Current Eye Research Growth factors in amniotic membrane 173

0271-3683/00/2003-0173 $15.00
2000, Vol. 20, No. 3, pp. 173–177 © Swets & Zeitlinger

Growth factor mRNA and protein in preserved human amniotic

membrane

Noriko Koizumi, Tsutomu Inatomi, Chie Sotozono, Nigel J. Fullwood, Andrew J. Quantock and Shigeru Kinoshita

Department of Ophthalmology, Kyoto Prefectural University of Medicine, Japan; the Division of Biology (NJF),
Lancaster University, Lancaster, UK; and the Department of Optometry and Vision Sciences (AJQ), Cardiff University,
Cardiff, UK
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Abstract
Purpose. To investigate the expression of growth factor Keywords: growth factor; amniotic membrane; cornea;
mRNA and the level of growth factor protein in preserved ELISA; RT-PCR
human amniotic membrane (AM).
Methods. RT-PCR was used to examine the expression of
Human amniotic membrane (AM) has been used in a vari-
mRNA for eight growth factors (EGF, TGF-α, KGF, HGF,
ety of surgical procedures to promote epithelialization and
bFGF, TGF-β1, -β2, -β3) and two growth factor receptors
prevent tissue adhesion.1 The first reported application of AM
(KGFR and HGFR) in human AM preserved at –80°C for
in ophthalmic surgery was in 1940 when De Rötth used live
For personal use only.

one month. In addition, ELISAs were used to measure the

protein concentrations of seven growth factors (EGF, TGF-
α, KGF, HGF, bFGF, TGF-β1, -β2) in preserved human
corneas and in AM both with and without amniotic epithe-
lium.
Results. RT-PCR revealed that human AM expresses mRNA
for EGF, TGF-α, KGF, HGF, bFGF, TGF-β1, -β2, -β3, KGFR
and HGFR, while ELISAs showed that it contains EGF, TGF-
α, KGF, HGF, bFGF, TGF-β1, -β2. AM without amniotic
epithelium also contains all seven growth factors examined,
however, in this tissue the protein levels of EGF, KGF, HGF
and bFGF were found to be significantly lower than in na-
tive AM.
Conclusions. Preserved human AM expresses mRNAs for a
number of growth factors and contains several growth fac-
tor proteins that might benefit epithelialization after AM
transplantation. High levels of EGF, KGF, HGF and bFGF
in AM with amniotic epithelium as compared to AM with-
out amniotic epithelium suggest an epithelial origin for these
growth factors. We feel that EGF, KGF and HGF in par-
ticular might play important roles in ocular surface wound
healing after AM transplantation.
Correspondence: Noriko Koizumi MD, Department of Ophthalmol-
ogy, Kyoto Prefectural University of Medicine, Kawaramachi-
hirokoji, Kamigyo-ku, Kyoto 602-0841, Japan, Tel: 81-75-251-5578,
Fax: 81-75-251-5663, E-mail: nkoizumi@eye.ophth.kpu-m.ac.jp
fetal membrane containing both amnion and chorion to re-
pair a symblepharon; the procedure, however, met with only
limited success.2 More recently, Kim and Tseng have shown
that, when used to treat experimental ocular surface injuries
in rabbits, AM transplantation is successful in maintaining
corneal clarity 40% of the time.3 Moreover, in the last few
years Tsubota, Tseng, Tsai and their co-workers have dem-
onstrated the beneficial effects of AM transplantation when
performed in conjunction with limbal grafting.4–8
The precise reason why a graft of AM aids healing after
ocular surface reconstructive surgery remains unclear, how-
ever, it is possible that AM contains factors that promote
normal epithelialization. For example, one can envisage situ-
ations where the AM’s basement membrane might provide
a substrate that facilitates easy epithelial migration. We know
that, like cornea, the basement membrane of AM contains
collagen types IV and V and laminin,9–10 however, its com-
position does seem to more closely resemble the basement
membrane of conjunctiva than that of cornea. 11 Another
mechanism by which AM transplantation might affect ocular
surface reepithelialization is via the action of growth fac-
tors. Some researchers have started to investigate this pos-
sibility,12–13 but as yet the identities and relative levels of
growth factors in AM have not been ascertained. To help
remedy this, we used RT-PCR to determine the expression
in preserved AM of mRNA for several different growth fac-
tors. We also used ELISAs to measure the concentrations
of a number of growth factor proteins in AM. Furthermore,

