The expression of vascular endothelial growth …...2 The expression of vascular endothelial growth factor, angiopoietin-1 and angiopoietin-2 in adenomyosis and adenomyosis under levonorgestrel

1

The expression of vascular endothelial

growth factor, angiopoietin-1 and

angiopoietin-2 in adenomyosis and

adenomyosis under levonorgestrel impact

Joo Hyun Park

Department of Medicine

The Graduate School, Yonsei University

2

The expression of vascular endothelial

growth factor, angiopoietin-1 and

angiopoietin-2 in adenomyosis and

adenomyosis under levonorgestrel impact

Directed by Professor Byung Seok Lee

The Master's Thesis submitted to the Department of Medicine

the Graduate School of Yonsei University

in fulfillment of the requirements for the degree of Master of

Medicine

Joo Hyun Park

December 2008

3

This certifies that the Master's Thesis of

Joo Hyun Park is approved.

[Signature]

------------------------------------

Byung Seok Lee: Thesis Supervisor

[Signature]

------------------------------------

Hyeon Joo Jeong: Thesis Committee Member#1

[Signature]

------------------------------------

Bo Yon Lee: Thesis Committee Member#2

The Graduate School

Yonsei University

December 2008

4

ACKNOWLEDGEMENTS

I tribute my gratitude towards Professor Byung Seok Lee for

providing guidance as a mentor for my thesis to be submitted

for a master in science.

I would also like to thank all my professors at the

Department of Obstetrics and Gynecology at Yonsei

University College of Medicine, who have inspired me

during my training years.

I pay honor to the Lord, the creator of all sciences, the

principles of which we will always endeavor to discover but

will never understand fully.

5

<TABLE OF CONTENTS>

ABSTRACT ...........................................................................................1

I. INTRODUCTION ..................................................................................3

II. MATERIALS AND METHODS ...........................................................7

1. Patient selection..............................................................................7

2. Immunohistochemical staining.......................................................8

A. Antisera......................................................................................8

B. Immunohistochemical procedure...............................................9

C. Analysis of immunohistochemical staining .............................10

3. Statistical Analysis .......................................................................10

III. RESULTS........................................................................................... 12

1. Vascular endothelial growth factor.............................................13

2. Angiopoietin-1 ............................................................................19

3. Angiopoietin-2 ............................................................................25

IV. DISCUSSION .................................................................................... 31

V. CONCLUSION ................................................................................... 36

REFERENCES......................................................................................... 37

ABSTRACT (IN KOREAN) ..............................................................41

6

LIST OF FIGURES

Figure 1. The analysis of H-scores for vascular endothelial growth factor

expression .....................................................................................15

Figure 2. The immunolocalization of vascular endothelial growth factor .. 16

Figure 3. The analysis of H-scores for angiopoietin-1 expression ................21

Figure 4. The immunolocalization of angiopoietin-1 ....................................22

Figure 5. The analysis of H-scores for angiopoietin-2 expression ................27

Figure 6. The immunolocalization of angiopoietin-2 ....................................28

LIST OF TABLES

Table 1. Demographic factors of the study samples .................................. 12

7

The expression of vascular endothelial growth factor, angiopoietin-1

and angiopoietin-2 in adenomyosis and adenomyosis under

levonorgestrel impact

Joo Hyun Park



(Directed by Professor Byung Seok Lee)

Although adenomyosis is a common gynecologic cause of

dysmenorrhea, menorrhagia and subsequent anemia, the etiology and

pathophysiology of the disease is still obscure. The infiltration of

endometrium into the adjacent myometrium is the key feature of

adenomyosis and an alteration of the angiogenic potential of the

endometrium may be the culprit of the evolution of ectopic endometrial

implants in adenomyosis as in endometriosis. The purpose of this study

is to investigate on the expressions of vascular endothelial growth

factor (VEGF), angiopoietin-1 (ANGPT-1) and angiopoietin-2

(ANGPT-2) in the ectopic and eutopic endometrium of adenomyosis

tissues and the role of levonorgestrel-intrauterine system (LNG-IUS)

insertion on these factors. The experimental materials consisted of 38

hysterectomy samples confirmed as adenomyosis with or without

LNG-IUS insertion, whereas 12 normal endometrium without any

significant uterine abnormalities were used as controls. The tissues

were stained with polyclonal VEGF, ANGPT-1 and ANGPT-2

8

antibodies and assessed semiquantitatively. Both the ectopic and

eutopic endometrium in adenomyosis displayed increased levels of

VEGF, ANGPT-1 and ANGPT-2 compared with the control

endometrium. Both the ectopic and eutopic endometrium under the

impact of LNG-IUS expressed decreased levels of all three factors

compared to that of adenomyosis. No differences in VEGF, ANGPT-1

and ANGPT-2 levels were observed in the LNG-IUS inserted eutopic

endometrium compared to control endometrium.

