Invest Clin 64(4): 482 - 494, 2023 https://doi.org/10.54817/IC.v64n4a5

Corresponding author: Lihong Chen. No. 20, Chazhong Road, Fuzhou, Fujian 350005, China. Phone: +86-

13506986776. E-mail: whcclh@163.com. Lie Zheng. No. 20, Chazhong Road, Fuzhou, Fujian 350005, China. Pho-

ne: +86–13905918829. E-mail: zelie@sina.com

Effects of SU5416 on angiogenesis

and the ERK-VEGF/MMP-9 pathway

in rat endometriosis.

Danyang Zhao1, Qiufang Bao1, Lihong Chen1,2,3 and Lie Zheng1,2

1Department of Obstetrics and Gynecology, the First Affiliated Hospital, Fujian Medical

University, China.

2Department of Gynecology, National Regional Medical Center, Binhai Campus

of the First Affiliated Hospital, Fujian Medical University, China.

3Fujian Key Laboratory of Precision Medicine for Cancer, the First Affiliated Hospital,

Fujian Medical University, China.

Keywords: angiogenesis; endometriosis; ERK-VEGF/MMP-9 pathway; SU5416.

Abstract. SU5416 is a small molecule vascular endothelial growth factor

(VEGF) receptor signal transduction inhibitor, which can block the VEGF re-

ceptor autophosphorylation and inhibit receptor tyrosine kinase signal trans-

duction, thereby reducing VEGF activity. However, there are few reports about

the correlation of SU5416 to the occurrence and angiogenesis in endometrio-

sis. In this study, we observed the effects of VEGF receptor inhibitor SU5416

on angiogenesis in endometriosis in rats. Thirty female specific-pathogen-free

Sprague-Dawley rats were randomly divided into sham operation group (SOG),

model group (MG), and SU5416 group (n=10 for each group). In the SOG, only

the uterus was cut and sutured, and endometriosis models were established in

the MG and SU5416 group by autologous transplantation. The SU5416 group

was injected with 15 mg/kg SU5416 intraperitoneally, and the SOG and MG

were intraperitoneally injected with an equal volume of normal saline for 6

weeks. The volume of ectopic lesions was lower in the SU5416 group at 42

d postoperatively than in the MG (p<0.05). The proportion of CD31-positive

cells in the endometrial tissue of the SU5416 group was lower than that of

the MG (p<0.05); angiopoietin-1 (Ang-1), angiopoietin-2 (Ang-2), laminin-5γ2

(LN-5γ2) and phosphorylation of ERK (P-ERK), VEGF, matrix metalloproteinase

(MMP)-2, and MMP-9 protein expressions were lower in the SU5416 group than

in the MG (p<0.05). VEGF receptor inhibitor SU5416 can inhibit endometrio-

sis angiogenesis and reduce inflammatory response in rats, and its mechanism

of action may be related to the down-regulation of the ERK-VEGF/MMP-9 path-

way expression.

Role of SU5416 in angiogenesis in rat endometriosis 483

Vol. 64(4): 482 - 494, 2023

Efecto del SU5416 sobre la angiogenesis y la via ERK-VEGF/

MMP-9 en la endometriosis de ratas.

Invest Clin 2023; 64 (4): 482 – 494

Palabras clave: angiogénesis; endometriosis; vía ERK-VEGF/MMP-9; SU5416.

Resumen. SU5416 es un inhibidor de la transducción de señales del re-

ceptor del factor de crecimiento endotelial vascular (VEGF), una molécula pe-

queña, capaz de bloquear la autofosforilación del receptor VEGF e inhibir la

transducción de señales de la tirosina quinasa del receptor, reduciendo así la

actividad del VEGF. Sin embargo, existen escasos informes acerca de la co-

rrelación entre SU5416 y la aparición y angiogénesis de la endometriosis. En

este estudio, hemos observado los efectos del inhibidor del receptor del VEGF,

SU5416, sobre la angiogénesis en la endometriosis en ratas. Treinta ratas Spra-

gue-Dawley hembra, libres de patógenos específicos, fueron divididas aleatoria-

mente en un grupo de operación simulada (SOG), un grupo de modelo (MG)

