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Increased expression of TGF-β protein in the lesional skins of melasma

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Increased expression of TGF-β protein in the

lesional skins of melasma patients following
treatment with platelet-rich plasma

Eman R. M. Hofny, Mahmoud Rezk Abdelwahed Hussein, Alaa Ghazally,

Asmaa M. Ahmed & Amira A. Abdel-Motaleb

To cite this article: Eman R. M. Hofny, Mahmoud Rezk Abdelwahed Hussein, Alaa Ghazally,
Asmaa M. Ahmed & Amira A. Abdel-Motaleb (2019): Increased expression of TGF-β protein in
the lesional skins of melasma patients following treatment with platelet-rich plasma, Journal of
Cosmetic and Laser Therapy, DOI: 10.1080/14764172.2019.1668016

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JOURNAL OF COSMETIC AND LASER THERAPY
https://doi.org/10.1080/14764172.2019.1668016

Increased expression of TGF-β protein in the lesional skins of melasma patients

following treatment with platelet-rich plasma
Eman R. M. Hofnya, Mahmoud Rezk Abdelwahed Hussein b
, Alaa Ghazallya, Asmaa M. Ahmedb, and Amira A. Abdel-
Motaleba
a
Department of Dermatology, Venereology and Andrology, Faculty of Medicine, Assuit University Hospitals, Assuit, Egypt; bDepartment of
Pathology, Faculty of Medicine, Assuit University Hospitals, Assuit, Egypt

ABSTRACT ARTICLE HISTORY

Background: Melasma is a common acquired facial hyperpigmented skin disorder. Platelet-rich plasma Received 15 May 2018
(PRP) is autologous plasma containing higher than normal platelets concentrations. Recently, PRP has Revised 9 April 2019
been used as a therapeutic modality in melasma with significant clinical improvement, possibly due to Accepted 11 September 2019
its abundant contents of growth factors such as TGF-β. The latter represents a natural multifunctional KEYWORDS
polypeptide that negatively regulates melanocyte differentiation and therefore reduces skin hyperpig- Platelet rich plasma;
mentation. To date, the expression pattern of TGF-β protein in skin of melasma patients following PRP melasma; therapy; TGF-β
injection is unknown. Here we hypothesize that “injection of PRP in the lesional skin of melasma patients
is associated with alterations of TGF-β protein expression”.
Patients and Methods: The study included 20 adult patients with melasma. Autologous PRP was
delivered into the lesional skin either through microneedling or as intradermal microinjections. TGF-β
protein expression was immunohistochemically examined in the perilesional and lesional skins before
and after PRP treatment and in the healthy skins of nine volunteers (control group).
Results: TGF-β protein was expressed within the epidermis, dermal adnexal structures, vascular endothe-
lium, nerves and arrector pili muscle fibers of the healthy skins (control group), perilesional and lesional skins
of melasma patients before and after treatment with PRP. Before treatment with PRP, the expression ofTGF-β
protein in the lesional (1.26 ± 0.41) and perilesional (1.68 ± 0.51) skins of melasma patients were significantly
lower than that in the healthy skins (2.26 ± 0.37, p value<.05). After treatment with PRP, the expression of
TGF-β protein was significantly increased in the lesional (2.15 ± 0.44) skin of melasma patients.
Conclusions: Our study provides the first indication about increased TGF-β protein expression in skin of
melasma patients after PRP treatment. The alterations of TGF-β protein in skin of melasma patients not
only support its roles in the development of this condition but also have some therapeutic ramifications.

