October 1986
Vol. 27/10
Investigative Ophthalmology
& Visual Science
A Journal of Dosic and Clinical Research
Articles
Maintenance of Corneal Endothelial Cell Shape by
Prostaglandin E2: Effects of EGF and Indomethacin
Arthur H. Neufeld, Marcia M. Jumblarr, Eriko D. Markin, and Goil M. Raymond
Confluent, cultured, rabbit corneal endothelial cells maintain a polygonal shape which is characteristic
of these cells in vivo. When cultured in the presence of EGF (10 ng/ml) and/or indomethacin (1.0 MM),
the endothelial cells have markedly different shapes at confluency. By morphometry, untreated cells
are polygonal and have a maximum axis of 33 n; EGF treatment causes a spindle-shaped elongation to
48 M and indomethacin treatment causes a stellate-shaped elongation to 48 fi. There is a slight increase
in cell density. When cells are cultured in the presence of both drugs, elongation is more pronounced to
a fibroblastic appearing cell population, with maximum axes of 60 n and more, but no additive increase
in cell density. Continuity of cell borders is often lost. Corneal endothelial cells cultured in the presence
of EGF, indomethacin, and PGE2 (0.5 Mg/ml) maintain their polygonal shape; PGF2o is not effective at
reversing the drugs' effects. Untreated and EGF-treated cells sythesize and release substantial quantities
of PGE2 (2-4 ng/104 cells). Indomethacin completely inhibits PGE2 synthesis. It is concluded that PGE2
maintains the polygonal cell shape of the corneal endothelium in vitro and, perhaps, in vivo. The elongated
forms of the cell may be related to migration and important in wound closure. Invest Ophthalmol Vis
Sci 27:1437-1442, 1986
The corneal endothelium, a single layer of hexagonal
cells lining the posterior surface of the cornea, is critical
for the maintenance of transparency of the tissue. The
cell layer is avascular, lacks innervation, and is bathed
on its apical face by aqueous humor. In the aging human, the density of corneal endothelial cells gradually
decreases and the remaining cells retain their polygonal
shape while becoming wider and thinner to maintain
a permeability barrier.' When a wound or discontinuity
occurs in this tissue, the cells bordering the defect become elongated, migratory, and, perhaps, synthesize
new extracellular matrix material.2'3 These events can
be studied in vivo and in vitro.2"7 Studies of the events
of wound closure in tissue culture systems, even using
rabbit cells which, unlike human cells, are mitotically
active, allow independent observation of changes in cell
shape, migration, metabolism, and proliferation.4"7
Previous studies have indicated that epidermal
growth factor (EGF) promotes mitosis and elongation
in cultured rabbit corneal endothelial cells.5 EGF-induced elongation is reversible, independent of EGFstimulated mitosis, and similar to the elongation of
migratory cells adjacent to experimental endothelial
injuries. Apparently EGF exerts its effect via a specific
receptor and can increase phosphotidyl inositol turnover and subsequent synthesis of arachidonic acid metabolites.8 Furthermore, in the bovine aortic endothelium, exogenous arachidonic acid induces cellular
elongation.9
We designed a series of experiments to characterize
the morphologic responses of cultured corneal endothelial cells to EGF, via pathways that would be dependent and independent of the synthesis of arachidonic acid metabolites. Our findings indicate that endogenous synthesis of prostaglandin E2 is necessary for
the maintenance of the normal, polygonal endothelial
cell shape and that, if this pathway is blocked, an extraordinary change in the shape of the cells occurs
which is potentiated by EGF.
From the Ophthalmic Pharmacology Unit, Eye Research Institute,
and the Department of Ophthalmology, Harvard Medical School,
Boston, Massachusetts.
This work was supported in part by USPHS grant EY-05767.
Submitted for publication: January 16, 1986.
Reprint requests: Arthur H. Neufeld, PhD, Eye Research Institute,
20 Staniford Street, Boston, MA 02114.
1437
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INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / October 1986
1438
Materials and Methods
Tissue Culture
Pieces of Descemet's membrane with attached endothelial cells were isolated from albino rabbit corneas,
incubated in Dispase II (1 unit/ml in calcium and
magnesium-free Hanks buffer) for 60 min at 37 °C, and
then triturated vigorously with a flame-polished Pasteur
pipette to suspend clumps of endothelial cells. Cells
were collected by centrifugation (200 X g, 5 min, 20 °C),
suspended in control culture medium (Medium-199
with 50 ng/m\ gentamicin and 10% fetal bovine serum)
that was supplemented with 25 ng/ml fibroblast growth
factor, inoculated into 35 mm wells, and incubated at
37°C in humidified 5% CO 2 . In general the cells from
three animals (six corneas) were combined and used
to inoculate a six-well plate. The following day, the
medium was changed and Descemet's membrane fragments were discarded.
