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INTRODUCTION
Numerous peptide growth factors are supposed to influence
angiogenesis, and thereby wound healing and tissue
regeneration. The healing process is particularly interesting in
two closely related tissues: muscle and tendon.
Muscle usually heals remarkably well due to highly
developed vasculature network and existence of satellite cells
and muscle derived stem cells. Tendons, which are during
development rich in cells, metabolically active and containing
high number of blood vessels (1), are eventually maturing to
hypocellular, hypovascular and hyponeural structures (2-4).
Tendons do not heal as well as muscle, but the healing process
in both is very complex and under regulation of many different
factors such as vascular endothelial growth factor (VEGF),
fibroblast growth factor (FGF), transforming growth factor beta
(TGF-β), tumor necrosis factor (TNF) and NO (5, 6).
Pentadecapeptide BPC 157 is a peptide effective in muscle
and tendon healing (7-12), applied alone, without any carrier.
In tendon and muscle healing its functional, biomechanical,
and pathohistological beneficial effect is accompanied by
angiogenic action. BPC 157 may directly protect endothelium
(13), influence NO-system, counteract the effect of both NOS-
inhibitor and NO-precursor (14, 15) as well as over expression
of endothelin (16). BPC 157 has a particular wound healing
effect, also in response to vessel injury and coordination of the
expression of multiple genes involved in the pathogenesis of
vascular disease; it stimulates the expression of the early
growth response 1 (EGR-1) gene and expression of the EGR-1
repressor, nerve growth factor 1-A binding protein-2 (nab2)
(17). From these results BPC 157 shows a particular
angiogenic/angiomodulatory potential, worthy of further
investigation.
In this study, we correlated the angiogenic effect of BPC 157
with VEGF expression using in vitroand in vivo models.
MATERIALS AND METHODS
BPC 157
Synthetic pentadecapeptide BPC 157,
GEPPPGKPADDAGLV, M.W. 1419, (Diagen, Ljubljana,
Slovenia) is a part of protein sequence isolated from gastric juice
and after high pressure liquid chromatography purification
theprotein is obtained with 99% purity, having 1-des-Gly peptide
as impurity. It is very stable and applied dissolved in sterile
saline, without any additional carriers.
Cell culture
As an angiogenesis model TCS CellWorks AngioKit (TCS
CellWorks, Buckingham, United Kingdom) was used. Following
manufacturer's instructions we treated cells with pure medium
(TCS CellWorks, Buckingham, United Kingdom), or with
VEGF as positive control (2 ng/ml), Suramin as negative control
(20 uM) and BPC 157 in two different final concentrations (10
ug/ml and 2 ug/ml). Medium was changed on the first, fourth,
JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2009, 60, Suppl 7, 191-196
www.jpp.krakow.pl
L. BRCIC1, I. BRCIC2, M. STARESINIC3, T. NOVINSCAK3, P. SIKIRIC3, S. SEIWERTH1
MODULATORY EFFECT OF GASTRIC PENTADECAPEPTIDE BPC 157
ON ANGIOGENESIS IN MUSCLE AND TENDON HEALING
1Institute of Pathology, University of Zagreb Medical School, Zagreb, Croatia; 2Clinical Department of Pathology and Cytology,
University Hospital Center Zagreb, Zagreb, Croatia; 3Department of Pharmacology, University of Zagreb Medical School,
Zagreb, Croatia
Angiogenesis is a natural and complex process controlled by angiogenic and angiostatic molecules, with a central role
in healing process. One of the most important modulating factors in angiogenesis is the vascular endothelial growth
factor (VEGF). Pentadecapeptide BPC 157 promotes healing demonstrating particular angiogenic/angiomodulatory
potential. We correlated the angiogenic effect of BPC 157 with VEGF expression using in vitro (cell culture) and in vivo
(crushed muscle and transected muscle and tendon) models. Results revealed that there is no direct angiogenic effect of
BPC 157 on cell cultures. On the other hand, immunohistochemical analysis of muscle and tendon healing using VEGF,
CD34 and FVIII antibodies showed adequately modulated angiogenesis in BPC 157 treated animals, resulting in a more
adequate healing. Therefore the angiogenic potential of BPC 157 seems to be closely related to the healing process in
vivo with BPC 157 stimulating angiogenesis by up-regulating VEGF expression.
