Vitiligo is a common disease reported to affect approximately 1% of the population worldwide, irrespective of skin color or ethnic origin. It is characterized by depigmented patches that affect the patient cosmetically and psychologically 1. The exact etiology of vitiligo is unknown, but four main theories exist to explain it: the autoimmune hypothesis, the neural hypothesis, the self-destruct hypothesis, and the growth factor defect hypothesis. It has been found that melanocytes in lesions from vitiligo patients showed ‘defective growth and passage capacities.’ The researchers then noted that the growth defects of these melanocytes were partially corrected by adding a growth factor to their culture additionally suggesting that growth defects may be part of the pathology of vitiligo 2.
Previous studies demonstrated that the basic fibroblast growth factor (bFGF) elaborated by keratinocytes in vitro sustains melanocyte growth and survival. They suggested that keratinocyte-derived bFGF is a natural growth factor for normal human melanocytes in vivo, and this melanocyte mitogen increases after irradiation with ultraviolet B. This mitogen (bFGF) is inhibited by neutralizing antibodies to bFGF and also by a synthetic peptide that blocks the binding of bFGF to its receptor 3–8.
This study was designated to investigate the expression of bFGF in vitiligo by measuring its level in the tissues of involved and noninvolved skin of vitiligo patients and comparing these levels with normal controls.
Patients and methods
The study was conducted on 15 cases of vitiligo. The inclusion criteria were stable vitiligo vulgaris with no topical or systemic treatment at least 6 weeks before the study. Ten age-matched and sex-matched healthy volunteers were enrolled as controls. Clinical assessment was performed for each patient, and the involved body surface area was evaluated by the Rule of Nines. For each patient, two 3-mm-punch skin biopsy specimens were taken, one from the lesion and the other from the noninvolved skin (2 cm away from the lesion). For each control, a 3-mm-punch skin biopsy specimen was taken. All biopsy specimens were taken from sun-protected areas of the skin, either from the patients or controls. All these biopsy specimens were subjected to bFGF assessment.
The bFGF was measured in skin specimens that were homogenized in 1 ml lysis buffer for protein extraction; the buffer contained 0.0625 mol/1 Tris buffer (pH 6.8), 2% SDS, 3% 2-mercaptoethanol, 10% glycerol, 10 μg/ml aprotinin, and 1 mmol/1 phenyl methyl sulfonyl fluoride (SIGMA, Minnieapolis, St Louis, USA). After cell lysis, the homogenate was centrifuged at 8000 rpm for 20 min at 4°C. The supernatant was kept frozen at −70°c till the analysis of bFGF. bFGF was examined in the supernatant using the enzyme-linked immune sorbent assay according to the manufacturer’s recommendations. The kit was supplied by Quantaquin R&D system (USA) 9.
Descriptive statistics were performed for all variables of the study. For the quantitative variables, the range, mean, ±SD, and ±SEM were calculated. For categorical variables, absolute counts and percentages were generated.
A comparison of quantitative data was tested using the ‘Student t-test’ to compare two groups and the ‘paired t-test’ to compare the affected and the unaffected sites in the same group. A comparison of categorical data was performed using the χ2-test. A correlation study to establish the relationship between different variables was conducted using the Pearson correlation coefficient ‘r.’ The P-value was considered significant when it was less than or equal to 0.05. The statistical program used was SPSS, version 14 (SPSS Chicago, Illinois, USA).
The study was conducted on 15 cases of stable vitiligo vulgaris and 10 age-matched and sex-matched healthy controls. The clinical data of the patients are illustrated in Table 1. The controls were three men (30%) and seven women (70%). Their age ranged from 27 to 65 years and the mean age was 41.80±12.20 years.
The levels of bFGF in the lesional skin of vitiligo, the noninvolved skin of vitiligo patients, and the controls are illustrated in Table 2 and Figs 1 and 2.
Comparisons between the level of bFGF in the lesional skin of vitiligo patients and the controls showed that there was a high statistically significant difference, the levels being higher in the controls than in the patients (P-value <0.001). Comparisons between the involved and the noninvolved skin in vitiligo patients showed that there was a high statistically significant difference, the level of bFGF being higher in the noninvolved skin than in the involved skin of vitiligo (P-value <0.001). Comparisons between the level of bFGF in the noninvolved skin of vitiligo patients and normal control showed a statistically significant difference, the levels being higher in the control than in the noninvolved skin of vitiligo patients (P-value <0.001).
