VEGF upregulates VEGF receptor-2 on human outer root sheathcells and stimulates proliferation through ERK pathway

Wei Li • Zhong-Fa Lu • Xiao-Yong Man • Chun-Ming Li •

Jiong Zhou • Jia-Qi Chen • Xiao-Hong Yang •

Xian-Jie Wu • Sui-Qing Cai • Min Zheng

Received: 29 January 2012 / Accepted: 6 June 2012 / Published online: 16 June 2012

� Springer Science+Business Media B.V. 2012

Abstract Vascular endothelial growth factor (VEGF) is a

key regulator of physiological and pathological angiogen-

esis. The biological effects of VEGF are mediated by

receptor tyrosine kinases. VEGF receptor-2, the primary

receptor for VEGF, is thought to mediate most functional

effects. In this study, we examined the expression and roles

of VEGF receptor-2 on human outer root sheath cells

(ORS). The expression of VEGFR-2 was determined at

mRNA and protein levels by reverse transcription-poly-

merase chain reaction (RT-PCR) and Western blot.

Localization of VEGFR-2 in ORS cells was detected by

immunofluorescence. The effect of VEGF on ORS cell

proliferation was determined by MTT assays. Our data

showed the expression of VEGFR-2 on ORS cells at both

mRNA and protein levels. Immunostaining for VEGFR-2

demonstrated strong signal on cultured ORS cells. Exoge-

nous VEGF165 stimulated proliferation of ORS cells and

upregulated expression of VEGFR-2 in a dose-dependent

manner. Moreover, VEGF165 induced phosphorylation of

VEGFR-2, PLC-c1, PKC-a, MEK, and p44/42 MAPK

(ERK1/2) in a time-dependent manner. Taken together,

human ORS cells express functional VEGF receptor-2 and

exogenous VEGF165 upregulates expression of VEGFR-2

and stimulates proliferation of ORS cells via VEGFR-2

mediated ERK signaling pathway.

Keywords VEGF � VEGFR � Outer root sheath �Proliferation � ERK � Hair follicle

Abbreviations

VEGF Vascular endothelial growth factor

VEGFR Vascular endothelial growth factor receptor

ORS Outer root sheath

DPC Dermal papillae cells

MAPK Mitogen-activated protein kinase

ERK Extracellular signal regulated kinase

JNK C-Jun N-terminal protein kinase

PI3K Phosphatidylinositol-3-kinase

PLC-c Phospholipase C-cPKC-a Protein kinase C-aDKSFM Defined keratinocyte serum-free medium

Introduction

Hair follicle, the only organ that undergoes cyclic trans-

formations for entire life in the mammalian body, consists

of epidermal layers as well as dermal compartments

including dermal papilla [1]. Epidermal layers are grouped

into outer root sheath (ORS), inner root sheath (IRS) and

hair shaft. The ORS cells are morphologically similar to

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11033-012-1725-6) contains supplementarymaterial, which is available to authorized users.

W. Li � Z.-F. Lu � X.-Y. Man � J. Zhou � J.-Q. Chen �X.-J. Wu � S.-Q. Cai � M. Zheng (&)

Department of Dermatology, Second Affiliated Hospital,

Zhejiang University School of Medicine, 88 Jiefang Road,

Hangzhou 310009, Zhejiang, China

e-mail: minz@zju.edu.cn

C.-M. Li

Department of Dermatology, Second Affiliated Hospital,

Nanchang University School of Medicine,

Nanchang 330000, China

X.-H. Yang

Department of Dermatology, Zhejiang Provincial Hospital

of Traditional Chinese Medicine, Hangzhou 310009, China

123

Mol Biol Rep (2012) 39:8687–8694

DOI 10.1007/s11033-012-1725-6

the basal keratinocytes of the interfollicular epidermis and

play an important role during hair cycle, hair growth and

healing of skin wounds [2–4].

Vascular endothelial growth factor (VEGF, commonly

referred to VEGF-A), which belongs to a family of growth

factors, including VEGF-B, VEGF-C, VEGF-D and pla-

cental growth factor (PLGF) in mammals, is a key regu-

lator of vessel formation during embryonic development,

wound healing and maintaining vessel homeostasis in adult

organisms [5, 6]. The biological effects of VEGF are

mainly mediated through receptor tyrosine kinases (RTK),

known as VEGF receptor-1, -2 and -3 (VEGFR1-3). The

predominant receptor for VEGF, VEGFR-2, is thought to

mediate most functional effects such as cell proliferation,

migration and survival [6–9].