Key words: angiogenesis, pentadecapeptide BPC 157, cell culture, healing, muscle, tendon, vascular endothelial growth factor
seventh and ninth day. Cell cultures were cultivated in usual
conditions on 37°C and 5% CO2. After 11th day cultures were
fixed and tubules were visualized using CD31 antibody (TCS
CellWorks, Buckingham, United Kingdom). Using digital
camera, tubules were transferred into ISSA- special software for
morphometrical analysis (Vamstec, Zagreb, Croatia).
Angiogenic effect was evaluated by counting the number of the
tubule branching on 5 visual fields (microscope objective x 20)
(modification of Jones RA, 18).
Animal models
All experimental protocols were approved by the Ethics
Committee at the University of Zagreb School of Medicine.
Three different, already established and published models were
used (10-12). Animals used were male Wistar Albino rats,
weighting from 280 to 320 g. Six animals per each experimental
group and time interval were used. Animals were anaesthetized
prior to any trauma. Animals were treated either with BPC 157
(10 ug/kg) dissolved in saline, or with equivalent volume of
saline alone (5 ml/kg).
1. Muscle crush injury
Right hind limbs were shaved, and using special system a
force of 0.727 Ns/cm2was delivered to a maximum diameter of
gastrocnemius muscle complex 2 cm proximal to the insertion of
the Achilles tendon (12). The therapy was applied
intraperitoneally immediately after injury and once a day until
one day before sacrificing on 1st, 4th, 7th and 14th day after trauma.
2. Quadriceps muscle transection
The right quadriceps muscle was isolated and transected 1 cm
proximal to patella. The therapy was applied intraperitoneally 30
minutes after injury and once a day until one day before
sacrificing on 4th, 14th, 21st and 28th day after trauma (11).
3. Achilles tendon transaction
Skin incision in the length of 3 cm was performed above the
right Achilles tendon which was transected 0.5 cm proximal to
calcaneus's insertion. After transection, only skin was sutured.
The therapy was applied intraperitoneally 30 min after surgery
and once a day until one day before sacrificing on 1st, 4th, 7th, 10th
and 14th day after transection (10).
Pathohistological analysis
After sacrificing the respective tissue was dissected, fixed
in buffered formalin (pH 7.4) for 24 hours and embedded in
paraffin using standard procedures. For pathohistological
evaluation transversal sections of crushed muscle, longitudinal
section of transected muscle and longitudinal section of
transected tendon were used. Tissue samples were cut
semiserially, stained with hematoxylin and eosin, or
immunohistochemically for FVIII, CD34 and VEGF (Dako,
Glostrup, Denmark) using Dako Autostainer and
manufacturer's protocols. Vascular elements on hematoxylin
and eosin stained slides as well as immunohistocemically
positive elements were examined in a blinded fashion using
hot-spot assessment and ISSA program (V
AMSTEC, Zagreb,
Croatia). From the area of maximal tissue damage detected
using semiserial sections, five high power fields were
randomly selected for analysis.
Stastistical analysis
For analysis of distribution normality in vivo and in vitro
acquired data Kolmogorov-Smirnov test was used. Since no
group had normal distribution Mann-Whitney U test was applied.
Statistical analysis was performed using SPSS 11.5 for Windows
and all values of p<0.05 were considered statistically significant.
RESULTS
Cell culture
The number of branching points of newly formed tubular
structures was without significant difference (p>0.05) between
cell cultures treated with pure medium or with BPC 157 in both
concentrations (10 ng/ml and 2 ng/ml). As expected, VEGF as
positive control had proangiogenic effect, as opposed to negative
control Suramin which practically completely inhibited
branching (Fig. 1).
Animal models
1. Muscle crush
After crush injury (Fig. 2) in control animals angiogenic
response with all tested parameters (number of VEGF, CD34 and
FVIII positive elements (data obtained on hematoxylin-eosin stain
were equal to FVIII presentation and are not presented))
consistently reached its peak at day 7. Increase in CD34 and VEGF
positive elements was obvious since 2 hours, while FVIII showed
192
Fig. 1. The number of branching tubules in cell cultures
demonstrated the absence of positive and negative angiogenic
effect of BPC 157 in in vitroconditions.
