The Role Of P311 In Wound Angiogenesis And Its Possible Mechanism

BackgroundSkin wounds are one of the most common clinical issues,and burns,wounds,surgery and various medical diseases can cause skin wounds.Wound repair,especially the repair of refractory wounds,still faces many clinical challenges.In the United States alone,the direct medical cost per year for refractory wound treatment exceeds $25 billion.Although more research has been focusing on skin wound repair in recent years,the mechanism is not completely clear.Wound angiogenesis is one of the key aspects of wound repair.Wound angiogenesis mainly includes steps such as vascular basement membrane degradation,vascular endothelial cell migration and budding growth,lumen formation,local needs adjustment and stable maturation.The whole process involves a variety of cells and cytokines.Microvascular endothelial cells are the main functional cells involved in this process.They form new blood vessels by migration,proliferation,sprouting,etc.under the precise regulation of various cytokines and signalling pathwaysVEGF(now expressed as VEGFA)is the most important pro-angiogenic factor in wound healing.VEGF and its mediated signalling pathways are involved in various processes of angiogenesis.VEGFR2 is the most important binding receptor for VEGF and is capable of transducing all known effects of VEGF.It is a VEGFR that plays an important role in angiogenesis.VEGF binds to VEGFR2 and phosphorylates VEGFR2 Tyr1173(phosphorylation of Tyr1175 in humans)in mice.Phosphorylation at this site is critical for ERK1/2 activation.It was found that if the site was mutated to phenylalanine(Tyr1173F),endothelial progenitor cell differentiation was inhibited during embryonic development,leading to early embryonic death,which is consistent with the performance after knocking out the VEGFR2 gene.P311 is a highly conserved intracellular multifunctional protein between species.Although P311 does not belong to any known protein family,studies have found that P311 protein plays an important role in tissue fibrosis,nervous system diseases,tumor infiltration,blood pressure maintenance,and tissue regeneration.Recently our research team found that P311 plays an important role in wound healing.P311 can enhance the activity of Rho A and Rac1 in epidermal stem cells to improve the migration ability of epidermal stem cells,then to promote wound re-epithelialization.In addition,P311 can promote the transdifferentiation of epidermal stem cells to myofibroblast-like cells through the TGF?1/Smad signalling pathway to promote the repair of burn wounds.As wound angiogenesis is one of the key aspects of wound repair,this study investigated the effects of P311 on the biological function of dermal microvascular endothelial cells and its role in wound angiogenesis based on bioinformatics predictions that P311 may be involved in angiogenesis.The mechanism of P311 in the regulation of wound angiogenesis was further explored from the perspective of activation of VEGFR2/Erk1/2 signaling pathway and regulation of VEGF expression.Part 1 Exploring P311 protein interaction network and identifying its biological functionsMethod:1.Bioinformatics prediction of P311's functionsP311 and its interacting proteins identified by yeast two-hybrid assay constituted the original P311-containing network.Then the initial P311-containing network was merged with the human PPI network dataset to form new PPI network dataset.The new PPI network dataset were partitioned by OCG to obtain reconstructed P311-containing networks.Through DAVID analysis of the biological processes involved in the constituents of each P311-containing network to predict the possible fuction of P311.2.The role of P311 in inflammatory response during wound healingP311 wild-type mice and P311 knockout mice were used to construct full-thickness excisional skin wound models.The recruitment of inflammatory cells to the wound was observed by HE staining.The CD14 and CD16 m RNA levels in wounds were detected by real-time quantitative PCR.Flow cytometry was performed to detect the F4/80 positive cells in wound.3.Effect of P311 on the proliferation of mouse fibroblastsMouse fibroblasts were transfected with P311 recombinant adenoviral vector to express P311.The effect of P311 on the proliferation of fibroblasts was detected by CCK8;the effect of P311 on cell cycle was detected by flow cytometry.4.Effect of P311 on coagulation function after burnA superficial second degree burn mouse model was made with P311 wild type(WT)mice or P311 knockout(KO)mice,and Thromboelastography(TEG)was then used to monitor