Our results suggest that the enhanced expressions of VEGF, ANGPT-1

and ANGPT-2 may play a role in the pathogenesis of adenomyosis and

the LNG-IUS may decrease menstrual blood loss by suppressing the

pathways mediated by these angiogenic factors.

--------------------------------------------------------------------------------------

Key words: vascular endothelial growth factor, angiopoietin, adenomyosis,

levonorgestrel

9

The expression of vascular endothelial growth factor, angiopoietin-1

and angiopoietin-2 in adenomyosis and adenomyosis under

levonorgestrel impact

Joo Hyun Park



(Directed by Professor Byung Seok Lee)

I. Introduction

Adenomyosis is characterized by the infiltration of the endometrium into the

myometrium, which belongs to the spectrum of endometriosis. Adenomyosis

is a major cause of dysmenorrhea and menorrhagia during the reproductive

years, and thus it is a worthy endeavor to attempt to reveal the

pathophysiology of the disease. A unique characteristic of the endometrium to

undergo rapid growth and degeneration on a monthly basis requires dynamic

vascular remodeling, which presumably may also act as the causative factor in

the pathogenesis of adenomyosis and endometriosis. Abundant angiogenic

factors and adhesion molecules are known to be present in the endometrium

and the differential expression of these factors may play a role.1

Derangements in the angiogenic regulation of the endometrium is a possible

cause for the abnormal recruitment and proliferation of the endometrium into

10

the adjacent or distant tissues. Endometriotic lesions tend to be surrounded by

well vascularized tissues and peritoneal fluid from women with endometriosis

display increased angiogenic activity. Functional disturbances associated with

adenomyosis, such as menorrhagia have been known to be associated with a

marked increase in the vascular density of the endometrial stroma.2 However,

only a limited number of descriptions exist on the factors which mediate

angiogenesis within the endometrium and how these are altered in

endometriosis.

The vascular endothelial growth factor (VEGF) is the most extensively

described angiogenic factor of the endometrium. The VEGF is a glycoprotein,

specifically promoting the growth and activation of vascular endothelial cells,

increasing vascular permeability and promoting the expression of several

proteolytic enzymes involved in angiogenesis.3 The VEGF is predominantly

involved in the cyclic growth of vasculature in the endometrium with each

menstrual cycle. Under normal physiologic conditions, VEGF is expressed

limitedly in tissues and is secreted by activated endothelial cells with tumor

derived growth factors and under adverse stimuli such as hypoxia,

inflammation and trauma. VEGF is also over-expressed in the abnormal

endometrium and previous observations show an increased expression within

the ectopic endometrium of endometriosis.4

The cytokine angiopoietin-1 (ANGPT-1) enhances the actions of other

11

angiogenic factors, mainly the VEGF. Secreted by periendothelial support

cells, ANGPT-1 binds to the receptor tunica interna cell kinase-2 (TIE-2),

which is a member of the tyrosine kinase family.5 ANGPT-1 is necessary for

the maturation of new vessels, playing a stabilizing role, maintaining cell to

cell interactions and inhibiting apoptosis.6,7 Without ANGPT-1, formation of

vessels by VEGF stimulus soley, result in leaky, immature, poorly functioning

vessels. ANGPT-1 is expressed in both endothelial and nonendothelial cell

lines, expressed in the endometrial glandular, stromal and periendothelial

cells.1

On the other hand, angiopoietin-2 (ANGPT-2) is a natural antagonist of

ANGPT-1, binding competitively to the same receptor, the TIE-2.8 Under the

influence of ANGPT-2, vessels are destabilized, where cell to cell, cell to

matrix connections become loose, and the basement membrane and the peri-

endothelial support cells become disassociated from the endothelium.9

Homeostatic conditions of healthy endometrium are maintained by ANGPT-1

whereas localized expression of ANGPT-2 seems to represent a temporary

exit from quiescence and the initiation of vascular remodeling. The co-

expression of VEGF and ANGPT-2 stimulates the re-entry into the cell

division cycle, resulting in vascular proliferation. Without any mitogenic

signals from VEGF, the endothelial cells are more likely to undergo apoptosis,

eventually leading to the regression of vessels.