y un grupo de SU5416 (n=10 en cada grupo). En el SOG, solo se realizó una

incisión en el útero y se suturó, mientras que en los grupos MG y SU5416 se es-

tablecieron modelos de endometriosis mediante trasplante autólogo. Al grupo

SU5416 se le inyectaron 15 mg/kg de SU5416 por vía intraperitoneal, y tanto

el SOG como el MG recibieron una inyección intraperitoneal de un volumen

igual de solución salina normal durante 6 semanas. El volumen de lesiones ec-

tópicas fue menor en el grupo SU5416 a los 42 días después de la operación en

comparación con el MG (p<0,05); la proporción de células CD31 positivas en

el tejido endometrial del grupo SU5416 fue inferior a la del MG (p<0,05); las

expresiones de las proteínas angiopoyetina-1 (Ang-1), angiopoyetina-2 (Ang-2),

laminina-5γ2 (LN-5γ2) y la fosforilación de ERK (P-ERK), VEGF, metaloprotei-

nasa de matriz (MMP)-2 y MMP-9 fueron menores en el grupo SU5416 que en

el MG (p<0,05). El inhibidor del receptor del VEGF, SU5416, puede inhibir la

angiogénesis de la endometriosis y reducir la respuesta inflamatoria en ratas, y

su mecanismo de acción puede estar relacionado con la regulación a la baja de

la expresión de la vía ERK-VEGF/MMP-9.

Received: 22-02-2023 Accepted: 08-09-2023

INTRODUCTION

Endometriosis (EMs) is a benign mor-

phological manifestation, but the biological

behaviors such as implantation invasion, ag-

gressive growth and distant metastasis are

similar to those of malignant tumors, which

can induce painful intercourse, dysmenor-

rhea, chronic pelvic pain, and infertility up

to 25%-35% 1,2. The specific etiology of this

disease has not been elucidated, and it is

mostly thought to be related to genetic fac-

tors, endometrial implantation, retrograde

menstruation, implantation, and immune

regulation 3,4. However, both endometrial

implantation and menstrual reflux implanta-

tion depend on adequate blood supply, so an-

giogenesis plays a key role in the occurrence

and development of EMs.

484 Zhao et al.

Investigación Clínica 64(4): 2023

Vascular endothelial growth fac-

tor (VEGF) is an autocrine and paracrine

growth factor that can improve vascular

permeability, damage the tight junctions of

vascular endothelial cells, induce endothe-

lial cell proliferation, and promote extracel-

lular fluid accumulation, vascular leakage,

and neovascularization 5,6. Matrix metallo-

proteinase-9 (MMP-9), one of the important

protein hydrolases in the family of MMPs,

disrupts basement membrane integrity and

promotes neovascularization and vascular

endothelial cell outgrowth, thus playing an

important role in the ectopic implantation,

adhesion, and growth of endometrial cells

7. Therefore, downregulation of VEGF and

MMP-9 expression is particularly critical in

inhibiting angiogenesis and blocking sig-

naling in vascular endothelial cells. SU5416

is a small molecule VEGF receptor signal-

ing inhibitor, which can block VEGF recep-

tor autophosphorylation, inhibit receptor

tyrosine kinase signaling, and reduce VEGF

activity 8. It was found that SU5416 in a rat

pulmonary hypertension model reduced

pulmonary inflammatory response, inhibit-

ed intimal proliferation of small pulmonary

arteries, and promoted pulmonary vascular

remodeling 9. However, there are few reports

on the relevance of SU5416 on the develop-

ment of EMs and angiogenesis worldwide.

The objective of this study was to analyze

the effects of SU5416, a VEGF receptor

inhibitor, on angiogenesis and ERK-VEGF/

MMP-9 signaling pathway in EMs in rats.

MATERIAL AND METHODS

Experimental animals

The experiment procedures conformed

to the relevant requirements of the Regula-

tions of the People’s Republic of China on the

Administration of Laboratory Animals; 30 fe-

male specific-pathogen-free (SPF) Sprague-

Dawley (SD) rats weighting 180-200 g were

purchased from (purchased from Beijing

Viton Lihua Laboratory Animal Technology

Co., Ltd; animal Use License), Animal Use Li-

cense No.: SYXK (Beijing) 2018-0015, Labo-

ratory Animal Production License No.: SCXK

(Beijing) 2018-0022. Rats were housed at a

temperature of (24±1) °C, relative humid-

ity of 50%, and noise<80 db. The researcher

changed the bedding, and cleaned and disin-

fected the rat cages regularly.

