A NOVEL ANGIOGENESIS INHIBITOR BEVACIZUMAB
INDUCES APOPTOSIS IN THE RAT ENDOMETRIOSIS MODEL
Soysal D1,*, Kızıldağ S2,*, Saatlı B1, Posacı C1, Soysal S3, Koyuncuoğlu M4, Doğan ÖE1
*Corresponding Author: Sefa Kızıldağ, Ph.D., Dokuz Eylül University, Faculty of Medicine, 35340 İnciralti,
İzmir, Turkey. Tel.: +90-2324124613. Fax: +90-2322590541. E-mail:sefa.kizildag@deu.edu.tr
page: 73
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MATERIALS AND METHODS
Thirty-five female Wistar albino mature rats at 8
weeks, weighing 200-250 g, were used for the study.
Animals were housed in steel cages with free access
to food and water. The temperature of the environment
was controlled within 24 °C and 12-hour light/dark
cycles were maintained. The experimental procedures
were approved by the institutional review board of
Dokuz Eylül University, İzmir, Turkey. The induction
of endometriosis was conducted by the method
described by Vernon and Wilson [12]. All rats were
anesthetized, using ketamine hydrochloride at a dose
of 40-80 mg/kg, and 5-10 mg/kg of xylazine hydrochloric
was administered intraperitoneally. The abdomen
was opened through a 5 cm midline incision. The
left uterine horn was ligated at both the uterotubal
junction and the cervical end, and removed. The excised
horn was immersed in sterile saline solution,
the endometrium was exposed by bisecting along its
antimesenteric axis and 5 × 5 mm sections were cut.
These explants were then anchored onto the peritoneum
on the left side of the ventral abdominal wall
close to an artery by 5-0 polypropylene sutures. Abdominal
layers were closed anatomically, using 3-0
polyglactin 910 sutures and the animals were allowed
to recover from anesthesia. Three rats died in the first
week of the initial surgery. Three weeks after the
initial surgery, each rat was anesthetized and a midventral
laparotomy was performed to determine the
attachment and viability of endometrial explants. The
surface areas of the explants were measured (length
× width). Twenty-nine of the 32 rats (90.6%) had viable
and well vascularized endometrial explants in the
second laparotomy. Twenty-nine rats were randomly
divided into three groups. The GnRH agonist group
was treated with a single subcutaneous injection of
GnRH analog, leuprolide acetate depot formulation
(1 mg/kg body weight) (Lucrin Depot; Abbott Laboratories,
Abbott Park, IL, USA). The Bevacizumab
group was given a single intraperitoneal injection
of bevacizumab (2.5 mg/kg body weight) (Avastin;
Genentech/Roche, San Francisco, CA, USA). The
Control group received saline solution as placebo by
gastric lavage. Three weeks after treatment, a third
laparotomy was performed. After a midline incision,
the endometrial explants were measured and the level
of the adhesions were scored macroscopically by the
method defined by Linsky et al. in 1987 [13]. The severity of the adhesions was scored as follows: 0
point no resistance to separation, 0.5 point partial
resistance to separation, 1 point sharp dissection was
needed. The extension of the adhesions was scored as:
0 point no adhesions; 1 point 25.0% of traumatized
area; 2 points 50.0% of traumatized area; 3 points total
involvement. The total grade was additive, giving a
range of adhesion scores from 0 to 4, which represented
both extent and severity of adhesions. These
measurements and evaluations were made by one
operator blinded to the study. For histologic examination
and apoptosis assays, endometriotic explants
were excised. A small part of endometrial explant
was kept for polymerase chain reaction (PCR) study
and the remainder was fixed in 10.0% formalin. The
formalin-fixed endometriotic foci were embedded in
paraffin blocks, sectioned at ~5 mm thickness (four
sections per sample), stained with hema-toxylin-eosin
and CD10 (a 94 kDa zinc-dependent cell membrane
metalloprotein that participates in the postsecretory
processing of neuropeptides) immunohistochemistry
and examined under a light microscope (Figure 1).