Seven days post-culture, the confluent endothelial
cells were subcultured by incubation in 1.0 ml trypsinversene solution (0.5 g/L trypsin 1:250 and 0.2 g/1
EDTA) for 15 min, collected by centrifugation (200
X g, 5 min, 20°C), triturated, suspended in control
culture medium, and plated out in experimental media
at 2 X 104 cells per well in each well of 24-well tissue
culture plates. Experimental media consisted of control
medium (B) plus the addition of 10 ng/ml epidermal
growth factor (BE), or 10"6 M indomethacin (BI), or
both (BEI). Where indicated, prostaglandin E2 analogue
was added to the control and experimental media in
increasing concentrations from 0.005 /ug/ml to 0.5 fig/
ml; similarly, PGF2a analogue was tested. In some experiments, arachidonic acid (0.05 pM to 5.0 fiM) was
added to EGF and/or indomethacin medium. The effects of EGF plus suprofen (10~5 M), flurbiprofen (10~5
M) and dexamethasone (10~6 M) were also examined.
Cell Density
Seven days post-subculture, 0.5 ml trypsin-versene
solution was added to the wells for 45 min to detach
growing cells. Detachment was confirmed by phase
contrast microscopy. Following trituration, cell number
was determined using a Coulter Counter.
Morphology
To quantitate the induced morphological effects at
7 days post-subculture, the cells were fixed in situ with
10% neutral buffered formalin and stained with full
strength Giemsa. Photomicrographs were then taken
Vol. 27
using a Leitz Wetzlar light microscope. The photographed cells were outlined in several randomly selected
constant area fields using a Zeiss Videoplan 2 Digitizer.
The associated software, MOP-Videoplan Image
Analysis Systems by Kontron Bildanalyse, was used to
calculate cell area, perimeter (Per) and maximum axis
(Amax). All equipment was from Rainin Instruments
(Woburn, MA).
Radioimmunoassay of PGE2
Prostaglandin E2 concentrations in various experimental media were determined using a commercially
available kit. Medium samples were taken from day 7
subcultures, extracted with ethylacetate, evaporated to
dryness, redissolved in assay buffer, and assayed according to the standard kit protocol.
Animals
New Zealand white rabbits used in this study were
cared for and treated in accordance with the ARVO
Resolution on the Use of Animals in Research.
Materials
Tissue culture multiplates were from Falcon (Oxnard, CA), medium 199, gentamicin, fetal bovine
serum, trypsin-versene, and Hank's balanced salt solution from GIBCO Laboratories (Grand Island, NY),
indomethacin, arachidonic acid, and dexamethasone
from Sigma Chemical Company (St. Louis, MO), epidermal growth factor from Collaborative Research
(Lexington, MA), endothelial mitogen fibroblast
growth factor from BioMedical Technologies Inc.
(Cambridge, MA), 16-16-Dimethyl prostaglandin E2
and prostaglandin F 2a Tromethamine from Boehringer
Mannheim (Indianapolis, IN), Na-flurbiprofen from
Allergan Pharmaceuticals (Irvine, CA), and suprofen
from Alcon Laboratories (Ft. Worth, TX). The PGE 2
radioimmunoassay kit was from Seragen (Boston, MA).
Results
Figure 1 demonstrates the appearance of confluent,
rabbit corneal endothelial cells after 7 days of subculture in the presence or absence of EGF and/or indomethacin. Under control conditions, in the absence of
added indomethacin or EGF, the cells are polygonal
and morphologically resemble native tissue (Fig. 1A).
As determined by morphometry, the frequency distribution of the maximum axes (Amax) of cells under
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No. 10
PROSTAGLANDIN E2 AND ENDOTHELIAL CELL SHAPE / Neufeld er QI.
1439
Fig. 1. Morphologic effects of EGF and/or indomethacin on rabbit corneal endothelial cells. Rabbit corneal endothelial cells were subcultured
and grown for 7 days to confluence. Cells grown in control media (A) are polygonal and resemble native endothelial tissue. When grown in the
presence of EGF (B) there is elongation producing spindle-shaped cells. In the presence of indomethacin (C) cells have assumed a stellate shape
with loss of continuity between cell borders. Cells grown in the presence of both EGF and indomethacin (D) elongate to the extent of appearing
fibroblastic. (X340)
control culture conditions is shown in Figure 2A. As
indicated in Table 1, the mean of the major axes of
the polygonal shaped cells in 33 ^ and the mean of the
perimeters (Per) is 95 p. For an ideal, perfectly round
cell, the ratio Per/Amax should be equal to TT (3.14).