M-pure medium; S-Suramin (negative control); V-VEGF
(positive control); B1-BPC 157 10 µg/ml; B2-BPC 157 2 µg/ml;
*- statistically significant difference (p<0.05)
an increase after day 4. On the other hand, angiogenic response
after BPC 157 application showed a shift toward left, implying at
the early interval (2h-4th day) increased blood vessels formation
(VEGF>CD34>FVIII) with gradual decrease in later period (4th -
14th day). VEGF reached its peak the 1st day, as the earliest
breakpoint, while CD34 and FVIII reached their peaks at 4th day.
2. Muscle transection
In concordance with the severity of injury (complete
transection) we should note that angiogenic response was
generally higher in both groups than after crush injury (measured
with the number of VEGF, CD34 and FVIII positive elements),
but without a consistent peak (Fig. 3). Interestingly, curves in
both groups are of the same shape, but with the statistically
significant difference- higher numbers of positive vascular
elements in the BPC 157 treated group. The angiogenic activity
in both groups was obviously prolonged. The highest activity in
this model demonstrated CD34 positive elements with earliest
peak at 4th day, followed by VEGF (peak at 14th day) and FVIII
(peak at 21 day).
3. Transected Achilles tendon
After tendon transection (Fig. 4), in both groups, angiogenic
response demonstrated with all tested parameters (the number of
VEGF, CD34 and FVIII positive elements) consistently reached
its peak already at day 4. In control group the numbers in the first
time periods remained within relatively low values, but toward
the end reaching higher values than BPC 157 animals. The
number of FVIII positive elements showed a second peak only in
control animals, retaining this number of vascular structures till
the end. BPC 157 treated animals showed pronounced decrease
in all parameters after their peak, resulting in low number of
positive vascular elements at the end of investigated period.
DISCUSSION
This study evaluated the angiogenic potential of BPC 157. In
early post-injury periods, BPC 157 therapy induced a prominent
increase of angiogenesis in rats with transected Achilles tendon or
quadriceps muscle and in rats with crushed muscle. This was
consistently visualized with different endothelial cell antigens,
FVIII (involved in platelet adhesion and aggregation, present on
endothelial cells of mature blood vessels) and CD34 (involved in
leukocyte adhesion and endothelial cell migration during
angiogenesis, present on capillary endothelial cells), as well as
with VEGF presentation (main factor in angiogenesis, expressed
on endothelial cells, mitogen for vascular endothelial cells).
Generally, BPC 157 increased the number of VEGF, CD34 and
FVIII positive vascular elements, and angiogenic response was
193
Fig. 2. Results of immunohistochemical
analysis after muscle crush injury,
demonstrating positive angiomodulatory
effect of BPC 157.
Legend: N- number of positive elements,
full line- BPC 157 treated animals;
broken line- controls; time-time after
injury; *- statistically significant
difference (p<0.05).
regularly augmented and shifted toward the left. On the other
hand, results obtained in in vitroconditions, using human
endothelial cells, showed that there is no direct angiogenic effect
of BPC 157 on cell cultures. The angiogenic potential of BPC 157
seems to be closely related to the healing process in vivo with BPC
157 stimulating angiogenesis by up-regulating VEGF expression.
In general, angiogenesis, the formation of new vessels from
pre-existing ones, consists of an orderly sequence of events
triggered by growth factors secreted from the surrounding
hypoxic tissue (19). It is a complex process controlled by
angiogenic and angiostatic molecules resulting in ideal
revascularization of the wound bed, and providing oxygen,
nutrients, and inflammatory cells to the newly
growing/regenerating as well as tumor tissue (20, 21).