Received on June 28, 1999; revised and accepted on October 22, 1999

99111.p65 173 2/1/00, 1:04 PM

 174 N. Koizumi et al.
Figure 1. Ethidium-bromide-stained gel of RT-PCR products for eight growth factors (epidermal growth factor (EGF), transforming growth
factor-α (TGF-α), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), basic fibroblast growth factor (bFGF), and the transforming
growth factors (TGF)-β1, -β2, and -β3) and two growth factor receptors (KGF receptor (KGFR) and HGF receptor (HGFR)) in preserved
human amniotic membrane.
Curr Eye Res Downloaded from informahealthcare.com by Deakin University on 10/12/14
to examine whether growth factors were contained more in
the amniotic epithelial cells or the membrane itself, we meas-
ured the growth factor concentrations in AM from which the
epithelial cells had been removed. For reference, we also
measured the growth factors in preserved human cornea,
although we are aware of the difficulty in directly compar-
ing the concentrations of growth factors extracted from two
For personal use only.
different tissues.
With proper informed consent, human AM (n = 13) was
obtained at the time of Cesarean section. Under sterile condi-
tions it was washed three times in phosphate buffered saline
(PBS) containing antibiotics (0.005% ofloxacin), once in 50%
glycerol / DMEM and then stored at –80°C in 50% glycerol
/ DMEM. One month later, total RNA was extracted by the
use of TRIZOL (Life Technologies, Gaithersburg, MD), after
which it was subjected to reverse-transcription following a
previously described method.14 Oligonucleotide primers for
epidermal growth factor (EGF), transforming growth factor-
α (TGF-α), keratinocyte growth factor (KGF), KGF receptor
(KGFR), hepatocyte growth factor (HGF), HGF receptor
(HGFR) , basic fibroblast growth factor (bFGF), and the
transforming growth factors (TGF)-β1, -β2, and -β3 were
synthesized as described previously.14–18 All ten primer pairs
were designed to include at least one intron so as to exclude
PCR products amplified from residual genomic DNA. Am-
plification (35 cycles consisting of 1 min at 94°C for dena-
turation, 1 min at 60°C for annealing, and 1 min at 72°C for
extension) was performed with a DNA thermal cycler (Perkin-
Elmer Cetus, Norwalk, CT), and the reaction mixture (7 µl)
was electrophoresed on a 1.5% agarose gel with a primer
set for β-actin used to confirm RNA isolation and cDNA
synthesis.19 RNAs isolated from SV40 large T antigen-trans-
fected human corneal epithelial cells (HCE)20 and mouse 3T3
fibroblast cells (3T3)21 were used as positive controls. The
RT-PCR detected mRNA for all eight growth factors exam-
ined in this experiment (EGF, TGF-α, KGF, KGFR, HGF,
HGFR, bFGF, TGF-β1, TGF-β2 and TGF-β3; Fig. 1). In
contrast, none of the negative controls were amplified. These
99111.p65 174
data clearly demonstrate that AM preserved with its epithe-
lial cells intact has the potential to produce a wide range of
growth factors.
Next, we wanted to know whether the growth factor pro-
teins themselves were present in AM. To this end, we used
ELISAs to quantify seven of the growth factors (EGF, TGF-
α, HGF, KGF, bFGF, TGF-β1 and TGF-β2) whose mRNA
we found in preserved AM. To further investigate the pos-
sible source of these growth factors, we also studied AM
from which the epithelium had been removed by incubation
for two hours in 0.02% EDTA (37°C) followed by physical
removal with a cell scraper (Nunc A/S, Roskilde, Denmark).
For comparison we also assayed the seven growth factors
in seven human corneas (Rocky Mountain Eye Bank, Den-
ver, CO) that had been preserved at –80°C in Optizol GS
(Chiron, Claremont, CA). For the ELISAs, tissue samples
were frozen in liquid nitrogen, then smashed and homog-
enized in 1.6 ml of PBS. Growth factor levels were mea-
sured using commercially available ELISA systems (EGF,
TGF-β1, TGF-β2, KGF, HGF, bFGF, R&D System, Min-
neapolis, MN; TGF-α Oncogene Research Products, Cam-
bridge, MA).
The concentrations of the growth factors in AM with and
without epithelial cells and in cornea are given in Table 1.
As we can see, AM contains all seven growth factors whose
mRNAs were detected by RT-PCR. We also note that in our
experiments the levels of EGF, HGF, and KGF are higher
in AM with amniotic epithelium than in cornea (Fig. 2),
however, we should interpret this with caution because of
the possibility of extraction differences in different tissues.
Potentially, the most important result of this set of experi-
ments is the finding of significantly higher levels of EGF,
KGF, HGF and bFGF in AM containing amniotic epithe-
lium when compared to acellular AM.
The fact that three mitogenic growth factors – EGF, HGF
and KGF – were found predominantly in amniotic epithe-
lium (Fig. 2) is of considerable interest with regard to the
possible mode of action of AM transplantation. EGF is sup-
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 Growth factors in amniotic membrane 175

Table 1. Concentrations of 7 growth factors’ protein measured by ELISA (pg/mg,

w/w)