We previously demonstrated that VEGF and VEGFR-2

are not only expressed in normal human epidermis, but also

regulate the functions of epidermal keratinocytes such as

enhancing proliferation and migration of keratinocytes,

decreasing the adhesion ability of keratinocytes [10–12].

Kozlowska et al. [13] had displayed expression of VEGF in

various compartments of the human hair follicle a decade

ago. However, little is known as to whether VEGFR-2 is

expressed by hair follicle and its potential roles up to now.

Recently we have shown that VEGF and VEGFR-2 are

both expressed in human anagen hair follicles including

outer root sheath and dermal papillae by immunofluores-

cence [14]. In the work presented here, we give further

evidence for the expression of functional VEGFR-2 in

human outer root sheath cells. Moreover, we show here

that VEGF up-regulated expression of VEGFR-2 and

stimulated proliferation of ORS cells through VEGFR-2

mediated extracellular signal regulated kinase (ERK) sig-

naling pathway.

Materials and methods

Chemicals and reagents

Dispase, trypsin, defined keratinocyte serum-free medium

(KSFM) supplemented with keratinocyte growth factor

(KGF), fetal bovine serum (FBS), and Trizol were obtained

from Gibco and Invitrogen (Invitrogen, Auckland, USA).

Monoclonal mouse anti-human VEGFR-2 antibodies were

purchased from R&D Systems (MAB3571, Minneapolis,

MN, USA) and Santa Cruz Biotechnology (SC-6251, Santa

Cruz, CA, USA). Rabbit anti-human phospho-VEGFR-2

(Tyr1175), phospho-PLC-c1 (Tyr783), PLC-c1, phospho-

PKC-a (Thr638/641), PKC-a, phospho-MEK (Ser217/221),

MEK, p44/42 MAP kinase antibodies and mouse anti-

human phospho-p44/42 MAPK (Thr202/Tyr204) antibody

were obtained from Cell Signaling Technology (Beverly,

MA, USA). Mouse anti-human GAPDH was from Kang-

Chen Bio-tech (Shanghai, China). Horseradish peroxidase–

linked anti-mouse IgG was from Jackson ImmunoResearch

Laboratories (West Grove, PA, USA). Polyclonal rabbit

anti-mouse immunoglobulins/FITC was from DakoCyto-

mation (Denmark). VEGF165 from Chemicon International

Inc. (Temecula, CA, USA); Moloney murine leukemia

virus (MMLV) reverse transcriptase and RNase inhibitor

from Fermentas (Amherst, NY, USA); cocktail protease

inhibitors from Roche Diagnostics (Indianapolis, IN,

USA); propidium iodide (PI) and mouse IgG from serum

from Sigma-Aldrich (St. Louis, MO, USA). Primers were

synthesized by Sangon (Shanghai, China), and purified

PCR products were directly sequenced by Genscript

(Nanjing, China).

Isolation and culture of ORS cells from human scalp

specimens

This study conformed to the principles outlined in the

Declaration of Helsinki and was approved by Zhejiang

University Institutional Review Board. Normal human

scalp specimens (n = 16, including 9 females and 7 males,

aged 18–50) were obtained as excess tissues from subjects

with informed consent, who were undergoing cosmetic

surgery without systemic disease. The specimen was cut

into small pieces and put into 0.5 % dispase for overnight

incubation at 4 �C. Hair follicles were extruded from the

specimen and then further incubated in 0.25 % trypsin with

0.03 % EDTA at 37 �C for 30 min. Trypsin activity was

neutralized by adding fetal bovine serum (FBS). To obtain

ORS cell suspension, the isolated cells were filtrated

through nylon gauze (200 lm mesh) and ORS cells were

washed twice at 500 g for 5 min prior to resuspension in

complete defined keratinocyte serum-free medium

(DKSFM). ORS cells were plated into 25 cm2 culture flask

(Corning, NY, USA), maintained at 37 �C in a humidified

atmosphere containing 5 % CO2. Passages 2 to 4 were used

in all experiments. HUVECs, expressing VEGFR-2 and

serving as a positive control, were obtained from isolated

umbilical veins by a standard method [10].