Introduction greater than in the peripheral blood is thought to be enough to

have therapeutic effects in skin rejuvenation (7), in the treatment
Melasma is an acquired facial hyperpigmentation disorder
of pigmentary disorders (such as melasma and periorbital hyper-
common among females (1). It is characterized by brown
pigmentation) (8–10) and in wound healing (11,12). The thera-
spots with irregular outlines that mostly affect the face. It
peutic efficacy of PRP is based on the premise that the α-granules
has a bad impact on the patient’s life because facial appear-
of the platelets contain abundance of growth factors such as
ance is important for self-perception and interactions with
platelet-derived growth factor, transforming growth factor beta
others (2). Melasma may have centrofacial, mandibular and
(TGF-β 1, 2), insulin-like growth factor (11), adhesion molecules,
malar patterns (3). It has diverse histological features includ-
integrins and several cytokines (10). These growth factors bind to
ing epidermal hyperpigmentation, dermal solar elastosis, dis-
their cognate receptors expressed over the cutaneous epithelial
ruption of the basement membrane, increased vascularization,
and mesenchymal stem cells, fibroblasts, endothelial cells, and
and an increased number of mast cells (4,5). In melasma, the
keratinocytes and therefore inducing signal-transduction path-
melanocytes are functionally active and therefore produce
ways with subsequent expression of genes and the release of
more melanin because they contain more cytoplasmic orga-
proteins involved in melanogenesis and tissue repair (10,11).
nelles such as dendrites, mitochondria, Golgi bodies and
The therapeutic efficacy of PRP is reasoned to its abundant
rough endoplasmic reticulum as compared to healthy skin (6).
contents of growth factors including TGF-β. The latter is
Platelet-rich plasma (PRP) is prepared by centrifugation and
a multifunctional natural polypeptide with three major iso-
represents an autologous plasma containing higher than normal
forms namely TGF-β1, β2, and β3 that signal through type
platelets concentrations. Although no accord on what is the best
I and type II TGF-β receptors (13).TGF-β is released as an
concentration, platelet concentration that is three to seven times
inactive form from the platelets α-granules of PRP and then

CONTACT Mahmoud R. Hussein frcpath17@gmail.com Diploma in Dermatopathology, Professor of Pathology, Consultant Pathologist/Dermatopathologist,
Department of Pathology, Assiut University Hospitals, Assiut, Egypt
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/ijcl.
© 2019 Taylor & Francis Group, LLC.
2 E. R. M. HOFNY ET AL.
become activated by the proteolytic cleavage. In the skin,
TGF-β family regulates several cell functions including cell
proliferation, differentiation, and melanogenesis (14). Several
observations support a role for TGF-β in regulating melano-
genesis. In the spontaneously immortalized mouse melano-
cyte cell line, TGF-β1 significantly repressed melanin
synthesis (melanogenesis) through several mechanisms such
as delayed extracellular signal-regulated kinase activation (6).
TGF-β1 can reduce the activity of tyrosinase, tyrosinase-
related proteins and the promoter of the microphthalmia
transcription factor (MITF). TGF-β can delay the activation
of extracellular signal-regulated kinase. Also, TGF-β can
repress the expression of paired-box homeotic gene (PAX),
which regulate UV-induced melanogenesis (15). Recent stu-
dies have indicated clinical improvement in skin of melasma
patients following PRP injection, possibly due to its abundant
contents of growth factors such as TGF-β. These studies also
proposed the use of PRP as a new therapeutic modality in
melasma. However, to date the underlying mechanisms of the
clinical improvement is unknown (8,9). Whether PRP injec-
tion would be associated with alterations in the levels of TGF-
β is not yet elucidated.
To the best of our knowledge, the expression of TGF-β
protein in the skin of melasma patients following PRP injec-
tion has not been investigated to date. Here we hypothesize
that “PRP injection in the lesional skin of melasma patients is
associated with alterations of TGF-β protein expression”. We
carried out this investigation to test our hypothesis and to
examine these issues.
Patients and methods
The study was approved by the Institutional Ethics and
Research Committee at the Faculty of Medicine, Assuit
University. All participants signed an informed consent before
being enrolled in the study.
Inclusion criteria included: 20 eligible melasma patients,
above 18 years old, who were willing to participate in the
study and to give an informed consent.
Exclusion criteria included: pregnant females, those on oral
contraceptives, positive history of keloids, hypertrophic scars,
recurrent herpes infection, and hemorrhagic blood diseases.
Positive medical history for systemic chemotherapy, antiplate-
let agents or anticoagulation therapy.
Tissue specimens
Twenty female patients with melasma and nine healthy volun-
teers were enrolled in the study. Before PRP treatment, patients
were asked to avoid the intake of any type of medications (local
or systemic) for 14 days and sun exposure for 24 h.
The patients were injected by PRP every 4 weeks for 3
times. PRP was prepared following Na et al. (16) with
approximately 10 ml of the patient’s blood was withdrawn
in a tube containing an anticoagulant (ethylenediaminetetraa-
cetic acid, EDTA) that was then subjected to two centrifuga-
tion steps 10 min each, the first was at 160 g then the second
rotation was at 400 g, the buffy coat (enriched with platelets)
was then aspirated and activated with calcium chloride.
Transdermal delivery of PRP was carried out on the right
side of the face through microneedling using dermapen with
2 mm needle length. While on the left side microinjections of
PRP into the dermis separated by 1 cm apart, using a 100 U/
mL insulin syringe with a 4 mm mesoneedle was performed.
Punch skin biopsies (2 mm) were obtained from lesional/
melasma skins before PRP treatment and 1 month after the
last session (20 patients) (17), perilesional skin (10 patients)
and the facial skin of age and sex-matched health volunteers
(nine volunteers). The tissue specimens were fixed in forma-
lin, processed and embedded in paraffin for routine histology
and immunostains.
Immunohistochemistry
Immunostainings was performed following other groups (18).
Four microns sections of the formalin-fixed paraffin-embedded
blocks were prepared. For immunostaining, sections were depar-
affinized and hydrated. Endogenous peroxidase activity was
blocked using 3% hydrogen peroxide. Heat-induced antigen
retrieval was done by immersing the slides in a container contain-
ing preheated sodium citrate buffer solution (pH 6.0) and main-
tained at a sub-boiling temperature (between 95 and 98)°C for 20
min on a hot plate. The slides were allowed to cool at room
temperature for 30 min. Incubation of the slides with sufficient
amounts of the primary polyclonal anti-rabbit antibody (TGF-β3
(III), sc83, Santa Cruz biotechnology, CA, USA) at dilution of 1:25
at room temperature overnight average at 4°C (according to the
manufacturer, it can detect precursor and mature TGFβ1, TGFβ2,
and TGF3 proteins). A secondary staining system (EnVision™
FLEX, K8000, DAKO, USA) was used according to the manufac-
turer instructions. Human appendicular tissue was used as posi-
tive control (19)
Positive and negative controls: The positive controls con-
sisted of human vermiform appendix (mucosal epithelium and
lymphocytes). A similarly stained appendicular tissue sections
(but with omission of the primary antibody) served as negative
controls. The positive and negative controls were positive and
negative, respectively, indicating the validity of our results.
Histological and immunohistochemical evaluation
The H&E and immunostained slides were examined by two
pathologists (Dr. Mahmoud Hussein and Dr. Asmaa Ahmed).
The expression pattern of TGF-β protein (control healthy skin,
perilesional and lesional skin) was independently evaluated and
reported as staining intensity following other groups (18). The
results were scored as follows: 0 for negative staining, 1+ for
weak staining, 2+ for moderate staining intensity, and 3+ for
strong positive staining. The findings in lesional skin of melasma
patients were reported and compared to healthy skin (18).
Statistical analysis
Data entry and data analysis were done using SPSS version 19
(Statistical Package for Social Science). Data were presented as
number, mean and standard deviation. Mann–Whitney test
was used to compare quantitative variables between two
groups in case of non-parametric data. Wilcoxon Signed
 JOURNAL OF COSMETIC AND LASER THERAPY 3

