The Expression And Function Of VEGFR-2on Hair Follicle Epidermal Cells

Vascular endothelial growth factor (VEGF) has been investigated for its angiogenic behavior extensively in physiological and pathological conditions1-3which is a family including VEGF-A,-B,-C,-D, and-E, and placental growth factor (P1GF). VEGF-A, also named VEGF, is knowed as the most important factor in the family, includes different VEGF isotypes such as VEGF121, VEGF145, VEGF165, VEGF189, VEGF206according to the different slicing pattern during VEGF transcription course, and VEGF165is the most effective one of them4.VEGF family promotes the proliferation of endothelial cells, angiogenesis and vascular permeability by binding to VEGF receptors3’5which belong to the receptor tyrosine kinase (RTK) family (VEGFR-1, VEGFR-2, VEGFR-3) and non-tyrosine kinase transmembrane receptors (NRPs, including NRP-1and NRP-2). VEGFR-2is the major receptor for endothelial cell function6’7. Recently, VEGFR-2was found to be expressed not only on the endothelial cells but also on other non-endothelial cells8-11, such as retinal progenitor cells, neuronal cells. VEGFR-2was also detected on hair follicle keratinocytes, sebaceous glands and sweat glands. Confirmation of the biology activity of VEGFR-2expression on hair follicle keratinocytes will be helpful to clarify mechanisms of hair follicle regulation.ObjectiveOur aim in this paper is to confirm the expression of VEGFR-2on hair follicle keratinocytes and investigate whether VEGF secreted by DPC not only acts in an paracrine pattern by binding to VEGFR-2on themselves to promote angiogenesis but also is involved in regulating hair follicle keratiniocytes directly. Furthermore, we will investigate the expression on hair follicle bulge cells and its roles in the proliferation, adhesion, migration on bulge cells and hair cycling regulation. By the way, the downstream signal expression of VEGFR-2will be investigated.Methods1. Detect the expression of VEGFR-2during the hair cycling in mice by immunofluorescence test. By using double immunofluorescence techniques, the paper discusses the relationship between VEGFR-2and the mark of proliferation, differentiation, apoptosis in hair follicles.2. RT-PCR、Western-blot and indirect immunofluorescent staining were used to determine the mRNA and protein expression of the VEGFR-2on hair follicle bulge cells. MTT assays were used to determine the effect of the VEGF165on the proliferation and adhesion abilities of hair follicle bulge cells. Migration assays were used to determine the effect of the VEGF165on the migration of hair follicle bulge cells. Neutralizing antibody to VEGFR-2, MAB3572, was used to determine the role of VEGFR-2on proliferation、migration and adhesion of hair follicle bulge cells induced by VEGF16. The effect of VEGF165at25ng/ml on phosphorylation of VEGFR-2, P38, C-Jun, ERK1/2, Artemis (serine516) were detected by western-blot in hair follicle bulge cells pretreated with or without MAB3572at0.5ug/ml.3. RT-PCR were used to determine the Artemis (serine516) mRNA expressed in hair follicles. Artemis (serine516) expression on mouse hair follicle during hair cycling and on human scalp were also observed by indirect immunofluorescent staining. Indirect immunofluorescent co-staining was used to determine the effect of Artemis (serine516) on the proliferation, apoptosis and differentiation of hair follicle keratinocytes.Results1. VEGFR-2was expressed on human hair follicle, and varied on mouse hair follicle during hair cycling.2. VEGFR-2was co-stained with K16、K14、Bax、Bcl-2, Ki67、p-catenin、 integrin β1on hair follicle. Furthermore, VEGFR-2was also co-stained with K10and involucrin on hair cortex, but the relationship reversed out of cortex.3. The mRNA and protein expression of the vascular endothelial growth factor receptors-2(VEGFR-2) on hair follicle bulge cells were confirmed by RT-PCR. Western-blot and indirect immunofluorescent staining. VEGF165enhanced VEGFR-2expression on hair follicle bulge cells, and neutralizing antibody to VEGFR-2(MAB3572) reversed it.4. VEGF165enhanced the proliferation, heterogeneity adhesion and migration of hair follicle bulge cells, and inhibited homogeneous adhesion.5. Neutralizing antibody to VEGFR-2(MAB3572) reversed proliferation, adhesion and migration activity of hair follicle bulge cells induced by VEGF165.6. Neutralizing antibody to VEGFR-2(MAB3572) reversed promotion of integrin β1expression and inhibition of Lgr6expression on cultured hair follicle bulge cells. 7. Neutralizing antibody to VEGFR-2(MAB3572) reversed up-regulation of the K16、K19、K14expression by VEGF165on cultured hair follicle bulge cells.8. Neutralizing antibody to VEGFR-2(MAB3572) reversed down-regulation of the K10、lnvolucrin expression by VEGF165on cultured hair follicle bulge cells.9. VEGF165induced the phosphorylation of VEGFR-2, c-Jun, ERK1/2and p38in cultured hair follicle bulge cells, which were inhibited by pretreating with neutralizing antibody to VEGFR-2(MAB3572).10. VEGF165prmote the the phosphorylation of Artemis at serine516in cultured hair follicle bulge cells, which were inhibited by pretreating with neutralizing antibody to VEGFR-2(MAB3572).11. Artemis (serine516) was expressed on mouse hair follicle varied during hair cycling. Artemis (serine516) expressed on upper part of human anagen hair follicle, and also expressed on sweat glands and sebaceous glands.12. Artemis (serine516) was co-stained with K16on hair follicle, but reversed with K10、p-P53、Bax、bcl-2、K14and K10.And c-myc, p21express located near the Artemis (serine516), signal representation is consistent with the trend of the Artemis (serine516) expression.Conclusions1. The identified VEGFR-2is expressed by hair follicle epidermal cells.2. VEGFR-2is involved in proliferation, adhesion, differentiation and migration of hair follicle bulge cells.3. VEGFR-2is involved in hair cycling regulation.4. VEGFR-2functions in hair follicle partly through Artemis phosphorylation.