Indirect immunocytochemistry assay

Indirect immunocytochemistry assays were performed as

described previously [10, 11]. Briefly, cells were seeded on

coverslips in 24-well culture plates. When cells were

60–70 % confluence, the coverslips were then fixed with

4 % paraformaldehyde buffer for 20 min at room temper-

ature. After incubation in sodium citrate buffer (10 mM,

pH 8.5) at 95 �C for 20 min, the coverslips were perme-

abilized at room temperature for 15 min with phosphate

buffered saline (PBS) containing 0.1 % Triton-100, and

8688 Mol Biol Rep (2012) 39:8687–8694

123

incubated with 10 % normal rabbit serum for 1 h at room

temperature to prevent nonspecific binding. Fixed cover-

slips were then incubated with antibodies against VEGFR-

2 (mouse anti-human, sc-6251; Santa Cruz Biotechnology,

CA, USA) overnight at 4 �C, followed by fluorescein iso-

thiocyanate-conjugated rabbit anti-mouse secondary anti-

body (Aako Cytomation; Dako, Glostrup, Denmark)

diluted 1:40 with 10 % rabbit serum in PBS incubated for

2 h at room temperature in the dark. Coverslips were

counterstained with propidium iodide mounting medium

(Sigma-Aldrich, St Louis, MO, USA) to visualize the

nuclei and analyzed by fluorescence microscopy (Olympus,

Tokyo, Japan). For each case, a negative control incubated

with nonimmune mouse IgG (Sigma-Aldrich, St Louis,

MO, USA) was included.

Reverse transcription and polymerase chain reaction

(RT-PCR)

Total RNA was extracted from VEGF treated or untreated

ORS cells using Trizol, according to the manufacturer’s

instructions. Total RNA was reverse-transcribed into first

strand cDNA in a total reaction volume of 20 ll, as

described previously [10]. The following parameters were

used for PCR: 95 �C for 5 min, followed by 36 cycles of

95 �C for 1 min, 60 �C (for VEGFR-2) or 55 �C (for CD31

and GAPDH) for 45 s, and 72 �C for 45 s, and ending with

a full extension cycle of 72 �C for 10 min. In each

experiment, negative control (using total RNA as the

template) was included and GAPDH served as an internal

control. Sequence identity of PCR product was confirmed

by direct sequencing. Primers were designed based on

published cDNA sequences as follows:

VEGFR-2 (NM_002253).

50-GACGGACAGTGGTATGGTT-30 (forward).

50-CCGAGTCAGGCTGGAGAAT-30 (reverse).

CD31 (BC051822).

50-CTTCGCGGATGTCAGCACCAC-30 (forward).

50-CCTCAACGGGGAATTCCAGTATCA-30 (reverse).

GAPDH (NM_002046).

50-TGAAGGTCGGAGTCAACGG-30 (forward).

50-TGGAAGATGGTGATGGGAT-30 (reverse).

Western blot analysis

Western blot were carried out as previously described [10,

11]. Briefly, ORS cells grown to 80–90 % confluence were

washed twice in ice-cold PBS, scraped, and centrifuged.

The pellet was incubated for 30 min in modified RIPA

lysis buffer then centrifuged. Protein concentrations were

measured using the QuantiPro BCA assay kit (Sigma-

Aldrich). Total cellular protein was separated by sodium

dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-

PAGE) and then transferred to PVDF membrane (Hybond-

P, Amersham). Blots were blocked for 60 min at room

temperature in PBS containing 1 % Tween-20. After rins-

ing 3 times for 10 min with PBS containing 0.05 %

Tween-20, the membrane was probed with the respective

primary antibody overnight at 4 �C in PBS containing 1 %

Tween-20. Blots were then washed 4 times (5 min each) in

PBS containing 0.05 % Tween-20 and incubated for 2 h

with horseradish peroxidase–conjugated goat polyclonal

anti-mouse IgG antibody (1:5000; Jackson). The mem-

brane was washed 5 times (5 min each) with 0.05 %

Tween-20-PBS, and the immunoreactive bands were

detected using enhanced chemiluminescent (ECL) plus

reagent kit before exposure for at least 3 min to Kodak film

(Kodak, Rochester, NJ, USA). Mouse monoclonal anti-

GAPDH (diluted 1:5000) was used as a loading control.

HUVECs were used as a positive control in all panels.

Effect of VEGF165 on ORS cell proliferation

Normal human ORS cells were plated in 96-well plates at

5,000 cells/well in defined KSFM and cultured for 24 h.