Rank Test was done to compare quantitative variables
between before and after treatment. Spearman correlation
was done to measure correlation between quantitative vari-
ables. p value considered statistically significant when p< .05.
occasional mast cells, solar elastotic fibers. The infundibular
epithelium, hair follicles, sebaceous and sweat glands and the
arrector pili muscle fibers were unremarkable. The vascular
endothelium was swollen. The prelesional skins were unre-
markable. A summary of these changes is shown in Figure 2.

Results
The clinical characteristics of the patients and healthy controls
are shown in Table 1. A significant to excellent clinical improve-
ment of the melasma skin lesions was observed in most of the
patients following PRP treatment as shown in Figure 1.
Prominent basal cell hyperpigmentation in the lesional
skin of melasma patients (before PRP treatment)
Before treatment, the lesional skin (the melasma patient)
showed orthokeratosis, hyperpigmentation of the basal cell
layer with scatters of melanocytes among the basal cell kera-
tinocytes ranging (one melanocytes/4 – 5 keratinocytes),
capillary dermal ectasia, melanin incontinence, scanty mela-
nophages, mild perivascular lymphohistiocytic infiltrate with
Amelioration of basal cell hyperpigmentation in the skin
of melasma patients following PRP treatment
Following PRP injection, the epidermis was essentially unre-
markable with amelioration or even absence of basal cell
hyperpigmentation as compared to skin of melasma patients
before PRP treatment. Melanin incontinence and melano-
phages were essentially absent. Other histological features
following PRP treatment included orthokeratosis, capillary
dermal ectasia, solar elastosis and perivascular lymphohistio-
cytic infiltrate (no mast cells were noted). The infundibular
epithelium of the hair follicles, the sebaceous and sweat
glands, the arrector pili muscle fibers and the vascular
endothelium were unremarkable. A summary of these
changes is shown in Figure 2.

