의의의학학학 석석석사사사학학학위위위 논논논문문문

TTThhheeeVVVaaassscccuuulllaaarrrCCChhhaaarrraaacccttteeerrriiissstttiiicccsssooofffMMMeeelllaaasssmmmaaa

아아아 주주주 대대대 학학학 교교교 대대대 학학학 원원원

의의의 학학학 과과과

김김김 은은은 형형형

TTThhheeeVVVaaassscccuuulllaaarrrCCChhhaaarrraaacccttteeerrriiissstttiiicccsssooofffMMMeeelllaaasssmmmaaa

by

En Hyung Kim

A Dissertation Submitted to The Graduate School of Ajou University

in Partial Fulfillment of the Requirements for the Degree of

MASTER OF MEDICAL SCIENCES

Supervised by

Hee Young Kang, M.D., Ph.D.

Department of Medical Sciences

The Graduate School, Ajou University

February, 2007

김김김은은은형형형의의의 의의의학학학 석석석사사사학학학위위위 논논논문문문을을을 인인인준준준함함함...

심심심사사사위위위원원원장장장 강강강 희희희 영영영 인인인

심심심사사사위위위원원원 이이이 은은은 소소소 인인인

심심심사사사위위위원원원 김김김 유유유 찬찬찬 인인인

아아아 주주주 대대대 학학학 교교교 대대대 학학학 원원원

222000000666년년년 111222월월월 222222일일일

Acknowledgement

이 논문을 완성할 때까지 지도와 격려를 아끼지 않으셨던 지도교수이신

강희영 선생님께 먼저 진심으로 감사 드립니다. 또한 많은 조언을 베풀어

주신 이은소 선생님과 김유찬 선생님께도 감사의 마음을 전해 드립니다.

그리고 연구가 진행되는 동안 도움을 주신 피부과학 교실원 여러분과

실험이 진행되는 동안 조직 염색과 분석을 도와주신 김영배 선생님께 감사

드립니다.

언제나 힘이 되어주시고 사랑과 위로를 베풀어주셨던 양가 부모님들과

동생에게 감사의 마음을 전하고, 항상 옆에서 따뜻한 격려를 아끼지 않은

남편에게 마음속 깊은 사랑을 전합니다. 또한 지금까지 저의 모든 것을

가능케 하신 하나님께 영광을 드립니다.

i

- ABSTRACT -

The vascular characteristics of melasma

Background: Melasma is developed or aggravated by UV radiation, sex

hormone, and genetic predisposing factors, however the exact mechanism of

pathogenesis is not yet fully understood. Previous studies indicated that dermal

environment such as fibroblasts may have an important role on the development

of melasma. Also, it was suggested that interactions between the altered

cutaneous vasculature and melanocytes may have an influence on the

development of pigmentation.

Purpose: The purpose of this study was to investigate the vascular characteristics

of melasma and expression of VEGF, a major angiogenic factor, and its receptor

in lesional melasma skin.

Material and Methods: Erythema intensity was quantified by the increase of

the a* parameter using a colorimeter. 2 mm punch biopsy skin samples were

obtained from lesional and non-lesional facial skin of 50 Korean women with

melasma. The immunohistochemistry was taken to determine the expression of

factor VIIIa and VEGF in melasma.

Results: The values of a* was significantly higher in the melasma lesion than

that of perilesional normal skin. Computer-assisted image analyses of factor

ii

VIIIa-stained sections revealed a significant increase of both the number and the

size of dermal blood vessels in the lesional melasma skin. In lesional melasma

skin, the expression of VEGF was significantly increased and melanocytes were

positively stained with VEGFR-2.

Conclusion: These data suggest that increased vascularity is one of the major

findings in melasma and VEGF may be a major angiogenic factor for altered

vessels in melasma. The relationship between vessel proliferation and

melanogenesis needs further research.

