cell biochemistry and function

Cell Biochem Funct 2004; 22: 373378.

Published online 24 August 2004 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1027/cbf.1121
Stimulation of growth factor synthesis in skin wounds using
tissue extract (G-90) from the earthworm Eissenia foetida
M. Grdisa*
1
, M. Popovic
2
and T. Hrzenjak
2
1
Division of Molecular Medicine, Rudjer Boskovic Institute, Bijenicka 54, Zagreb, Croatia
2
Department of Biology, Faculty of Veterinary Medicine, Heinzelova 53, Zagreb, Croatia
Growth factors are biologically-active mediators that bind to specic receptors on target cells and regulate genes involved in
cell growth, wound healing and regeneration. In the case of wound healing, a proper wound dressing is needed to cover the
wound area, protect the damaged tissue, and if possible to activate cell proliferation and stimulate the healing process. In this
study we examined the efcacy of a glycolipoprotein tissue homogenate extract from Eisenia foetida (G-90) to activate
signal transduction pathways, leading to wound healing. We measured the activation of EGF and FGF in healthy skin, in
wounds with physiological healing and in wounds treated with G-90. The activation of EGF and FGF was measured during
the rst 24 h of wound healing under both physiological conditions and treatment with G-90. In both cases an increased
concentration of EGF and FGF was observed 6 h after wounding. In comparison with healthy skin, the concentration of
EGF increased 10-fold and FGF ve-fold in wounds treated with G-90 (10 ng ml
1
). Healing in physiological conditions
resulted in a two-fold increase of EGF and 1.5-fold of FGF. Copyright # 2004 John Wiley & Sons, Ltd.
key words earthworm; G-90; growth factors; EGF; FGF
INTRODUCTION
The earthworm (phylum Annelida, family Lumbrici-
dae) is one of the rst organisms in the evolutionary
tree that possesses immunological recognition and
memory.
1,2
They regenerate amputated parts if the
nervous system is intact.
3
The segmented earthworms
body cavity is lled with coelomic uid. Many inves-
tigations on earthworms have been made on their coe-
lomic uid. Coelomocytes, leukocytes that are present
within the coelomic uid, are thought to be responsi-
ble for the observed effects as they secrete proteins
and glycoproteins that act as opsonins, agglutinins,
lysins and a certain factor, which resembles a factor
that inhibits the migration of macrophages.
4
It is well documented that coelomic uid possesses
numerous biological activitieshemolytic and agglu-
tinative
5,6
as well as bacteriolytic and bacteriostatic.
7,8
It can act on mammalian cell growth in vitro, indepen-
dent of cell type, either as a cytotoxic or mitogenic
agent.
6
In addition, coelomic uid acts as a mitogen
for murine and human lymphocytes in vitro.
9
However,
the presence of Eisenia foetida coelomic uid in cul-
tures of chicken broblasts and guinea-pig leukocytes
can be toxic.
6
Besides its mitogenic and cytotoxic
effects, bacteriostatic and bacteriolytic activities
important for their life in soil have also been observed.
8
All biological activities found in coelomic uid are
derived from the earthworm coelomocytes. Therefore
we established a method for preparing an earthworm
tissue homogenate. This macromolecular mixture
with glycolipoprotein characteristics has been named
G-90.
10
G-90 increased the proliferation of different cell
cultures in vitro, depending on the concentration and
G-90 is neither a mutagen nor a carcinogen.
10
It was
shown that G-90 contains insulin-like growth factors
(IGF) with molecular masses between 14 and
95 kDa. SDS-PAGE analysis separated these into six
fractions, antigenically similar to insulin that induced
Received 16 June 2003
Revised 29 July 2003
Copyright #2004 John Wiley & Sons, Ltd. Accepted 30 July 2003
* Correspondence to: Dr M. Grdisa, Division of Molecular
Medicine, Rudjer Boskovic Institute, Bijenicka 54, 10 001 Zagreb,
Croatia. Tel: 385 1 456 11 10. Fax: 385 1 456 10 10.
E-mail: grdisa@rudjer.irb.hr
cell proliferation in vitro.
11
G-90 also contains immu-
noglobulin-like molecules. By afnity chromatogra-
phy a 45-kDa fraction named G-90/4, has been
isolated. This fraction stimulated cell proliferation at
nanogram quantities, but in higher amounts (micro-
grams) it caused cell lysis. Using immunohistochem-
ical analysis, it was shown that G-90/4 acts as an
adhesion molecule. It is involved in signal transduc-
tion pathways for the synthesis of biologically active
molecules.
12
G-90 exhibited strong brinolytic and
anticoagulative activities. From the G-90 mixture,
two tyrosine-like serine peptidases (PI) and (PII), hav-
ing molecular masses of 34 and 23 kDa respectively,
have been isolated. Fibrinolytic and mitogenic activity
of PI is remarkably stronger than PII.
13
In euglobuli-
nic assays, G-90 recognized serum from patients with
malignant tumours
14
and in blood coagulation assays
it distinguished cardiopathies from malignancy.
15
G-
90 also exhibits strong antioxidative
16
and antibacter-
ial activities.
In our experiments we treated skin wounds made
on the backs of mice with G-90. We measured the
appearance of EGF and FGF in the wounds during
the healing period.
MATERIALS AND METHODS
Earthworms and mice
Earthworms, Eisenia foetida (Annelida, Oligochaeta,
Lumbricidae), were used for isolation of the biologi-
cally active mixture G-90.
NIH Mice (Immunological Institute, Zagreb, Croa-
tia), males aged 20 days and weighing 20 0.23 g
were used. They were kept in separate cages at 23