Drugs, reagents and instruments

VEGF receptor inhibitor SU5416

(Shanghai Hengfei Biotechnology Co., Ltd.,

China), Two-steps IHC detection kits for rat

tissues (Shanghai Qi Ming Biotechnology

Co., Ltd., China), Western blot electropho-

resis instrument (Bio-Rad Inc., USA), BCA

protein concentration assay kit (Beijing

Solaibao Technology Co. Ltd., China), He-

matoxylin Eosin Staining Kit (Wuhan PhD

Bioengineering Co., Ltd., China), angiopoi-

etin (Ang)-1, Ang-2, laminin-5γ2 (LN-5γ2),

phosphorylation of ERK (P-ERK), VEGF,

MMP-2, MMP-9, GAPDH antibodies (CST

Biotechnology Co., Ltd., USA), Horserad-

ish Peroxidase (HRP)-Labeled Goat Anti-

Rabbit Immunoglobulin (Ig) G (Solepow

Technology Co., Ltd., China), VEGF, MMP-

9, and GAPDH primers (synthesized by

Sangon Biotech (Shanghai) Co., Ltd.),

fluorescent quantitative PCR kit (Lot. No.

639519, Takara Bio Inc., Japan), Trizol kit

(Thermo Fisher Science, USA), RNA extrac-

tion kit (Nanjing Novozymes Biotechnology

Co., Ltd., China), reverse transcription kit

(Genecopoeia, Inc., USA), 5415D high-

speed centrifuge (Eppendorf, Germany),

and CX21 optical microscope (Olympus,

Japan), Light Cycler 2.0 Real-time PCR in-

strument (Roche Equipment Ltd., Switzer-

land), -80℃ ultra-low temperature refriger-

ator (SANYO, Japan), enzyme immunoassay

analyzer (Shanghai Kunke Instruments Co.,

Ltd., China), surgical instruments (Beijing

Youcheng Jiaye Biotechnology Ltd., China),

VEGF, MMP-9, GAPDH primers (Shanghai

Bioengineering Co., Ltd., China), vernier

calipers (Shanghai YuYan Scientific Instru-

ments Co., Ltd., China), etc.

Role of SU5416 in angiogenesis in rat endometriosis 485

Vol. 64(4): 482 - 494, 2023

METHODS

Model establishment and grouping

Thirty female SPF SD rats were random-

ly divided into sham operation group (SOG),

model group (MG), and SU5416 group

(n=10 for each group). In the SOG, only the

uterus was cut and sutured. The EMs mod-

els were established in the MG and SU5416

group by autologous transplantation, that

is, the rats were anesthetized by intraperi-

toneal injection of 10% chloral hydrate at a

dose of 300 mg/kg, and the rats were placed

in the supine position and fixed on the oper-

ating table, disinfected. A 2 cm incision was

made in the middle of the lower abdomen,

the uterus and endometrium were separat-

ed, and two 5mm × 5mm fragments were

taken from the left uterine horn, which were

quickly transplanted into the rat mesenteric

artery with abundant blood vessels. The ab-

dominal cavity was washed and sutured layer

by layer, and the postoperative anti-infec-

tion was performed for 3 days. The success

criteria for model establishment: the graft

was opened 14 d postoperatively, and the

volume of the graft was observed visually to

be increased, with a light red, round or oval

vesicle with internal fluid accumulation, and

the surface was covered with a large number

of blood vessels and closely adhered to the

surrounding tissues. The SU5416 group was

injected intraperitoneally with 15 mg/kg

SU5416 twice a week, and the sham opera-

tion and MGs were injected intraperitoneally

with equal volume of saline for 6 weeks. Ev-

ery procedure was approved by the Animal

Care and Use Committee of the First Affili-

ated Hospital of Fujian Medical University.

Morphology of normal and ectopic

endometrial tissue and volume of ectopic

lesions in rats

At 42 d after surgery, morphological

changes of endometrial tissue in rats were

observed under optical microscope; at 14 d

and 42 d after surgery, the width and length

of ectopic lesions were measured using ver-

nier calipers, and the volume of lesions was

calculated = length×width2×0.5.