Key Words: melasma, angiogenesis, vascular endothelial growth factor

iii

TABLE OF CONTENTS

ABSTRACT i

TABLE OF CONTENTS iii

LIST OF FIGURES v

LIST OF TABLES vi

I. INTRODUCTION 1

II. MATERIALS AND METHODS 4

A. Subjects 4

B. Methods 4

1. Biopsies 4

2. Human melanocyte culture 4

3. Colorimetric measurement 5

4. Immunohistochemistry 6

5. Immunocytochemistry 7

6. Image analysis 8

7. Statistical analysis 9

III. RESULTS 10

A. Erythema intensity in melasma patients 10

B. Immunohistological evaluation of vascularity in melasma patients 13

C. Immunohistological evaluation of VEGF in melasma patients 16

D. Immunohistological evaluation of VEGFR-2 in melasma patients 17

iv

IV. DISCUSSION 20

V. CONCLUSION 25

REFERENCES 26

v

LIST OF FIGURES

Fig. 1. Pronounced telangiectatic erythema noticed on melasma lesion 6

Fig. 2A. The L value in melasma and perilesional normal skin 11

Fig. 2B. The b* value in melasma and perilesional normal skin 12

Fig. 2C. The a* value in melassma and perilesional normal skin 12

Fig. 3. Immunohistochemical staining for factor VIIIa 14

Fig. 4. Computer assisted morphometric analysis of factor VIIIa stained

sections 15

Fig. 5. Immunohistochemical staining for VEGF 17

Fig. 6. Cultured human melanocytes express VEGFR-2 18

Fig. 7. Immunohistochemical staining for VEGFR-2 19

vi

LIST OF TABLES

Table 1. Antibodies and their working dilutions 7

Table 2. Quantitative analysis of factor VIIIa staining 13

- 1 -

I. INTRODUCTION

Melasma is a common acquired symmetrical hypermelanosis on sun-

exposed areas of the skin. It is very common in women and in oriental persons.

The major etiological factors include genetic influences, exposure to ultraviolet

(UV) radiation, and sex hormones (Urabe et al., 1997). However, the

pathogenesis of melasma is not yet fully understood.

Previous report have demonstrated that lesional melasma skin show

more prominent solar elastosis compared with normal skin, suggesting that the

dermal change may influence the development of melasma (Kang et al., 2002).

Recently, it was suggested that the inflammation in the dermis from the

accumulated UV irradiation may be associated with activation of fibroblasts,

which result in the up-regulation of stem cell factor in melasma dermal skin

leading to increased melanogenesis (Kang et al., 2006). These researches have

given us a new insight on the pathogenesis of melasma that dermal environment

imay have an important role on the development of melasma.

Several studies have suggested that a connection between vessels and

cutaneous pigmentation could exist. Postinflammatory hyperpigmentation (PIH)

is more frequently noticed in ABNOM patients after laser treatment. In ABNOM

histopathologic finding often show pigment-bearing cells distributed in the

- 2 -

perivascular area. So the PIH may be due to proinflammatory cytokines released

from these vessels, which are capable of altering the pigmentary and the

inflammatory responses simultaneously (Lee et al., 2004). Also, it has been

reported that the topical plasmin inhibitor, tranexamic acid, is an effective

treatment for UV-induced hyperpigmentation (Maeda et al., 1998). And the

localized microinjection of tranexamic acid, improved melasma in vivo (Lee et

al., 2006). Tranexamic acid inhibits UV-induced plasmin activity in

keratinocytes by preventing the binding of plasminogen to the keratinocytes,

which ultimately results in less free arachidonic acid (AA) and a diminished

ability to produce prostaglandins (PG). Reduced production of PGs may lead to

decreased melanocyte tyrosinase activity (Maeda et al., 1998). Human

melanocytes may respond to angiogenic factors because normal human

melanocytes express functional vascular endothelial growth factor (VEGF)

receptors (Kim et al., 2005). These in vitro and in vivo findings suggest that

interactions between the altered cutaneous vasculature and melanocytes may

have an influence on the development of hyperpigmentation in the overlying

epidermis.

Clinically, melasma is characterized by hyperpigmented patches, but

additional distinguishing features like pronounced telangiectatic erythema

confined to the melasma lesions have been observed. So the vascular

characteristics of melasma lesions were investigated and then the expression of

- 3 -

VEGF, a major angiogenic factor of the skin, was examined in altered

vasculature of melasma. UV irradiation is known to upregulate VEGF

production in keratinocyte-derived cell lines, both directly through transcription

factor activation and indirectly through cytokine release (Mildner et al., 1999;

Kosmadaki et al., 2003). Many keratinocyte-derived signaling molecules and

cytokines, for which melanocytes express receptors, affect melanocyte survival,

migration, and melanogenesis (Gilchrest et al., 1998). Because of the physical

proximity of melanocyte to keratinocyte, and because of the embryologic origin

of melanocyte, we asked whether melanocyte may be a potential target for

keratinocyte derived VEGF.