Table 1. Clinical characteristics of the melasma patients and healthy controls. Decreased TGF-β protein expression in the perilesional
Melasma patients Healthy controls skin of melasma patients (before PRP treatment) as
No. n = 20 No. n = 9 compared to the healthy skins
Age: (years) In the appendicular tissue specimens (positive control), TGF-
Mean ± SD 32.75 ± 5.99 30.89 ± 4.3
Sex: β protein expression was seen as a strong cytoplasmic staining
Male 3 (3/20) 1 (1/9) in the lining mucosal epithelial cells and in the lymphocytes of
Female 17 (17/20) 8 (8/9)
Skin photo-type the lamina propria. The expression was also observed in the
Type III 5 (5/20) 2 (2/9) fibroblasts and endothelial cells lining the walls of the blood
Type IV 15 (15/20) 7 (7/9) vessels. Weak expression was noticed in the muscle fibers and
SD, standard deviation. nerve plexuses. A summary of these findings is shown in

Figure 1. Melasma patient before PRP treatment and 1 month after the last session.
Significant clinical improvement was observed in melasma lesions following PRP intradermal injection.
4 E. R. M. HOFNY ET AL.

Figure 2. Histological changes and TGF-β protein expression in the skin of healthy controls and melasma patients.
A-B) Before PRP treatment, there was basal cell layer hyperpigmentation (A, Scale bar, 10 µm) and dermal melanin incontinence and melanophages (B, Scale bar, 10
µm), C-D) PRP treatment was associated with amelioration of the basal cell layer hyperpigmentation (C, Scale bar, 10 µm) and lack of melanin incontinence or
melanophages (D, Scale bar, 10 µm), E: Strong cytoplasmic TGF-β protein in the epidermis of the healthy skin (all epidermal layers, Scale bar, 10 µm), F-G: Faint to
absent TGF-β protein in the perilesional (F) and lesional (G) skin of the melasma patient before PRP treatment (Scale bar, 10 µm) and H: A strong TGF-β protein in the
lesional skin of the melasma patient after PRP treatment (Scale bar, 10 µm).

Figure 1. In the healthy skins (control group), TGF-β protein
expression was observed throughout the epidermis, in the
dermal adnexal structures, vascular endothelium, nerves and
in the arrector pili muscle fibers. In the epidermis, the expres-
sion values were higher in the basal cell keratinocytes (2.33 ±
0.71) than in the other epidermal layers (spinous cell, granular
cell, and horny cell layers: 2.11 ± 0.60). The mean value of
TGF-β protein expression in the epidermis was 2.17 ± 0.60.
As compared to the healthy skin, the expression values of
TGF-β protein were lower in the perilesional skin. In the
epidermis, the values ranged from 1.60 ± 0.70 (prickle cells
keratinocytes) to 1.70 ± 0.68 (basal cell keratinocytes).
Similarly, the expression in the dermal adnexa, fibroblasts,
endothelial cells and lymphocytes were low as compared to
the healthy skin. A summary of these findings is presented in
Tables 2 and 3 and in Figures 2 and 3.
Decreased TGF-β protein expression in the lesional skin
of melasma patients (before PRP treatment) as compared
to both healthy and perilesional skin
We compared the expression patterns of TGF-β protein among
the healthy and lesional skin of melasma patients (before treat-
ment), and we found statistically significant variations, i.e.,
decreased TGF-β protein expression in the lesional skin before
treatment. In the lesional epidermis, TGF-β expression values
ranged from 1.05 ± 0.51 (spinous, granular and cornified cells)
to 1.30 ± 0.47 (basal cell keratinocytes). Similarly, the