The pathologist assessing the samples was blinded
to the treatment groups. The persistence of epithelial
cells in endometrial explants was semiquantitatively
evaluated as follows: 3, well-preserved epithelial layer;
2, moderately preserved epithelium with leukocyte
infiltrate; 1, poorly preserved epithelium (occasional
epithelial cells only) and 0, no epithelium. This evaluation
was based on a previous rat endometriosis study
[14]. All rats were sacrificed at the end of study.
Total RNA Extraction and cDNA Synthesis.
Total RNA was extracted from cells using High Pure
RNA isolation Kit according to the protocol provided
by Roche Diagnostics (Basel, Switzerland).
Complementary DNAs (cDNAs) were synthesized
from 2 mg of the total RNA with SuperScript First-
Strand Synthesis System for reverse transcription-
PCR (RT-PCR) according to the protocol provided
by Invitrogen (Carlsbad, CA, USA). The mixture was
incubated at 42 °C for 50 min., 72 °C for 15 min. After
the addition of 2 U RNase H, the PCR was per-formed
in a volume of 20 μL containing 2 μL cDNA.
Real-Time Reverse Transcription-Polymerase
Chain Reaction. Real-time PCR was carried out
using a Light Cycler® 2.0 instrument and FastStart
TaqMan Probe Master kit (both from Roche Diagnostics).
Reactions were performed in a 20 μL volume
with 5 pmol of each primer and 2 μL of cDNA
template derived from reverse-transcribed RNA of
the GnRH agonist group, Bevacizumab group and
Control group tissue cells. A hypoxanthine phosphoribosyl-
transferase (HPRT) housekeeping gene was
used as endogenous control and reference gene for
relative quantifications. Sequences of oligonucleotide
pri-mers were as follows: HPRT (F) 5’-GTG GAG
ATG ATC TCT CAA CT-3’, HPRT (R) 5’-ACA TGA
TTC AAA TCC CTG AAG-3’, BAX (F) 5’-AAG
AAG CTG AGC GAG T-3’, BAX (R) 5’-GCC CAT
GAT GGT TCT G-3’, CYC (F) 5’-TGG GTG ATG
TTG AGA AAG G-3’, CYC (R) 5’-TTT GTT CCA
GGG ATG TAC T-3’, BCL XL (F) 5’-GCT GGT
GGT TGA CTT TC-3’, BCL XL (R) 5’-GGA TGG
GTT GCC ATT GA-3’, BCL-2 (F) 5’-ACC TGA
CGC CCT TCA C-3’, BCL-2 (R) 5’-AGG TAC TCA
GTC ATC CAC-3’. The same thermal profile was
optimized for all primers: a pre incubation for 10
min. at 95 °C, followed by 40 amplification cycles of
denaturation at 95 °C for 10 seconds, primer annealing
at 59 °C for 5 seconds, and pri-mer extension at
72 °C for 10 seconds. Distilled water was included
as a no template control. Melting curves were derived
after 40 cycles by a denaturation step at 95 °C
for 10 seconds, followed by annealing at 65 °C for
15 seconds, and a temperature rise to 95 °C with a
heating rate of 0.1 °C/second and continuous fluorescence
measurement. Final cooling was performed
at 40 °C for 30 seconds. Melting curve analyses of
each sample were done using LightCycler Software
version 4.0.0.23 (Roche Diagnostics). The step of relative quantification was a fully automated
process done by the software, with the efficiency set
at 2 and the cDNA of untreated cells defined as the
calibrator. All experiments were done in triplicate.
Statistical Analyses. The statistical analyses
were performed using the Statistical Package for the
Social Sciences version 16.0 (SPSS Inc., Chicago,
IL, USA). Non normally distributed metric variables,
adhesion scores, histopathologic scores and gene expression
levels were analyzed by the Kruskal-Wallis
test and Mann-Whitney U-test. The mean surface
areas of the endometriotic explants between the same
group (before and after medical treatment) were analyzed
by Wilcoxon’s signed-rank test. p Values of
<0.05 were considered statistically significant. Values
were expressed as mean ± standard deviation (SD),
unless stated otherwise.analysis
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