Specimen collection

After 24h after the last administration,

3mL of tail vein blood was collected from

rats, left for 15min, centrifuged at 2000×g

for 15min, and the supernatant was stored

in an ultra-low temperature refrigerator at

-80℃. After anesthesia, the rats were exe-

cuted and dissected. In the MG and SU5416

group, ectopic endometrium was removed,

and in the SOG, normal endometrium was

removed. Each specimen was immediately

cut into three parts and rinsed with saline,

and 1g of tissue was cut off and stored in an

ultra-low temperature refrigerator at -80℃,

which were used for real-time quantitative

PCR and western blot assay. The remaining

endometrium tissue was fixed with 4% para-

formaldehyde, dehydrated in gradient alco-

hol, transparent in xylene, embedded in par-

affin, and routinely pathologically sectioned.

Immunohistochemical staining method

CD31 is a platelet endothelial cell ad-

hesion molecule expressed at the tight

junctions between endothelial cells, which

regulates the process of angiogenesis and

reflects the microvessel density (MVD), and

can therefore be used as a marker for the

measurement of microangiogenesis. In this

study, the number of CD31 positive cells in

endometrial tissues was mainly detected by

immunohistochemical detection. Paraffin

sections were dewaxed, soaked in alcohol

from high to low gradient, rinsed with dis-

tilled water, incubated with 0.3% H2O2 for 30

min, blocked with 5% serum for 2 h, incubat-

ed with CD31 primary antibody at 4℃ over-

night, rinsed with TBST, immunohistochem-

ical staining, rinsed with distilled water,

dehydrated in gradient alcohol, transparent

in xylene, and sealed. The staining was ob-

served using light microscopy, and any five

fields of view of each section were photo-

graphed. The number of CD31 positive cells

and the total number of cells were counted,

486 Zhao et al.

Investigación Clínica 64(4): 2023

and the proportion of CD31 positive cells

was calculated. Proportion of CD31 positive

cells = number of CD31 positive cells/total

number of cells × 100%.

Enzyme-linked immunosorbent assay

(ELISA)

The spare serum was taken, and the

levels of serum VEGF, MMP-9, interleukin

(IL)-1, IL-2, IL-6 and tumor necrosis factor

(TNF)-α were determined according to the

instructions of the ELISA kit. Blank wells

(no sample and enzyme reagents were added

to the blank control wells, the rest of the

procedure was the same), standard wells and

test sample wells were set respectively. Stan-

dard (50 μL) was accurately added on the

ELISA coated plate. Sample diluent (40 μL)

was added to the test sample wells, and then

10 μL of test sample was added (the final

sample dilution was five times). The sample

was added to the bottom of the well of ELISA

plate without touching the wall of the well,

and was gently shaken and mixed. After seal-

ing the plate with sealing film, the sample

was incubated at 37 °C for 30 min. The 30

times concentrated washing solution was di-

luted with 30 times distilled water for fur-

ther use. After carefully removing the seal-

ing film, the solution was discarded, and the

sample was shaken dry. Each well was filled

with washing solution, and after leaving for

30 s, the solution was discarded, repeating

this for 5 times and patting dry. Enzyme re-

agent (50 μL) was added to each well, except

blank wells. The plate was the sealed with

sealing film, and the sample was incubated

at 37°C for 30 min. After washing the plate

as the above method, Chromogen solution

A (50 μL) was added to each well first, and

then Chromogen solution B (50 μL) was add-

ed to each well, and the mixture was gently

shaken and mixed for chromogenic reaction

at 37 °C for 15 min. The stop solution (50

μL) was added to each well to stop the re-

action (at this time, the blue immediately

turned to the yellow). The blank well was set

to zero, and the optical density (OD) of each

well at 450 nm wavelength was measured.

Quantitative real-time fluorescence

(qRT-PCR)

Spare uterine tissue was taken and to-

tal RNA from uterine tisuue extracted ac-

cording to the instructions of Trizol kit and

total RNA extraction kit, and use reverse

transcription kit to reverse-transcribe the

total RNA into cDNA. Using the reverse tran-

scribed cDNA as a template, the expression

of VEGF and MMP-9 was detected by Real-

time PCR instrument. The total reaction vol-

ume was 20 μL, GAPDH was used as the in-

ternal reference, and the relative expression

of genes in each group was calculated by the

2-ΔΔCt method.