Cells were then treated with various concentrations of

VEGF165 at 0, 1, 10, 50, and 100 ng/ml in basal defined

KSFM and incubated for 48 h. At the end of the incuba-

tion, freshly prepared and filtered dimethylthiazol-diphe-

nyltetrazolium bromide (MTT) (Sigma, St Louis, MO,

USA) was added and the mixture was further incubated for

4 h. After the medium was removed, cells in each well

were dissolved with 100 ll DMSO, and optical density was

read at 570 nm with a 96-well plate ELISA reader (Bio-

Rad Instruments, CA, USA).

Statistical analysis

Results are expressed as mean ± SD. All determinations

were performed in triplicate and experiments were repeated

at least 3 times. One-way ANOVA was used to evaluate

significant differences. Statistical analysis were performed

by SPSS Software (V13.0, SPSS, USA), with a

P value \ 0.05 considered to be statistically significant.

Results

Human ORS cells expressed VEGFR-2 mRNA

and protein

The expression of VEGFR-2 mRNA was detected in cul-

tured human ORS cells from all three normal donors

(Fig. 1a). Direct sequencing for PCR product was identical

Mol Biol Rep (2012) 39:8687–8694 8689

123

to published sequences of human VEGFR-2 (NM_002253).

The expression of CD31 gene was detected in HUVECs but

not in any sample of cultured ORS cells, suggesting no

contamination of endothelial cells in cultured ORS cells

(Fig. 1a).

VEGFR-2 protein was determined by Western blot. The

bands of VEGFR-2 were at about 180 and 200 kDa (indi-

cating the glycosylation or phosphorylation of VEGFR-2,

Fig. 1b). Immunostaining for VEGFR-2 showed that strong

positive signal was distributed on the membrane and in the

cytoplasm of cultured ORS cells (Fig. 1c).

VEGF165 stimulated proliferation of cultured ORS cells

in a dose-dependent manner

VEGF165 at the concentration of 50 ng/ml significantly

increased proliferation of cultured ORS cells compared to

the untreated control (p \ 0.05) (Fig. 2). Doses lower than

50 ng/ml of VEGF tended to stimulate proliferation of

ORS cells without statistical significance.

VEGF165 upregulated the expression of VEGFR-2

on cultured ORS cells in a dose-dependent manner

To investigate whether VEGF affects the expression of

VEGFR-2, ORS cells were incubated with various

concentrations of VEGF165 at 0, 1, 10, 50 and 100 ng/ml in

basal defined KSFM for 24 h. We found that VEGF165

significantly upregulated expression of VEGFR-2 mRNA

and protein in dose-dependent manners (Fig. 3a, b).

Fig. 1 Expression of VEGFR-2 in cultured human ORS cells. a RT-

PCR analysis of VEGFR-2 and CD31 gene in HUVECs and cultured

ORS cells from 3 different healthy samples. NEG is a negative control

showing no amplification from potential genomic DNA contamina-

tion. GAPDH served as an internal control for different amplification

performed for respective genes. b Immunoblot analysis of VEGFR-2

in protein extracted from 3 different cultured ORS cells. The anti-

VEGFR-2 showed bands at about 180 and 200 kDa. GAPDH served

as a loading control for protein normalization. c Immunofluorescence

staining for VEGFR-2. Strong immunostaining signal for VEGFR-2

(green color) was observed on cultured ORS cells. d Staining with

non-immune mouse IgG as a negative control. The cellular nuclei

were stained with PI (red color). Scale bar: 50 lm. (Color figure

online)

Fig. 2 Effect of VEGF165 on cultured ORS cell proliferation. MTT-

based assays were performed to determine the effects of VEGF165 for

48 h treatment. VEGF165 stimulated proliferation of cultured ORS

cells in a dose-dependent manner. Data represent the mean values of

optical density measured in 5 wells for each treatment. The

experiment was repeated at least 3 times with similar results.

