell number or viability during the first 48 h of the culture. BB-94 at a concentration of 500 nM and 5 μM reduced the number of the growing cells [Figure 5] but did not affect the cellular viability during the 2 days incubation [Table 2]. There was no significant effect of the solvent DMSO on cell growth or viability.
|Figure 5: Human retinal pigment epithelial cell proliferation in the presence of different concentrations of Batimastat (dose response) on second postincubation day. Note, reduction in cell proliferation at a dose of 5 μM compared to controls (inhibitor-free and dimethyl sulfoxide containing media)|
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|Table 2: Proliferation and viability data for human retinal pigment epithelial cells grown in the presence and absence of BB-94|
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Effect of synthetic matrix metalloproteinases inhibitors on human retinal pigment epithelial-induced collagen gel contraction
HRPE cells incubated with collagen gels in the presence of the inhibitor BB-94 showed a dose-dependent decrease in their ability to contract the gel during the time course of the experiments, but gel was not completely contracted [Figure 2]e. At the higher doses of 500 nM and 5 μM, BB-94 demonstrated antienzymatic as well as antiproliferative effects, both of which may have had an effect in decreasing gel contraction [Figure 6].
|Figure 6: Human retinal pigment epithelial cell-mediated collagen gel contraction after incubation in the presence of Batimastat for 72 h. There is a statistically significant reduction in the amount of gel contraction in the presence of Batimastat at doses of 500 nM and 5 μM|
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| Discussion|| |
PVR is a modified wound healing process, which occurs as a result of cell-vitreous interaction. Many cells, singly or in combination, are thought to be involved in the induction of PVR. Retinal pigment epithelial cells, ,,,,,, retinal glial cells, ,,,,, fibroblasts, ,, and inflammatory cells (macrophages , and lymphocytes , ) are the most consistent finding in the periretinal membranes. This study has investigated the mechanism of collagen gel contraction by HRPE cells. The data have shown that HRPE cells cause contraction of Type I collagen gels in vitro in a dose and time-dependent manner and that the effects of the cells on the gel are to reduce the gel volume to approximately 50% of its original size. On the other hand, THP-1 cells failed to reduce gel diameter; however, it caused some disturbance to the gel structure (liquefaction). This confirms previous work showing that gels contraction by RPE cells is relatively less complete than the other contractile cells such as fibroblasts. 
The data in this study appear to support the view that gel collapse is related to intragel cellular invasion by cells and by their expression of MMPs. Evidence of gel collapse was noticed as lacunae which formed inside the gel. This phenomenon may have been due to cellular secretion of collagenases and gelatinases (MMPs-2 and 9) which cleave and dissolve the collagen fibrils. This effect was seen when THP-1 cells (which are known of their secretion of MMP enzymes) failed to contract the gel but caused areas of liquefaction inside the collagen matrices. Another essential role for MMPs is to pave the way for the HRPE cells to migrate from its basement membrane to the vitreous gel. As a result, destabilization of the gel permitted the surface sheets of adherent non-MMP expressing HRPE to induce contraction and shrinkage of the gel. The model used here mimics the in vivo condition of PVR in which RPE cells are known to migrate on the posterior surface of the partially detached vitreous and the retina, while probably relatively fewer cells actually migrate through the interstices of the vitreous. Thus, contraction of the vitreous gel may be achieved by external traction on the surface of the gel rather than complete internal collapse of the gel.
This hypothetical mechanism for the process of PVR of surface gel cell-mediated contraction with partial intragel lysis and destabilization is supported in part by the results of the MMP inhibitor studies. The MMP inhibitor BB-94 failed to completely prevent gel contraction even at higher doses, indicating that MMP inhibition is not required for gel contraction since the surface cells do not express detectable levels of MMPs. These results provide possible insights into the mechanism of gel contraction by cells generally and in particular have relevance for the clinical condition "PVR." MMPs are known to be present in the vitreous during PVR and have been implicated in its pathogenesis. However, it is not clear whether expression of MMPs would lead to greater or lesser gel contraction in PVR. Cells adhere to collagen either directly or via bridging ECM molecules such as fibronectin and vitronectin, and cell surface integrin receptors mediate these cell-matrix attachments. Cells migrate into three-dimensional-collagen matrices by making and breaking attachments to ECM molecules and cell surface and secreted proteolytic enzymes are important in this process including MMPs.  Thus, cells are considered to migrate through tissue matrices via pericellular proteolysis which is usually restricted to the vicinity of the cell and is rapidly induced and inhibited in the cell depending on the requirement for attachment versus forward movement. For low-density gel matrices such as the vitreous, gel contraction is the more likely outcome than gel collapse unless here is considerable production of proteolytic enzymes. Thus, the experiments performed here suggest that HRPE cells produce insufficient proteolytic enzymes including MMPs to shift the balance from gel contraction to gel collapse but produce sufficient enzymes to allow migration of the HRPE cells into the gel and weaken its structure.
| Conclusions|| |
Our study indicated that HRPE cells have the ability to invade the collagen gel, especially in the presence of the serum. The study also showed that MMPs secreted by the cells are selectively involved in HRPE invasion and collagen gel contraction. Both these processes of the proliferating and migrating cells may be implicated in the pathogenesis of similar conditions in vivo in which contraction of collagen gel is a feature, for example, proliferative vitreoretinopathy.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2]