Vascular Endothelial Growth Factor 165(VEGF165) Induces Differentiation Of Hair Follicle Stem Cells Into Endothelial Cells And Plays A Role In In Vivo Angiogenesis

Objective To improve the approaches to isolate,culture,proliferate and identify the rat hair follicle stem cells(rHFSCs)in vitro,to explore their adipogenic and osteogenic differentiation potentials and to determine the feasibility of rat hair follicle stem cells(rHFSCs)differentiating into endothelial cells in vitro and investigate the mechanism of Notch signaling in this process,then further clarify the induced cells in a role of the process of angiogenesis in vivo.Methods The cirri skin of one-week SD rat was cut in aseptic condition and digested by using intermixture of Dispase and type IVcollagenase enzyme,the bulge part of hair follicle was isolated under the microscope,the rHFSCs were cultured by gradient plus medium method and tissue explant adherence method,passaged by two-step enzyme digestion method and purified by collagen IV anchorage velocity-dependent separation method.Then observing their biological morphology;testing the proliferation and viability of different generation rHFSCs;observing the internal structure by transmission electron microscope;finally,identifing by conbined flow cytometry instrument detection with cell immunofluorescence staining.The third-generation HFSCs were cultured by osteogenic inductor and adipogenic inductor respectively,then identifying the related indexes.With 10 ng/ml and 20 ng/ml VEGF165 as main inducible factor dividing the concentration of cells into two groups,observing the induction of cell morphology respectively in 1,2,3 weeks without passage;detecting differentiation efficiency by Flow cytometry instrument detection and cell immunofluorescence staining;Then selecting the best inducible factor concentration and work time,the induced cells were identified tube formation assay in vitro and observed Weibel-Palade bodies under transmission electron microscope.The cells were inhibited with different concentrations of DAPT(0.5 and lmol/L)and then divided into induction group and inhibition group.These two groups were simultaneously treated for 1 week and the expression of CD31 and VE-cadherin were measured by using western blotting.The Dil-ac-LDL phagocytosis was also detected.Twelve 6-week-old nude mice were divided into three groups:Group A(PBS+Matrigel),Group B(VEGF165+Matrigel),and Group C(VEGFi65+rHFSCsGFP+Matrigel).The mixed Matrigel matrix for each group was injected into the abdomen of nude rats.After inoculation for 2 weeks,the matrix was harvested and specimens were obtained for morphological comparisons.Furthermore,hematoxylin&eosin(HE)staining,CD31 immunohistochemistry,CD31 fluorescence detection,and three-dimensional(3D)reconstruction of vessels and rHFSCsGFP under the two-photon microscope,were conducted to evaluate the role of rHFSCs in angiogenesis after in vivo induction.Results The isolated,culture-expanded and purified rHFSCs had good cloning capability and strong viability,with an S-shaped growth curve.Cells were primitive state under transmission electron microscope,The detection of flow cytometry instrument detection and immunofluorescence staining indicated integrin?1 integrin?6?CK15 were high expression,while CD31?VE-cadherin were weakly expression.1 week after adipogenesis induced,PPAR-y and C/EBPa increased the most obvious(t=-34.955,P=0.000;t=-20.266,P=0.000),and 2 week later,the cells with oil red staining showed positive,and the OD value is obvious differ significantly with control group(t=-114.641,P=0.001).2 week after osteogenesis induced,OPG and Runx2 increased the most obvious(t=-4.667,P=0.01:t=-17.332,P=0.000),and 14 day later,the cells with alizarin red staining showed positive,and two groups of OD values differ significantly(t=-14.078,P=0.001).Under the action of induced liquid,two groups of cells all changed from flat slabs of sample changes to the fully occupied by a long spindle cells from 1 week to 3 weeks.The outcome of CD31 and VE-cadherin detected by flow cytometry instrument detection and immunofluorescence staining indicated two kinds of concentration at the same time had no obvious difference,for 1 week high expression,2 weeks lower expression,3 weeks negative expression.10 ng/mlVEGF165 induced cells(lweek later)were showed tube formation assay positive,and observed Weibel-Palade bodies which is the unique structure of endothelial cells.After the suppression of the Notch signaling pathway,western blot analysis showed that protein expression of both CD31 and VE-cadherin were remarkably decreased,along with the obviously decreased phagocytosis capability of Dil-ac-LDL.Gross observation of in vivo samples showed that there was almost no vessel formation in Groups A and B;in contrast,comprehensive neovascularization was seen in Group C.In Group C,HE staining and CD31 immunohistochemistry showed high positivity,through immunofluorescence,3D reconstruction with blood vessels and rHFSCsGFP,the induced rHFSCsGFP were predominantly located at the periphery of the newly formed vessels,with a small amount on the internal vessel wall.Conclusion After the improvement,we can successfully establish the system of isolate,culture,proliferate and identify rHFSCs in vitro,prove its proliferation,and verify its multi-directional differentiation potential.Induction of rHFSCs in the presence of 10ng/mL VEGF165 for 1 week yielded vascular endothelial cells with typical characteristics.The Notch signaling pathway affected the efficiency of inducing vascular endothelial cells.After induction in vivo,rHFSCs promoted angiogenesis,thus accelerating host-derived neovascularization.rHFSCs could therefore be considered as an ideal cell source for vascular tissue engineering and cell transplantation in the treatment of ischemic diseases.