Studies On Pro-angiogenic Factors And Their Mechanisms In Mice Infected With Echinococcus Granulosus

Echinococcosis is an important zoonotic disease which causes serious threats to human health,the mechanisms of its pathogen's growth,development and pathogenesis are still not clear.Angiogenesis is critical for parasites to acquire the nutrients necessary for growth,maturity and breeding after infection,and it is mainly regulated by parasite-derived and/or host-derived angiogenic factor.But the pro-angiogenic activity of Echinococcus granulosus still need to be studied.Moreover,MDSCs(myeloid derived suppressor cells)can be enriched after E.granulosus infection,while the pro-angiogenesis of MDSCs from parasitic infection animal is not clear.The researches on pro-angiogenic factors and their mechanisms in mice infected with E.granulosus have implications for the studies about prophylaxis and treatment of echinococcosis.In the present study,mouse microRNA microarray was used to identified the differentially expressed microRNA in monocytic-type MDSC(M-MDSC)from mice infected with E.granulosus,and the effects of parasite-derived and host-derived materials on human umbilical vein endothelial cells into tubes were investigated in order to understand the angiogenesis caused by E.granulosus infection preliminarily.In addition,it is urgent to find new drugs for echinococcosis treatment.And ursolic acid,an anti-angiogenesis compound,distributes extensively,and inhibits angiogenesis through cell apoptosis induction,down-regulation of matrix metalloproteinase expression and other pro-angiogenic factors,etc.In the present study,the in vitro anthelmintic effects of ursolic acid on protoscoleces,germinal cells and metacestodes of E.granulosus and the in vivo efficacy in mice experimentally infected with E.granulosus,and its anti-angiogenic effects were investigated,in order to evaluate the potential of ursolic acid served as a drug for echinococcosis treatment.Part 1 Analysis of microRNA expression profile in monocytic-type MDSC derived from mice infected with Echinococcus granulosusTwenty-eight microRNAs expressed differentially in M-MDSC derived from E.granulosus infected mice were identified by mouse microRNA microarray in the present study,and 272 target genes were predicted in the intersection of three microRNA database of TargetScan,PITA and microRNAorg.These target genes were mainly involved in the Biological Process of intracellular protein transport,protein targeting to lysosom and protein transport,etc.,and mainly located at cytoplasm,neuronal cell body and membrane,etc.,and were mainly involved in the Molecular Function of protein binding,metal ion binding and SH3 domain binding,etc.And the differentially expressed microRNAs were mainly enriched in the signaling pathways of Endocytosis,Wnt signaling pathway and Axon guidance,etc.In addition,these microRNAs and their target genes were enriched in the signaling pathways of MAPK signaling pathway,Focal adhesion pathway,PI3K-Akt signaling pathway,cAMP signaling pathway,mTOR signaling pathway and TGF-beta signaling pathway which linked to immnunoregulation and angiogenesis through the analysis of DIANA-miRPath3.0.In addition,the expression of 8 microRNAs among 10 ones were randomly verified by quantitative PCR in three mice infected with E.granulosus and three normal mice,and it indicated the reliability of the microRNA microarray.Therefore,MDSCs derived from mice infected with E.granulosus may regulate the angiogenesis through their microRNAs.Part 2 Preliminary study on the in vitro pro-angiogenic activity of Echinococcus granulosus hydatid cyst and myeloid-derived suppressor cells from experimentally infected miceCD31 of high expression was observed in the host tissue of film surrounding hydatid cysts through immunohistochemical method,and the level of VEGF in serum from mice infected with E.granulosus(548.100 ±134.200 pg/ml)was higher than that from normal mice(76.950±2.760 pg/ml)by ELISA(t = 4.109,P = 0.001),The culture supernatant of hydatid cyst(F = 73.03,P<0.001)could significantly promote human umbilical vein endothelial cell into tubes,the total lengths of tube fragments of different groups stimulated with the culture supernatant of hydatid cyst,protoscolex and cyst fluid and RPMI-1640 were 13253± 169,7586± 1694,2838±410 and 7497± 709,respectively.And the supernatant of polymorphonuclear-type MDSC(PMN-MDSC)(4873 ± 1436)also could significantly promote human umbilical vein endothelial cell into tubes compared with RPMI-1640(3015 ±602)(P = 0.028),while no statistical difference with M-MDSC(3941 ± 1435)(P = 0.219),and no differences were found between M-MDSC and RPMI-1640(P = 0.243).In addition,The transcriptions of parasite-MMP-9(t =-11.654,P<0.001)and HMGB1(t = 6.433,P?0.003)were detected in cyst wall and protoscolex with differences in transcriptional levels,and the transcriptions of VEGF from both PMN-MDSC(t = 13.81,P<0.001)and M-MDSC(t = 12.98,P<0.001)was higher in infected mice than normal ones.Therefore,angiogenesis in parasitic sites in mice infected with E.granulosus may be attributed to some parasite-derived and host cell-derived pro-angiogenic molecules.Part 3 The efficacy of ursolic acid on Echinococcus granulosus in vivo and in vitroProtoscoleces(45.95 ± 5.30%)were killed through UA(40 ?g/ml)for 3 days in vitro,a considerably stronger and dose-dependent effect was on germinal cells,and the effect in metacestodes was detectable and resulting ultrastructural damage compared with the control protoscoleces cultured in 0.5%DMSO medium was confirmed by scanning and transmission electron microscopy(SEM and TEM).Moreover,the growth of the metacestodes reduced with oral administration of UA at 200 and 100 mg/kg in vivo(39.5%and 38.3%,respectively)with the evidences of ultrastructural damages revealed by SEM and TEM,although this effect was found to be statistically significant only at 200 mg/kg UA(P = 0.0178).In addition,UA could inhibit the proliferation of HUVEC in vitro significantly(F= 25.69,P<0.001),and down regulated the expression of CD31,the mRNA expression of parasite-MMP-9(t?3.380,P = 0.004)and HMGB1(t = 6.377,P<0.001),and mouse-MMP-9(t =8.746,P<0.001)and VEGF(t = 9.399,P<0.001)in vivo.Therefore,UA could suppress the growth of hydatid cysts through the direct role of anti-echinococcus cyst or the indirect activity of anti-angiogenesis.Conclusion1.MicroRNAs in MDSCs derived from mice infected with E.granulosus may play a role in angiogenesis drived by MDSCs through regulation of some relative signaling pathways.It is the fisrt discovery of the potential pro-angiogenic function of microRNA in MDSCs in parasitic infection models,and provides a molecular basis for studies on the angiogenic function of MDSCs at the microRNA level.2.Angiogenesis in mice infected with E.granulosus may be attributed to some parasite-derived and host cell-derived pro-angiogenic molecules.It is the fisrt discovery of the pro-angiogenic functions of Echinococcus granulosus and MDSCs derived from the mice infected with this parasite,and it provides some ideas for the researches on the mechanisms of angiogenesis in parasitic sites caused by E.granulosus infection from both parasite and host aspects.3.Ursolic acid as an angiogenesis inhibitor was firstly demonstrated as a potential anti-echinococcosis agent,and it could suppress the growth of hydatid cysts through the direct role of anti-echinococcus cyst or the indirect activity of anti-angiogenesis.It provides some ideas for the treatment of echinococcosis,and a scientific basis for ursolic acid becoming a drug candidate against echinococcosis.