Xian-Jie Wu

Expression and localization of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 in human epidermal appendages: a comparison study by immunofluorescence

Background.  Vascular endothelial growth factor (VEGF) promotes angiogenesis and plays important roles in neovascularization and development of tissues. VEGF receptors (VEGFRs) are high‐affinity receptors for VEGF and are originally considered specific to endothelial cells. We have previously shown that keratinocytes from human normal skin express VEGFRs. This poses the question of whether these receptors are also expressed by epidermal appendages, as epidermal appendages are lined with epithelial cells.

VEGF165 modulates proliferation, adhesion, migration and differentiation of cultured human outer root sheath cells from central hair follicle epithelium through VEGFR-2 activation in vitro.

BACKGROUND The functional state of vasculature is tightly controlled by vascular endothelial growth factor receptor-2 (VEGFR-2). Recent studies revealed that VEGFR-2 is expressed on hair follicle keratinocytes. OBJECTIVE We proposed to investigate its effect on proliferation, adhesion and migration of cultured human outer root sheath cells from central hair follicle epithelium. METHODS These studies were undertaken in vitro using human outer root sheath cells from central hair follicle epithelium, immunohistochemistry analysis, immunofluorescence microscopy, western blot analysis, MTT, trans well analysis, and RT-PCR. RESULTS Our results show that VEGFR-2 is expressed in these cells in vivo and in vitro. Furthermore, proliferation and migration of cultured human outer root sheath cells from central hair follicle epithelium is increased by VEGF165, while homotypic adhesion is decreased but heterotypic adhesion is increased. VEGF165 upregulates integrin β1 but dowregulates lgr6 expression. In addition, phosphorylation of VEGFR-2, Erk1/2, c-Jun and p38, are increased following VEGF165 treatment and these effects are reversed by a VEGFR-2 neutralizing antibody. CONCLUSION Our results suggest a role of VEGF/VEGFR-2 beyond angiogenesis in hair follicle regulation.

Role of VEGF Receptors in Normal and Psoriatic Human Keratinocytes: Evidence from Irradiation with Different UV Sources

Vascular endothelial growth factor (VEGF) promotes angiogenesis and plays important roles both in physiological and pathological conditions. VEGF receptors (VEGFRs) are high-affinity receptors for VEGF and are originally considered specific to endothelial cells. We previously reported that VEGFRs were also constitutively expressed in normal human keratinocytes and overexpressed in psoriatic epidermis. In addition, UVB can activate VEGFRs in normal keratinocytes, and the activated VEGFR-2 signaling is involved in the pro-survival mechanism. Here, we show that VEGFRs were also upregulated and activated by UVA in normal human keratinocytes via PKC, and interestingly, both the activated VEGFR-1 and VEGFR-2 protected against UVA-induced cell death. As VEGFRs were over-expressed in psoriatic epidermis, we further investigated whether narrowband UVB (NB-UVB) phototherapy or topical halomethasone monohydrate 0.05% cream could affect their expression. Surprisingly, the over-expressed VEGFRs in psoriatic epidermis were significantly attenuated by both treatments. During NB-UVB therapy, VEGFRs declined first in the basal, and then gradually in the upper psoriatic epidermis. VEGFRs were activated in psoriatic epidermis, their activation was enhanced by NB-UVB, but turned undetectable after whole therapy. This process was quite different from that by halomethasone, in which VEGFRs and phospho-VEGFRs decreased in a gradual, homogeneous manner. Our findings further suggest that UV-induced activation of VEGFRs serves as a pro-survival signal for keratinocytes. In addition, VEGFRs may be involved in the pathological process of psoriasis, and UV phototherapy is effective for psoriasis by directly modulating the expression of VEGFRs.

Activation of VEGFR-2 signaling in response to moderate dose of ultraviolet B promotes survival of normal human keratinocytes.

