Journal of Dermatological Science (2007) 46, 111—116

www.intl.elsevierhealth.com/journals/jods

The vascular characteristics of melasma

En Hyung Kim, You Chan Kim, Eun-So Lee, Hee Young Kang *

Department of Dermatology, Ajou University School of Medicine, 5 Wonchon-Dong, Yeongtong-Gu,Suwon 443-721, South Korea

Received 27 October 2006; received in revised form 24 January 2007; accepted 24 January 2007

KEYWORDSMelasma;Angiogenesis;Vascular endothelialgrowth factor

Summary

Background: The pathogenesis of melasma is not yet fully understood. Previousstudies indicate that dermal environment such as fibroblasts may have an importantrole in the development of melasma. Recently, it has been suggested that interactionsbetween the cutaneous vasculature and melanocytes might have an influence on thedevelopment of pigmentation.Objectives: We investigated the vascular characteristics in melasma lesions. Theexpression of vascular endothelial growth factor (VEGF), a major angiogenic factor ofthe skin, was also investigated in melasma.Methods: Erythema intensity was quantified by the increase of the a* parameterusing a colorimeter. Skin samples were obtained from lesional and non-lesional facialskin of 50 Korean women with melasma. Immunohistochemistry was performed todetermine the expression of factor VIIIa-related antigen and VEGF in melasma.Results: The values of a* was significantly higher in the melasma lesion than that ofperilesional normal skin. Computer-assisted image analyses of factor VIIIa-relatedantigen-stained sections revealed a significant increase of both the number and thesize of dermal blood vessels in the lesional skin. There was significant relationshipbetween the number of vessels and pigmentation in melasma. The expression of VEGFwas significantly increased in melasmaConclusions: These data suggest that increased vascularity is one of themajor findingsin melasma. VEGF may be a major angiogenic factor for altered vessels in melasma.# 2007 Japanese Society for Investigative Dermatology. Published by Elsevier IrelandLtd. All rights reserved.

* Corresponding author. Tel.: +82 31 219 5190;fax: +82 31 219 5189.

E-mail address: hykang@ajou.ac.kr (H.Y. Kang).

0923-1811/$30.00 # 2007 Japanese Society for Investigative Dermadoi:10.1016/j.jdermsci.2007.01.009

1. Introduction

Melasma is a common acquired symmetrical hyper-melanosis on sun-exposed areas of the skin. It is verycommon in oriental women. The major etiologicalfactors include genetic influences, exposure to

tology. Published by Elsevier Ireland Ltd. All rights reserved.

112 E.H. Kim et al.

ultraviolet (UV) radiation, and sex hormones [1].However, the pathogenesis of melasma is not yetfully understood.

Previous report have demonstrated that lesionalmelasma skin show more prominent solar elastosiscompared with normal skin, suggesting that thedermal change may influence the development ofmelasma [2]. Very recently, we suggested that der-mal inflammation induced by accumulation of UVirradiation may be associated with activation offibroblasts, which result in the up-regulation of stemcell factor in melasma dermal skin leading toincreased melanogenesis [3]. These studies havegiven us new insights on the pathogenesis of mel-asma, in particular, that the dermal environmentmay have an important role in the development ofmelasma.

Recent studies have suggested that a connectionbetween vessels and cutaneous pigmentationcould exist. Human melanocytes may respond toangiogenic factors because normal human mela-nocytes express functional vascular endothelialgrowth factor (VEGF) receptors [4]. Also, it hasbeen reported that the topical plasmin inhibitor,tranexamic acid, is effective in the treatmentof UV-induced hyperpigmentation [5]. Localizedmicroinjection of tranexamic acid, improved mel-asma in vivo [6]. Tranexamic acid prevents thebinding of plasminogen originating from endothe-lial cells, to keratinocytes, which is thought to be apossible mechanism for melasma treatment [5].These in vitro and in vivo findings suggest thatinteractions between the altered cutaneous vas-culature and melanocytes may have an influenceon the development of hyperpigmentation in theoverlying epidermis.

A major clinical characteristic feature of mel-asma is hyperpigmented patches, but we haveobserved that melasma patients have additionaldistinguishing features like pronounced telangiec-tatic erythema confined to the melasma lesionalskin. Therefore, in the present study, we haveinvestigated the vascular characteristics of mel-asma lesions. Our data confirmed that melasmalesions show significant increase in vascularity. Wethen investigated the expression of VEGF, a majorangiogenic factor of the skin, in altered vasculatureof melasma lesions.