Western blot

The spare remaining uterine tissue was

taken, followed by the BCA method for pro-

tein quantification, 12% SDS-polypropylene gel

electrophoresis, wet transfer to PVDF mem-

brane, treatment with a closure solution for

two h at room temperature in a shaker, pri-

mary antibody (1:500 dilution) (Ang-1, Ang-2,

LN-5γ2, P-ERK, VEGF, MMP-2, MMP-9, GAPDH)

was added, incubated overnight at 4 °C, and the

membrane was washed three times with TBST

for 15 minutes each time. HRP-labeled Goat

Anti-Rabbit IgG secondary antibody (1:2000

dilution) was added, incubated for two hours

at room temperature, and the membrane was

washed three times with TBST for 15 minutes

each time. Enhanced chemiluminescence

(ECL) reagents were used to develope the

color and quantitative analysis was performed

using Image J software. GAPDH (1:1000 dilu-

tion) was used as the internal reference, and

the ratio result indicated the relative concen-

tration of the target protein.

Statistical analysis

Statistical Package for the Social Sci-

ences (SPSS) 24.0 statistical analysis soft-

ware was used, and the measurement data

conforming to normal distribution were ex-

Role of SU5416 in angiogenesis in rat endometriosis 487

Vol. 64(4): 482 - 494, 2023

pressed as

sx ±

and compared using one-

way analysis of variance (ANOVA) while least

significant difference (LSD)-t test was used

for two-way comparison, p<0.05 was consid-

ered statistically significant difference.

RESULTS

Volume of ectopic lesions

The volume of ectopic lesions in the MG

and SU5416 group at 42 days after operation

was higher than that at 14 days after opera-

tion (p<0.05), but there was no significant

difference in the volume of ectopic lesions

between the MG and SU5416 group at 14

days after operation (p>0.05); the volume

of ectopic lesions in the SU5416 group was

lower than that in the MG at 42 days after

operation (p<0.05), indicating that SU5416

could effectively inhibit the increase in the

volume of ectopic lesions in rats (Fig. 1).

Morphological changes of normal and

ectopic endothelial tissues in rats under

light microscopy

Since the rats in the sham-operation

group did not develop EMs and there was no

presence of ectopic endometrial tissue, the

comparison was made between normal endo-

metrial tissue and ectopic endometrial tissue

from the other groups. At 42 days after opera-

tion, the normal endometrial epithelial cells

and glandular epithelium of the rats in the

sham-operation group were arranged in a co-

lumnar shape with a complete structure, and

the glands, blood vessels and interstitial cells

of the lamina propria were neatly arranged

and structurally complete. The ectopic en-

dometrial epithelial cells and the glandular

epithelium of the rats in the MG were high

columnar; the endometrial structure was cir-

cular sawtooth closed, the numbers of lamina

propria glands, stromal cells and blood vessels

were large, and the nucleus oval was deeply

stained. The ectopic endometrial epithelial

cells of the SU5416 group showed atrophic

changes, the structure of some epithelial

cells is incomplete, the lamina propria gland

epithelial cells are incomplete, the mesen-

chymal cells become smaller, the number of

blood vessels is reduced and the arrangement

is disordered (Fig. 2).

CD31-positive cell expression

in endometrial tissue

Since the rats in the sham-operation

group did not develop EMs and there was no

presence of ectopic endometrial tissue, the

comparison was made between normal endo-

metrial tissue and ectopic endometrial tissue

from the other groups. The staining of CD31

positive cells and the proportion of positive

cells were detected by immunohistochemi-

cal method, which could show the changes

of MVD of endometrium. The proportion of

MVD of ectopic endometrial tissue (that is,

CD31-positive cell expression) in the MG

and SU5416 group was higher than that of

normal endometrial tissues in the SOG (p <

0.05); The proportion of CD31-positive cell

Fig. 1. Comparison of ectopic lesion volume in

each group.

