The Role Of HMGB1-induced Migration Of Endothelial Progenitor Cells In Wound Neovascularization And Molecular Mechanism

Background:The skin wounds caused by burns trauma and ischemic diseases such as diabetes are problems in clinical treatment.Delayed healing of wounds eventually leads to hypertrophic scars,which lead to different degrees of dysfunction of the body.Neovascularization is one of the key steps in wound healing.Effective wound repair requires abundant neovascularization in newly formed granulation tissue to maintain nutritional supply an d extracellular matrix deposition in the wound area.The growth of neovascularization plays an important role in the formation of granulation tissue,improving microcirculation,reducing infection and the promoting the healing of chronic refractory of infection wounds or deep burn wounds.At present,the refractory skin ulcer is still a challenge for burn and plastic surgery.Thus,to further illuminate the growth theory of refractory skin ulcerous and promoting their early repair is the key of the study in the field of injury.Endothelial progenitor cells(EPCs)are the key cellular effectors in the angiogenesis of injured wounds.Angiogenesis begins with the mobilization of EPCs,which proliferates and forms new blood vessels.EPCs contribute to the re-endothelialization and enhancement of angiogenesis after ischemia or tissue injury,thus accelerating the healing of ischemic limbs or wound healing.However,the specific molecular mechanism of EPCs involved in neovascularization has not determined.According to the existing research results,many chemokines may be involved in the activation,mobilization,homing and migration of bone marrow-derived stem cells to damaged site or ischemic tissues.HMGB1 is a ubiquitous intranuclear DNA binding protein,mainly as a non-histone,stable nucleosome,involved in DNA transcription,replication,repair and so on.Recent studies have confirmed that HMGB1 can actively be released or secreted from necrotic cells and inflammatory cells stimulated by LPS,TNF-?,IL-1,and entered extracellular space,as an inflammatory factor,causing inflammation.Subsequent studies found that besides inducing inflammation,high mobility group box1 protein(HMGB1)secreted to cells also has chemokine-like functions and is a universal regulator of cell migration.Previous studies have suggested that various inflammatory factors produced in the wound play an important role in inducing cell migration to the wound site and promoting wound repair.Combined with the chemotactic effect of HMGB1,whether the elevation of HMGB1 level can play a chemotactic cytokine role in EPC migration,has not been studied much.Previous studies have shown that HMGB1 can promote the migration and proliferation of mesangial vascular cells from fetal and adult rat vascular-related stem cells through the endothelial cell barrier.Recent studies have also confirmed that integrins are involved in the homing of EPCs to the active sites of neovascularization and promote their neovascularization.Based on the above evidence s,we speculate that HMGB1 released from wound inflammation may also be one of the important molecules inducing EPCs migration in addition to the chemokines such as SDF-1 and vascular endothelial growth factor(VEGF)in the process of EPCs migration and differentiation to skin wounds.The important receptor of HMGB1 is advanced glycation end product receptor(RAGE).RAGE is widely expressed on different cell surfaces,which is the transmembrane protein of immunoglobulin.RAGE is the natural receptor of HMGB1.When combined with RAGE,HMGB1 can activate many signal transduction molecules in the signaling pathway.At present,it is believed that signal pathways involved in EPCs migration are related to MAPK,PI3 K,and NF-KB.The main process of cell migration is the polarization of cells caused by activation of external signals,which leads to the formation of synapses or extension of pseudopodia along the direction of cell migration.Cytoskeleton is a protein system involved in many important activities such as cell deformation,cell movement and cell migration.PI3 K signaling pathway plays an important role in many biological processes,such as cell growth and apoptosis and EPCs migration induced by Chemokines.PI3 K inhibitor can block integrin activation and integrin-dependent adhesion,migration and exudation of EPC from human peripheral blood through chemokine-induced integrin activation.PI3K/Akt signaling pathway plays an important role in the directional migration of EPCs induced by VEGF or simvastatin,a lipid-lowering drug,and increases the synthesis of NO in EPCs.We speculate that extracellular recombination by binding to RAGE,thus causing cell movement and migration.In conclusion,we speculate that HMGB1 may be involved in the intracellular PI3K/Akt/eNOS signaling pathway during the induction of EPCs migration as a chemokine.HMGB1 may participate in the migration of EPCs by influencing the change of cytoskeleton structure.HMGB1 may play an important role in inducing EPCs to migrate to the wound,increasing the formation of new blood vessels in granulation tissue and promoting wound healing.Matirals and Methods:Part ? Changes of HMGB1 expression in skin wounds of mice and its effect on wound healingTo establish a mouse skin wound skin model.Western blot and qRT-PCR were used to detect the expression of HMGB1 in wound tissue of STZ-induced diabetic mice and normal mice.Flow cytometry was used to determine the difference of CD34 positive cells in peripheral blood between normal mice and hyperglycemic mice.HMGB1-adenovirus vector was used to treat the wound of mice,and its effect on wound healing of diabetic mice was observed by section staining.Part ? The effects of HMGB1 on the biological characteristics and chemotaxis of cultured EPCs in vitroEndothelial progenitor cells were isolated by gradient centrifugation from mice bone marrow and identified by flow cytometry and cell fluorescence.Western blot,qRT-PCR and immunofluorescence test were used to measure the expression of RAGE on the surface of EPCs.CCK8 and ELISA were used to detect the effects of HMGB1 on the differentiation,proliferation and paracrine function in EPCs.Angiogenesis