The Expression Of Angiogenin In Human Hair Follicles And Its Role In Regulating Hair Growth

BackgroundHair follicle(HF)is a highly complex appendage of the skin,which consists of the mesenchymal components including dermal papilla and dermal sheath and the epithelial components such as outer root sheath and inner root sheath.Each hair follicle undergoes a cycle of growth(anagen phase),regression(catagen phase)and resting(telogen phase).Numerous growth factors,cytokines,hormones and the corresponding receptors are involved in regulating the hair growth and hair cycle.It has been proved that the angiogenesis around the hair follicle is hair-cycle dependent,with significant vascularization observed during the anagen phase,and blood vessel degeneration and disappearance during the catagen phase.As such, enhanced vascularization of the hair follicle has been shown to promote hair growth and to increase the diameter of both hair follicles and hair shafts.In contrast,the inhibition of perifollicular angiogenesis can delay the hair follicle entering into anagen.Therefore,perifollicular vasculature plays an important role in regulating the hair cycle and hair growth.The regulation of perifollicular angiogenesis is complex and it involves multiple vasoactive and angiogenic factors.Vascular endothelial growth factor(VEGF),also named as vascular permeability factor or vasculotropin,is recently identified to be a kind of angiogenic factor derived from the hair follicle.Previous investigations indicated that VEGF was highly expressed in the dermal papilla,dermal sheath and outer root sheath of the anagen hair follicles,while VEGF decreased rapidly during the catagen and telogen.It has been demonstrated that VEGF can promote the proliferation and migration of the dermal papilla cells(DPCs),and can induce the angiogenesis around the hair follicle.In addition,VEGF can enhance the local nutrition supply by increasing the permeability of the endothelial cells.It is also found that VEGF can stimulate protease synthesis.Together,VEGF is an important stimulator for hair growth and its role is not confined to inducing the perifolliclar angiogenesis.Human angiogenin,a 14 kDa polypeptide,is composed of 123 amino acids and is found to be a member of the ribonuclease superfamily. Angiogenin was initially purified from serum-free media conditioned by growth of a human adenocarcinoma cell line HT-29 based on its ability to initiate vascularization in the chicken embryo chorioallantoic membrane. Angiogenin is also expressed in a number of other tumors,including prostatic carcinoma,malignant melanoma and hepatocellular,as well as in normal tissues such as human testis and endometrium.In addition, angiogenin is a component of normal human plasma and follicular fluid. Its widespread expression in human tissues suggests that angiogenin may have other roles in addition to promoting angiogenesis.However,up to date,the expression of angiogenin in human hair follicles and the role of angiogenin in hair growth have not been described previously.ObjectivesTo investigate the expression of angiogenin in human hair follicles and therefore determine the effects of aniogenin on hair growth in vivo and in vitro.MethodsPartâ… :The isolation of human hair follicles and the culture of human dermal papilla cells(DPCs)and outer root sheath(ORS) kerationocytesIntact human hair follicles were dissected under the microscope,and the DPCs and ORS keratinocytes were isolated and cultured by the two-step enzymic methods.Partâ…¡:The expression of angiogenin in human hair folliclesRT-PCR and immunofluorescence analyses were applied to detect angioenin mRNA and angiogenin protein expression respectively in human hair follicles.In addition,ELISA was used to determine the concertration of angigenin protein in the culture medium of the human hair follicle,which was then compared with the hair follicle growth. Partâ…¢:The expression of angiogenin in human DPCsRT-PCR was used to investigate angiogenin mRNA expression in human DPCs.Western blot was applied to determine angiogenin protein expression,which was further confirmed by immunofluorescence analysis.Angiogenin in the conditioned medium of cultured DPCs was detected by ELISA.Partâ…£:The expression of angiogenin mRNA in depilation induced murine hair cycleAnagen was induced in the back skin of the mice in the telogen phase of the hair cycle by melted wax/rosin.At 0,1,3,5,8,12,17,19,20, 25 days after depilation,mice were sacrificed and their back skin was harvested perpendicular to the paravertebral