There was a statistically significant positive correlation between the levels of bFGF in the involved skin and the noninvolved skin in the same patient.
There was no significant correlation between the levels of bFGF and the age of the patients (P-value >0.05), the percent of body surface area involved (P-value >0.05), and the disease duration (P>0.05).There was no statistically significant relation between the mean level of bFGF and the sex of the patients (P-value >0.05), the site (P-value >0.05), and the symmetry of the lesion (P-value >0.05) in vitiligo. No relation could be established with regard to the level of bFGF in different patient subgroups concerning the laterality and the skin type of the patients because of the small number of the patients studied.
Several studies have addressed the role of peripheral blood and lesional cytokine and growth factor expression in patients with vitiligo. One of the important growth factors that was suggested to play a role in vitiligo is bFGF, because of its mitogenic effect on human melanocytes 10,11.
bFGF RNA transcripts have been shown to be expressed at various stages of human melanocyte progression and were detected in tissues from dermal nevi, primary melanomas, and metastatic melanomas. Interestingly, the expression level of bFGF RNA transcripts detected decreased with increasing progression toward malignant tissues 12. Inhibition of cell growth and proliferation of melanoma cells have been observed with the use of bFGF-neutralizing antibodies and with antisense oligonucleotides to bFGF 13.
It is believed that during repigmentation of vitiligo, inactive melanocytes in the outer root sheath of the hair follicle become activated, proliferate, and migrate into the depigmented skin. However, the mechanisms controlling melanocyte migration remain to be elucidated. Melanocyte chemokinetic movement was induced by bFGF, stem-cell factor, endothelin-1, and leukotriene C4. It is believed that these factors may be effective in stimulating vitiligo repigmentation by inducing the proliferation and migration of hair follicle outer root sheath melanocytes into the depigmented epidermis 14.
It has been suggested that, in vivo, besides direct stimulation, ultraviolet B light stimulates melanocytes indirectly through neighboring irradiated keratinocytes by an increased bFGF production. Melanocytes could be exposed to bFGF through direct contact with keratinocytes and through the extracellular matrix deposited by neighboring keratinocytes 6. It has been found that bFGF significantly enhanced the migration of melanocytes and p125FAK expression in melanocytes. Herbimycin A, a potent p125FAK inhibitor, effectively abolished bFGF-induced melanocyte migration, which indicates that p125FAK plays an important role in the signal transduction pathway of melanocyte migration induced by bFGF 15. Also, a reduction in the level of the antiapoptotic Bcl-2 was involved in the execution of apoptosis induced by transforming growth factor-β1 in normal melanocytes cultured on collagen gel, and FGF-2 can prevent transforming growth factor-β1 from causing this reduction 16. From all these findings, we can suggest that bFGF has a major influence on melanocyte growth, proliferation, movement, and migration. Therefore, one question remains to be elucidated. Does bFGF play a role in the pathogenesis of vitiligo? If yes, what is its role?
In this study, we measured the level of bFGF in the involved and the noninvolved skin of vitiligo patients. We found lower levels of bFGF in vitiliginous lesions compared with noninvolved sites. The level of bFGF is lower in both the involved and the noninvolved skin of vitiligo patients compared with the controls. This might highlight the role of bFGF in the pathogenesis of vitiligo.
The level of bFGF in the noninvolved area of the skin in patients with stable vitiligo vulgaris was intermediate between the vitiliginous lesions and the healthy controls, which might represent a transition state until vitiligo develops. This could give rise to a question. Could the level of bFGF be used as a predictor of the development of new lesions in vitiligo patients or on genetically predisposed individuals?
Our results are in agreement with Horikawa et al.14, who found that four mitogens including bFGF stimulated melanocyte migration, thus indicating that this factor may play a role in vitiligo healing. However, these results are contradictory to the results by Ozdemir et al.17, who found a high level of bFGF in the vitiliginous skin blister fluid and the serum fluid compared with healthy controls, but they could not find a difference between bFGF levels in the blister fluid from pigmented and nonpigmented skin. Actually, we could not find a cause for these contradictory results. It might be because of the different methods used, as we measured bFGF directly from the tissues, whereas Ozdemir et al.17 measured it in the blister fluid induced in the vitiligo lesions.
