Study On The Roles Of Sulfated Escherichia Coli K5 Polysaccharide Derivatives In Endothelial Progenitor Cells Differentiation And Tumor Angiogenesis

As components of heparan sulfate proteoglycan(HSPG),glycosaminoglycan(GAG)/ heparan sulfate(HS)polysaccharide chains could facilitate numerous signaling events by interacting with multiple growth factors,chemokines,and receptors.For example,it is not only involved in stem cell differentiation and angiogenesis,but also involved in tumor progression.Thus,it is believed that new drug development with HS as a potential target can achieve significant progress.Currently it has been proven that specific HS mimetics could promote stem cell differentiation with similar function,and exogenous soluble HS mimetics could inhibit tumor progression and tumor angiogenesis by act as competitive inhibitor of membranous HS.Bone marrow-derived endothelial progenitor cells(EPC)are considered critical factors during angiogenesis,for they not only incorporate into the endothelium and differ into mature endothelial cells(EC)to provide structural support to nascent vessels,but also promote the angiogenic process via paracrine secretion of pro-angiogenic growth factors,thus it could apply in pre-vascularization process in tissue engineering and in clinical treatment.However,EPC obtained in vitro are mainly derived from mononuclear cell(MNC)that separated from bone morrow or peripheral blood,and its differentiation depends heavily on the induction effect of the culture environment.Moreover,it has been proved that EPC also playes an essential role during tumor progression by proving tumor angiogenesis.Thus,inhibition of EPC behavior might be a useful anti-tumor strategy.For its similar carbohydrate backbone,Escherichia coli K5 capsular polysaccharide was used to synthesize HS mimetics,or sulfation derivatives,through chemical modification.However,neither the effect of these sulfation derivatives on the differentiation of MNC to EPC nor their effect on the EPC behavior has never been reported.Thus,this study evaluated the role of sulfation derivatives in EPC differentiation and tumor angiogenesis through these two aspects.The main results obtained in this dissertation were listed as follows:(1)Three kinds of sulfation derivatives were obtained from K5 capsular polysaccharide,including N-sulfated mimetic KNS,O-sulfated mimetic KOS and N,O-sulfated mimetic KNOS.The degree of substitution(DS)of K5 capsular polysaccharide was 0,and the DS of above three kinds of sulfation derivatives was 0.635,0.324 and 1.107,respectively.Compared with the composed of K5 polysaccharide(GlcA-GlcNAc),KNS was nearly composed of GlcA-GlcNS;the GlcA-GlcNAc6 S and GlcA2S-GlcNAc6 S content was increased in KOS;for KNOS,the existence of GlcA-GlcNS6 S and GlcA2S-GlcNS6 S further confirms its structure.(2)KOS and KNOS could promote MNC differentiate into early EPC(eEPC)through enhancing Notch signaling,and the promotion effect of KNOS was greater,whereas other samples have no obvious effect,indicating existence of O-sulfate group was required for this effect and the existence of O-and N-sulfate groups was more effective in exhibiting promotion activity.However,all the sulfation derivatives showed no effect during the process of MNC differentiate into late EPC/outgrowth endothelial cell(OEC).These results suggested that the promotion bioactivity of sulfation derivatives associated with its sulfation position,and they played different roles in different applications.(3)In normal cultural environment,KOS and KNOS significantly impaired the proliferation,migration,tube formation and secretion of pro-angiogenesis factors of EPC,whereas other samples have no obvious effect.(4)In tumor environment,TEM(Tumor endothelial markers)expression of EPC was heavily enhaced,and EPC behaviors,including proliferation,migration and pro-angiogenesis factors gene expression,was significantly enhanced,indicated tumor envioroment has alignant induction effect on EPC.However,KNOS still showed inhibition effect,indicating sulfation at both O-and N-site was essential to impair EPC behavior in tumor environment.The results showed KNOS have potential effect to inhibit tumor angiogenesis.(5)In tumor environment,KNOS could impaired the the effect of CXCL12 on CXCR4 internalization,Then,the downstream signaling,such MAPK,FAK and PI3K/AKT pathway,was also disturbed.Finally lead to the suppress of EPC behavior,which negative affect the tumor angiogenesis.