ability was measured by tube formation test in vitro.The EPCs migration induced by different concentrations of HMGB1 was detected by scratch test and cell migration(Transwell)test in vitro.Part ? Molecular mechanisms of EPCs migration induced by HMGB1The effects of different signal pathway inhibitors LY294002(PI3K inhibitor),PD98059(ERK inhibitor),L-NAME(eNOS inhibitor)on the migration of EPCs induced by HMGB1 were detected by migration test and scratch test.The expression of PI3 K,Akt and ERK proteins and their phosphorylation process were measured by Western blot.Laser confocal microscopy was performed after F-actin fluorescence staining in EPC.The changes of cell morphology and cytoskeleton structure were observed.Results:Part ?1.The results showed that the expression of HMGB1 protein in the wound of normal mice had significantly increased on the first day of wound formation.In DM group,the expression of HMGB1 protein in wounds was significantly lower than that in normal mice.2.The time of wound healing in diabetic mice was longer than in normal mice.At the same time,the number of CD34 positive cells in peripheral blood was significantly lower than in normal mice.3.The using of exogenous HMGB1 on the wounds of diabetic mice resulted in shorter healing time.The number of granulation tissue formation and small vessel formation was better than that of DM group.Part ?1.Flow cytometry showed that adherent cells expressed CD133(49.6%)?CD34(90.03%)and VEGFR-2(76.48%).The number of double positive cells expressing Dil-ac-LDL and FITC-UEA-1 was over 95%.The results showed that these adherent cells accorded with the characteristics of EPC.2.After stimulating EPCs with different concentrations of HMGB1,the results showed that HMGB1 up-regulated the expression of RAGE protein and mRNA in EPC on a concentration-dependent manner.3.The results of CCK8 showed that HMGB1 could promote the proliferation of EPCs in a certain concentration range(1-50ng/mL),but the effect was significantly weakened after using Anti-RAGE-Ab.4.It was found that HMGB1 in a concentration range enhanced the secretion of SDF-1 by EPCs.5.The facts that HMGB1 may promotes EPCs angiogenesis ability was confirmed by tube formation test.6.The results of cell migration and scratch test showed that EPCs migration increased with the increase of HMGB1 concentration,indicating that HMGB1 could promote the migration of EPCs in a concentration-dependent manner.Part ?1.Cell migration and scratch test were used to detect EPC migration induced by HMGB1 with different signal pathway inhibitors.The results showed that HMGB1 could significantly increase the EPCs migration.Anti-RAGE antibody significantly inhibited EPCs movement induced by HMGB1.Meanwhile,the migration of EPC induced by HMGB1 pretreated with LY294002 was blocked.However,PD98059 had no effect on HMGB1-induced cell motility.2.HMGB1-RAGE participates in Akt and ERK phosphorylation.HMGB1(100 ng/mL)significantly up-regulated the expression of p-Akt,while LY294002 and anti-RAGE antibody significantly inhibited this behavior.The addition of PD980059 and anti-RAGE antibody down-regulated expression of p-ERK,suggesting that HMGB1-RAGE also participated in the phosphorylation of ERK signaling pathway.3.The expression of p-eNOS protein in EPCs treated with HMGB1 was up-regulated,but the process was inhibited by the addition of anti-RAGE antibody and L-NAME.4.The supernatant of cell culture medium was collected for detection.The results showed that the production of NO in EPCs treated with HMGB1 increased,while the content of NO decreased significantly after adding Anti-RAGE antibody and L-NAME(1mM).5.HMGB1 also participates in the phosphorylation of ERK signaling pathway.At the same time,HMGB1 is dependent on concentration and time.It was found that HMGB1 activated ERK signaling pathway had no significant effect on EPCs migration.This result shows that although HMGB1 participates in the phosphorylation of ERK,but its role is not manifested in cell migration and needs further study to confirm.6.Fluorescence staining of F-actin in EPCs showed that HMGB1 activated PI3K/Akt signaling pathway to regulate F-actin levels in EPCs.But this process was weaken by LY294002 and anti-RAGE antibody.Conclusion:1.HMGB1 and CD34 positive cell was significantly reduced in the wounds of diabetic mice.Exogenous HMGB1 promoted wound healing and increased neovascularization density and granulation tissue formation in diabetic mice.2.Exogenous HMGB1 up-regulated the expression of RAGE in EPCs,and it was concentration-dependent.Within a certain concentration range,HMGB1 promotes the proliferation and paracrine function of EPCs.At the same time,it contributes to the ability of angiogenesis and cell migration in vitro.Meanwhile,these functions were positively correlated with RAGE.MEK/ERK signaling pathway has no significant effect on HMGB1-induced EPC migration,although HMGB1-RAGE activates ERK phosphorylation.Therefore,signaling pathway involved in HMGB1-RAGE mediated cell migration may depend on cell type and MAPK/ERK may also participate in angiogenesis through other methods.These potential mechanisms need to be confirmed in further experiments.3.HMGB1-RAGE signal cascade caused the activation of PI3K/Akt/eNOS signaling pathways,then induced the production of NO and deformation of cytoskeleton,lead to EPCs migration.This indicates that PI3K/Akt signaling pathway is closely related to vascular endothelial cell migration and angiogenesis.RAGE may be a more effectively receptor that HMGB1-induced EPCs communicate via PI3K/Akt signal pathway migrates.These results are helpful to elucidate the molecular mechanism of HMGB1 inducing EPCs migration and reveal that the HMGB1-RAGE dependent PI3K/Akt/eNOS pathway is a potential therapeutic target for wound healing.4.Although HMGB1 participates in phosphorylation of ERK,JNK and P38 protein,but MEK/ERK signaling pathway has no significant effect on EPCs migration induced by HMGB1.Therefore,signal pathways involved in HMGB1-RAGE mediated cell migration may depend on cell type,and MAPK/ERK may also participate in angiogenesis through other methods.These potential mechanisms need to be confirmed in further experime nts.