the coagulation profile of blood after burn.Results:1.Bioinformatics prediction of P311's functionsP311 may have 14 new functions,of which P311 plays the most reliable role in angiogenesis,inflammation,cell proliferation and coagulation.2.The role of P311 in inflammatory response during wound healingThe number of inflammatory cells in the wound of P311 knockout mice was significantly lower than that of P311 wild type mice.The levels of CD14 and CD16 m RNA in the wounds of P311 knockout mice were significantly lower than those in P311 wild type mice.3.Effect of P311 on the proliferation of mouse fibroblastsP311 promotes the proliferation of mouse fibroblasts.The proportion of cells in the S phase of fibroblasts with high expression of P311 was significantly higher than that of th e control group(25.16±4.92% vs 36.68±3.56%,p<0.05).4.Effect of P311 on coagulation function after burnComparing the P311 WT-burn mice to the P311 KO-burn mice,except for the clot formation time(R 8.850 ± 1.541 vs.7.733 ± 1.210 min,p = 0.57),other parameters(Angle,MA,and G)were all significantly different(p < 0.05)In summary,we reconstructed P311-containing PPI networks and predicted that P311 might have 14 new functions,of which P311 plays the most reliable role in angiogenesis,inflammation,cell proliferation and coagulation with the networks.The role of P311 in inflammatory response,cell proliferation and coagulation was preliminarily confirmed by biological experiments in this section.The role of P311 in angiogenesis is systematically studied in the following sections.Part 2 The role of P311 in wound angiogenesisMethods:1.Dermal microvascular endothelial cells express P311 and their expression is enhanced under inflammatory stimulation conditions.Immunohistochemistry and immunofluorescence staining were conducted to detect the expression of P311 in human wounds and mouse wounds.Mouse dermal microvascular endothelial cells were isolated and cultured by Percoll separation and CD31 magnetic bead separation.Immunofluorescence and flow cytometry were performed to detect the expression markers of endothelial cells on the isolated cells.Immunofluorescence and real-time quantitative PCR were used to detect the expression of P311 in isolated and cultured microvascular endothelial cells under steady state and inflammatory stimuli(L-1? stimulation).2.Effect of P311 on the cell biological function of dermal microvascular endothelial cellsThe effect of P311 knockout on the migration of dermal microvascular endothelial cells was examined by the in vitro scratch wound migration model.The effect of P311 knockout on angiogenesis of dermal microvascular endothelial cells was detected by in vitro tube formation assay.The effect of P311 knockout on angiogenesis in dermal microvascular endothelial cells was examined by matrigel plug assays.3.The role of P311 in angiogenesis in mouse woundsP311 wild-type mice and P311 knockout mice were used to construct full-thickness excisional skin wound models.Gross and HE staining were used to observe wound healing,re-epithelialization and new granulation and microvessels in P311 wild-type mice and P311 knockout mice.The expression of CD31,VEGF and TGF?1 in the wound and normal skin of mice were detected by immunohistochemical staining and Western Blot.ELISA was u sed to detect expression of VEGF and TGF?1 in the wound and normal skin of mice.Results:1.Dermal microvascular endothelial cells express P311 and their expression is enhanced under inflammatory stimulation conditions.1.1 Wound microvascular endothelial cells express P311Immunohistochemical staining showed that some P311 positive cells appeared in the vascular-like structure of human skin wounds.Furthermore,immunofluorescence staining showed that v WF positive cells also expressed P311 in granulation tissue i n mice.That meant vascular endothelial cells expressed P311.1.2 Successful isolation and culture of dermal microvascular endothelial cellsThe isolated cultured cells were characterized by a characteristic "cobblestone" morphology and high expression of v WF,CD31 and CD34.1.3 Dermal microvascular endothelial cells express P311Immunofluorescence results showed that mouse dermal microvascular endothelial cells expressed P311,but the expression level was not high,and the expression was enhanced after stimulation with 10 ng/ml IL1?.Realtime PCR results showed that the transcription level of P311 in mouse dermal microvascular endothelial cells was significantly increased after 10 ng/ml IL1? stimulation(p<0.01).2.Effect of P311 on the cell biological function of dermal microvascular endothelial cells2.1 