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Analysis of Extracellular Matrix Synthesis During Wound
Healing of Retinal Pigment Epithelial Cells
Eye Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195 USA
of Ophthalmology, Osaka University Medical School, Osaka, Japan
extracellular matrix; growth factors; immunohistochemistry; retinal pigment
epithelium; wound healing
To investigate changes in retinal pigment epithelial (RPE) cells during wound
healing, we evaluated the deposition of newly synthesized extracellular matrix (ECM) over time
during wound healing in rat RPE cultures. We also estimated the effect of growth factors on the
healing rate and ECM synthesis. After preparing rat RPE cell sheet cultures, we made round 1-mm
defects in the cultures. Fibronectin, laminin, and collagen IV synthesis were evaluated with
immunocytochemistry every 12 hours after wounding. S-phase cell distribution was analyzed every
12 hours by 5-bromodeoxyuridine uptake. We added either platelet-derived growth factor (PDGF),
epidermal growth factor (EGF), or transforming growth factor- b2 (TGF-b2) to cultures at
concentrations of 1, 10, and 100 ng/mL and immunocytochemically analyzed the effects on ECM and
estimated the rate of wound closure. Although approximately 50% closure was achieved 24 hours
after wounding, fibronectin deposits first appeared at that time. Laminin and collagen IV were first
detected at 36 hours and fibronectin staining had extended toward the wound center. S-phase cells
were distributed in concentric rings that moved centripetally over time and corresponded to the
leading edge of the area stained with anti-ECM antibodies. TGF-b2 enhanced ECM deposition, but
EGF and PDGF did not. TGF-b2 decreased the healing rate in a dose-dependent manner, whereas
PDGF promoted wound closure. EGF enhanced closure at the highest concentration only. In
summary, wound healing in RPE may be initiated when cells at the wound edge slide or migrate
toward the wound center, which is followed by cell proliferation and then ECM synthesis. ECM
components may be produced in a specific sequence during healing. TGF-b2 may promote RPE cell
differentiation, and PDGF may enhance proliferation during wound healing of the RPE. Microsc.
Res. Tech. 42:311–316, 1998. r 1998 Wiley-Liss, Inc.
Age-related macular degeneration (AMD) is the leading cause of irreversible severe visual loss in people
over the age of 50 in the Western hemisphere (Bressler
et al., 1988). Recent advances in vitreous surgery have
made it possible to treat submacular disorders, including subretinal neovascularization and submacular hemorrhage associated with AMD (de Juan and Machemer,
1988; Kamei et al., 1996a; Lambert et al., 1992; Thomas
et al., 1992). Submacular surgery, however, causes local
debridement of the retinal pigment epithelium (RPE)
(Berger and Kaplan, 1992; Das et al., 1992; Grossniklaus
et al., 1992; Thomas et al., 1992). Although the RPE
defect is eventually healed by migration and proliferation of the RPE cells adjacent to the damaged area (Del
Priore et al., 1995; Heriot and Machemer, 1992; Valentino et al., 1995), characteristics specific to the RPE
are lost to some degree (Del Priore et al., 1988; Grisanti
and Guidry, 1995; Hergott et al., 1989; McKechnie et
al., 1988; Opas, 1991), and such damage may induce
photoreceptor death and increase the incidence of recurrent neovascularization after the surgery. Therefore,
repairing RPE damage may be crucial for recovery of
visual function. However, little is known about changes
in RPE cells during wound healing.
Basement membrane, or extracellular matrix (ECM),
is important in wound healing (Choi, 1994; Herrick et
al., 1992; Yamakawa et al., 1988). Most kinds of cells
synthesize ECM. Retinal pigment epithelial cells produce ECM, including the ECM components fibronectin,
laminin, elastin, heparan sulfate proteoglycan, and
collagen types I, III, and IV in vivo and in vitro
(Campochiaro et al., 1986; Li et al., 1984; Newsome et
al., 1988; Turksen et al., 1984). Cytokines are one of the
factors regulating gene expression in RPE cells (Ando
et al., 1995; Opas and Dziak, 1989; Osusky et al., 1994).
Synthesis of ECM during wound healing, however, is
not well understood in RPE cells.
In this study, we investigated the deposition of newly
synthesized ECM over time during wound healing in
rat RPE cell sheet cultures and compared it with the
distribution of proliferating cells. We also examined the
influence of growth factors on the healing rate and
ECM synthesis.
Contract grant sponsor: Nippon Eye Bank Association.
*Correspondence to: Motohiro Kamei, M.D., The Eye Institute (FFb 33),
Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195 USA.