As demonstrated in Table 1, under control conditions,
this ratio is close to IT.
In the presence of EGF, the corneal endothelial cells
become elongated. Within 7 days, the endothelial cells
are spindle-shaped (Fig. IB) at confluence and have a
significantly greater distribution of major axes (Fig. 2B),
although there is little or no effect on cell density (Table
1). The ratio Per/Amax decreases significantly (Table
1), indicating loss of rotundness.
In the presence of indomethacin, the cells are elongated at confluence with a stellate shape (Fig. 1C).
Continuity of cell borders is often lost. The distribution
(Fig. 2C) and mean of the major axes increases (Table
1), and the Per/Amax ratio of 2.6 indicates loss of the
polygonal shape.
As seen in Figure ID, when corneal endothelial cells
are cultured in the presence of EGF and indomethacin,
marked elongation occurs to the extent of producing
rod-shaped cells at confluence that bear little resemblance to their sister cultures grown under control conditions. The cells are fibroblastic in appearance and
often form swirling patterns of dense populations. The
mean of the major axes of these cells is approximately
twice (60 n) that of control cells (Table 1), and the
frequency distribution of Amax of these cells is skewed
markedly to higher values (Fig. 2D). The decreased
ratio Per/Amax to 2.5 is characteristic of an elongated
cell. Under these conditions, cell density is approximately one-third greater than cells grown under control
conditions. Potentiation of EGF-induced elongation
also occurs with 10~5 M suprofen and, to a lesser extent,
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1440
INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / October 1986
Vol. 27
B
10 20 30 40 50 60 70 80 90 100 1?0 1&) 1&) 140
0 10 20 30 40 50 60 70 80 90 100 110 120130140
Amax
10 20 30 40 50 60 70 80 90 100 110 120 130 140
Fig. 2. Frequency distribution of maximum axes of
cells grown in the presence or
absence of EGF and/or indomethacin. The major axes
(Amax), determined by morphometry, of subcultured
cells grown to confluence in
control media (A), in the
presence of EGF (B), in the
presence of indomethacin (Q,
and in the presence of both
EGF and indomethacin (D).
0 10 20 30 40 50 60 70 80 90 100 110120 130 140
Amax
with equimolar flurbiprofen; dexamethasone has no
effect (data not shown).
The addition of increasing concentrations of a synthetic analogue of prostaglandin E2 causes inhibition
of the elongation of Amax when cells are grown in the
presence of EGF and indomethacin (Fig. 3). Cells
grown in the presence of EGF, indomethacin, and 0.5
Mg/ml PGE 2 analogue are indistinguishable from cells
grown in the absence of EGF and indomethacin. The
addition of increasing doses of PGF 2a has no effect on
the elongation of Amax produced by the combination
of EGF and indomethacin (data not shown). When
added in the absence of EGF, arachidonic acid had no
effect on cell shape in the presence or absence of indomethacin (data not shown).
Synthesis of PGE 2 by cultured corneal endothelial
cells was measured by growing cells in the presence
and absence of EGF and/or indomethacin (Table 2).
Under control conditions, cultured corneal endothelial
cells synthesize and release substantial quantities of
endogenous PGE 2 . Synthesis of PGE 2 is unaffected by
the addition of EGF. Indomethacin totally inhibits
PGE2 synthesis in control cultures and cultures to
which EGF has been added.
Discussion
We have found that indomethacin causes marked
elongation of corneal endothelial cell shape which is
(um)
greatly enhanced by EGF. Other cyclooxygenase inhibitors are also effective but less potent; the rank order
of potency appears to be: indomethacin > suprofen
> flurbiprofen. The inactivity of dexamethasone is
consistent with a suggested lack of nuclear steroid receptors in these cells.10
When the analogue of prostaglandin E2 is included
in the culture medium, the marked effect of EGF and
indomethacin to induce elongation does not occur.
This demonstrates that, if the cells are exposed to prostaglandin E 2 , they retain their normal, polygonal shape.
Furthermore, under control conditions, when polygonal morphology is evident, corneal endothelial cells
in culture endogenously synthesize prostaglandin E 2 ,
as we have shown. We conclude that, when corneal
endothelial cells are growing in culture under conditions which favor their normal, polygonal shape, synthesis of prostaglandin E2 occurs and is necessary for
maintenance of this shape. The presence of indomethacin blocks the formation of PGE 2 and the cells become
stellate in shape.