P311 knockout weakens the vascular tube formation ability of dermal microvascular endothelial cells in vitroIn tube formation assay,after P311 knockout,the number of nodes in the tubular structure formed by dermal microvascular endothelial cells was significantly reduced(p<0.01),and the total length of tubular structures was significantly shorter(p<0.01).2.2 P311 knockout weakens the migration ability of dermal microvascular endothelial cells in vitroIn a scratch wound migration model,after P311 knockout,the coverage of dermal microvascular endothelial cells was significantly decreased at each observation time point(p<0.01).P311 knockout attenuates angiogenesis in dermal microvascular endothelial cells in vitro?2.3 P311 knockout weakens the vascular tube formation ability of dermal microvascular endothelial cells in vivoThe number of endothelial cell,which invaded into the plugs of P311 KO mice,was significantly lower compared with that of P311 WT mice(p<0.01).3.The role of P311 in angiogenesis in mouse wounds3.1 Wound healing and re-epithelializationGross observation showed that the percentage of wound area of P311 WT mice on day 3,5 and 7 after injury was all significantly less than the percentage of wound area of P311 KO mice.The wounds of P311 WT mice healed on average 6.2 days and the wounds of P311 KO mice healed on average 7.8 days(p<0.05).HE staining showed that on the 3rd and 5th day after injury,the length of neo-epithelium in P311 KO mice was significantly shorter than that in P311 WT mice(p<0.05).3.2 Wound granulation and microvascular cellsHE staining showed that the thickness of new granulation tissue in the wound of P311 KO mice was thinner than that of P311 WT mice,and the number of visible microvessels in the granulation tissue of P311 KO mice was less than that in the new granulation tissue of P311 WT mice.(p<0.05).Immunohistochemical staining showed that the number of new blood vessels(CD31 positive cells)in P311 KO wound mice was significantly lower than that in P311 WT wound mice.The results of Western Blot showed that the CD31 protein level in P311 WT mice was significantly higher than that in P311 KO mice(P<0.01).3.3 P311 knockout inhibits wound VEGF and TGF?1 expressionImmunohistochemical staining showed that the expression of VEGF and TGF?1 in P311 KO wound was significantly lower than that in P311 WT wound.The results of Western Blot showed that the levels of VEGF and TGF?1 in wound tissue of P311 WT mice were significantly higher than those of P311 KO mice VEGF and TGF?1(P<0.01);the ELISA results showed that the concentration of VEGF and TGF?1 in P311 KO wound tissue homogenate supernatant was significantly lower than that of P311 WT wound.In summary,we report for the first time that P311 affects angiogenesis in wound healing by affecting the biological function of dermal microvascular endothelial cells.Part 3 Mechanism of P311 in promoting wound angiogenesisMethods:1.Effect of high expression of P311 on the biological function of dermal microvascular endothelial cellsThe effect of high expression of P311 on the migration of dermal microvascular endothelial cells was examined by the in vitro scratch wound migration model.The effect of high expression of P311 on angiogenesis of dermal microvascular endothelial cells was detected by the in vitro tube formation assay.The effect of high expression of P311 on angiogenesis gene expression in dermal microvascular endothelial cells was detected by n Counter gene expression profile analysis system and real-time quantitative PCR.2.P311 promotes angiogenesis via VEGFR2/ERK1/2 signalling pathwayThe effect of high expression of P311 on the expression of VEGFR2 and ERK1/2 protein and phosphorylation was detected by Western blot.The si RNA-VEGFR2 and ERK1/2 inhibitor SCH772984 were used to detect that P311 promotes blood vessel formation by activating VEGFR2 and Erk1/2.3.P311 regulates the expression of VEGFP311 was strongly expressed in dermal microvascular endothelial cells by Ad-P311.The expression of VEGF in microvascular endothelial cells was detected by Western Blot and real-time quantitative PCR.The level of VEGF protein in microvascular endothelial cell culture supernatant was detected by ELISA.4.Molecular mechanism of P311 regulating VEGF expressionThe VEGF promoter dual luciferase reporter vector was constructed,and the effect of P311 on the activity of VEGF promoter was detected by luciferase reporter assay.The effect of P311 on the methylation of VEGF promoter Cp G island was detected by bisulfite sequencing PCR.The effect of P311 on the stability of VEGF m RNA was detected by