12

The levonorgestrel intrauterine system (LNG-IUS) is a contraceptive device

that releases a daily dose ranging from 10 to 20mcg of levonorgestrel in a

continous manner, which is accepted as a reliable alternative to surgery in

women with adenomyosis having secondary dysmenorrhea, menorrhagia and

anemia. Previous reports have elaborated on the effectiveness of the LNG-

IUS in improving the hematologic indices and inducing symptomatic relief of

menorrhagia and dysmenorrhea in adenomyosis.10

The objective of this study is to investigate on the expressions of VEGF,

ANGPT-1 and ANGPT-2 in adenomyosis tissues, endometrium of no

significant gynecologic diseases, and how the LNG-IUS affects the expression

of these angiogenic factors, to possibly reveal the angiogenic alterations

present in adenomyosis and whether the improvement secondary menorrhagia

induced by the LNG-IUS is associated with the suppression of angiogenic

activity.

13

II. Materials and Methods

1. Patient Selection

Patients enrolled in the study were recruited from a group of patients

receiving hysterectomy or endometrial curettage during the period of March

2005 to February 2008 at the Department of Obstetrics and Gynecology,

Yonsei University College of Medicine. The specimens were examined for

pathological diagnosis by experienced pathologists at Yonsei University

College of Medicine.

20 hysterectomy specimens confirmed as adenomyosis, without other

coexisting gynecological pathologies, 18 adenomyosis specimens with

histories of levonorgestrel intrauterine system (LNG-IUS) (Mirena®,

20mcg/day release) insertion and 12 normal endometrial specimens were

selected for analysis. Only those in whom the LNG-IUS were removed intra-

operatively or immediately before hysterectomy were selected. The reasons

for LNG-IUS removal and a subsequent hysterectomy were irregular spotting,

abdominal cramping and mal-odorous vaginal discharge. For the selection of

normal endometrial specimens, those with normal preoperative sonographic

findings and normal CA-125 levels were chosen. For the 18 patients with

adenomyosis with histories of LNG-IUS insertion, the LNG-IUS were left

indwelled until the day of the surgery. The demographic factors were

14

reviewed for retrospective analysis. Those with concomitant gynecologic

pathologies other than adenomyosis, history of smoking, hormone therapy,

recent intrauterine device insertion other than LNG-IUS, significant medical

illness and body mass index of greater than 25kg/m2 were excluded from the

study. The sections with clearly visible endomyometrial junctions were

selected from the adenomyosis specimens to allow comparison of eutopic and

ectopic endometrial tissue within the same patient. Hysterectomy was

performed either through a laparotomy or laparoscopy and endometrial

curettage was performed for the sampling of the control endometrium.

On all samples, immunohistochemical analysis was performed for VEGF,

ANGPT-1 and ANGPT-2.

2. Immunohistochemical staining

A. Antisera

Polyclonal antibodies of the following properties were used as the primary

antisera: goat anti-VEGF 1:20, goat anti-ANGPT-1 1:20 and goat anti-

ANGPT-2 1:100 (R&D Systems, Minneapolis, MN, USA). As the secondary

antibody, the ChemMateTM, DAKO EnVisionTM Detection Kit (Dako

15

Cytomation, Carpinteria, CA, USA) was used and diaminobenzidine (DAB)

was used as the chromogen.

B. Immunohistochemical procedure

The tissue sections on the microslides were deparaffinized with xylene,

hydrated in serial dilutions of alcohol and immersed in 3% H2O2 to quench the

endogenous peroxidase activity. For antigen retrieval, the sections were then

microwaved for 15 minutes in Tris-EDTA buffer (pH 9.0) supplemented with

distilled water. The sections were incubated for 90 minutes at an ambient

temperature with the primary antibodies (VGEF, ANGPT-1, ANGPT-2).

After rinsing with the washing buffer, further incubation was performed using

the biotin-free polymeric horseradish peroxidase (HRP)-linker antibody

conjugate system (Dako Cytomation, Carpinteria, CA, USA) for 30 minutes at

an ambient temperature. The slides were washed and developed with the

chromogen for 10 minutes. The slides were counter stained with Meyer's

hematoxylin, and then mounted with a synthetic mountant for examination.

16

C. Analysis of immunohistochemical staining

The immunohistochemical sections were examined under a light microscope

at 100 and 200 power fields. To analyze the stained sections in a semi-

quantitative manner, the HSCORE = Pi (i + 1) was evaluated, where i is the

intensity of staining on a scale of 0, 1, 2 or 3, Pi is the percentage of the area

stained for each intensity varying from 0 to 100%, and 1 is a correction for

optical density. The intensity of 0 represents negative staining, 1 represents

minimal antigenicity, 2 represents moderate antigenicity without a describable

pattern of distribution and 3 represents strong antigenicity with a membranous

pattern of distribution. An average of 30 endometrial glands were examined

for the estimation of the percentage of each intensity scale. The examinations

were performed by three independent observers in a blinded fashion, where

two were experienced pathologists and one was a gynecologist. Only the data

from the two pathologists were included in the statistical analysis.