Table 2. TGF-β protein expression in the skin of the healthy controls and melasma patients.
Healthy skin (control) Peri-lesional skin
n=9 n = 20
Epidermis
Mean ± SD 2.17 ± 0.60 1.63 ± 0.68
p value1 0.059
p value2
p value3
Dermis
Mean ± SD 2.31 ± 0.38 1.71 ± 0.50
p value1 0.007*
p value2
p value3
Entire skin
Mean ± SD 2.26 ± 0.37 1.68 ± 0.51
p value1 0.015*
p value2
p value3
1: Comparison with healthy control skin
2: Comparison with peri-lesional skin
3: Comparison with lesional skin before treatment
*: p value (<0.05) statistically significant
expression values in the dermal structures were significantly
low ranging from 1.25 ± 0.44 (vascular endothelium) to 1.40 ±
0.60 (sweat gland epithelium). A summary of these data is
presented in Tables 2 and 3 and in Figures 2 and 3.
Increased TGF-β protein expression in the lesional skin of
melasma patients following PRP treatment
We found a considerable increase in the TGF-β protein
expression in the lesional skin of melasma patients following
PRP treatment, not only in the epidermal layers but also in
the dermal structures. These expression values were higher
than those of the perilesional skin and were nearly close to
those of the healthy skin. In the epidermis of the treated
lesional skin, TGF-β protein expression ranged from 2.05 ±
0.61 (spinous cells) to 2.25 ± 0.44 (basal cell keratinocytes). In
the dermis, these values ranged from 2.15 ± 0.59 (nerves) to
2.25 ± 0.64 (follicular epithelium). The differences between
some TGF-β protein expression values in the treated lesional
skin were statistically significant when compared to values in
the lesional skin before treatment. A summary of these find-
ings is shown in Tables 2 and 3 and in Figures 2 and 3.
Discussion
The promising therapeutic effects of PRP in the treatment of
pigmentary disorders such as melasma and periorbital hyperpig-
mentation (8–10) is based on its abundant contents of growth
factors; each possibly represents a part of PRP treatment success
(9,20,21). However, our understanding of underlying molecular
changes associated with its beneficial therapeutic effects in mel-
asma is lacking (8,9). In this investigation, our hypothesis believes
that TGF-β1 relates to the improvement of melasma with altera-
tion of TGF-β1 protein expression. To our knowledge, our ther-
apeutic trial is the first systematic study investigating the
expression pattern of TGF-β following the application of PRP
injection in face of melasma patients. Our study clearly elucidate
the following findings: i) amelioration of basal cell hyperpigmen-
tation in the skin of melasma patients following PRP treatment, ii)
decreased TGF-β protein expression in the lesional skin of
JOURNAL OF COSMETIC AND LASER THERAPY 5
Lesional skin before treatment Lesional skin after treatment
n = 20 n = 20
1.11 ± 0.46 2.10 ± 0.54
0.000* 0.499
0.046* 0.033*
0.000*
1.34 ± 0.45 2.18 ± 0.53
0.000* 0.152
0.040* 0.120
0.000*
1.26 ± 0.41 2.15 ± 0.44
0.000* 0.360
0.040* 0.021*
0.000*
melasma patients (before PRP treatment) as compared to both
healthy and perilesional skin and iii) an increased TGF-β protein
expression in the lesional skin of melasma patients following PRP
treatment.
Amelioration of basal cell hyperpigmentation in the skin
of melasma patients following PRP treatment
In normal skin, the melanocytes are scattered among the basal
cell keratinocytes along the dermoepidermal interface. They
produce and export melanin to the neighboring kertainocytes
forming a sort of camouflage around them upon UV exposure
(22). Here, we observed epidermal hyperpigmentation in the
skin of melasma patients before PRP treatment. The hyper-
pigmentation is due to the effects of the solar damage which
stimulates the dermal fibroblasts to produce melanogenic
cytokines, including stem cell factor and hepatocyte growth
factor resulting in hyperpigmentation of the overlying epider-
mis (4). UV irradiation also elevates the levels of matrix
metalloproteinase which degrade collagens resulting in basal
vaculopathy and the basal basement membrane disruption
(23). Increased vascularization is associated with increased
stem cell factor levels. Increased numbers of dermal mast
cells and their histamine release are common findings in
skin of melasma patients. Histamine induce the proliferation
and migration of melanocytes (melanogenesis) (4).
In our investigation, we observed an amelioration or even
absence of hyperpigmentation of the basal cell layer of the skin
of melasma patients following PRP treatment. This finding
concurs with previous studies (10,24,25). Alshami et al. exam-
ined the therapeutic effects of three sessions of autologous PRP
injections into the periorbital area as well as the whole face in 50
patients with periorbital hyperpigmentation. The results were
documented by digital photography. Excellent, significant,
moderate and mild improvement were reported in two, six, 23
and 19 patients, respectively (10). Similarly, Mehryan et al.
examined the outcome of intradermal PRP injection (a single
session) in 10 patients with periorbital dark circles and crow’s
feet. The effects of PRP injections included improvement in
infraorbital color homogeneity but without significant changes
6 E. R. M. HOFNY ET AL.