Received 27 July 1997; accepted in revised form 1 April 1998
Retinal Pigment Epithelial Cell Sheet Culture
and Wounding
This study was conducted in accordance with the
Association for Research in Vision and Ophthalmology
Statement for the Use of Animals in Ophthalmic and
Vision Research. Sheet cultures of rat RPE cells were
prepared by a procedure modified from a previously
described method (Kamei et al., 1996b). Briefly, eyes
from 7–10-day-old Sprague-Dawley rats were incubated in 0.1% proteinase K (Merck, Darmstadt, Germany) at 37°C for 8 minutes and then for 10 minutes in
culture medium, which consisted of a mixture of equal
volumes of Dulbecco’s modified Eagle’s medium and
Ham’s F-12 (both, Nikken Bio Medical Laboratory,
Kyoto, Japan) supplemented with 10% fetal bovine
serum (FBS; HyClone Laboratories, Logan, Utah). Then,
after bisecting the whole eye posterior to the ora
serrata, the sclera and choroid were peeled away from
the neural retina, preserving a sheet of RPE cells
attached to it. The isolated tissue composed of the
neural retina and adherent RPE was placed on a
culture plate with culture medium with the RPE side
down. A 2-chamber plastic slide (Lab-Tek, Nunc Inc.,
Naperville, IL) was used as a culture plate. One hour’s
incubation at 37°C allowed the RPE sheet to detach
spontaneously from the retina as a sheet and weakly
attach to the culture plate with the apical-microvilli
side up.
After the RPE sheets were incubated for 24 hours to
allow adequate adhesion to the culture plates, round
defects 1 mm in diameter were made by pressing a
trephine on the sheet culture. One to several defects per
sheet were made according to sheet size so that the
defects were separated by 1 mm. Six defects were
analyzed in each experiment.
Immunocytochemical Analysis of Synthesized
Extracellular Matrix
To evaluate the distribution of fibronectin, laminin,
and collagen IV, the cultures were examined immunocytochemically every 12 hours after wounding. After
rinsing away the culture medium with phosphatebuffered saline (PBS, pH 7.4), the culture plates were
filled with 50% methanol and 50% acetone and the cells
were fixed for 10 minutes. The cells then were incubated overnight at 4°C with the appropriate primary
antibody: monoclonal mouse anti-fibronectin antibody
(MAB1940, Chemicon International, Inc., Temecula,
CA), monoclonal mouse anti-laminin antibody (A2–
20018, Amresco, Solon, OH), or polyclonal rabbit antihuman collagen IV antibody, which has cross-reactivity
with rat (PC10760, Progen Biotechnik GmbH, Heidelberg, Germany). These primary antibodies were used at
a dilution of 1:500 in 0.1 M PBS containing 3% bovine
serum albumin (BSA) and 0.3% Triton X-100. After
overnight incubation, all specimens were labeled with
fluorescein isothiocyanate-conjugated goat anti-mouse
IgG (Jackson Co., St. Louis, MO) or anti-rabbit IgG
(Sigma Chemical Co., St. Louis, MO) diluted 1:1000
with the PBS-BSA solution, and the cells again were
incubated at 4°C overnight. The specimens were examined under an epifluorescent microscope (BX-50, Olympus, Tokyo, Japan).
S-Phase Cell Analysis
To investigate the distribution of proliferating cells
during wound healing, cells in the S-phase were identified every 12 hours using 5-bromodeoxyuridine (BrdU)
incorporation and immunocytostaining. Ten µM BrdU
(Sigma) was added to the culture medium 9 hours
before fixation. Immunocytochemical staining using
the same procedures described above was performed
using monoclonal anti-BrdU antibody (Becton Dickinson Immunocytometry Systems, San Jose, CA) as the
primary antibody. The nuclei were counterstained with
0.04 µg/mL propidium iodide.
Growth Factor Supplementation
After the RPE sheets were incubated in the medium
with 10% FBS for 24 hours, they were rinsed twice with
serum-free medium and then incubated under serumfree conditions for 24 hours to eliminate the influence of
growth elements in the serum. Then 1-mm round
defects were made in the sheet, the medium was
replaced with medium supplemented with 0.1% FBS,
and one of the three following recombinant human
growth factors was added: platelet-derived growth factor-BB (PDGF-BB; Genzyme, Cambridge, MA), epidermal growth factor (EGF; Toyobo, Osaka, Japan), or
transforming growth factor- b2 (TGF-b2; Genzyme).
These growth factors were applied at concentrations of
1, 10, and 100 ng/mL. For controls, RPE sheets were
treated in the same way, but no growth factors were
added. Immunocytochemistry was performed for the
ECM as described above and the rate of wound closure
was estimated.