3. Statistical Analysis

The statistical analysis was conducted with the Statistical Package for Social

Science (version 10.0; SPSS, Inc., Chicago, IL). The Student’s t-test was

17

performed to assess the differences in the H-scores of VEGF, ANGPT-1 and

ANGPT-2 among each group. A p-value of less than 0.05 was considered

statistically significant.

18

III. Results

No statistically significant difference was observed for age, BMI and parity

among the adenomyosis, the adenomyosis-LNG-IUS and the control groups.

The mean duration of LNG-IUS insertion was 5.43±2.5 months. No statistical

difference was present for the expression of VEGF, ANGPT-1 and ANGPT-2

according to the menstrual phase (proliferative versus secretory) in

adenomyosis, LNG-IUS inserted adenomyosis tissues and the controls. (Table

1)

Table 1. Demographic factors of the study samples

Adenomyosis Adenomyosis-

LNG-IUS

Control

(n=20) (n=18) (n=12)

Age (years) 42±9.8 40±8.4 39±11.3

BMI (kg/m2) 23.8±2.4 22.9±3.2 22.1±2.2

Parity 3±1.9 3±1.6 2±1.1

Ratio of Proliferative:Secretory

Phase

11:9 8:10 7:5

Duration of LNG-IUS insertion

(months)

- 5.43±2.5 -

19

1. Vascular endothelial growth factor

All 50 tissues sections stained positive for VEGF. VEGF was expressed in a

membranous pattern of distribution, mainly along the endometrial glandular

cell cytoplasm, the endothelial and periendothelial tissues and some discrete

expressions were observed within the stromal tissue. After the insertion of

LNG-IUS, there was an overall decrease in the microscopic density and size

of the endometrial glands, decreasing the overall expression of VEGF as well.

When the VEGF expressions were compared between the eutopic and the

ectopic endometrium in adenomyosis, the ectopic endometrium displayed

significantly elevated levels (319±35.84 vs 280.36±32.37, p<0.05). As for

adenomyosis with LNG-IUS, no significant difference was observed between

the eutopic and ectopic endometrium (151.43±31.19 vs 166.43±27.47,

p>0.05). (Fig 1A, 2A, B)

VEGF expression was significantly lower in the adenomyosis-LNG-IUS

group in both the eutopic (151.43±31.19 vs 280.36±32.37, p<0.05) and

ectopic endometrium (166.43±27.47 vs 319±35.84, p<0.05), compared to the

adenomyosis group. (Fig 1B, 2A, B, C, D)

20

The expression of VEGF was significantly increased in the eutopic

endometrium of adenomyosis (280.36±32.37 vs 150.91±24.68, p<0.05)

whereas no difference was observed for the eutopic endometrium of

adenomyosis-LNG-IUS group when compared with the control endometrium

(151.43±31.19 vs 150.91±24.68, p>0.05). (Fig 1C, 2A, C, E)

21

Fig 1. The analysis of H-scores for vascular endothelial growth factor

expression

0000

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1111 2222

eutopiceutopiceutopiceutopic

ectopicectopicectopicectopic

0000

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adenoadenoadenoadeno

controlcontrolcontrolcontrol

adeo-LNGadeo-LNGadeo-LNGadeo-LNG


P=0.8143

Comparison with control

*

* *

0000

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adeno-LNGadeno-LNGadeno-LNGadeno-LNG

* AAAA BBBB

CCCC *

VEGF H-scores: A. 1. Comparison between eutopic and ectopic endometrium of

adenomyosis. 2. Comparison between eutopic and ectopic endometrium of LNG-

IUS inserted adenomyosis. B. 1. Comparison between eutopic endometrium of

adenomyosis and LNG-IUS inserted adenomyosis. 2. Comparison between

ectopic endometrium of adenomyosis and LNG-IUS inserted adenomyosis. C. 1.

Comparison between eutopic endometium of adenomyosis and control

endometrium. 2. Comparison between eutopic endometium of LNG-IUS inserted

adenomyosis and control endometrium.

22

Fig 2. The immunolocalization of vascular endothelial growth factor

A

B

←←←←

←←←←

23

C

D

↑↑↑↑

↘↘↘↘

←←←←

24

E

Fig 2. Anti-VEGF immunostaining: A. Adenomyosis-eutopic endometrial

tissue; arrow: strong staining within the endometrial glandular cytoplasm. B.

