The Function And Mechanism Between Dermal Mesenchymal Stem Cells And Psoriatic Angiogenesis Mediated By EDIL3

Objective:Psoriasis is a chronic,recurrent,inflammatory skin disorder that is associated with both a physical and psychological burden.The disease is characterized by the hyperplasia of epidermis,inflammatory cell infiltration into dermis and epidermis,dilatation of dermal capillaries and angiogenesis.The dilatation of dermal capillaries and angiogenesis played distinct and important roles in psoriasis.The abnormal vascular network is associated with increased endothelial cells(ECs)survival,proliferation,adhesion,migration,angiogenesis and permeability in psoriatic lesions.The abnormal functions of ECs are caused by inflammatory molecules,pro-angiogenic and anti-angiogenic mediators in local microenvironment.Mesenchymal stem cells(MSCs),alternatively termed as multipotent mesenchymal stromal cells,could secrete cytokines by autocrine or paracrine,promoting the proliferation,adhesion and migration of ECs and maintaining vascular stability.Our group has successfully isolated dermal MSCs(DMSCs)from skin in psoriatic patients.The m RNA and protein expression of epidermal growth factor-like repeats and discoidin I-like domains 3(EDIL3)in psoriatic DMSCs is upregulated,but whether EDIL3 play unique role in pathological processes of psoriasis is a lack of understanding.EDIL3 is expressed in ECs during early embryogenesis,ischemia and tumor vascular tissues.EDIL3 is identified to be a switch that initiates angiogenesis through inducing cell adhesion,migration and inhibiting the apoptosis of vascular endothelial cells(VECs)and play an important role in maintaining the morphology and stability of neovascularization,which is regulated by integrin-FAK signal pathway.We assumed that the local angiogenesis of skin is regulated by psoriatic DMSCs through EDIL3 mediated integrin signaling pathway,which are involved in the pathogenesis of psoriasis.In conclusion,this study will provide a theoretical basis for explaining the microvascular abnormalities of pathogenesis and a new target of antiangiogenic therapy of psoriasis in future.Methods:1.Human umbilical vein endothelial cells(HUVECs)were isolated from human umbilical and identified by CD31 marker.DMSCs derived from skin tissues and was identified by differentiational abilities and marker of cells.To further confirm the identification of cells,DMSCs at passage 3 were induced to differentiate into osteoblasts,adipocytes and chondrocytes.In this study,we adopted lentivirus infection to regulate the EDIL3 expression on DMSCs in healthy and psoriasis in vitro.After transfection,the m RNA and protein of EDIL3 were analyzed by real time quantitative polymerase chain reaction(RT-q PCR)and western blot.We divided HUVECs into five groups according to DMSCs co-cultured with them.The details of groups were as follows:Control group:HUVECs were cultured in the lower chamber alone.C-DMSCs group:DMSCs from healthy volunteers were co-culture with HUVECs.C-DMSCs^EDIL3-high group:DMSCs from healthy volunteers with over-expressed EDIL3 co-cultured with HUVECs.P-DMSCs group:DMSCs from psoriasis co-cultured with HUVECs.P-DMSCs^EDIL3-low group:DMSCs from psoriasis with low-expressed EDIL3 co-cultured with HUVECs.After co-culture for 48 hours,the proliferative potential of HUVECs was assessed by cell counting kit-8(CCK8).2.After co-culture,all HUVECs were harvested to detect the characteristics and the protein of EDIL3-related integrin signaling pathway.To evaluate the adherent ability of HUVECs,the assay of attachment of DMSCs to HUVECs was conducted.Adherent cells were quantified by counting the average number of cells in five different field of each well using LSCM and analyzed with Image Pro Plus software.Wound scratch assay and transewll migration assay were used to assess the motility and directional migration ability of HUVECs.In vitro,the tube formation of ECs was assessed using Matrigel matrix(mimics the natural basement membrane matrix of ECs).The mesh numbers of tube structures were measured and the results were used to evaluate the tube formation ability of HUVECs.The protein of EDIL3-related integrin signaling pathway was detected by western blot.3.To further explore the role of EDIL3 in psoriasis in vivo,we detected the epidermis thickness and microvessel density in IMQ-induced mouse model through injecting EDIL3 or NS.At day 4,half of mice were sacrificed and the samples were collected for next experiments.At day 7,remainder mice were sacrificed and skin samples were collected.Skin biopsies were processed for paraffin sections,which were then stained with hematoxylin and eosin(H&E)to measure the epidermal thickness and conduct the histopathological examinations.The immunofluorescent(IF)staining of mice skin biopsies for CD31was performed to assess the microvessel densities.Results:1.In present study,we successfully isolated DMSCs and HUVECs.Co-culture with DMSCs all showed a positive effect on the proliferation of HUVECs.The proliferation of HUVECs co-cultured with C-DMSCs^EDIL3-highand P-DMSCs was none significante unaltered compared to the C-DMSCs.However,compared with P-DMSCs group,P-DMSCs^EDIL3-low group showed a slight reduction in proliferation of HUVECs.2.Compared with the control,co-cultured with DMSCs resulted in an increased adherent,migration and tube formation ability of HUVECs.Our results showed that compared with the C-DMSCs,in C-DMSCs^EDIL3-high and P-DMSCs group HUVECs adhesion,migration and tube formation were increased,and the protein expression of integrin?v?3??5?1 and MEK-ERK signaling pathways were upregulated.What's more,P-DMSCs treatment with si-EDIL3 inhibited this adhesion,migration and tube formation compared with the P-DMSCs.We confirmed that in vitro DMSCs-derived-EDIL3 involved in the adhesion,migration and tube formation of ECs via integrin-FAK/MEK/ERK signaling.3.Histological analysis showed that epidermal thickness was markedly increased in IMQ treated mice compared with that vaseline treated.And IMQ+EDIL3-induced psoriasis-like lesion in mouse model resulted in higher epidermis thickness compared with control.These results showed EDIL3 accelerated the pathological progress of IMQ-induced psoriasis mice.In addition,compared to that at day 4,EDIL3-injected skins exhibited more increased blood vessel density at day 7,and EDIL3 caused a time dependent increase of angiogenesis.Together,these data suggested that the upregulation of EDIL3 can promote microvascular formation in development of psoriasis.Conclusions:1.Our study demonstrated DMSCs from healthy and psoriatic skin showed the characteristics of MSCs and all promoted the proliferation of HUVECs.2.We demonstrated that DMSCs-derived-EDIL3 induced the adhesion,migration and angiogenesis of HUVECs through enhanced the FAK protein associated with integrin?v?3 and?5?1 and FAK phosphorylation.And these enhancements correlated with the activation of the MEK/ERK signaling pathway in ECs.The over-expressed EDIL3in DMSCs induced the activation of the signaling pathway that can be inhibited by knockdown of EDIL3 expression via si-RNA in P-DMSCs,in accordance with the inhibiting the capabilities of adhesion,migration and angiogenesis of ECs.3.Our studies found that the epidermis thickness and microvessel density were both elevated in the IMQ-induced psoriasis-like mouse models.The hyperplasia of the epidermis and maintenance may depend partly on abnormal elongation of microvascular.The results suggested that EDIL3 accelerated the process of psoriasis through promoting ECs to change into activated phenotype and induce elongation of microvascular.