C
and 50% humidity. Food and water were always
available. The animals were handled according to
European standards Directive for the Protection of
Vertebrate Animals used for Experimental and other
Purposes (86/609/EEC).
17,18
Under inhalatory anaesthesia, mice were wounded
under sterile conditions on the lumbal-sacral region.
The wound was elliptically shaped with an area about
23.5 mm
2
. The mice were divided into three groups,
with 20 animals per group: A, non-treated wounds,
negative control; B, wounds treated with 25 ml of
G-90 (10 mg ml
1
); C, wounds treated with 25 ml of
G-90 (10 ng ml
1
). The outow from the wounds
was collected every 6 h (in the rst 24 h). Within
the group, each sample was obtained by pooling the
outow from ve mice (total 15 0.09 ml) and mea-
suring the amounts of growth factors (dot blot,
ELISA).
Methods
Glycolipoprotein extract (G-90) was prepared from
the tissue homogenate of earthworms Eisenia foetida
and Lumbricus rubellus (Annelida, Oligocheta, Lum-
bricidae) according to the methods decscribed by
Hrzenjak et al.
10
Dot blot. Experiments were carried out according to
the method described by Towbin and Gordon.
19
A
15-ml aliquot of each sample was loaded onto a nitro-
cellulose membrane (Immobilon PVDF, Millipore,
Bradford, USA) along with 5 ml (2 ng ml
1
) of stan-
dard (EGF or FGF). After blotting with 5% non-fat
milk, the membrane was incubated overnight with
primary Ab (mouse monoclonal to EGF and FGF;
Sigma, St. Louis, USA), then washed with TBS-10
and incubated for 2 h with secondary Ab (goat anti
mouse IgG-alkaline phosphatase). The spots were
visualized by staining with BCIP (15 ng ml
1
) and
NBT (30 ng ml
1
).
ELISA. This technique was used to determine the con-
centration of growth factors in new-formed tissue,
according to Cochrane (a protocol from Genzym).
20
The samples were prepared as follows: newly-formed
wound tissue was taken from sacriced mice and one
sample (2 g of tissue) was collected from ve mice in
each of the groups examined. The samples were
extracted with xylene (1 ml, 10 min on ice), then
homogenized in 1 ml of buffer (2 M NaCl, 50 mM
Na
2
HPO
4
, pH 7.4, 2.5 mM EDTA, 15 mM EACA ("-
amino-n-capronic acid), 10 mM NEM (N-ethylmalei-
mide)) for 5 min and centrifuged for 15 min at
3900 g and 4

C. The supernatants (100 ml) were used

for analysis. Mouse monoclonal antibodies to EGF
and FGF (2 ng ml
1
) were used. The absorbance at
450 nm (alkaline phosphatase) was measured and
the concentration of growth factors was determined
from a standard curve obtained with commercial
EGF and FGF.
Immunohistochemistry. The concentrations of EGF
and FGF in tissue sections were determined by immu-
nohistochemistry.
21
Parafn tissue sections (8 mm)
were treated with xylene and ethanol according to
the protocol. The slides were incubated overnight with
primary Abs (mouse monoclonal to EGF and FGF,
5 mg ml
1
) at 4