Adenomyosis-ectopic endometrial tissue; arrow: strong staining within the

endometrial glandular cytoplasm. C. LNG-IUS inserted adenomyosis-eutopic

endometrial tissue; arrows: weak staining within the endometrial glandular

cytoplasm. D. LNG-IUS inserted adenomyosis-ectopic endometrial tissue;

arrow: weak staining within the endometrial glandular cytoplasm. E. Control

endometrial tissue; arrow: weak staining within the endometrial glandular

cytoplasm. (A-E: original magnification X100)

←←←←

25

2. Angiopoietin-1

All 50 tissue sections showed positive antigenicity for ANPGT-1. The

expression of ANGPT-1 was strongest in the endometrial glandular cell

cytoplasm, however, ANGPT-1 was widely distributed throughout the stroma,

endothelial and periendothelial cells as well. Such pattern of distribution for

angiopoietin-1 was clearly different from that of VEGF and ANGPT-2 where

antigenicity was fairly limited, to the glandular cytoplasm and endothelial

tissues.

When the ANGPT-1 expressions were compared between the eutopic and the


significantly elevated levels (340±21.93 vs 272.86±48.58, p<0.05). As for

adenomyosis with LNG-IUS, the ectopic endometrium displayed an increased

mean level of ANGPT-1 comprared to the eutopic endometrium

(212.14±11.50 vs 161.43±50.47, p<0.05). (Fig 3A, 4A, B)

ANGPT-1 expression was significantly lower in the adenomyosis-LNG-IUS

group in both the eutopic (161.43±50.47 vs 272.86±48.58, p<0.05) and

ectopic endometrium compared to the adenomyosis group (212.14±11.50 vs

340±21.93, p<0.05). (Fig 3B, 4A, B, C, D)

26

The expression of ANGPT-1 was significantly increased in the eutopic

endometrium of adenomyosis (272.86±48.58 vs 169.09±25.48, p<0.05)


adenomyosis-LNG-IUS group when compared to the control endometrium

(161.4±350.47 vs 169.09±25.48, p>0.05). (Fig 3C, 4A, C, E)

27

Fig 3. The analysis of H-scores for angiopoietin-1 expression

0000

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0000

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0000

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P=0.3355

*

* *

BBBB

* *

ANGPT-1 H-scores: A. 1. Comparison between eutopic and ectopic endometrium

of adenomyosis. 2. Comparison between eutopic and ectopic endometrium of

LNG-IUS inserted adenomyosis. B. 1. Comparison between eutopic endometrium

of adenomyosis and LNG-IUS inserted adenomyosis. 2. Comparison between

ectopic endometrium of adenomyosis and LNG-IUS inserted adenomyosis. C. 1.




*p<0.05

AAAA

CCCC

28

Fig 4. The immunolocalization of angiopoietin-1

A

B

←←←← aaaa

←←←← bbbb

↑↑↑↑

←←←← aaaa

bbbb

←←←← c c c c

←←←← a a a a

29

C

D

↑↑↑↑

aaaa

bbbb

a a a a

↙↙↙↙ ↘↘↘↘

←←←← b b b b

30

E

Fig 4. Anti-ANGPT-1 staining: A. Adenomyosis-eutopic endometrial tissue;

a: strong staining within the endometrial glandular cytoplasm. b: strong

staining within the vascular endothelial cells. B. Adenomyosis-ectopic

endometrial tissue; a: strong staining within the endometrial glandular

cytoplasm. b: diffuse staining within the stromal tissue c: strong staining

within vascular endothelial cells. C. LNG-IUS inserted adenomyosis-eutopic

endometrial tissue; a: weak staining within the endometrial glandular

cytoplasm. b: diffuse staining within the stromal tissue. D. LNG-IUS inserted

adenomyosis-ectopic endometrial tissue; a: weak staining within the

endometrial glandular cytoplasm. b: weak to moderate staining within

vascular endothelial cells. E. Control endometrial tissue; a: faint staining

within the endometrial glandular cytoplasm b: weak to moderate staining

within vascular endothelial cells. (A-E: original magnification X100)

a a a a →→→→

←←←← b b b b

31

3. Angiopoietin-2

All 50 tissue sections stained positive for ANGPT-2. The distribution of

ANGPT-2 was similar to that of VEGF, where the strongest antigenicity was

observed in a membranous pattern of distribution along the glandular

epithelial cytoplasm, periendothelial and endothelial cells, whereas discrete,

focal staining was observed within the stroma.