Rate of Wound Closure
Phase-contrast micrographs were taken every 8 hours
after wound formation and the area of wound remaining was measured using a computerized area analyzer
(Micro Computer Imaging Device, Imaging Research
Inc., Ontario, Canada). The ratio of the wound area
remaining was compared to the initial 1-mm wound
area to evaluate the rate of wound healing.
Statistical Analysis
The wound healing rate with each growth factor at
each concentration was compared with controls and the
effects were analyzed by using two-way repeatedmeasures ANOVA. Significance was accepted at P ,
Deposition of Extracellular Matrix and
Distribution of S-Phase Cells Over Time
Although approximately 50% closure was achieved
24 hours after wounding and complete closure achieved
at 48 hours (Fig. 1, column 1), fibronectin deposits were
first apparent at 24 hours (Fig. 1, column 3), and
laminin and collagen IV were detected at 36 hours (data
not shown). At 48 hours, fibronectin staining (Fig. 1,
column 3) occurred more centrally than laminin and
collagen IV staining (Fig. 1, columns 4 and 5). Fibronectin was detected only in the area covered with regenerated RPE cells, whereas laminin and collagen IV were
present in both wounded and unwounded areas. The
entire wound area except a central zone was covered
Fig. 1. First column: Phase-contrast photomicrographs of the
wounds in RPE cell sheet cultures 24, 48, 72, and 96 hours after
wounding. Approximately 50% closure was achieved at 24 hours. The
defect was completely covered with migrating and proliferating cells
at 48 hours although the cells were spindle-shaped. The cell population became denser and the cells became more polygonal over time.
Second column: Micrographs of RPE cells in the wound stained for
anti-BrdU antibody. S-phase cells that showed positive signals were
first recognised at 24 hours as a circle at the peripheral zone of the
wound and then moved centripetally, forming concentric circles over
time after wounding. Few nuclei stained at 96 hours; those that did
were scattered uniformly over the sheet. Third through fifth columns:
Immunofluorescent micrographs of the wounds stained with antifibronectin (FN, 3rd column), laminin (LN, 4th column), and collagen
VI (Col IV, 5th column) antibody. Fibronectin was first observed at 24
hours, but laminin and collagen VI were not stained yet. Laminin and
collagen IV staining was recognised 48 hours after wounding and, at
that time, fibronectin staining occurred more centrally than other
ECM staining. The entire wound area except a central zone is covered
with deposits of these ECM components at 72 hours and completely
covered at 96 hours. Distribution of BrdU staining (2nd column)
corresponded to the leading edge of the area staining positively for
ECM. Original magnification, 10x.
with deposits of these ECM components at 72 hours
(Fig. 1, columns 3–5). Fibronectin stained in a filamentlike manner, and collagen IV and laminin stained
diffusely. It took 96 hours for these ECM constituents to
accumulate over the entire wound area.
Anti-BrdU antibody staining revealed S-phase cells
that formed concentric circles according to the time
elapsed since wounding (Fig. 1, column 2). The first
circle to appear was the largest, located at the peripheral zone of the wound at 24 hours after wounding;
subsequently, smaller circles appeared centripetally. At
84 hours, the innermost circles were located at the
center of the wound as a cluster (data not shown) and
this BrdU-positive zone corresponded to the leading
edge of the area staining positively for ECM (Fig. 1,
columns 3–5). Few additional nuclei stained at 96
hours, and those that did were scattered uniformly over
the sheet. Consequently, cell proliferation to repair
wounds 1 mm in diameter ceased by 96 hours after
when compared with a control sheet (Fig. 2D) under an
epifluorescent microscope, whereas cultures supplemented with PDGF or EGF (Fig. 2B,C) did not show an
apparent increase in deposition of these ECM components. Cultures incubated with EGF or PDGF showed a
filamentous pattern of deposition that differed from the
control and TGF-b2-supplemented cultures.
Compared to controls, TGF-b2 decreased the healing
rate at all three concentrations (P 5 0.03, 0.003, 0.0003
at 1, 10, 100 ng/mL, respectively), whereas PDGF
promoted wound closure at concentrations of 10 and
100 ng/mL (P 5 0.77, 0.002, 0.002 at 1, 10, 100 ng/mL,
respectively) (Fig. 3). The healing rate was not significantly affected at EGF concentrations of 1 and 10
ng/mL but was enhanced at 100 ng/mL (P 5 0.72, 0.69,
0.02 at 1, 10, 100 ng/mL, respectively).