Table 3. TGF-β protein expression in the epidermal layers, adnexal epithelium, and dermal structures.
Healthy skin (control) Peri-lesional skin Lesional skin before treatment Lesional skin after treatment
n=9 n = 10 n = 20 n = 20
Mean ± SD Mean ± SD Mean ± SD Mean ± SD
Basal cells 2.33 ± 0.71 1.70 ± 0.68 1.30 ± 0.47 2.25 ± 0.44
p value1 0.064 0.001* 0.570
p value2 0.090 0.017*
p value3 0.000*
Prickle cells 2.11 ± 0.60 1.60 ± 0.70 1.05 ± 0.51 2.05 ± 0.61
p value1 0.096 0.000* 0.801
p value2 0.028* 0.072
p value3 0.000*
Granular cells 2.11 ± 0.60 1.60 ± 0.70 1.05 ± 0.51 2.05 ± 0.61
p value1 0.096 0.000* 0.801
p value2 0.028* 0.072
p value3 0.000*
Cornified cells 2.11 ± 0.60 1.60 ± 0.70 1.05 ± 0.51 2.05 ± 0.61
p value1 0.096 0.000* 0.801
p value2 0.028* 0.072
p value3 0.000*
Dermal fibroblasts 2.33 ± 0.50 1.70 ± 0.48 1.35 ± 0.49 2.15 ± 0.59
p value1 0.017* 0.000* 0.446
p value2 0.075 0.048*
p value3 0.000*
Follicular epithelium 2.33 ± 0.71 1.50 ± 0.53 1.30 ± 0.47 2.25 ± 0.64
p value1 0.015* 0.001* 0.713
p value2 0.292 0.005*
p value3 0.000*
Sweat gland epithelium 2.56 ± 0.73 2.00 ± 0.82 1.40 ± 0.60 2.25 ± 0.55
p value1 0.122 0.001* 0.145
p value2 0.040* 0.386
p value3 0.000*
Vascular endothelium 2.33 ± 0.50 1.70 ± 0.48 1.25 ± 0.44 2.15 ± 0.49
p value1 0.017* 0.000* 0.364
p value2 0.020* 0.027*
p value3 0.000*
Nerves 1.89 ± 0.60 1.70 ± 0.48 1.35 ± 0.49 2.15 ± 0.59
p value1 0.483 0.024* 0.275
p value2 0.075 0.048*
p value3 0.000*
Sebaceous glands 2.67 ± 0.50 1.70 ± 0.48 1.35 ± 0.49 2.15 ± 0.59
p value1 0.002* 0.000* 0.030*
p value2 0.075 0.048*
p value3 0.000*
Lymphocytes 2.22 ± 0.44 1.70 ± 0.48 1.35 ± 0.49 2.15 ± 0.59
p value1 0.030* 0.001* 0.794
p value2 0.075 0.048*
p value3 0.000*
Arrector pili muscle fibers 2.11 ± 0.60 1.70 ± 0.48 1.35 ± 0.49 2.15 ± 0.59
p value1 0.122 0.003* 0.866
p value2 0.075 0.048*
p value3 0.000*
1: Comparison with healthy control skin
2: Comparison with peri-lesional skin
3: Comparison with lesional skin before treatment
*: p value (<0.05) statistically significant

in melanin contents (24). The amelioration of hyperpigmenta-
tion following PRP treatment may be reasoned to an increase in
skin volume making the skin more glowing. The volemic
increase is due to the formation of blood vessel (under the
effects of platelet-derived growth factor), collagen and compo-
nents of the extracellular matrix, including hyaluronic acid. The
latter increases skin tone and volume (25,26).
Decreased TGF-β protein expression in the lesional skin
of melasma patients (before PRP treatment) as compared
to both healthy and perilesional skin
In normal skin, TGF-β is expressed by the keratinocytes,
fibroblasts, endothelial cells and some dermal mesenchymal
and inflammatory cells (27–29). We found decreased expres-
sion values of TGF-β protein in both prelesional and lesional
skin of the patients with melasma as compared to the health
control skins. This decrease may be reasoned to sun exposure-
related suppression of TGF-β production both at transcrip-
tional (mRNA) and translational (protein) levels. The reduced
TGF-β expression is associated with amelioration of its inhi-
bitory effects on the cutaneous melanocytes, with subsequent
enhancement of melanogenesis (melanin production), epider-
mal hyperpigmentation and the development of melasma.
Our proposition is supported by several experimental obser-
vations. Exposure to UV irradiation is associated with sup-
pression (15,30) or even cessation (22) of TGF-β expression at
the transcriptional and protein levels both in the epidermal
keratinocytes and in the fibroblasts. In the absence of UV
radiation, TGF-β is released by the keratinocytes, which
induces Smad signaling in melanocytes to repress paired-box
homeotic gene (PAX3) and therefore block melanocyte