When the ANGPT-2 expressions were compared between the eutopic and the


significantly elevated levels (311.43±53.44 vs 280.74±42.10, p<0.05). As for

adenomyosis with LNG-IUS, no significant difference was observed between

the eutopic and ectopic endometrium (164.29±36.22 vs 190.71±16.18,

p>0.05). (Fig 5A, 6A, B)

ANGPT-2 expression was significantly lower in the adenomyosis-LNG-IUS

group in both the eutopic (164.29 ±36.22 vs 280.74±42.10, p<0.05), and

ectopic endometrium (190.71±16.18 vs 311.43±53.44, p<0.05) when

compared with the adenomyosis group. (Fig 5B, 6A, B, C, D)

The expression of ANGPT-2 was significantly increased in the eutopic

endometrium of adenomyosis (280.74±42.1 vs 149.09±27.37, p<0.05),

32


adenomyosis-LNG-IUS group when compared with the control endometrium

(164.29±36.22 149.09±27.37, p>0.05). (Fig 5C, 6A, C, E)

33

Fig 5. The analysis of H-scores for angiopoietin-2 expression

0000

50505050

100100100100

150150150150

200200200200

250250250250

300300300300

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1111 2222



0000

50505050

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1111 2222



0000

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*

* *

*

ANGPT-2 H-score: A. 1. Comparison between eutopic and ectopic endometrium

of adenomyosis. 2. Comparison between eutopic and ectopic endometrium of LNG-

IUS inserted adenomyosis B. 1. Comparison between eutopic endometrium of

adenomyosis and LNG-IUS inserted adenomyosis. 2. Comparison between ectopic

endometrium of adenomyosis and LNG-IUS inserted adenomyosis. C. 1.




*p<0.05

AAAA BBBB

CCCC

34

Fig 6. The immunolocalization of angiopoietin-2

A

BBBB

←←←← a a a a

bbbb

↓↓↓↓

←←←← →→→→

35

C

D

←←←←

→→→→

36

E

Fig 6. Anti-ANGPT-2 staining: A. Adenomyosis-eutopic endometrial tissue;

a: strong staining within the endometrial glandular cytoplasm. b: moderate

staining within the vascular endothelial cells. B. Adenomyosis-ectopic

endometrial tissue; arrows: strong staining within the endometrial glandular

cytoplasm. C. LNG-IUS inserted adenomyosis-eutopic endometrial tissue;

arrow: weak staining within the endometrial glandular cytoplasm. D. LNG-

IUS inserted adenomyosis-ectopic endometrial tissue; arrow: weak staining

within the endometrial glandular cytoplasm. E. Control endometrial tissue;

arrow: weak staining within the endometrial glandular cytoplasm. (A-E:

original magnification X100)

←←←←

37

IV. Discussion

The effort to explore the underlying pathophysiology of adenomyosis and

therefore find the possible ways to overcome complications caused by this

common illness in women of their reproductive years, has been a major

challenge for gynecologists. Adenomyosis is a benign disease, characterized

by a peculiar trait, which is the infiltration of endometrium into adjacent and

even extrapelvic tissues11. Such characteristics somewhat resemble that of

malignant tumors, and in light of such quality, aberrations in normal

angiogenic regulation is a possible mechanism in the evolution and

progression of the disease. Yet, little has been excavated on the role of

angiogenic factors on menstrual cycle regulation, not to mention on the

abnormal proliferation of ectopic endometrium in endometriosis. The level of

angiogenic factors are thought to be relatively constant throughout the

menstrual cycle12. Among these angiogenic factors, the expression of VEGF

in normal and abnormal endometrium has been explored most extensively.

VEGF is expressed in the human endometrium in all phases of the menstrual

cycle and elevated during menstrual flow, presumably as a result of hypoxia13.

A number of articles have shown that VEGF is produced in an estrogen

dependent manner14. Midluteal estrogen peak is known to have impact on the

increase in VEGF levels, but the independent action of progesterone has been

38

studied to a lesser extent. Controversy exists as to whether VEGF levels are

increased in the eutopic and ectopic endometrial tissues in endometriosis

where scientists have described that the growth factor exist in higher levels

only in the ectopic endometrium and that VEGF is increased even in the

eutopic endometrium of women with endometriosis15,16.

The angiopoietins are younger members of the family of angiogenic factors

and few articles elaborate on their contributions to normal menstrual cycles

and in endometriosis. Through mRNA analysis, ovarian endometriomas were

claimed to have increased expressions of ANGPT-1 and ANGPT-217. This

current article is the first to analyze the histologic pattern of distribution for

ANGPT-1, ANGPT-2 and VEGF in adenomyosis tissues.