line.Total RNA was extracted from the skin with TRIZOL Reagent,and semi-quantitative RT-PCR was performed to assess angiogenin mRNA expression in murine skin phased at different hair cycle.Partâ…¤:The effects of angiogenin local injection on the skin angiogenesis and the hair cycle in vivoAnagen was induced in the back skin of mice in the telogen phase of the hair cycle by melted wax/rosin.At day 0 after depilation,mice were administered a dorsal intradermal injection of 1μg recombinant human angiogenin dissolved in 100μL PBS containing 0.1%BSA(0.1% BSA-PBS)one daily for 7 days(totally 7μg),and then sacrificed on day 10.Control mice were injected with 100μL 0.1%BSA-PBS.The skin color of the injection site and the blood vessel of the reverse side were observed and photographed on day 10.The skin sample from the injection site was embedded for cryosection and then was stained with HE for further analyses of the skin thickness and the percentage of hair follicles in anagenâ…¥.Partâ…¥:The effects of aniogenin on human hair follicle growth in vitroHuman occipital hair follicles in the anagenâ…£stage were isolated and cultured in vitro.Recombinant human angiogenin was then added to the cultures in the absence or the presence of 0.8μg/mL anti-human angiogenin antibody,for final concentrations in the range of 0-200ng/mL. At days 0 and 6,the length of each hair follicle was measured under the microscope,enabling calculation of the 6-day elongation rate.Partâ…¦:The effects of angiogenin on the proliferation of human DPCs in vitroDPCs were seeded on 96-well plates and were then cultured in 150μl serum-free DMEM for 24 hours before adding recombinant human angiogenin.Recombinant human angiogenin(0-200ng/mL final)in the absence or the presence of 0.8μg/mL anti-human angiogenin antibody were added to the cultures and were then incubated for 48 hours.MTT assay was used to detect cell proliferation,and flow cytometry was applied to analyze cell cycle.In addition,pEGFP-ANG,pEGFP-C2 and the transfected medium containing neither pEGFP-ANG nor pEGFP-C2 were transfected into human DPCs respectively by Lipofectamin^TM2000. After 48h cultures,MTT was performed to quantitate cell prolifertation.Partâ…§:The effects of angiogenin on the proliferation of human ORS keratinocytesORS keratinocytes were seeded on 96-well plates and were then cultured in 150μl serum-free keratinocytes medium for 24 hours before adding recombinant human angiogenin.Recombinant human angiogenin (0-200 ng/mL final)in the absence or the presence of 0.8μg/mL anti-human angiogenin antibody were added to the cultures and were then incubated for 48 hours.Cell proliferation was quantified by MTT assay.ResultsPartâ… :The isolation of human hair follicles and the culture of human dermal papilla cells(DPCs)and outer root sheath(ORS) kerationocytesHuman DPCs and ORS keratinocytes were successfully isolated and cultured.Partâ…¡:The expression of angiogenin in human hair folliclesAngiogenin mRNA was found in human hair follicles by RT-PCR analysis.After amplification with angiogenin-specific primers,we detected the PCR product of approximately 352bp,which was then confirmed by gene sequencing.Importantly,in human anagenâ…£hair follicle,the dermal papilla as well as connective tissue sheath could also express angiogenin,while follicular epithelium including hair matrix, inner and outer root sheath did not show angiogenin deposition.And,in hair follicle cultures,single cultured hair follicle can also secrete angiogenin.Angiogenin amounts and hair follicle growth length reach the maximum during the second two days,which both declined rapidly within the third two days.Partâ…¢:The expression of angiogenin in human DPCsRT-PCR analysis showed that angiogenin mRNA was expressed in cultured DPCs.The PCR product,approximately 352bp,was demonstrated to be angiogenin by gene sequencing.Western blot analysis of human DPC lysates with an angiogenin-specific antibody revealed a single band of 14 kDa.Also,we detected high levels of angiogenin in the cytoplasm of cultured DPCs,but not in DPCs nuclei.After 48 hours incubation in serum-free DMEM,DPCs can secrete an average of 85.499±3.153 pg/10⁵ cells angiogenin in the culture media.Partâ…£:The expression of angiogenin mRNA in the depilation induced murine hair cycleWax/rosin can successfully induce homogeneous anagen development in C57BL/6 mice,which was accompanied by the alternation of the skin color range from pink and grey to black,and then from black and grey to pink finally.Transcripts for angiogenin(265bp) were