Conditions less than ideal (e.g. hypoxia, wound fragments
misadaptation, infection) tend to hamper this process resulting in
scar formation or delayed healing, and probably induce VEGF
formation by more than one mechanism (22). Our study
demonstrated in all investigated models of tendon and muscle
injury that BPC 157 induces higher VEGF and CD34 positivity,
preceding the increase in actual number of blood vessels as
demonstrated on HE and FVIII stains. Such a particular activity
in angiogenesis and healing is concordant with the previous
evidence that BPC 157 may directly protect endothelium (13),
influence NO-system, counteract the effect of NOS-inhibitor and
NO-precursor (14, 15), as well as over expression of endothelin
(16). Beside the stimulation of expression of the EGR-1 gene,
BPC 157 also stimulated expression of nab2 (17). Coordinated
regulation of this transcription factor and its repressor suggests
that this system may play a role in maintaining vascular
homeostasis. It is possible that BPC 157 - nab2 interaction is part
of a feedback mechanism which serves to regulate EGR-1-
mediated gene transcription. The BPC 157 effects on rats with
crushed muscle, transected muscle and tendon suggest
appropriate angiogenic response that results in better healing
(10-12). We assessed the angiogenic response in connection to
different extent of tissue damage (muscle transection vs. muscle
crush) and different tissue healing capacity (muscle vs. tendon).
The detrimental consequence of muscle transection outweighs
crush injury, and after quadriceps muscle transection more
VEGF and CD34 positive elements were present than after blunt
trauma. Consistently, after Achilles tendon transection the same
parameters remained far below the values noted after muscle
transection.
However, after either muscle or tendon had been completely
transected, the commonly negligible tissue repair clearly shows
that without therapy the described angiogenic response may still
be inadequate. In tendon transection model control animals had
two peaks of FVIII positivity, announced by the higher number
of CD34 and VEGF positive elements on the seventh day, in
comparison to the BPC 157 animals. As previously published,
there is no longer visible detachment between Achilles tendon
194
Fig. 3. Results of immunohistochemical
analysis after muscle transection,
demonstrating positive angiomodulatory
effect of BPC 157.
Legend: N- number of positive elements,
full line- BPC 157 treated animals;
broken line- controls; time- time after
transection; *- statistically significant
difference (p<0.05).
ends in BPC animals after seventh day, while it disappears only
after tenth day in control group (10). This space is filled with
granulation tissue with active angiogenic process. Although
angiogenesis is very important for tendon healing, prolonged
angiogenesis may result in prolonged/impaired healing and leads
to chronic tendon disease. Thus, regardless of particularities in
angiogenic response(s), different tissues and different healing
stage, a more generalized and more powerful angiogenic
response induced by BPC 157, with adequately improved
angiogenesis leads consistently to better healing conditions and
thereby eventually to more adequate healing.
It is important to stress that the positive influence of BPC
157 on healing can not be attributed to angiogenesis alone, and
that one should bear in mind its effects on inflammatory
reaction where it decreases leukotriene B4, tromboxan B2 and
myeloperoxidase concentration in injured tissues (23).
Tkalcevic et al. have recently demonstrated a better modulatory
effect on granulation tissue in excisional wounds in genetically
modified diabetic mice db/db in comparison to PDGF-BB
(which is the only approved medication for diabetic ulcers
treatment) (17). Interestingly, PDGF in wounds directly induces
VEGF-A mRNA (24). Tkalcevic et al. also showed that in in
vitroconditions BPC 157 stimulates mRNA EGR-1 (17) having
the same effect as VEGF in cell cultures (25, 26). Interactions
of BPC 157 with the same growth factors and cytokines are
under investigation.
In conclusion, we have demonstrated that, although BPC
157 doesn't have any direct angiogenic effect in cell culture, it
has a positive angiomodulatory effect in animal models of
muscle and tendon healing, resulting in faster and better healing,
which could be helpful in further therapy development.
Conflict of interests: None declared.
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Fig. 4. Results of immunohistochemical
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R e ce iv e d : October 15, 2009
A c ce pt e d : December 11, 2009
Author's address: Prof. Sven Seiwerth, M.D., Ph. D.,
Institute of Pathology, University of Zagreb Medical School,
Salata 10, 10000 Zagreb, Croatia; Phone: +385 1 4566977;
Fax: +385 1 4921151; E-mail: seiwerth@mef.hr
196