- 4 -

II. MATERIALS AND METHODS

A. Subjects

We examined 50 Korean patients with newly diagnosed melasma who

attended the Department of Dermatology, Ajou University Hospital (Suwon,

Korea), between January 2000 and December 2005. In each patient, the

diagnosis of melasma was confirmed by medical history and physical

examination as well as characteristic histologic findings.

B. Methods

1. Biopsies

Two millimeter punch biopsies from lesional and perilesional normal

appearing (usually within 1 cm from lesion) skin was done under local

anesthesia (1% lidocaine). Tissues were prepared for light microscopic study by

10% formalin fixation.

2. Human melanocyte culture

Normal human melanocytes were obtained from adult foreskin. After

separating single cells by vortex, they were plated in 100 mm culture dishes, and

maintained in a F-12 medium, supplemented with 10% fetal bovine serum

(FBS), 1% penicillin/streptomycin, 6.5 ug/ml basic fibroblast growth factor (b-

- 5 -

FGF), 50 mg/ml tetradecanoyl phorbol acetate (TPA), 24 ug/ml 3-isobutyl-1-

methylxanthine (IBMX), and 100 ng/ml cholera toxin. The culture medium was

changed every three days. The cells were cultured in an incubator at 37°C with

0.5% CO2.

3. Colorimetric measurement

The lesional melasma and perilesional normal skin (usually within 1 cm away

from the lesional border) of 30 melasma patients were evaluated. The subjects

had a wash-out period for at least 2 weeks for bleaching products, UV light

therapy and topical retinoids and at least 3 months for lasers, dermabrasion, and

chemical peeling. The intensity of erythema was measured by skin reflectance

with a tristimulus color analyzer (Chromameter CR300, Minolta, Japan) and

expressed in the L*a*b* system. This system allows a color to be quantified

according to 3 axes: white-black or lightness (L*), red-green or chrome (a*) and

yellow-blue or hue (b*). The a* parameter was taken as the measure of redness.

4. Immunohistochemistry

Four micrometer paraffin-embeded sections of both lesional and control

skin were mounted on Polysine microscope slide (Menzel-Glaser, Germany)

coated with 0.1% poly p-lysine. Tissues were deparaffinized and rehydrated by

sequential immersion in xylene, graded concentrations of ethanol, and distilled

- 6 -

water. They were incubated for 30 min at room temperature in a solution of

0.5% hydrogen peroxidase in methanol to quench endogenous peroxidase

activity, followed by washing three times in Tris-buffered saline (TBS, 0.1

mol/L, pH 7.4, Dako, Carpinteria, CA). They were subsequently incubated in

0.05% trypsin in TBS for 18 min at 48 °C (factor VIIIa) or boiled in 10 mM

citrate buffer, pH 6.0 for 15 minutes followed by cooling at room temperature

for 30 minutes (VEGF, VEGFR-2). After washing three times in TBS, they were

flooded with a protein-blocking agent (PBA; Immunon, Pittsburgh, PA) for 10

min at room temperature. Excess PBA was drained and the primary antibodies

were applied to the tissue sections. These antibodies include factor VIIIa, VEGF,

VEGFR-2 (Table 1). The slides were then incubated for 1 h at room temperature

and for 30 min at 37 °C in a humid chamber. Following three washes in TBS,

sections were incubated for 30 min at room temperature while being flooded

with a biotinylated universal secondary antibody reagent (Santa Cruz, Santa

Cruz, CA). The slides were then washed in TBS, followed by incubation in

streptavidin peroxidase reagent (Immunon) for 30 min. After washes in TBS,

sections were incubated in 3-amino-9-ethylcarbazole (AEC) (Biomeda Corp.,

Foster City, CA) for 10 min. The sections were counterstained with

haematoxylin modified solution (Merck, Darmstadt, Germany) and mounted in

an aqueous mounting medium (Biomeda, Foster City, CA).

- 7 -

5. Immunocytochemistry

Melanocytes grown on Lab-Tek® chambers were washed three times in

Tris-buffered saline (TBS, 0.1 mol/L, pH 7.4, Dako, Carpinteria, CA). After

washing, primary antibodies were applied to the tissue sections. These

antibodies include VEGF, VEGFR-2 (Table 1). The slides were then incubated

for 1 h at room temperature and for 30 min at 37 °C in a humid chamber.