Effect of Growth Factors on ECM Deposition
and Healing Rate
TGF-b2 remarkably enhanced deposition of fibronectin (Fig. 2A), laminin, and collagen IV (data not shown)
This study revealed a time lag between ECM deposition and wound closure. Wound healing may be initiated when cells at the wound edge slide or migrate
toward the center of a round defect, which is followed by
cell proliferation and then synthesis of ECM.
We observed BrdU-positive S-phase cells at 24 hours
but not at 12 hours after wounding, although we
Fig. 2. Epifluorescent micrographs of the wound stained with
anti-fibronectin antibody 60 hours after wounding. TGF-b2 added in
the culture medium (A) appears to enhance deposition of fibronectin
when compared with the no-treatment control (D). Cultures incubated
with PDGF (B) or EGF (C) show a filamentous pattern of deposit that
differs from the control and TGF-b2-supplemented cultures. Original
magnification, 20x.
previously reported (Kamei et al., 1996b) in the same
wound healing model that about 20% of closure is
obtained at 12 hours after wounding. This finding
suggests that cell spreading or sliding from the wound
edge accomplishes the initial 20% of wound closure
without proliferation. Considering that a circle 900 µm
in diameter corresponds to 81% of a 1-mm round defect,
an initial 20% wound closure at 12 hours means that an
approximate 50-µm wide peripheral zone is covered
with spreading cells. We thus can say that cells covering an approximate 50-µm wide peripheral zone at 12
hours had not yet proliferated. This observation is
consistent with a previous report using organ culture
and proliferating cell nuclear antigen (PCNA) staining
that found RPE cells in wounds narrower than 125 6 48
µm did not express PCNA (Hergott and Kalnins, 1991).
Thus, the process of wound healing may be initiated by
cell sliding or migration at the wound edge; cell proliferation then follows.
A BrdU-positive signal observed as a ring near the
wound edge at 24 hours moved centripetally, forming
concentric circles until 84 hours. ECM staining did not
occur centrally beyond these concentric BrdU-positive
circle at any time point. In other words, the BrdUpositive zone constituted the leading edges of ECM
deposits. Thus, ECM synthesis may follow cell proliferation during RPE wound healing, but studies of mRNA
expression that include in situ hybridization are needed
to confirm this observation.
Fibronectin synthesis preceded that of laminin and
collagen IV. Deposits of fibronectin were first apparent
at 24 hours after wounding, and laminin and collagen
IV were first seen at the peripheral zone of the wound at
36 hours when fibronectin staining had begun to extend
toward the center of the wound. This sequence of events
suggests that ECM components are produced in a
certain order during wound healing.
We selected the TGF-b2, PDGF, and EGF cytokines
for several reasons. TGF-b upregulates the synthesis of
ECM components in the RPE (Ando et al., 1995; Osusky
et al., 1994) and many other kinds of cells (Ignotz and
Massague, 1986; Vollberg et al., 1991), and it is the only
growth factor that inhibits cell proliferation under in
vitro conditions (Massague, 1990) and that is clinically
applied in macular hole surgery as a promotor of wound
healing in RPE or glial cells (Glaser, 1992). As an
autocrine stimulator of growth in RPE, PDGF may play
an essential role in retinal wound repair (Campochiaro
et al., 1994). EGF enhances wound healing in various
types of epithelial cells, including RPE cells (Leschey et
al., 1990), and clinically is applied to persistent corneal
ulcer (Scardovi et al., 1993). Although the growth
factors used in this study were recombinant human
proteins, we decided to apply them to rat cells because a
healing rate. This result is consistent with previous
studies that report that TGF-b upregulates the synthesis of ECM components and inhibits cell proliferation in
vitro. In contrast to TGF-b2 and controls, both PDGF
and EGF produced fibronectin deposits in a filamentous
pattern and did not increase ECM deposition, but did
promote wound closure.
In conclusion, these findings suggest that TGF-b2
promotes differentiation of RPE cells, and PDGF enhances their proliferation during wound healing of the
RPE. Quantitative analysis of ECM deposition and
proliferating cells is required to establish the validity of
this mechanism.
We are indebted to Dr. Atsushi Hayashi for valuable
advice on the experimental design and technique,
Michelle Secic for statistical analysis, and Cassandra
Talerico for manuscript preparation.
Fig. 3. Wound closure rate in the retinal epithelial cell sheet
cultures incubated with various kinds and doses of growth factors.
Compared to control cells (no growth factor added), TGF-b2 decreased
the healing rate at all three concentrations, whereas PDGF promoted
wound closure at concentrations of 10 and 100 ng/mL. EGF enhanced
closure only at 100 ng/mL.
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