Through immunohistochemical staining, our results demonstrate that

ANGPT-1, ANGPT-2 and VEGF expression is enhanced within tissue

sections for ectopic endometrial tissues in adenomyosis and significantly

suppressed with LNG-IUS insertion. When the normal endometrium, eutopic

and ectopic endometrium in adenomyosis are compared, the expressions of

VEGF, ANGPT-1 and ANGPT-2 are strongest in the infiltrated ectopic

endometium, followed by the eutopic endometrium in adenomyosis and

control endometrium. When the LNG-IUS is inserted in adenomyosis, the

levels of these three factors are all suppressed within the eutopic and ectopic

39

endometrium. When the control endometrium and eutopic endometrium of

adenomyosis with and without LNG-IUS insertion are compared, significant

increases in VEGF, ANGPT-1 and ANGPT-2 are observed for the eutopic

endometrium in adenomyosis whereas a difference is not evident between the

eutopic endometrium of LNG-IUS inserted adenomyosis and the control

endometrium. Such findings suggest that in adenomyosis, an increase in

angiogenic activity may be an essential feature in the pathophysiology of the

disease and that the LNG-IUS has a suppressive effect.

Within the tissue sections, ANGPT-1 show a diffuse pattern of distribution in

almost all cell types of the endometrial and myometrial layers, although it is

most prominently stained, in a membranous pattern of distribution, in the

endometrial glandular and the vascular endothelial cells. On the other hand,

the VEGF and ANGPT-2 have a more localized pattern of distribution,

showing strong antigenicity mainly in the cytoplasm of the endometrial

glandular epithelium, in a membranous pattern of distribution. The VEGF and

ANGPT-2 were assumed to be co-expressed in a non-experimental article1,

and interestingly, positively staining areas of these two angiogenic factors

coincided with each other according to our immunohistochemistry results as

well. However, the pattern and extent of distribution of ANGPT-1 do not

coincide with that of ANGPT-2 and VEGF.

40

Previously, it has been postulated that VEGF expression is increased by

progesterone stimulation with increased capillary endothelial proliferation as

well as enlargement and dilatation of superficial vessels18,19. On the other

hand, other reports have shown that the increase in VEGF, resulting in leaky

dysfunctional blood vessels is evident only in those with clinically evident

bleeding symptoms and also that a decrease in VEGF level is observed even

in those with irregular bleeding after LNG-IUS insertion20,21. Such

inconsistent findings imply that the expression of VEGF may vary according

to the duration of LNG-IUS insertion. Thus, it would be worthy to stratify the

patients according to the duration of LNG-IUS insertion, but the number of

patients should be increased for such analysis. According to our study, with an

overall decrease in the density and size of endometrial glands, VEGF

antigenicity, within the eutopic and ectopic endometrial glandular cytoplasm,

also seems to be decreased with LNG-IUS insertion. Regarding the

angiopoietins, this study is also the first to elaborate on their alterations with

LNG-IUS insertion in adenomyosis. As with VEGF, the ANGPT-1 and

ANGPT-2 expressions are also decreased along the endometrial glandular

cells and in the vascular endothelial cells. Such in vivo observations on the

expressions of VEGF, ANGPT-1 and ANPGT-2 imply that an alteration of

vascular remodeling may be a component involved in the evolution of

adenomyosis, providing the milieu for the growth and survival of ectopic

41

implants within the myometrium. The LNG-IUS is known to significantly

decrease menstrual blood flow within 3 months and improve iron deficiency

anemia during the first year of insertion for those with dysfunctional uterine

bleeding and menorrhagia secondary to adenomyosis22,23. Although the

clinical effects of LNG-IUS have been reported extensively, little has been

described on the underlying histologic or molecular changes associated with

the decrease in blood flow. Our observations suggest that the LNG-IUS has a

suppressive effect on the angiogenic activity in adenomyosis.

This current study verifies the distribution of VEGF, ANGPT-1 and ANGPT-

2 in a semi-quantitative manner within tissue sections. Although quantitative

protein analysis would be a more objective means to quantify the

overexpression of angiogenic factors in adenomyosis, the extraction of

endometrial tissue from myometrium is tedious, unlike the ectopic

endometrium from ovarian endometriomas or peritoneal implants, where a

clean extirpation would yield in almost pure ectopic endometrial tissue. Since

adenomyosis is characterized by islands of endometrial implants within the

myometrium, the composition of the cell types is quite heterogeneous and a

precise comparison of the protein expression between the ectopic and eutopic

endometrium is difficult if not impossible. Microdissection of ectopic

endometrial implants is one way to obtain ectopic endometrial tissues within

42

the myometrium or cell flowcytometry could be utilized to discard the

myometrial cells, but may be quite unreliable in terms of quality control, and

the yield would be low. Therefore, in order to analyze the expression of

VEGF, ANGPT-1 and ANGPT-2 in adenomyosis, immunohistochemical

staining followed by a semiquantitative assay and analysis was chosen as the

reasonable method.