(*p \ 0.05: compared to untreated control)

8690 Mol Biol Rep (2012) 39:8687–8694

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VEGF165 induced phosphorylation of VEGFR-2,

PLC-c1, PKC-a, MEK, and p44/42 MAPK (ERK1/2)

in cultured ORS cells

50 ng/ml of VEGF165 phosphorylated VEGFR-2, which

has intrinsic kinase activity. Phosphorylation of VEGFR-2

reached peak level at 15 min and regressed to the basal

level at 120 min (Fig. 4a). Phosphorylation of PLC-c1 was

elevated by VEGF165 treatment and reached the maximum

level at 15 min, sustained at 30 min, then gradually went

back to the basal level (Fig. 4b). Phosphorylation of PKC-awas found at 5 min and peaked at 15 min, then regressed to

the baseline (Fig. 4c). The phosphorylation of MEK also

reached maximal level at 15 min and maintained at

30 min, then returned to the basal level (Fig. 4d).

Enhanced ERK1/2 phosphorylation by VEGF165 was found

at 5 min and peaked at 15 min, and the activation ceased

after 60 min (Fig. 4e).

Discussion

Hair follicle is known for its cyclic transformations from

stages of rapid growth (anagen) to relative quiescence

(telogen), then to apoptosis-driven regression (catagen) and

back to anagen again [1, 3]. Accompanied with stages of

hair cycle is perifollicular vascularization, which promotes

hair growth and increases hair follicle and hair size [1, 15].

Yano and colleagues [15] reported that perifollicular

angiogenesis is temporally and spatially correlated with

upregulation of VEGF expression by ORS cells, but the

mechanism of VEGF enhancing hair growth is still

obscure. During hair cycle, both epidermal and dermal

layers undergo cyclic and dramatic changes in prolifera-

tion, differentiation and apoptosis. Central to the hair fol-

licle growth are epithelial–mesenchymal interactions,

which involve many molecular mediators such as fibroblast

growth factor (FGF), transforming growth factor (TGF)-b,

and vascular endothelial growth factor (VEGF) etc. [1–3,

16, 17], but how the dermal papillae cells (DPCs) exert

their power over epithelial layers such as ORS cells

remains unclear.

VEGF receptors were initially thought to be expressed

exclusively in endothelial cells [6, 8, 9], but recently,

growing studies have found expression of VEGF receptors

in other cell types and tissues [10, 11, 18–20]. VEGFR-2,

the primary receptor of VEGF, is implicated in all aspects

of normal and pathological vascularization and regulates

endothelial cell proliferation, migration, differentiation and

survival as well as vessel permeability and dilation [6, 8,

21]. Our previous work has demonstrated that VEGF

receptors are expressed in normal human epidermal

keratinocytes, and through activating VEGFR-2, VEGF

stimulates proliferation and migration of epidermal

Fig. 3 Effect of VEGF165 on mRNA and protein levels of VEGFR-2

in cultured ORS cells. ORS cells were cultured to 80–90 % confluent

in growth medium, then switched to basal defined KSFM with various

concentrations of VEGF165 (0, 1, 10, 50, and 100 ng/ml) for 24 h

before harvest. a RT-PCR analysis of VEGFR-2 mRNA expression in

cultured ORS cells in response to different concentrations of

VEGF165; GAPDH served as an internal control. The columnar

section was relative quantitative analysis of VEGFR-2 at mRNA

levels, which was shown after normalization to the endogenous

control GAPDH. b Western blot analysis of VEGFR-2 in cultured

ORS cells exposed to VEGF165; GAPDH served as a loading control

for protein normalization. The columnar section was relative quan-

titation of VEGFR-2 protein levels, which were shown after

normalization to the endogenous control GAPDH. Bars represented

mean ± SD in triplicate (*p \ 0.05; **p \ 0.01: compared to

untreated controls)

Mol Biol Rep (2012) 39:8687–8694 8691

123

keratinocytes [10]. Based on these important roles of

VEGF receptor-2, we hypothesized that VEGF receptor-2

might also be expressed by hair follicle, as which is lined

with epithelial cells.

A decade ago, Kozlowska et al. [13] detected VEGF

expression in human hair follicle. Recently, our study has

shown both VEGF and VEGFR-2 are expressed in human

anagen hair follicle including outer root sheath and dermal

papillae [14]. Furthermore, ORS cells were isolated from

normal human scalp hair follicles and detected by RT-PCR

and western blot analysis which showed the expression of

VEGFR-2 in cultured human ORS cells at mRNA and

protein levels (Fig. 1a, b). And intense immunostaining

signal for VEGFR-2 was observed on ORS cells (Fig. 1c).