Following three washes in TBS, sections were incubated for 30 min at room

temperature while being flooded with a biotinylated universal secondary

antibody reagent (Santa Cruz, Santa Cruz, CA). The slides were then washed in

TBS, followed by incubation in streptavidin peroxidase reagent (Immunon) for

30 min. After washes in TBS, sections were incubated in AEC (Biomeda Corp.,

Foster City, CA) for 20 min. The sections were counterstained with

haematoxylin modified solution (Merck, Darmstadt, Germany) and mounted in

an aqueous mounting medium (Biomeda, Foster City, CA).

Table 1. Antibodies and their working dilutions

Specificity/ Antibody Mono / Poly Source Working

Dilution

Factor VIIIa Rabbit polyclonal clone Immunon, Pittsburgh, PA 1:100

VEGF Rabbit polyclonal clone Santa Cruz, Santa Cruz, CA 1:50

VEGFR-2 Rabbit polyclonal clone Santa Cruz, Santa Cruz, CA 1:50

- 8 -

6. Image analysis

A CCD camera (CCD-IRIS, Sony, Tokyo, Japan) mounted on a

microscope (Olympus BX50F, Olympus Optical Co., Tokyo, Japan) was

connected to an IBM personal computer. The image signals taken by the

personal computer were evaluated using Image Pro Plus Version 4.5 (Media

Cybertics Co., Silver Spring, MD, U.S.A.). The image analysis was performed

on a representative area of each specimen. In factor VIIIa staining, the number

of vessels mm-2, the average vessel size and the relative area occupied by blood

vessels were measured in the dermis of each slide section, in an area within

100 µm distance from the epidermal–dermal junction (Yano et al., 2004). Each

measurement was evaluated under constant magnification (x100). In VEGF and

VEGFR-2 staining, VEGF and VEGFR-2 expression was evaluated under

constant magnification (x200). The stained area per epidermal area (SA EA-1)

was measured in lesional and perilesional skin. All morphometric procedures

were performed by manually tracing the borders of the epidermis and rete ridges,

and epidermal areas that contained hair follicles were excluded from this tracing.

For each frame, the tracing was repeated three times and the mean was used for

evaluation; all morphometric measurements were performed by the same person.

- 9 -

7. Statistical analysis

Data were expressed as mean ± standard deviation. A possibility value

of less than 0.05 was considered as statistically significant. SPSS 11.0 statistics

program was used for analysis.

- 10 -

III. RESULTS

A. Erythema intensity in melasma patients

On physical examination, melasma patients exhibited pronounced

telangiectatic erythema within lesions of melasma (Fig. 1). The values of L was

lower in melasma lesion (mean value of Rt. and Lt. cheek area, 59.26±2.93 and

59.10±2.46, mean ± SD) than that of perilesional normal skin (mean value of Rt.

and Lt. cheek area, 63.38±2.49 and 62.65±2.89, p<0.01) suggesting that

melasma lesions are darker than normal perilesional skin (Fig. 2A).

There was no difference in the values of b* between melasma lesion

(mean value of Rt. and Lt. cheek area, 18.93±1.83 and 19.09±1.94, mean ± SD)

and perilesional normal skin (mean value of Rt. and Lt. cheek area, 19.88±1.98

and 19.76±2.02) (Fig. 2B).

The values of a* was higher in the melasma lesion (mean value of Rt.

and Lt. cheek area, 12.92±1.96 and 12.78±2.19, mean ± SD) than that of

perilesional normal skin (mean value of Rt. and Lt. cheek area, 10.24±1.35 and

10.53±1.73, p<0.01), suggesting that erythema, in other words, vascularity is

increased in melasma lesions (Fig. 2C).

- 11 -

다른 환자

Fig. 1. Pronounced telangiectatic erythema noticed on melasma lesion.

Fig. 2A. The L* value in melasma and perilesional normal skin.

- 12 -

Fig. 2B. The b* value in melasma and perilesional normal skin.

Fig. 2C. The a* value in melasma and perilesional normal skin.

- 13 -

B. Immunohistological evaluation of vascularity in melasma patients

To investigate objectively whether vascularity is increased in melasma

lesions, we performed immunohistochemistry for factor VIIIa (n=50). We found

increased numbers of enlarged blood vessels in the melasma lesion (Fig. 3B), as

compared with perilesional normal skin (Fig. 3A). Computer-assisted image

analyses of factor VIIIa-stained sections revealed a significant, 16.28% increase

(P <0·05) in vessel size (Fig. 4A), 33.89% increase (P < 0·01) in vessel density

(Fig. 4B), resulting in a 68.75% increase (P <0.01) in the cutaneous area

covered by blood vessels (Fig. 4C) in melasma skin.