Mounting evidence indicates that signaling via VEGF receptors (VEGFRs) extends beyond blood vessel formation. Recently, VEGFRs are also found to be constitutively expressed in keratinocytes and epidermal appendages. Here, we show that the expression of VEGFRs (including VEGFR-1, VEGFR-2, and NRP-1) was significantly enhanced by moderate dose of ultraviolet B (UVB) in normal human keratinocytes and epidermis. The elevated expression of VEGFRs by UVB was independent of autocrine stimulation by their natural ligand, VEGF, but mainly mediated through hypoxia and oxidative stress. Moderate dose UVB also promoted tyrosine phosphorylation of VEGFR-1 and VEGFR-2, this effect was again VEGF independent. Both α and δ isoforms of protein kinase C (PKC) were required for UVB-induced phosphorylation of VEGFR-1, but only the δ isoform was required for VEGFR-2 phosphorylation. The phosphorylation of VEGFRs or isoforms of PKC was completely inhibited by PP2, a specific inhibitor for Src family kinases (SFKs), indicating that SFKs are upstream of PKC and VEGFRs. Moderate dose UVB-induced VEGF exerted an anti-apoptotic effect for keratinocytes, whereas high dose UVB-induced VEGF played as an inflammatory factor. Of note, neutralization of VEGFR-2 but not VEGFR-1 exacerbated UVB-induced cell death and reduced survival of keratinocytes. Furthermore, VEGFR-2 neutralization inhibited the activation of ERK1/2 and Akt by UVB, suggesting that VEGFR-2 signaling was involved in the pro-survival mechanism via ERK1/2 and PI3-K/Akt pathway. Taken together, we demonstrate for the first time that VEGFR-2 signaling is activated and promotes survival of keratinocytes under moderate dose of UVB irradiation.

Expression of decorin throughout the murine hair follicle cycle: hair cycle dependence and anagen phase prolongation

Decorin is a prototypical member of the small leucine‐rich proteoglycan (SLRP) family, which is involved in numerous biological processes. The role of decorin, as a representative SLRP, in hair follicle morphogenesis has not been elucidated. We present our initial findings on decorin expression patterns during induced murine hair follicle (HF) cycles. It was found that decorin expression is exclusively restricted to the epidermis, outer root sheath and sebaceous glands during the anagen phase, which correlates with the upregulation of decorin mRNA and protein expression in depilated murine dorsal skin. Furthermore, we used a functional approach to investigate the effects of recombinant human decorin (rhDecorin) via cutaneous injection into HFs at various murine hair cycle stages. The local injection of rhDecorin (100 μg/ml) into the hypodermis of depilated C57BL/6 mice at anagen delayed catagen progression. In contrast, rhDecorin injection during the telogen phase caused the premature onset of anagen, as demonstrated by the assessment of the following parameters: (i) hair shaft length, (ii) follicular bulbar diameter, (iii) hair follicle cycling score and (iv) follicular phase percentage. Taken together, our results suggest that decorin may modulate follicular cycling and morphogenesis. In addition, this study also provides insight into the molecular control mechanisms governing hair follicular epithelial–mesenchymal interactions.

VEGF upregulates VEGF receptor-2 on human outer root sheath cells and stimulates proliferation through ERK pathway.

Vascular endothelial growth factor (VEGF) is a key regulator of physiological and pathological angiogenesis. 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-polymerase 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. Exogenous 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-γ1, PKC-α, 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.

Purse-string suture for round and oval defects: a useful technique in dermatologic surgery.