Shows that there was no significant difference

in the volume of ectopic lesions between the model

group and SU5416 group at 14 days after operation,

while the volume of ectopic lesions in the SU5416

group was lower than that in the model group at

42 days after operation. Note: MG: model group;

SG: SU5416 group. Compared within the group at

14 days after operation, &&&p<0.001; compared

with the model group at 42 days after operation,

###p<0.001.

488 Zhao et al.

Investigación Clínica 64(4): 2023

expression of ectopic endometrial tissues

was lower in the SU5416 group than in the

MG (p<0.05), which showed that SU5416

could effectively reduce the MVD of endome-

trial tissue in rats (Fig. 3).

Endometrial angiogenesis-related protein

expression

Ang-1, Ang-2, and LN-5γ2 protein ex-

pression of ectopic endometrial tissues in

the MG and SU5416 group were higher than

those of normal endometrial tissues in the

SOG (p<0.05); Ang-1, Ang-2, and LN-5γ2

protein expression in ectopic endometrial

tissues of the SU5416 group were lower than

that of the MG (p<0.05), indicating that

SU5416 could effectively inhibit endometri-

al angiogenesis-related protein expression in

rats (Fig. 4).

VEGF, MMP-9

The serum VEGF and MMP-9 levels

and the expression of VEGF messenger RNA

(mRNA) and MMP-9 mRNA in ectopic endo-

metrial tissues of the MG and SU5416 group

were higher than those of normal endome-

trial tissues in SOG (p<0.05); The serum

VEGF and MMP-9 levels and the expression of

VEGF mRNA and MMP-9 mRNA in ectopic en-

dometrial tissues of the SU5416 group were

lower than those of the MG (p<0.05), which

showed that SU5416 could effectively inhibit

the serum expression of VEGF and MMP-9

and endometrial tissues of rats (Fig. 5).

ERK-VEGF/MMP-9 pathway-related

protein expression

The protein expression of P-ERK, VEGF,

MMP-2 and MMP-9 of ectopic endometrial

tissues in the MG and SU5416 group were

higher than those of normal endometrial tis-

sues in the SOG (p<0.05); The protein ex-

pression of P-ERK, VEGF, MMP-2 and MMP-9

in ectopic endometrial tissues of the SU5416

group were lower than that of the MG (p<

0.05). It is evident that SU5416 can effec-

tively inhibit the activation of ERK-VEGF/

MMP-9 pathway in rats (Fig. 6).

Inflammatory factors

The levels of serum IL-1, IL-2, IL-6 and

TNF-α were higher in the MG and SU5416

group than in the SOG (p<0.05); The lev-

els of serum IL-1, IL-2, IL-6 and TNF-α were

Fig. 2. Morphological changes of normal and ectopic endometrial tissues of rats under light microscopy (200×).

Shows that there was no endometriosis lesion in the sham-operation group, so the comparison was

made between normal endometrial tissue and ectopic endometrial tissue from the other groups. At 42

days after operation, the normal endometrial epithelial cells and glandular epithelium of the rats in the

sham-operation group were arranged in a columnar shape with a complete structure. The ectopic endo-

metrial epithelial cells and the glandular epithelium of the rats in the model group were high columnar;

the endometrial structure was circular sawtooth closed, the numbers of lamina propria glands, stromal

cells and blood vessels were large, and the nucleus oval was deeply stained. The ectopic endometrial

epithelial cells of the SU5416 group showed atrophic changes.

Role of SU5416 in angiogenesis in rat endometriosis 489

Vol. 64(4): 482 - 494, 2023

Fig. 3. Comparison of CD31-positive cell expression in endometrial tissues of rats in each group.

Shows that there was no endometriosis lesion in the sham-operation group, so the comparison was

made between normal endometrial tissue and ectopic endometrial tissue from the other groups. (A)

Staining of CD31-positive cells detected by immunohistochemistry (200×); (B) proportion of CD31-

positive cell expression. Note: Compared with the sham operation group, ***p<0.001; Compared with

the model group, ###p<0.001. Proportion of CD31-positive cell expression = number of CD31 positive

cells/total number of cells × 100%.

Fig. 4. Comparison of endometrial an-

giogenesis-related protein ex-

pression.

