의의의학학학석석석사사사학학학위위위논논논문문문repository.ajou.ac.kr/bitstream/201003/1343/1/... ·...
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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)