To investigate potential effects of VEGF165 on cultured

ORS cells, we performed MTT-based assays which dem-

onstrated that exogenous VEGF165 could stimulate prolif-

eration of cultured ORS cells in a dose-dependent manner

(Fig. 2). Consistent with this, exogenous VEGF165 upreg-

ulated expression of VEGFR-2 at both mRNA and protein

levels in dose-dependent manners (Fig. 3).

The signaling pathways of VEGFR-2 have been well-

investigated in endothelial cells [6–8, 22–24]. MAPK is the

key player in VEGF-induced physiological and

pathological effects including proliferation, migration, sur-

vival and permeability [7, 8]. P38 MAPK has been impli-

cated in VEGFR-2 mediated migration [7, 25], and PI3 K/

Akt pathway is involved in VEGFR-2 mediated migration

and survival [6, 26, 27]. The signaling pathway of cell pro-

liferation induced by VEGFR-2 is extracellular signal reg-

ulated kinase (ERK) pathway, while other proliferation

signaling pathways such as SAPK/JNK, Jak/Stat are not

involved in VEGFR-2 mediated proliferation [7, 8, 21, 23,

24]. Through binding with VEGFR-2, VEGF induces auto-

phosphorylation of VEGFR-2, and then PLC-c1 binds to

phosphorylated Tyr1175 of VEGFR-2 and is tyrosine-

phosphorylated [8, 22]. Phosphorylated PLC-c1 activates

PKC-a by the generation of diacylglycerol and increased

concentration of intracellular calcium [8, 23]. The activated

PKC-a stimulates the mitogen-activated protein kinase

(MAPK)/extracellular-regulated kinase-1/2 (ERK1/2) cas-

cade and mediates the downstream proliferation effect. As

shown in Fig. 4, VEGF stimulated proliferation of cultured

ORS cells via the above VEGF/VEGFR-2/PLC-c1/PKC-a/

MEK/ERK1/2 signaling pathway.

The above data confirm our hypothesis that functional

VEGFR-2 is expressed on human ORS cells and play

important roles in hair follicle, which would improve our

Fig. 4 VEGF165 induced phosphorylation of VEGFR-2(a), PLC-

c1(b), PKC-a(c), MEK(d) and p44/42 MAPK (ERK1/2) (e) in

cultured ORS cells. Serum-starved ORS cells were either untreated or

treated with 50 ng/ml of VEGF165 for 5, 15, 30, 60 or 120 min.

Phosphorylation of VEGFR-2, PLC-c1, PKC-a, MEK and ERK1/2 in

cultured ORS cells responded to VEGF165 in a time-dependent

manner. The histogram below the WB bands show the relative

intensity of phosphorylation of indicated proteins. GAPDH served as

loading control for protein normalization

8692 Mol Biol Rep (2012) 39:8687–8694

123

understanding of hair follicle development, growth, cycling

and the role of hair follicle during wound healing.

Yano et al. [15] proposed a paracrine mechanism of

VEGF by which the proliferative epithelial compartment of

hair follicle induces enhanced vascular support to meet

highly increased nutritional needs during anagen phase, but

our findings indicate that it is not the whole story. Since

ORS cells express functional VEGFR-2, there also exists

an autocrine mechanism. VEGF derived from hair dermal

papilla cells [28], and/or from ORS cells themselves [14,

15], could exert its effects through activating VEGFR-2

expressed on ORS cells. In addition, functional VEGFR-2

expressed on ORS cells provides a novel target of treating

hair disorders such as androgenetic alopecia, the follicles of

which express much less VEGF than normal follicles [29].

Similarly, the key role of ORS cells playing during the

healing of skin wounds [30] may also be mediated by

VEGF/VEGFR-2. More detailed analysis of VEGF/VEG-

FR-2 expressed on ORS cells need further study.

In summary, the present work shows that the human

ORS cells express functional VEGF receptor-2. Exogenous

VEGF upregulates expression of VEGFR-2 and stimulates

proliferation of cultured ORS cells via VEGFR-2 mediated

ERK signaling pathway. Our findings suggest an autocrine

signaling loop in ORS cells and that molecular targeting of

VEGF/VEGFR-2 may be a useful and novel strategy for

treatment of hair disorders.

Acknowledgments This work was supported by grants from the

National Natural Science Foundation of China (NSFC) (No.

81171497, 81171496, 81171521) and Qianjiang Talent Program

2009R10045.

Conflict of interests The authors declare that there is no conflict of

interest.

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