Shows that (B) Ang-1, (C) Ang-

2, and (D) LN-5γ2 protein expressions

in endometrial tissues of the SU5416

group were lower than those of the

model group. Note: Ang-1: angiopoi-

etin-1; Ang-2: angiopoietin-2; LN-

5γ2: laminin-5γ2. Compared with the

sham operation group, ***p<0.001;

Compared with the model group,

###p<0.001.

490 Zhao et al.

Investigación Clínica 64(4): 2023

Fig. 5. Comparison of VEGF and

MMP-9 expression in rats in

each group.

Shows that the protein ex-

pression levels of serum (A) VEGF

and (B) MMP-9 and the mRNA ex-

pression levels of (C) VEGF and

(D) MMP-9 in endometrial tissue

of the SU5416 group were lower

than those of the model group.

Note: VEGF: vascular endothe-

lial growth factor; MMP-9: matrix

metalloproteinase-9. Compared

with the sham operation group,

***p<0.001; Compared with the

model group, ###p<0.001.

Fig. 6. Comparison of ERK-VE-

GF/MMP-9 pathway-rela-

ted protein expression.

Shows that the expres-

sion levels of (B) P-ERK, (C)

VEGF, (D) MMP-2, and (E)

MMP-9 proteins in the endo-

metrial tissue of the SU5416

group were lower than those of

the model group. Note: P-ERK:

phosphorylation of ERK; VEGF:

vascular endothelial growth

factor; MMP-2: matrix meta-

lloproteinase-2; MMP-9: matrix

metalloproteinase-9. Compa-

red with the sham operation

group, **p<0.01, ***p<0.001;

Compared with the model

group, ###p<0.001.

Role of SU5416 in angiogenesis in rat endometriosis 491

Vol. 64(4): 482 - 494, 2023

lower in the SU5416 group than in the MG

(p<0.05), which showed that SU5416 could

effectively reduce the inflammatory response

in rats (Fig. 7).

DISCUSSION

The etiology and mechanism of EMs

are complex and diverse. The implantation

theory of reverse flow of menstrual blood

proposed by Sampson in 1921 is the most

supported, that is, the exfoliated endome-

trial fragments flow back into the pelvic and

abdominal cavity with the menstrual blood,

and are implanted in the ovary and adjacent

pelvic and abdominal cavity, causing a local

inflammatory response in the peritoneum,

leading to the establishment of a local mi-

croenvironment and angiogenesis, thereby

providing a continuous angiogenesis stimu-

lus for vascular remodeling 10-13. However,

either theory involves neovascularization

and degradation and reconstruction of the

extracellular matrix. The reason for the ini-

tiation of pathological angiogenesis lies in

the unbalanced regulation of vascular inhib-

itory factors and promoting factors, among

which the increase of pro-angiogenic factors

such as VEGF and matrix metalloproteinase

(MMP) is the main cause 14. Therefore, anti-

angiogenesis is of great importance in the

prevention and treatment of EMs. SU5416

is a lipid-soluble small molecule VEGF re-

ceptor signal transduction inhibitor, which

can block the interaction between VEGF

and its receptor and inhibit angiogenesis 15.

However, there are no reports on the effect

of SU5416 in EMs. In this experiment, an

EMs model was established by autologous

transplantation. The results showed that

compared with the MG, the volume of ecto-

pic lesions, the proportion of CD31 positive

cells and the level of serum inflammatory

factors in the SU5416 group were reduced

Fig. 7. Comparison of serum inflam-

matory factor levels in rats

of each group.

Shows that the expression

levels of (A) IL-1, (B) IL-2, (C)

IL-6 and (D) TNF-α in endometrial

tissue of the SU5416 group were

lower than those of the model

group. Note: IL-1: interleukin-1;

IL-2: interleukin-2; IL-6: inter-

leukin-6; TNF-α: tumor necrosis

factor-α. Compared with the sham

operation group, ***p<0.001;

Compared with the model group,

###p<0.001.

492 Zhao et al.

Investigación Clínica 64(4): 2023

at 42 days after operation. Membrane epi-

thelial cells showed atrophic changes, mes-

enchymal cells became smaller, and the

number of blood vessels decreased. It can

be seen that SU5416 can reduce the volume

of ectopic lesions and prevent the inflam-

matory response by inhibiting cell prolifera-

tion and angiogenesis.