real-time quantitative PCR.The untranslated region(UTR)dual luciferase reporter vector was constructed and the activity of P311 on VEGF UTR was detected by luciferase report er assay.Results:1.Effect of high expression of P311 on the biological function of dermal microvascular endothelial cells1.1 High expression of P311 enhances vascular tube formation ability of dermal microvascular endothelial cells in vitroIn tube formation assay,the number of nodes in the tubular structure formed by dermal microvascular endothelial cells increased significantly after P311 expression(p<0.01),and the total length of tubular structures became significantly longer(p<0.01).1.2 High expression of P311 enhances the migration ability of dermal microvascular endothelial cells in vitroIn a scratch wound migration model,after P311 was highly expressed,the coverage of dermal microvascular endothelial cells increased significantly at each observatio n time point(p<0.01).1.3 High expression of P311 up-regulates the expression of pro-angiogenic genes in dermal microvascular endothelial cellsThe results of real-time quantitative PCR showed that after expression of P311 in dermal microvascular endothelial cells,CCL2 expression was up-regulated to 3.66-fold(p<0.05),CXCL8 expression was up-regulated to4.58(p<0.05),FGFR3 expression was up-regulated to 2.36(p<0.05),and IL6 expression was up-regulated to 2.55.(p<0.05),PTGS1 expression was up-regulated to 29.58(p<0.05),and VEGF expression was up-regulated to 2.94(p<0.05).2.P311 promotes angiogenesis via the VEGFR2/ERK1/2 signaling pathway2.1 High expression of P311 promotes phosphorylation of VEGFR2 and ERK1/2After high expression of P311,the phosphorylation levels of VEGFR2 and ERK1/2 in dermal microvascular endothelial cells increased to 3.62 and 2.17 times,respectively,compared to the control level.2.2 si RNA-VEGFR2 inhibits the phosphorylation of VEGFR2 and ERK1/2 and blocks the promotion of angiogenesis by P311After si RNA-VEGFR2,the phosphorylation level and total protein expression of VEGFR2 in microvascular endothelial cells were significantly decreased(p<0.05),and the phosphorylation level of Erk1/2 was significantly decreased(p<0.05).The promo ting effect of P311 on angiogenesis was significantly inhibited.(p < 0.05).2.3 SCH772984 inhibits the phosphorylation of Erk1/2 and blocks the promotion of angiogenesis by P311After using SCH772984,the phosphorylation level of Erk1/2 in microvascular endothelial cells was significantly decreased(p<0.05),and the promoting effect of P311 on angiogenesis was significantly inhibited.(p < 0.05).3.P311 regulates the expression of VEGFReal-time quantitative PCR showed that VEGF m RNA levels in microvascular endothelial cells were significantly increased after high expression of P311(p <0.05).Western Blot results showed that VEGF protein levels in microvascular endothelial cells were significantly increased after high expression of P311(p < 0.01);ELISA resul ts showed that after high expression of P311,the VEGF content in microvascular endothelial cell culture supernatant was significantly increased(p<0.01).4.Molecular mechanism of P311 regulating VEGF expression4.1 P311 may enhance promoter activity to promote VEGF transcription by binding to the VEGF promoter region from-2000 to-1550The dual luciferase reporter assays showed that P311 can bind to the VEGF promoter region from-2000 to-1550.Bisulfite sequencing results showed that P311 had no effect on Cp G island methylation of VEGF promoter.4.2 P311 induces translation of VEGF protein by binding to VEGF m RNA 3'-UTR and 5'-UTR and increasing its activity.Real-time quantitative PCR results showed that high expression of P311 had no effect on the stability of VEGF m RNA;dual luciferase reporter assay showed that P311 binds to VEGF m RNA 3'-UTR and 5'-UTR and increases its activity to induce translation of VEGF protein.In summary,P311 binds to the VEGF promoter-2000 to-15050 region to increase promoter activity and promote VEGF transcription,while P311 binds to VEGF m RNA 5'-UTR and 3'-UTR and enhances their activity to promote VEGF translation.Then VEGF activates VEGFR2/Erk1/2 signalling pathway to promote angiogenesis.Conclusion:This study demonstrates for the first time that P311 works as a novel key molecule for promoting wound angiogenesis.P311 may bind to the-2000 to-1550 region of VEGF promoter to increase its activity and promote VEGF transcription.On the other hand,P311 can bind to 5'-UTR and 3'-UTR of VEGF m RNA to promote VEGF translation.Moreover,it was identified that VEGF activates VEGFR2/Erk1/2 signalling pathway to promote angiogenesis.