The Exploration Of Apo(a) On Angiogenesis Ability Of Mouse Bone Marrow-derived EPCs And Its Mechanisms

At present, there are no very effective therapeutic measures for the treatment ofischemic peripheral vascular diseases which has a serious harming to human health.The discovery of endothelial progenitor cells (EPCs) opens up the possibility ofdeveloping angiogenesis therapies because EPCs are precursor cells of endothelialcell, can proliferate, and differentiate into mature endothelial cells. Apolipoprotein(a)[(apo(a)], an important component of lipoprotein (a)[Lp(a)], has been considered tobe an independent risk factor for peripheral vascular disease. Some studies havereported the effect of Lp(a) on EPCs biological functions, but we can not determinewhether Lp(a) impact EPCs proliferation and differentiation because of the twoopposite experimental evidences. Lp(a) level is not influenced by the environment,diet and medication, and its plasma concentration is mainly controlled by the rate ofapo (a) de novo biosynthesis. Furthermore, apo(a) can effect vascular pathological andphysiological function independent of the apoB-100or Lp(a). But the exactmechanism of apo(a) affecting EPCs angiogenesis is unclear.In the present study, we has isolated mouse bone marrow-derived EPCs,investigated the effect of apo(a) on EPCs angiogenesis in vitro and in vivo andexplored its mechanism. The main research results and methods are as follows:Part1Apo (a) affects angiogenesis of mouse bone marrow-derived EPCsObjective: To study the effect of apo(a) on mouse bone marrow-derived EPCs migration, adhesion, homing and angiogenesis abilities.Methods: EPCs were isolated from the bone marrow of apo(a) transgenic mice andwild-type litter mates. These cells were cultured with or without apo(a) beforetransplantation. Hindlimb ischemia models were surgically induced in mice, whichthen received an intravenously injection of3×10~5EPCs. At3,7and14days post EPCtransplantation，the adhesion, migration abilities and capillary density in calf muscleswere assessed. Then, EPCs and muscle tissues were harvested and the expression ofP/E selectin、PGSL-1、CXCR4、VEGF, etc. was detected by RT-PCR and westernblot.Results: Results showed EPCs could take up Dil-AcLDL and bind to FITC UEA-1.The number of EPCs expressing CD133+/VEGFR-2+,CD133+/CD34+and vWF was73.43%,72.12%and1.0%, respectively. Apo(a) inhibited EPCs adhesion ability in adose dependent manner. Addition of0.2μg/ml apo(a), inhibition of94.2%comparedwith control group was observed (P<0.01, n=3); at a concentration of15μg/ml apo(a), EPCs almost completely expressed apoptosis. An in vitro migration experimentalso produced the similar results. After7days cultured, tubule-like formation onmatrigel gels was impaired92.1%treated with10μg/ml apo(a) and almostcompletely eliminated at the concentration of15μg/ml(P<0.01, n=3); RT-PCRexamination disclosed a high expression of P/E-selectin in the endothelium ofischemic tissues compared with normal hindlimb tissues (P<0.05, n=3) and a downregulated expression of PGSL-1on apo (a)-treated EPCs detected by western blot (P<0.05, n=3). Histological examination revealed reduced local accumulation of EPCsand a decreased number of capillary ECs in apo (a) transgenic mice compared withcontrol group (P<0.05, n=8).Conclusion: Apo (a) impaired the abilities of EPCs adhesion, migration and homing,and resulted in an inhibition of EPCs angiogenesis. Part2The study on the mechanisms of apo(a) effecting EPCsangiogenesisObjective: To explore the mechanisms of apo(a) inhibits EPCs angiogenesis.Methods: EPCs were harvested to extract total RNA, then the expression of Notchreceptors, ligands and LOX-1receptors and the greatest time-effect, dose-effectrelationships of apo(a) on them were detected by RT-PCR; Immunoprecipitationmethod was applied to analyze the effect of apo(a) on the receptor-ligands binding,RT-PCR to analyze the expression of downstream genes.Results: Results indicated that only Notch2receptor, JAG-1and JAG-2ligandswere observed on EPCs, all of them had the highest expression after EPCs weretreated by10μg/ml apo(a) for24h; apo(a) could promote the binding ofNotch2-JAG-2,up-regulate the expression of RBPJ、HES、HEY genes (P<0.05,n=3)and down-regulate the expression of CXCR4、PGSL-1(P<0.05, n=3), but therewas no change in VEGF expression. The binding of Notch2-JAG-1was undetectableby immunoprecipitation and the expression of CXCR4、PGSL-1、VEGF had nosignificant differences after LOX-1receptor was blocked.Conclusion: Apo (a) could promote Notch2-JAG-1binding, up-regulate theexpression of RBPJ、 HES、 HEY genes and down-regulate the expression ofCXCR4、PGSL-1, inhibit EPCs adhesion,migration,homing and angiogenesisabilities. LOX-1was involved in the effect of apo (a) on EPCs angiogenesis.