found in the mouse skin samples of all hair cycle stages,which were also confirmed by gene sequencing.After normalized against GAPDH, angiogenin exhibited an expression pattern in the sense that show a peak in late anagen and decline subsequently.Aniogenin transcript increased slowly in the early anagen,then rose rapidly during the mid to late anagen, and reached the maximum at anagenâ…¥,followed by a rapid decrease in the early catagen and finally returned to the initial(telogen)level by late catagen.Partâ…¤:The effects of angiogenin local injection on the skin angiogenesis and the hair cycle in vivoAngiogenin or vehicle solution(0.1%BSA-PBS)was injected intradermally for seven consecutive days(total 7μg),and the mice were sacrificed on day 10.Skin color of the angiogenin injected site did not show significant difference from that of vehicle solution injected site. However,in the reverse side of the resected skin sample,increased branches of blood vessels,which not completely confining to the injection site,could be clearly observed in the angiogenin injected mouse skin,suggesting that angiogenin administration can initiate skin vascularization.Histologically,when compared with the vehicle solution injected mice,the angiogenin injected mice showed a significant increase in skin thickness(p＜0.05).By quantitative histomorphometry,the difference became obvious when the percentage of hair follicles in anagenâ…¥in the skin of angiogenin and vehicle solution injected mice was analyzed statistically(p＜0.05).Partâ…¥:The effects of aniogenin on human hair follicle growth in vitroWe found that angiogenin ranging from 25 to 200ng/mL significantly increased hair follicle elongation in a dose-dependent manner.Hair follicle growth following treatment with 25ng/mL angiogenin was significant as compared with that of vehicle-treated controls(p＜0.001).In contrast,angiogenin less than 25ng/mL did not affect hair follicle growth. And,neutralizing antibody against angiogenin can completely block the stimulation effects of exogenously added angiogenin,suggesting that recombinant angiogenin can promote human hair follicle elongation in vitro.Partâ…¦:The effects of angiogenin on the proliferation of human DPCs in vitroUsing the MTT assay,we found that recombinant human angiogenin stimulated the proliferation of human DPCs in vitro.Angiogenin less than 12.5ng/mL resulted in no change in DPCs proliferation,however, angiogenin ranging from 12.5-200ng/mL caused a significant increase in DPCs proliferation as compared with vehicle-treated controls(p＜0.001), with the maximal proliferation observed with the doses of 50ng/mL and 100ng/mL.Importantly,in the presence of 0.8μg/mL anti-human angiogenin antibody,DPCs keratinocytes proliferation stimulated by exogenous angiogenin could be completely neutralized.Flow cytometry showed that 12.5-200ng/mL recombinant human angiogenin can significantly increase the S%and the cell proliferation index(p＜0.05).In addition,transfection of pEGFP-ANG into DPCs can significantly promote cell proliferation as compared with the controls(P＜0.05).Partâ…§:The effects of angiogenin on the proliferation of human ORS keratinocytesMTT assay showed that recombinant human angiogenin can stimulate the proliferation of human ORS keratinocytes in vitro. Angiogenin less than 3.13ng/mL had no effects on the ORS keratinocytes proliferation,but 3.13-200ng/mL angiogenin could significantly stimulate the proliferation of ORS keratinocytes(p＜0.001),with the optimal mitogenic activity obtained at the concentration of 12.5ng/mL. Importantly,in the presence of 0.8μg/mL anti-human angiogenin antibody, ORS keratinocytes proliferation stimulated by exogenous angiogenin could be completely neutralized.Conclusions1.Angiogenin is a newly angiogenic factor derived from the hair follicle and it is located at the dermal papilla and dermal sheath.Further, angiogenin amount in the conditioned medium of the cultured hair follicle is related to the growth length of the hair follicle.2.Cultured human DPCs can also express and secrete angiogenin, and angiogenin is presnet at the cytoplasm.3.The expression of angiogenin mRNA is hair-cycle dependent during the murine hair cycle in vivo,which exhibits an expression pattern in the sense that show a peak in late anagen and decline subsequently.4.Angiogenin can stimulate hair growth and regulate the hair cycle through the following mechanisms:(1).induce local vascularization;(2).induce anagen development;(3).promote DPCs proliferation;(4).stimulate ORS keratinocytes proliferation.