Background: Round and oval skin wounds, like facial pigmented nevi after excision, are traditionally sutured linearly for closure, leaving significant scars, which greatly influences their appearance. Objective: This article provides an overview of our experience using intradermal purse-string suture for round and oval defects in the faciocervical region to ascertain whether purse-string suture closure for such defects can result in good functional and cosmetic outcomes. Methods and Results: We present 63 cases with different skin lesions in the faciocervical area. The defects of the lesions after excision were closed using intradermal purse-string suture. The wounds showed good final healing without obvious adverse events postoperatively. The result of scar assessment using the Vancouver Scar Scale was satisfying, with a total score of only 1.11. The final cosmetic appearance of the healed wounds seemed to be excellent and acceptable as they were always smaller than the original defects, with minimal scarring. Conclusion: Purse-string suture enabled us to repair small, circular wounds easily after excision of skin lesions. It is an excellent alternative to other reconstructions and a rapid, simple method to close skin defects with minimal scarring, achieving an excellent long-term cosmetic and functional outcome.

Haploinsufficiency caused by a nonsense mutation in NCSTN underlying hidradenitis suppurativa in a Chinese family

Hidradenitis suppurativa (HS) is a chronic disease of follicular occlusion. It involves the axilla, groin, perianal and perineal regions, and is characterized by recurrent draining sinuses, skin abscesses and disfiguring scars. Loss‐of‐function mutations in the genes encoding γ‐secretase have been identified as a cause of HS. We collected skin samples from three patients with HS from a Chinese family carrying a NCSTN mutation (c.1258C>T (p.Q420X)) and three unrelated healthy controls (HCs). Expression level of nicastrin in skin tissue and cultured keratinocytes and fibroblasts of patients and HCs was determined by real‐time quantitative PCR and western blotting. We found that the mRNA and protein levels of nicastrin were significantly reduced in the whole skin, epidermis, dermis, and cultured keratinocytes and fibroblasts compared with HCs. Therefore, we conclude that haploinsufficiency of the NCSTN gene caused by the nonsense mutation c.1258C>T (p.Q420X) contributes to the occurrence of HS in this family.

Expression and localization of Artemis serine 516 phosphorylation in human scalp skin

Artemis phosphorylation at serine 516 (Ser516) has important regulatory functions in the repair of radiation‐induced DNA damage, V(D)J recombination, p53‐dependent apoptosis and cell cycle control. Accordingly, Artemis mutations can lead to Omenn syndrome, which is associated with human radiosensitive severe combined immunodeficiency syndrome and alopecia. In this study, we investigated the expression of Ser516 phosphorylation of Artemis in the epidermis and epidermal appendages in normal human scalp skin. Immunofluorescence analysis revealed Ser516 phosphorylation of Artemis in the upper and middle portion of anagen hair follicle [including outer root sheath (ORS), inner root sheath but not stratum basale], hair matrix, sebaceous glands (secretory and ductal portions), eccrine sweat glands (secretory and ductal portions) and epidermis (stratum basale and stratum granulosum), respectively. Artemis phosphorylation at Ser516 was most prominent in ORS keratinocytes. Therefore, we suggest that phosphorylation of Artemis at Ser516 could be involved in regulation of human epidermal appendages.

Morroniside regulates hair growth and cycle transition via activation of the Wnt/β-catenin signaling pathway

Hair loss is characterized by a shortened hair anagen phase and hair follicles (HF) miniaturization. Morroniside is the most abundant iridoid glycoside extracted from Cornus officinalis and has various bioactivities in different cell functions and tissue regeneration. In this study, we investigated the effects and the underlying mechanism of morroniside on hair growth and regulation of HF cycle transition. Morroniside treatment significantly enhanced outer root sheath cell (ORSC) proliferation and migration in vitro. Additionally, morroniside upregulated Wnt10b, β-catenin and lef1. The enhanced ORSC proliferation and migration due to morroniside treatment were partly rescued by a Wnt/β-catenin signaling inhibitor, DKK1. Furthermore, in a hair-induced mouse model, morroniside injection accelerated the onset of anagen and delayed HF catagen, as shown by histological examination. Immunohistochemical analyses revealed that Wnt/β-catenin signaling pathway expression was upregulated in the HFs. These findings suggest that morroniside regulates HF growth and development partly through the Wnt/β-catenin signaling pathway and may be a potential treatment for hair loss.