C. Then followed by incubation with

secondary Ab (goat anti mouse IgG) for 1 h and incu-
bation with FITC-avidin for 45 min at room tempera-
ture. Finally, the slides were washed with PBS and
preserved in glycerol. The whole visual eld was
374 m. grdisa ET AL.
Copyright # 2004 John Wiley & Sons, Ltd. Cell Biochem Funct 2004; 22: 373378
scanned and analysed by Image Analyser, SFOR
(VAMP, Zagreb, Croatia). The uorescence is directly
proportional to the amount of bound avidin.
Western blot. After separation of proteins by SDS-
PAGE and transferring them to nitrocellulose mem-
branes, the concentrations of EGF and FGF were
determined by specic antibodies: mouse monoclonal
Ab to EGF 2 ng ml
1
(Sigma, St. Louis, USA); mouse
monoclonal Ab to FGF 2 ng ml
1
(UBI, Lake Placid,
USA).
22
After incubation with secondary Ab
(2 ng ml
1
) and washing with TBS-10, the membranes
were stained with NBT and BCIP (Sigma, St. Louis,
USA) reagents.
Statistical analyses. The MannWhitney and t-test
were used for all results. The analyses were performed
on software ANOVA.
RESULTS
Inuence of G-90 on the expression of EGF and FGF
During physiological wound healing, the concentra-
tions of growth factors increased in the rst 24 h.
The concentration of growth factors was measured
by immunohistochemistry in the wound tissue and
by dot blot and ELISA on the uid which owed
from the wounds. The results of the dot blot are
shown in Figure 1 (A, B). G-90 does not contain
FGF or EGF, according to our methods of measure-
ment. In wounds with physiological healing, the
expression of EGF increased within 18 h and FGF
within 24 h after initiation of the experiments. If the
wounds were treated with G-90 (10 ng ml
1
), the
expression of EGF was increased within 6 h and
FGF within 12 h after initiation of the experiments.
At higher concentrations of G-90 (10 mg ml
1
) the
expression of both growth factors was increased by
12 h after initiation of the experiments.
Concentrations of EGF and FGF in wound
samples were determined by ELISA (Figure 2). The
concentration of EGF in healthy mouse skin is
5.1 0.23 ng ml
1
. In the rst 24 h the concentration
of EGF increased by 1445% in physiological wound
healing (control). After 6 h its concentration was
5.9 0.27 ng ml
1
and after 24 h 9.2 0.28 ng ml
1
.
Treatment of wounds with G-90 resulted in a signi-
cant increase in EGF. At a concentration of 10 ng ml
1
G-90, the expression of EGF increased by 28%
(7.0 0.3 ng ml
1
) within 6 h and by 110%
(55.2 0.28 ng ml
1
) in 24 h. A higher concentration
of G-90 (10 mg ml
1
) induced increases of 17% in 6 h
and of 81% in 24 h, compared with the control group.
Similar observations were made for FGF. The results
are shown in Figure 3. The concentration of FGF
in healthy skin is 4.7 ng ml
1
. Six hours after wound-
ing, the concentration of FGF increased by 5%
(4.9 0.28 ng ml
1
) and after 24 h by 33% (7.2
0.19 ng ml
1
), during physiological healing (control).
When the wounds were treated with G-90, 10 mg ml
1
,
FGF increased by 17% (5.6 0.36 ng ml
1
) within 6 h
and by 60% (11.6 0.27 ng ml
1
) at 24 h. The opti-
mum effect was achieved at a concentration of
10 ng ml
1
, when the expression of FGF increased
by 22% (6.1 0.35 ng ml
1
) at 6 h, whereas at 24 h
it had increased by 80% (22.5 0.23 ng ml
1
).
Figure 1. Detection of EGF (A) and FGF (B) in the outow from
the wounds by dot blot using mouse monoclonal Ab to EGF and
FGF. 1, G-90 (10 mg ml
1
); 2, EGF or FGF (2 ng ml
1
); 3,
physiological healing; 4, treatment with G-90 (10 mg ml
1
); 5,
treatment with G-90 (10 ng ml
1
)
Figure 2. Effect of G-90 on the synthesis of EGF during treatment
of wounds. The concentration of EGF was determined in healthy
skin, in wounds with physiological healing and in wounds treated
with G-90. Results are recorded as mean value SD obtained from
three separate experiments with four samples in each experiment
(for details see Methods)
g-90 growth factors 375
Copyright #2004 John Wiley & Sons, Ltd. Cell Biochem Funct 2004; 22: 373378
Immunohistochemical detection of EGF and FGF
The expression of EGF and FGF in wound tissue
sections was measured by immunohistochemistry
24 h after wounding (Figure 4). The intensity of
uorescence was measured with an Image Analyser.
The concentration of EGF in control wounds was
0.43 0.71 ng ml
1
(Figure A1) and FGF 0.39
0.65 ng ml
1
(Figure B1). But in the wounds treated
with G-90 (10 ng ml
1
) signicant increases of both
growth factors were observed. EGF increased by
96% (9.87 0.54 ng ml
1
; Figure A2) and FGF by
78% (2.13 0.87 ng ml
1
; Figure B2) in comparison
with control wounds.
DISCUSSION
Up to now, our studies of G-90 have indicated that this
substance contains a mixture of macromolecules with
different biological activities. Obviously, the relation
between macromolecules in G-90 is in a balance
which maintains physiological conditions essential
for cell proliferation. Stimulation of cell proliferation