Table 2. Quantitative analysis of factor VIIIa staining.

Vessel density (mm-2) Vessel size (um-2) Vessel area (mm-2)

normal lesion normal lesion normal lesion

Mean±SD 76.75 ± 38.24 102.77 ± 56.35 63.85 ± 18.19 74.23 ± 26.65 1.54 ± 1.09 2.60 ± 1.90

p-value 0.008 0.025 0.001

- 14 -

Fig. 3. Immunohistochemical staining for factor VIIIa revealed enlarged and

elongated blood vessels in the upper dermis of melasma lesion (B), as compared

with perilesional normal skin (A). (original magnification x200)

- 15 -

(A) (B)

(C)

Fig. 4. Computer assisted morphometric analysis of factor VIIIa stained sections

revealed a significant increase in vessel size (A), vessel density (B) and the

relative area covered by blood vessels (C), in melasma lesion as compared with

- 16 -

perilesional normal skin.

C. Immunohistological evaluation of VEGF in melasma patients

To examine whether the increased angiogenesis in melasma lesions is

associated with accentuated expression of VEGF, we examined the expression

of VEGF protein using immunohistochemistry.

In melasma lesions staining with antibodies against VEGF revealed

distinctly positive immunoreactivity in keratinocytes, whereas in perilesional

normal skin only weakly positive immunoreactivity was noticed (Fig. 5). The

mean ± SD SA EA-1 of perilesional normal skin samples were 0.137 ± 0.132,

and that of lesional melasma skin samples were 0.270 ± 0.237. This difference

was statistically significant (P = 0.034). In addition, dermal blood vessels, and

fibroblasts were also faintly stained. However, the staining pattern of was

similar between perilesional normal skin and melasma lesional skin in papillary

and reticular dermis.

- 17 -

Fig. 5. Immunohistochemical staining for VEGF revealed more immuno-

positive and uniformly stained epidermis in melasma lesions (B) compared to

perilesional normal skin (A). (original magnification x200)

D. Immunohistological evaluation of VEGFR-2 in melasma patients

VEGF is known to bind to several identified receptors, including the

transmembrane tyrosinase kinase receptors VEGF receptor-1 (VEGFR-1, Flt-1),

VEGF receptor-2 (VEGFR-2, flk-1/KDR), and the cell-surface non-tyrosine

kinase receptor neuropilin-1 (NP-1). Among these VEGFR-2 is known to be

induced by UV. We first investigated whether cultured human melanocytes

expressed VEGFR-2. Cultured human melanocytes were positively stained for

the receptor VEGFR-2 (Fig. 6).

We then examined the lesional melasma and perilesional normal skin to

see whether UV increased the expression of VEGFR-2 in melasma lesions.

VEGFR-2 staining of lesional melasma and perilesional normal skin showed

- 18 -

positivity for keratinocytes, melanocytes and endothelial cells (Fig. 7). The

mean ± SD SA EA-1 of perilesional normal skin samples were 0.028 ± 0.029,

and that of lesional melasma skin samples were 0.040 ± 0.047. Although the

density was increased in melasma lesional skin there was no statistical

difference (p=0.31).

Fig. 6. Immunohistochemical staining of cultured human melanocytes express

VEGFR-2. (original magnification x 400)

- 19 -

Fig. 7. Immunohistochemical staining for VEGFR-2 revealed positively stained

keratinocytes and melanocytes in melasma lesion (B) and perilesional normal

skin (A). (x200)

- 20 -

IV. DISCUSSION

Melasma is defined as a light to dark brown or slate, irregular

hypermelanosis of the face. It develops slowly and is usually symmetric.

Although it may occur in men, it is far more common in women, particularly

those of Hispanic origin (Sanchez et al., 1981) and Asians. Dark skinned races

that live in India, Pakistan, and the Middle East tend to develop this problem in

the first decade of life (Pathak et al., 1986), but onset in patients of most other

races is at puberty or later. The condition may last for many years, with relapses

during the summer and relative remissions during the winter.