2. Materials and methods

2.1. Patients

We examined 50 Korean women with newly diag-nosed melasma, which was determined by physical

examination and confirmed by histological examina-tion. Informed written consent was obtained fromeach patient prior to skin sampling. This study wasapproved by the ethical committee of Ajou Univer-sity Hospital.

2.2. 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 sub-jects had a wash-out period for at least 2 weeks frombleaching products, UV light therapy and steroidcontaining triple agents and a wash-out period forat least 3 months from lasers, dermabrasion, andchemical peeling. The intensity of erythema wasmeasured by skin reflectance with a tristimuluscolor analyzer (Chromameter CR300, Minolta,Japan) and expressed in the L*a*b* system. Thissystem allows a color to be quantified accordingto three axes: white—black or lightness (L*), red—green or chrome (a*) and yellow—blue or hue (b*).The a* parameter was used as the measurement ofredness.

2.3. Immunohistochemistry and imageanalysis

Skin biopsy specimens were obtained from 50 mel-asma patients. Two-millimeter punch biopsies fromlesional melasma and perilesional normal facial skin(usually within 1 cm away from the lesional border)were obtained from each patient under localanesthesia. Fifty pairs of tissue were prepared forroutine and immunohistopathological examinationby 10% formalin fixation. Melanin pigment was visua-lized with the Fontana-Masson stain, performed bythe usual methods without an eosin backgroundstain. The amount of pigmentation was measuredby the ratio of pigmented area to measured epider-mal area (PA/EA) in lesional melasma and perile-sional normal skin. The measurement was doneunder constant magnification at 200�.

Paraffin-embedded sections (4 mm) of bothlesional and perilesional normal skin were processedfor immunohistochemistry as described earlier [7]. Amonoclonal antibody to factor VIIIa-related antigen(ImmunonTM, Pittsburgh, PA, U.S.A.) was diluted1:100 and incubated for 20 min at 48 8C. At 100�magnification, the number of vessels mm�2, theaverage vessel size and the relative area occupiedby blood vessels were determined in the dermis ofeach slide section. Themeasurement was performedin an area within 100 mm distance from the epider-mal—dermal junction [8]. Amonoclonal antibodies toVEGF (Santa Cruz Biotechnology, Inc., Santa Cruz,

Vascular characteristics of melasma 113

CA,U.S.A.)was diluted1:50 and incubated for 20 minat 48 8C.Thestainedareaperepidermal area (SA/EA)was measured in lesional and perilesional skin. Eachmeasurement was evaluated under constant magni-fication. For each frame, the tracing was repeatedthree times and the mean was used for evaluation.The image was analyzed using Image Pro Plus Version4.5 (Media Cybertics Co., MD, U.S.A.).

2.4. Statistics

Comparison of erythema intensity and vascularitybetween melasma lesional and perilesional normalskin was tested with two-sided unpaired Student’s t-test. The relationship between pigmentation andvascularity was tested with Pearson’s correlationcoefficients. A possibility value of less than 0.01was considered as statistically significant. SPSS11.0 statistics program was used for analysis.

3. Results

3.1. Erythema intensity in melasmapatients

On physical examination, melasma patients exhib-ited pronounced telangiectatic erythema withinlesions of melasma (Fig. 1). The values of a* washigher in the melasma lesion (mean value of Rt. andLt. cheek area, 12.92 � 1.96 and 12.78 � 2.19) than

Fig. 1 Pronounced telangiectatic erythema noticed onmelasma lesion.

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).

3.2. Immunohistological evaluation ofvascularity in melasma patients

To investigate whether vascularity is increased inmelasma lesions, we performed immunohistochem-istry for factor VIIIa-related antigen. We foundincreased numbers of enlarged blood vessels inthe melasma lesion (Fig. 2B), as compared withperilesional normal skin (Fig. 2A). Computer-assisted image analyses of factor VIIIa-related anti-gen-stained sections revealed a significant 16.28%increase in vessel size (Fig. 2C) and 33.89% increasein vessel density (Fig. 2D) in melasma skin comparedto perilesional normal skin. This resulted in an over-all increase of 68.75% in the cutaneous area coveredby blood vessels in melasma skin compared to peri-lesional normal skin (Fig. 2E).

As shown in Table 1, there was a positive relation-ship between number of vessels and pigmentation inmelasma lesional skin. However, no appreciablerelationship was found between pigmentation andvessel size or area.