In the present study, we could not find a statistical relationship between the level of bFGF and the age of the patients, the duration of the disease, the percent of surface area involved, the sex of the patients, and the site and symmetry of the lesion. No relationship could be established with regard to the laterality and the skin type of the patients because of the limited number of cases studied. This might encourage a large-scale research to study these relationships in the future.
Nonetheless, this study highlights the role that bFGF could play in the pathogenesis of vitiligo and has raised many questions that have to be answered in the future. Further studies are needed to analyze the role of various mitogens in the development of vitiligo. This may pave the way toward a better understanding of the pathogenesis of vitiligo to develop new modalities of treatment.
Conflicts of interest
There are no conflicts of interest.
1. Whitton M, Ashcroft D, Gonzales U. Therapeutic intervention for vitiligo
. J Am Acad Dermatol. 2008;59:713–717
2. Njoo M, Westerhof W. Vitiligo
: pathogenesis and treatment. Am J Clin Dermatol. 2001;2:167–181
3. Eisinger M, Marko O. Selective proliferation of normal human melanocytes in vitro in the presence of phorbol ester and cholera toxin. Proc Natl Acad Sci. 1982;79:2018–2022
4. Gilchrest B, Vrabel M, Szabo G. Selective cultivation of human melanocytes from new born and adult human skin. J Invest Dermatol. 1984;83:370–376
5. Halaban R, Ghosh S, Duray M, Kirkwood J, Lerner A. Human melanocytes cultured from nevi and melanomas. J Invest Dermatol. 1986;87:95–105
6. Halaban R, Longdon R, Birchal N, Cuono C, Baird A, Scott G, et al. Basic fibroblast growth factor
from human keratinocytes is a natural mitogen for melanocytes. J Cell Biol. 1988;107:1611–1619
7. Pitellkow K, Shipley G. Serum-free culture of normal human melanocytes: growth kinetics and growth factors. J Cell Physiol. 1989;140:565–576
8. Donatien P, Surleve-Bazeille J, Thody A, Taieb A. Growth and differentiation of normal human melanocytes in a TPA-free, cholera toxin-free, low-serum medium and influence of keratinocytes. Dermatol Res. 1993;285:385–392
9. Dell K, Williams L. A novel form of fibroblast growth factor receptor 2. Alternative splicing of the third immunoglobulin-like domain confers ligand binding specificity. J Biol Chem. 1992;267:21225–21229
10. Yu H, Chang K, Yu C, Li H, Wu M, Wu C, et al. Alterations in IL-6, IL-8, GM-CSF, TNF-α, and IFN-γ release by peripheral mononuclear cells in patients with active vitiligo
. J Invest Dermatol. 1997;108:527–529
11. Moretti S, Spallanzani A, Amato L, HAutmann G, Gallerani I, Fabiani M, et al. New insights into the pathogenesis of vitiligo
imbalance of epidermal cytokines at sites of lesions. Pigment Cell Res. 2002;15:87–92
12. Becker D, Meier C, Herlyn M. Proliferation of human malignant melanomas is inhibited by antisense oligodeoxynucleotides targeted against basic fibroblast growth factor
. EMBO J. 1989;8:3685–3691
13. Yamanishi D, Graham M, Florkiewicz R, Buckmeier J, Meyskens F. Differences in Basic Fibroblast Growth Factor
RNA and Protein levels in human primary melanocytes and metastatic melanoma cells. Cancer Res. 1992;52:5024–5029
14. Horikawa T, Norris D, Yohn J, Zekman T, Travers J, Morelli J. Melanocyte mitogens induce both melanocyte chemokinesis and chemotaxis. J Invest Derma. 1995;104:256–259
15. Wu CS, Elan CS, Chiou MH, Yu HS. Basic fibroblast growth factor
promotes melanocyte migration via expression of p125FAK
on melanocyes. Acta Derm Venereo. 2006;86:498–502
16. Von Willebrand M, Kohler K, Alanko T, Lahio M, Saksela O. FGF-2 blocks TGF-B1-mediated suppression of BCL-2 in normal melanocyte. Exp Dermatol. 2005;14:202–208
17. Ozdemir M, Yillar G, Wolf R, Yillar O, Unal G, Tuzun B, Tuzun Y. Increased basic fibroblast growth factor
levels in serum and blister fluid from patients with Vitiligo
. Acta Derm Venereol. 2000;80:438–439