Preliminary Study On The Injury Effect Of Lipoprotein (a)to Mouse Bone Marrow-derived Endothelial Progenitor Cells

OBJECTIVE：We preliminary study of the injury impact of lipoprotein (a)[LP(a)] as one of the most important risk factors of cardiovascular disease on mousebone marrow-derived endothelial progenitor cells (EPCs) and the possiblemechanism.METHODS: EPCs were treated of six groups,for five groups with differentconcentrations of Lp(a)(1,10,100,300and600μg/ml),and control groups of mediumwith200μmol/L EDTA. The isolation of EPCs by adherence culture and binding ofUlex europaeus agglutinin1(UEA-1), and uptake of acetylated low densitylipoprotein (Ac-LDL) were measured to determine EPCs. The abilities of survial andproliferation, migration activity and adherence were detected by the methods of MTT,transwell and gelatin adherence assays. EPCs were replated on the top of the ECM geland the total length of the tube structures in each photograph was measured usingAdobe Photoshop software7.0, and the size and counts of colony-forming units werephotographed with a microscope. mRNA and protein level were determined byRT-PCR and Western blot.With the techniques of immunohistochemistry to test theexpression and disposition situation of Nitric-Oxide Synthase（eNOS） protein. Reactiveoxygen species(ROS) were detemined by DCFH-DA staining and total Nitric-Oxide（NO） was measured with the Griess reagent.RESULTS:①Attachment technique could isolate EPCs from mus borrowmononunclear cells with purified highly over70%.②LP (a) dose-dependentlydecreased the surival of EPCs，incubation with LP (a) concentrations of100μg/mlpreliminaryly have impaired effect on EPCs，the impairment of300μg/ml LP (a) has rapid increased.③）It shows a significantly reduced migratory rate of EPCs aftertreatment with1μg/ml LP（a）,and there were decreased cells migration to the lowercompartment in response to10μ g/ml LP (a),more important,the count of themigratory cells was less than1/6of the control; when incubated at concentrations of100or300μg/ml, it was1/9or1/14of the control.④EPCs treated with LP (a)showed a dose-dependent decrease of adhesion to gelatin. Treatment of cells with1μg/ml LP (a) markedly decreased the number of adhesive cells（145.2±8.4/everyfield vs115.2±12.6/every field，P＜0.05，n=5. When EPCs were exposed to10or100μg/ml LP (a), the capability of adhesion had further descended, only couldachieve1/2or1/3of the control,after treated with300μg/ml, there was remarkablydescent to1/16of control.⑤Treatment with10μg/ml LP(a) impaired the ability ofEPCs to form tube structures.EPCs cultivanted with100μ g/ml had presenteddecreased tube formation and was less than1/4of control（36.34±1.54mm/everyfield vs8.76±0.62mm/every field，P＜0.001，n=5; and when incubated at300μg/mlof LP (a), the integrity tube structure was severely disrupted.⑥After treated with100μg/ml of LP（a）,not only decreasing the EPC colony–forming（10.2±1.3vs3.1±0.4，P＜0.01，n=5）but also inhibiting the growth of the colony–forming.⑦Thestudies on the mechanisms have revealed that EPCs incubated at100μg/ml of LP（a）,the expression of mRNA and protein of eNOS was reduced and a strikingdecrease of NO generation was observed at this concentration（36.35±3.91vs3.91±0.79，P＜0.001，n=3）. Assessment of ROS demonstrated that the production of ROShad significantly increased(1±0.12vs3.92±0.36，P＜0.05，n=5）after treated with1μg/ml LP（a）.When exposed with LP(a) at concentrations of10μg/ml, was15times compared with control; However, both the green fluorescence intensity and thecells number with fluorescence had increased significantly(Figure12D), futher,intensity was30times more than control and was8or2times equal to the groups of1or10μg/ml LP（a）.CONCLUSIONS：①L P（a） inhibit proliferation of EPCs dose dependly;②L P（a）inhibit magration of EPCs dose dependly;③L P（a） inhibit adhesion of EPCs dose dependly;④LP（a） inhibit vasogenesis of EPCs dose dependly;⑤LP（a）inhibitcolony–forming capacity of EPCs;⑥Injury effect of LP（a） are related todown-regulating expression of eNOS and release of NO, and up-regulating ROSrelease.