AM with cells AM without cells Cornea

(n = 13) (n = 13) (n = 9)

EGF 1.0 ± 0.6*‡ 0.1 ± 0.0* 0.1 ± 0.1‡

TGFα 0.7 ± 0.6 0.9 ± 1.0 1.3 ± 0.0
KGF 4.6 ± 2.8*‡ 2.7 ± 0.4* 2.0 ± 1.3‡
HGF 44.7 ± 33.7*‡ 5.3 ± 1.1* 9.4 ± 1.7‡
bFGF 5.6 ± 4.0*‡ 0.5 ± 0.6* 10.5 ± 3.8‡
TGFβ1 0.7 ± 0.0 0.6 ± 0.1 0.7 ± 0.1
TGFβ2 0.7 ± 0.1 0.7 ± 0.1 1.3 ± 0.4

* P < .05 for difference between AM with cells and AM without cells (Tukey’s HSD
test).
‡
P < .05 for difference between AM with cells and cornea (Tukey’s HSD test).
Curr Eye Res Downloaded from informahealthcare.com by Deakin University on 10/12/14

however, we think it likely that HGF is potentially the stron-

gest effector of corneal wound healing produced by amni-
otic epithelial cells (Table 1).
In contrast to the epithelially derived growth factors bFGF,
EGF, KGF, and HGF, the TGF-βs in these experiments were
found at almost the same levels in cellular AM, acellular AM
and cornea (Table 1). TGF-βs are known to increase syn-
thesis of protease inhibitors, up-regulate cell adhesion mol-
ecules, and suppress the synthesis of matrix-degrading pro-
For personal use only.

Figure 2. Comparison of the levels of growth factors in amni-
otic membrane with the levels in preserved cornea. The height of
each bar represent the growth factor protein levels in amniotic
membrane as a ratio of the growth factor levels in corneas. Sig-
nificantly high levels of EGF, KGF and HGF were detected in
amniotic membrane containing epithelium compared to preserved
cornea.
plied by the corneal epithelium and lacrimal gland,22–26 and
is one of the most effective mitogens for corneal epithelial
cell growth. In view of this, we speculate that the high ex-
pression of EGF in amniotic epithelium could be one of the
major reasons why the AM promotes ocular surface wound
healing after AM transplantation. Interestingly, our data also
show that, along with EGF, the amniotic epithelium produces
KGF and HGF, growth factors which are commonly produced
by mesenchymal cells such as corneal stromal fibroblasts.
On the corneal surface, these growth factors and their re-
ceptors in the corneal epithelium influence corneal wound
healing through the paracrine system.14, 27–33 It seems rea-
sonable, therefore, to assume that after AM transplantation,
corneal epithelial growth might be accelerated by KGF and
HGF produced by the amniotic epithelium. It is worth not-
ing that, as reported by Honma and associates, the growth-
promoting effect of EGF on corneal epithelial cells is about
ten times stronger than the effects of KGF and HGF at the
same concentrations.33 Judging from the results presented here,
tease, with the result that TGF-βs stimulate the synthesis and
deposition of extracellular matrix proteins.34–35 As such, TGF-
βs are considered to be the most influential growth factors
in the control of fibroblast activity during wound healing,
and TGF-βs which are detected in cornea and tears18,36 are
thought to play important roles in ocular surface homeosta-
sis. We cannot offer any information about the direct effect
of TGF-βs released by AM on ocular surface reconstruction
based solely on our results, however, it is noteworthy that
Tseng and colleagues recently reported that TGF-β expres-
sion by corneal fibroblasts cultivated on AM is suppressed
in vitro.13 It might be the case, then, that the reduction of
fibroblast overgrowth after AM transplantation depends more
on the suppression of TGF-β expression by corneal fibro-
blasts than on the direct effect of TGF-β released by AM.
Importantly, we found higher levels of certain growth fac-
tors (e.g. EGF, KGF, HGF and bFGF) in amniotic epithe-
lium than in amniotic stroma, suggesting that the amniotic
epithelium and amniotic stroma might have different influ-
ences on corneal re-epithelialization, and thus contribute
differently to the success of ocular surface reconstruction.
In view of this, we might be able to develop surgical proce-
dures that allow us to select a particular portion of the AM
for use in an individual patient depending on the nature of
the injury and the specific aim of the surgery.
Acknowledgments
Supported by the Japanese Ministry of Health and Welfare
and the Japanese Ministry of Education (10470365), the Kyoto
Foundation for the Promotion of Medical Science, the In-

99111.p65 175 2/1/00, 1:04 PM

 176 N. Koizumi et al.
tramural Research Fund of the Kyoto Prefectural University
of Medicine, and by a UK/Japan Joint Project grant from
the Royal Society (London).
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