The ERK signaling pathway is one of the

mitogen-activated protein kinase (MAPK)

pathways. Activated ERK can activate down-

stream targets such as the 90kD ribosomal

S6 protein kinase family (RSKs), and promote

the translocation of RSK1/2 and pERK1/

pERK2 into the nucleus, activate early and

immediate gene transcription, thereby regu-

lating cell survival, apoptosis, proliferation,

metabolism, transcription and other biologi-

cal behaviors 16,17. VEGF is one of the endo-

thelial cell-specific vascular-derived proteins,

which can promote fibrinogen exudation,

increase trophoblast, endometrial and meco-

nium permeability, promote endothelial cell

proliferation and subperitoneal vascular net-

work formation, thus inducing lesion growth

and endothelial implantation; Moreover,

VEGF can bind to relevant receptors on en-

dothelial cells and initiate paracrine mecha-

nisms via signaling pathways, which play an

important role in endothelial implantation

and placenta formation 18,19. MMP-9 can de-

grade the main components of extracellular

matrix such as collagen type IV, collagen V,

and gelatin, destroy the integrity of the base-

ment membrane, and promote the formation

of new blood vessels and the sprouting of

vascular endothelial cells 20. Meanwhile, the

degraded extracellular matrix protein frag-

ments could regulate apoptosis, migration,

and invasion of epithelial cells, leading to

invasion into other parts of the eutopic en-

dometrium 21. Chen 22 et al. found that the

ERK-VEGF/MMP-9 signaling pathway is close-

ly related to angiogenesis, and down-regulat-

ing the expression of this signaling pathway

can inhibit cell proliferation, invasion and

angiogenesis. Guo 23 et al. found that inhibi-

tion of extracellular ERK activation down-

regulated MMP-9 and VEGF expression and

signaling, thereby slowing down the rate of

vascular invasion and growth. Yilmaz 24 et al.

found increased expression of P-ERK, VEGF,

and MMP-9 proteins in rats with EMs, while

blocking cytokine binding to surface recep-

tors and intercellular signaling pathways

could control abnormal endometrial prolif-

eration and angiogenesis. The results of this

study revealed that the expression of P-ERK,

VEGF, MMP-2, and MMP-9 proteins in the en-

dometrial tissues of the MG was higher than

that of the SOG, which was consistent with

the above findings and again confirmed that

the activation of ERK-VEGF/MMP-9 signaling

pathway might be related to the development

of EMs. The expression of P-ERK, VEGF, MMP-

2, MMP-9 and Ang-1, Ang-2, LN-5γ2 proteins

were reduced in the endometrial tissues of

rats after SU5416 treatment, which showed

that the VEGF receptor inhibitor SU5416

could reduce the synthesis and secretion of

extracellular signal-regulated kinases and

inhibit the activation of ERK-VEGF/MMP-9

signaling pathway in ectopic endometrial tis-

sues, thus preventing angiogenesis and the

growth, implantation and adhesion of abnor-

mal proliferating cells in the pelvic and ab-

dominal peritoneum.

In summary, the VEGF receptor in-

hibitor SU5416 inhibited angiogenesis

and reduced inflammatory response in

rat EMs, and its mechanism of action may

be related to the downregulation of ERK-

VEGF/MMP-9 pathway expression. This ex-

periment confirmed the effects of SU5416

on angiogenesis, signaling pathway and

inflammatory response in rats with EMs,

providing new ideas for the clinical treat-

ment and the development of new drugs.

However, further in vivo experiments are

needed to verify the therapeutic effects

and specific mechanism of action of the

VEGF receptor inhibitor.

Role of SU5416 in angiogenesis in rat endometriosis 493

Vol. 64(4): 482 - 494, 2023

Conflicts of interest

There are no conflicts to declare.

Funding

None.

Authors ORCID’number

• Danyang Zhao: 0000-0002-0668-9000

• Qiufang Bao: 0009-0008-1944-7431

• Lihong Chen: 0000-0003-2514-2913

• Lie Zheng: 0000-0002-9457-9003

Authors Contribution

Each author has made an important

scientific contribution to the study and has

assisted with the drafting or revising of the

manuscript.

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