3.3. VEGF expression was increased inmelasma

To examine whether the increased angiogenesis inmelasma lesions is associated with increasedexpression of VEGF, we examined the expressionof VEGF protein using immunohistochemistry. Inmelasma lesions, distinctly positive immunoreactiv-ity against VEGF was noticed in keratinocyteswhereas perilesional normal skin only showed weakimmunoreactivity in keratinocytes (Fig. 3). The SA/EA of perilesional normal skin samples was0.137 � 0.132, and that of lesional melasma skinsamples was 0.270 � 0.237. This difference wasstatistically significant (P = 0.034). In addition, der-mal blood vessels, and fibroblasts were also faintlystained. However, the staining pattern of perile-sional and lesional skin was similar in papillaryand reticular dermis.

4. Discussion

We have demonstrated that a significant increase ofboth the number and size of dermal blood vessels inthe lesional skin is one of the major findings inmelasma. Interestingly, the increase in the numberof vessels was more prominent than the increase invessel size in lesional melasma skin. This finding

114 E.H. Kim et al.

Fig. 2 Immunohistochemistry for factor VIIIa-related antigen revealed enlarged and elongated blood vessels in theupper dermis (B), as compared with perilesional normal skin (A). Computer assisted morphometric analysis of factor VIIIa-related antigen stained sections revealed a significant increase in vessel size (C), vessel density (D) and the relative areacovered by blood vessels (E), as compared with perlesional normal skin.

suggests that the erythema noticed in melasmapatients could be due to angiogenesis as well astelangiectasia.

Angiogenesis, the formation of new blood vesselsfrom preexisting vessels, is restricted to the peri-follicular vasculature during the growth phase ofhair follicles in normal skin [9]. However, the skincan initiate a rapid angiogenic response duringwound healing and inflammation. For example,

Table 1 The relationship between epidermal pigmentation

Level of pigmentation Number ofvessels (mm�2)

Perilesional normal skin 0.031Lesional melasma skin 0.467 *

Each value represents Pearson’s correlation coefficient (r). There wnumber of vessels. *p < 0.01.

UVB irradiation of the skin induces an angiogenicswitch, associated with the up-regulation of proan-giogenic factors such as VEGF, basic fibroblastgrowth factor, and interleukin-8 [10—12]. In ourstudy, we found that VEGF expression was moreupregulated in melasma lesions compared to perile-sional normal skin. These results suggest that VEGFis a major angiogenic factor in vascular alterationnoticed in melasma.

and vascularity

Vesselsize (mm2)

Vesselarea (%)

0.179 0.1030.169 0.163

as a significant positive correlation between pigmentation and

Vascular characteristics of melasma 115

Fig. 3 Immunostaining with an antibody to VEGF. Theexpression of VEGF was significantly increased at lesionalepidermis (B) compared with perilesional normal skin (A).Original magnification 400�.

The significance of these increased vascularity inmelasma is unclear. It is well known that UV irradia-tion induces angiogenesis so the vascular alterationin melasma might be just a result of chronic UVaccumulation accompanying epidermal hyperpig-mentation. However, both lesional and perilesionalfacial skin had been exposed to chronic UV irradia-tion, but only lesional melasma skin showed pro-nounced vascular changes. Furthermore, thenumber of vessels had a positive relationship withpigmentation in melasma lesional skin. So, it ispossible to speculate that the increase in vascularitywas not only an epiphenomenon of UV damage butthat it may play an important role in the pathogen-esis of melasma by an increase in factors releasedfrom these proliferated vessels. However, althoughour patients were newly diagnosed, it should benoted that steroid containing triple reagent caninduce telangiectasia because people usually applytriple reagent for the treatment of melasma.

VEGF is known to stimulate the release of ara-chidonic acid and the phosphorylation and activa-tion of cytosolic phospholipase A2 [13]. It is possible

that the resulting metabolites from the arachidonicacid pathway may affect melanogenesis [14]. Thishypothesis is further supported by a report thatnormal human melanocytes express functional vas-cular endothelial growth factor receptors [4].Therefore VEGF could have a direct influence onmelanocyte behavior through its receptor. Also,blood vessels or endothelial cells modified by UVradiation may release cytokines and soluble factorslike plasminogen, which might be a possible cause ofhyperpigmentation in melasma [15]. The biologicalrole of cutaneous blood vessels in the pathogenesisof melasma remains an interesting topic for futurestudies.

In summary, we have shown that vascularity isincreased in melasma lesions. This present studymay offer future perspectives for pathogenesisand therapeutic consideration of melasma.

Acknowledgment

This work was supported by the Korea Science andEngineering Foundation through Chronic Inflamma-tory Disease Research Center Ajou University GrantR13-2003-019.

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