Figure 3. Mean TGF-β protein expression values in the epidermis, dermis and
entire skin.
In the healthy skin the mean value of TGF-β expression was 2.26 ± 0.37. The
expression in the perilesional skin was significantly lower (1.68 ± 0.51) than that
in the healthy skin (p value<.05). In the lesional melasma skin, TGF-β protein was
significantly downregulated as compared to both healthy and perilesional skins,
the mean expression was 1.26 ± 0.41. However, following treatment with PRP
the expression of TGF-β protein was significantly upregulated in the lesional
melasma skin. The mean value of expression was 2.15 ± 0.44. It approached the
mean expression in the healthy skin with no significant difference (p value>.05).
differentiation, i.e., TGF-β negatively regulates melanocytes.
TGF-β is the most important regulator of PAX-3 in adult
melanocytes (15,22). The protein encoded by paired-box
homeotic gene 3 is a key regulator of the microphthalmia-
associated transcription factor (MITF) in the melanocyte line-
age. PAX3 encodes a transcription factor crucial for melano-
cyte differentiation. Following UV irradiation, a Jnk/AP-1
pathway inhibits TGF-β, which together with a UV-induced
p53 pathway stimulate the differentiation of the cutaneous
melanocyte (15,22). Moreover, in vitro, TGF-β induces mela-
nocyte immaturity by downregulating MITF. The latter is
a transcriptional regulator of melanocyte differentiation, and
its downstream melanogenic genes. In vivo, activation of
TGF-β signaling contributes to the entry of the melanocyte
stem cells in the quiescent noncycling state (31).
Increased TGF-β protein expression in the lesional skin of
melasma patients following PRP treatment
Exposure to ultraviolet light is a major predisposing factor in
melasma (32,33). Several observations suggest that melano-
cytes, keratinocytes, and fibroblasts play role in the develop-
ment and relapse of melasma (34).In our investigation, we
found that TGF-β1 protein expression is increased in the
lesional skin of melasma patients following PRP treatment.
This upregulation of TGF-β1 protein expression was asso-
ciated with considerable clinical improvement. We propose
that the increased TGF-β protein expression following PRP
treatment is reasoned to enrichment of skin by growth factors
including TGF-β, released upon degranulation of α-granules
of the concentrated platelets in PRP. TGF-β is well known for
its autocrine stimulatory effects (35) and therefore, it is con-
ceivable to propose that TGF-β protein (one of PRP contents)
enhances the transcription of TGF-β gene to TGF-β mRNA
with subsequent increased TGFβ protein synthesis and inhibi-
tion of the melanocytes resulting in decreased melanin pro-
duction (melanogenesis) and clinical improvement of the
melasma (amelioration of epidermal hyperpigmentation).
JOURNAL OF COSMETIC AND LASER THERAPY 7
To conclude, here we provide the first indication about the
immunohistochemical expression of TGF-β protein, a major
constituent of plasma rich platelets, in melasma patients. Our
data provide a rich foundation for further investigations about
the therapeutic roles of PRP in melasma. For future research,
it is tempting to examine the upstream and downstream
molecules interacting with TGF-β in melasma such as tran-
scription of activating protein-1, p53, PAX3, MITF, tyrosinase
(TYR), tyrosinase-related protein 1.
Acknowledgments
This study was funded by the Research Grant Office, Quality assurance
Unit at Faculty of Medicine, Assiut University.
Funding
This study was funded by the Research Grant Office, Quality assurance
Unit at Faculty of Medicine, Assiut University, [Grant number: 15509].
ORCID
Mahmoud Rezk Abdelwahed Hussein http://orcid.org/0000-0001-
9366-2211
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