Figure 3. Synthesis of FGF in wounds treated with G-90 during
24 h. The concentration of FGF was determined in healthy skin, in
wounds with physiological healing and wounds treated with G-90.
Results represent mean value SD obtained from three separate
experiments with four samples in each experiment (for details see
Methods)
Figure 4. Immunohistochemical detection of EGF (A) and FGF (B) in wounds after 24 h (details in Methods). A
1
, B
1
, physiological wound
healing; A
2
, B
2
, wound healing with G-90 treatment (10 ng ml
1
)
376 m. grdisa ET AL.
Copyright # 2004 John Wiley & Sons, Ltd. Cell Biochem Funct 2004; 22: 373378
is only rarely caused by only one growth factor, such
as PDGF, FGF or bombesin.
23
Usually, two growth
factors are involved in stimulation of cell proliferation
(the factors of inhibition and progression). G-90 offers
three growth factors (belonging to the insulin family,
immunoglobulin superfamily and peptidases from the
serine family). PDGF was not detected in the G-90
mixture.
Growth factors such as EGF and FGF stimulate pro-
liferation in different types of cells. They provide the
inducer signal, and even in early embryonic develop-
ment, EGF and FGF are universal growth factors for
induction of mitosis.
24
This investigation has shown
the inuence of G-90 on induction of the synthesis
of EGF and FGF in the wounds. EGF stimulates the
proliferation of epithelial cells, contributing to more
rapid wound healing. It seems that FGF plays a role
in wound healing by promoting angiogenesis and
inducing the growth of broblasts. Increased expres-
sion of EGF preceded FGF, which is in accordance
with their possible functions during wound healing.
The increased proliferation of cells by means of
G-90 was observed in vitro as well as in vivo in the
process of wound healing. By treating wounds with
G-90 in microgram and nanogram quantities, incre-
ased concentrations of EGF and FGF were detected.
Wound healing is not only the result of cell prolifera-
tion, it is a complex biological process, including
remodelling of damaged tissue with different cells,
establishment of the extracellular matrix, vasculariza-
tion of tissue, as well as a formation of scars.
25
How-
ever, this is a regulated process that requires activation
by specic factors. It seems that G-90 can provide
excellent conditions for such stimulation. We would
like to believe that evolution has preserved the mem-
ories of factors in this medium (G-90) for differentia-
tion and regeneration of tissues and organs. To analyse
further and to discover examples of memory compo-
nents would be of benet to medicine.
The macromolecules in G-90 are products of earth-
worm coelomocytes. Thus for these macromolecules
in invertebrates there is probably a basic gene code
that has been preserved during evolution for as many
as 600 million years.
26
Thus macromolecules from
G-90 may be able to re-establish interactions with cor-
responding components of receptors on the cells of
organisms that are separated by enormous phyloge-
netic distances. The capacity of earthworms for
renewal of body components in the presence of certain
pre-growth factors may have been sustained during
evolution. Pre-growth factors perhaps have a broad
capability of action, and their basic code also exists
in mammals today. Insulin is an example which is a
product of a single gene in mammals, but in the inver-
tebrates it is a product of multiple genes.
27,28
Evolu-
tion of the basic code for the Ig-superfamily is well
known.
26
Throughout evolution, the basic function
of receptors and ligands has been conserved.
29
Similar
observations have been made with respect to G-90/4.
Using human epidermis as a model system, EGF,
FGF, IGF and PDGF were found to increase mitogen
activity and to stimulate repair of the epithelium.
30
Signicant results have been observed after treatment
of wounds (diabetes, ulcer, decubitus, burns, etc) with
growth factors (EGF, FGF, PDGF, IGF I). Their mito-
genic effects have been monitored by incorporation of
[
3
H]-thymidine.
31,32
A similar combination of growth
factors has been employed during periodontal and
gingival regeneration.
33,34
Treatment of wounds with
nanogram quantities of TGF-, resulted in signicant
differentiation of structures and a decrease in the size
of wound areas.
35,36
Signicant proliferation of epithelial cells detected
in wounds treated with G-90 by histological analysis
was probably a consequence of induced growth fac-
tors (EGF, FGF). From our results we conclude that
G-90 is a valuable mixture of biologically active
macromolecules, that in nanogram quantities increase
proliferation of cells, and may therefore accelerate
wound healing.
ACKNOWLEDGEMENTS
This work was supported by grants 053023 and P1104
from the Ministry of Science and Technology, Repub-
lic of Croatia.
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