In the presence of EGF, the corneal endothelial cells
must synthesize another product, which influences the
cells to elongate and become spindle-shaped. The action of the unknown product to favor the elongated
shape is potentiated in the presence of indomethacin
because synthesis of PGE 2 , which favors polygonal
shaped cells, is blocked. Thus, when the cyclooxygenase
pathway is inhibited, the cells become even more fibroblastic-like.
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No. 10
PROSTAGLANDIN E2 AND ENDOTHELIAL CELL SHAPE / Neufeld er ol.
1441
Table 1. Morphometric parameters in cultured corneal endothelial cells
00 00 00 DO
Growth Medium
Cells/mm2
Per (urn) t
Amax (nm)£
Per/A max
7r - Per/A max
303 ±31*
313 ±32
453 ±46
403 ± 23
94.9±2.4f
121.3±3.2
119.8 ±3.6
145.0 ±7.8
33.3±1.0t
47.7 ±1.4
47.5 ±1.7
60.3 ±3.1
2.90 ± 0.04
2.59 ±0.03
2.64 ± 0.04
2.46 ±0.08
0.24
0.55
0.50
0.68
All values are mean ± SEM.
* n = 9 — 13 cultured wells
+ n = 73 - 158 individual cells
Arachidonic acid is a common intermediate in this
scheme that is apparently available under control, culture conditions and made into prostaglandin E 2 . In
vivo, PGE 2 is synthesized by rabbit corneal endothelium." Arachidonic acid added to control media did
not cause alteration of the normal polygonal cell shape
and, thus, sufficient material must have already been
available for prostaglandin E2 synthesis. If EGF liberates arachidonic acid which is made into another product that favors elongation, then arachidonic acid added
to culture media containing indomethacin should have
E
<
PGE2 Concentration (ug/ml)
Fig. 3. Effect of exogenous PGE2 on cells grown in the presence
of both EGF and indomethacin. Subcultures of rabbit corneal endothelial cells were grown for 7 days in control media (B) and in the
presence of both EGF and indomethacin (BEI) with increasing concentrations of exogenous PGE2 analogue. The maximum axes
(Amax), as determined by morphometry, decreased as the concentration of PGE2 analogue increased, and the cells became less fibroblastic and more polygonal. Values are the mean ± SEM (n = 73
- 148).
i Per is the perimeter of each cell determined by digitized morphometry
£ Amax is the maximum axis of each cell determined by digitized morphometry
substituted for EGF. That this did not occur indicates
that the effect of EGF is not mediated via arachidonic
acid products, such as the lipoxygenase products generated following cryo-injury in vivo."
The spindle cell shape in the presence of EGF may
not necessarily be related to the stellate cell shape in
the presence of indomethacin. EGF may exert its effects
through a change in the extracellular matrix which the
cells secrete and on which they grow.6-7 Inhibition of
the synthesis of prostaglandin E2 causes a different cellular pattern which may not be related to changes in
the extracellular matrix, but, rather, internal, cytoskeletal changes. Nevertheless, with both EGF stimulation and cyclooxygenase inhibition, the cells become
extremely fibroblastic in appearance, forming typical
swirling patterns on the culture dish. Furthermore,
when indomethacin is present, neighboring cells often
lose continuity of their cellular borders, which probably
is reflected in increased permeability as compared to
the control, polygonal cell monolayer.
The corneal endothelium exists in vivo in a tissue
culture-like environment. Endothelial cells isolated
from rabbit corneas and grown in vitro are analogous
to cells in vivo; they proliferate readily in culture,
maintain a polygonal cell shape, synthesize a distinctive
extracellular matrix, and retain their characteristic /?adrenergic response pathway. 6712 We therefore suggest
extension of our in vitro findings. We hypothesize that
endogenous synthesis of prostaglandin E2 is required
to maintain the physiological, polygonal shape of the
corneal endothelium in vivo. In response to wounding,
synthesis of prostaglandin E2 decreases and an EGFlike mediator causes the cells to elongate. Elongation
Table 2. PGE 2 synthesis in cultured
corneal endothelial cells
Growth Medium
B
BE
BI
BEI
pg PGE2/l(f
Cells
2764 ± 5 2 0 *
3583 ±1010
12 ± 4
16±5
* Mean ± SEM of 3 seperate assays, each value determined in duplicate
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INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE / October 1986
may be a rate-limiting step in migration and extracellular matrix synthesis. Pharmacological intervention
in the synthesis of prostaglandin E2 may beneficially
promote the effects of endogenous or exogenous mediators in stressed corneas.
Key words: corneal endothelium, prostaglandin E2, epidermal
growth factor, indomethacin, rabbit
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