Clinically, melasma is characterized by hyperpigmented patches, but we

have observed that melasma patients have additional distinguishing features like

pronounced telangiectatic erythema confined to the melasma lesional skin.

Therefore, in the present study, we investigated the vascular characteristics of

melasma lesions. We have demonstrated that a significant increase of both the

number and size of dermal blood vessels in the lesional skin is one of the major

findings in melasma. Interestingly, the increase in the number of vessels was

more prominent than the increase in vessel size in lesional melasma skin. This

finding suggests that the erythema noticed in melasma patients was more likely

due to angiogenesis than telangiectasia.

The significance of these increased vascularity in melasma, and whether

- 21 -

they are a cause of epidermal hyperpigmentation, is unclear. It is well known

that UV irradiation induces angiogenesis so the vascular alteration in melasma

might just simply be a result of chronic UV accumulation accompanying

epidermal hyperpigmentation. However, both lesional and perilesional facial

skin had been exposed to chronic UV irradiation, but only lesional melasma skin

showed pronounced vascular changes. So, it is possible to speculate that the

increase in vascularity was not only an epiphenomenon of UV damage but that it

may play an important role in the pathogenesis of melasma.

Angiogenesis, the formation of new blood vessels from preexisting

vessels, is restricted to the perifollicular vasculature during the growth phase of

hair follicles in normal skin (Dvorak et al., 1995). However, the skin can initiate

a rapid angiogenic response during wound healing and inflammation. For

example, UVB irradiation of the skin induces an angiogenic switch, associated

with the up-regulation of proangiogenic factors such as VEGF, basic fibroblast

growth factor, and interleukin-8 (Kramer et al., 1993; Bielenberg et al., 1998;

Strickland et al., 1997).

But the relationship between angiogenesis and hyperpigmentation in

melasma lesions remains to be determined. First, photo-induced hormones,

growth factors, chemical mediators influence the function of melanocytes either

directly or indirectly (Morelli and Norris, 1993), increased release of these

factors from UV-altered blood vessels or endothelial cells might be a possible

- 22 -

cause of hyperpigmentation in melasma. Also, paracrine linkages among

keratinocytes, fibroblasts and melanocytes within the skin play important roles

in regulating epidermal melanization (Maeda et al., 1998). Many keratinocyte-

or fibroblast-derived signaling molecules and cytokines, for which melanocyte

express receptors, affect melanocyte survival, migration, and melanogenesis

(Gilchrest et al., 1998). The expression of these factors is frequently modulated

by stimuli of physiologic relevance to the skin, such as UV irradiation (Mildner

et al., 1999).

In our study, we have found that VEGF expression was more

upregulated in melasma lesions compared to perilesional normal skin. VEGF is

constitutively produced by keratinocytes in the skin. Its production is

upregulated in psoriasis, wound healing, and other states of increased skin

angiogenesis as well as by UV irradiation (Detmar et al., 2005; Lauer et al.,

2000; Gille et al., 2000). Keratinocyte-derived VEGF affects dermal blood

vessels, but to date it is not known whether VEGF affects the behavior of

epidermal cells. However it has been recently shown that other neuronal cells

express VEGFRs and that VEGF stimulates axonal outgrowth, promotes

neuronal cell survival, and plays a role in neural retinal development (Sondell et

al., 2000; Robinson et al., 2001; Gilchrest et al., 1998). Because of the physical

proximity of melanocytes to keratinocytes, and because of its embryologic

origin, melanocytes may be a potential target for keratinocyte derived VEGF.

- 23 -

Moreover, in a recent study it was reported that melanocytes expressed VEGF-1,

VEGF-2, and NP-1 (Kim et al., 2005). In this study we also found that

melanocytes were positively stained for VEGFR-2. But contrary to previous

finding that UV induced VEGFR-2 in melanocytes (Kim et al., 2005), we did

not find significant increase of VEGFR-2 expression in melasma lesions.

However VEGF, which is upregulated by UV irradiation (Yano et al., 2004),

downregulates the level of VEGFR-2 (Kim et al., 2005) in melanocytes and in

our study melasma lesions showed elevated expression of VEGF. So the overall

effect of UV irradiation on VEGF signaling in melanocytes is likely to be

complex.

The possible role of VEGF on melanocyte function is not yet known.