V. Conclusion

The expression of angiogenic factors VEGF, ANGPT-1 and ANGPT-2 are

enhanced in the ectopic endometrium compared with both the eutopic

endometrium of adenomyosis and the control endometrium. With the insertion

of LNG-IUS in adenomyosis, these factors are significantly suppressed in

both the eutopic and ectopic endometrial layers. Therefore, the alteration of

angiogenesis in adenomyosis is a possible underlying pathophysiologic

mechanism of adenomyosis and provides the evidence to support the

effectiveness of LNG-IUS as a treatment modality of menorrhagia in

adenomyosis patients.

43

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effects of angiopoietin-1 on endothelial cells. Microvasc Res 2002;

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13. Graubert MD, Ortega MA, Kessel B, Mortola JF, Iruela-Arispe ML.

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14. Herve MA, Meduri G, Petit FG, Domet TS, Lazennec G, Mourah S,

et al. Regulation of the vascular endothelial growth factor receptor

Flk-1/KDR by estradiol through VEGF in uterus. J Endocrinol 2006;

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18. Goodger AM, Rogers PA, Affandi B. Endometrial endothelial cell

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47

자궁선근증과 Levonorgestrel intrauterine system 삽입

자궁선근증 조직에서의 VEGF, angiopoietin-1 그리고

angiopoietin-2 발현분석

<지도교수 이병석>

연세대학교 대학원 의학과

박주현

자궁선근증은 가임기 여성에 있어 월경통, 월경곤란증 그리고

빈혈을 유발하는 주요 질환이나, 그 원인과 병태생리에

대해서는 밝혀진 것이 많지 않다. 자궁선근증은 자궁내막이

인접 자궁근층으로 침윤하는 소견을 특징으로 한다. 이를

위해서는 자궁내막의 혈관신생능력의 변이가 일어난다는

가설이 성립할 수 있는데 이에 대표적인 혈관신생인자들 즉

vascular endothelial growth factor (VEFG), angiopoietin-1

(ANGPT-1) 그리고 angiopoietin-2 (ANGPT-2) 등의 변이가

존재하지 여부가 매우 흥미롭다 할 수 있다. 따라서 본

연구의 목적은 자궁선근증의 내막과 근층내의 내막, LNG-

IUD를 삽입한 자궁선근증 조직에서의 내막과 근층내의 내막

그리고 대조군 내막간의 VEGF, ANGPT-1 그리고 ANGPT-2의

발현에 차이가 있는지를 분석하는 것이다.

자궁선근증 20예, LNG-IUS를 삽입한 자궁선근증 18예 그리고

자궁내막 생검상 이상소견이 없는 대조군 내막 12예에 대하여

48

면역조직화학염색법으로 VEGF, ANGPT-1 그리고 ANGPT-2의

발현을 분석하였다.

대조군과 비교했을 때 자궁선근증에서 근층내 그리고 내막

모두에서 VEGF, ANGPT-1 그리고 ANGPT-2 발현이 모두

증가하였고 자궁선근증 내에서 비교했을 때에도 근층내의

내막에서 위의 인자들의 발현이 모두 증가하는 것으로

나타났다. LNG-IUS를 삽입한 자궁선근증의 내막조직에서는

대조군과 비교하여 VEGF, ANGPT-1 그리고 ANGPT-2 모두

유의미한 차이를 보이지 않았고 자궁선근증과 LNG-IUS 삽입

자궁선근증의 내막과 근층내 내막간을 비교했을 때에는 LNG-

IUS 삽입군에서 세 인자 모두 유의미하게 억제되었다.

따라서 자궁선근증에 있어서 혈관생성인자인 VEGF, ANGPT-1

그리고 ANGPT-2의 발현이 증가해 있으며 이 현상은 LNG-IUS의

삽입에 의해 유의미하게 감소하는 것으로 나타났다.

---------------------------------------------------------

핵심되는 말: 자궁선근증, VEGF, angiopoietin-1,

angiopoietin-2

Documents

The expression of vascular endothelial growth …...2 The expression of vascular endothelial growth factor, angiopoietin-1 and angiopoietin-2 in adenomyosis and adenomyosis under levonorgestrel