VEGF is known to stimulate the release of arachidonic acid and the

phosphorylation and activation of cytosolic phospholipase A2 (Wheeler-Jones et

al., 1997). It is possible that the resulting metabolites from the arachidonic acid

pathway may affect melanogenesis (Abdel et al., 2006). This hypothesis is

further supported by the report that normal human melanocytes express

functional VEGF receptors (Kim et al., 2005). So, VEGF may have direct

influence on melanocytes through its receptor. Meanwhile Hepatocyte growth

factor is a potent stimulator of human melanocyte DNA synthesis and growth

and coadministration with VEGF revealed an additive effect in angiogenesis

(Matsumoto et al., 1991; Beilmann et al., 2004). It could be speculated that HGF

- 24 -

and VEGF may also have an additive effect during melanogenesis and the

increased release of these factors noticed in melasma could cause increased

hyperpigmentation as well as angiogenesis (Kim et al., 2005).

In summary, we found melasma lesions expressed more pronounced

erythema and computer-assisted image analyses of factor VIIIa-stained sections

revealed a significant increase in vessel size, vessel density and cutaneous area

covered by blood vessels in melasma skin. Such proliferation of vessels may be

due to VEGF, a major angiogenic factor of the skin, as we found expression of

VEGF was significantly increased at epidermis of lesional melasma skin

compared to perilesional normal skin. The relationship between angiogenesis

and melanogenesis is not yet certain however the vessel proliferation and

hyperpigmentation found in lesional melasma skin may be mediated by UV

induced VEGF and its receptor on melanocytes.

- 25 -

V. CONCLUSION

1. The values of a* was significantly higher in the melasma lesion than that of

perilesional normal skin showing erythema intensity is increased in lesional

melasma skin.

2. Computer-assisted image analyses of factor VIIIa - stained sections revealed

a significant increase in vessel size and vessel density resulting in a

significant increase in the cutaneous area covered by blood vessels in

lesional melasma skin.

3. The expression of VEGF was significantly increased at epidermis of lesional

melasma skin compared to perilesional normal skin.

4. The melanocytes in cultured human skin was positively stained by VEGFR-

2 but there was no significant difference in the expression of VEGFR-2 in

lesional melasma and perilesional normal skin.

These results suggest that increase of both the number and the size of dermal

blood vessels in the lesional skin is one of the major features for melasma. The

increased expression of VEGF in the lesional epidermis may play a role in the

mechanism of angiogenesis and pigmentation in melasma.

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- 국문요약 -

기미기미기미기미 병변의병변의병변의병변의 혈관적혈관적혈관적혈관적 특성특성특성특성

연구목적연구목적연구목적연구목적: 기미는 자외선, 여성호르몬 및 유전적 소인에 의해 유발 또는

악화 된다고 알려져 있으나 아직까지 그 기전은 확실히 밝혀져 있지 않다.

최근 연구에 따르면 진피의 환경이 기미 발생에 중요하다고 알려져 있으며,

혈관과 멜라닌 세포간의 상호 작용이 색소 형성에 영향을 줄 수 있을

것으로 생각되어지고 있다. 따라서 본 연구에서는 기미 병변의 혈관적

특성에 대해 관찰하였다.

재료재료재료재료 및및및및 방법방법방법방법: 기미로 진단된 환자 50명을 대상으로 병변의 홍조 정도를

colorimeter를 이용하여 측정하였고, 기미와 인접 정상피부에서 2mm

생검을 실시하여 Factor VIIIa, VEGF, VEGFR-2 염색을 실시 하였다.

결과결과결과결과: 기미환자에서 병변의 홍조 정도가 유의미하게 증가 되어 있었고,

Factor VIIIa 염색 소견상 혈관의 크기, 밀집정도 및 혈관이 상대적으로

차지하는 면적이 기미 병변에서 유의미하게 증가되어 있었다. 또한 VEGF

염색 소견상 기미 병변의 표피에서 VEGF의 표현이 유의미하게 증가 되어

있었으며, 병변의 멜라닌 세포는 VEGFR-2 염색에 양성 소견을 보였다.

결론결론결론결론: 기미 병변에서 색소 침착 외에 혈관의 증식 소견이 관찰되었으며,

피부의 주요 혈관 증식 인자인 VEGF가 이러한 증식에 중요한 역할을 할

것으로 생각되어진다. 향후 기미 병변에서 혈관 증식과 색소 침착 사이의

- 33 -

관계에 대해 추가 연구가 필요 할 것으로 생각된다.

핵심어: 기미, 혈관신생, 혈관내피세포성장인자(VEGF)