The Mechanism Of Bone Marrow Mesenchymal Stem Cells Aggravate Vascular Calcification

Part I Arterial tissues in vitro regulate the differentiation of bonemarrow mesenchymal stem cellObjective: To study the differentiation of bone marrow mesenchymal stem cellunder different calcification environments in artery.Methods: We made a vascular calcification model using warfarin, vitamin K1andvitamin D3.After Successfully made the model, we took normal SD rat arteries andcalcified arteries co-cultured with MSC, all were divided into three groups: normalgroup was normal artery with MSC, Calcified inducers group was calcified inducers with normal arterial and MSC, Calcification group was calcified arteries add withMSC. Each group was cultured for3weeks. On the10th day of the experiment, wedetected OPG protein secretion. After three weeks, we observed changes of cellmorphology, detected total protein content and alkaline phosphatase (ALP) activity,using Real-time PCR method detected the Ror2(receptor tyrosine kinase-like orphanreceptor2) mRNA expression.Results: Calcification group MSC spontaneously proliferated to osteoblast-likecells differentiation, Compared with normal group, the total protein content, ALPactivity of bone metabolism markers, Osteoprotegerin (OPG) were significantlyelevated, while Ror2mRNA expression was significantly lower. MSCs in calcifiedinducers group did not differentiated to osteoblast-like cells, compared with thenormal group, ALP activity was no significant difference, the total protein contentand OPG were increased, however, Ror2mRNA expression was lower than normalgroup. The Ror2mRNA expression of the calcification group was significantlylower than the calcified inducer Group.Conclusions: In vascular calcification environment, MSC proliferated intobone-like differentiation, while in normal vascular and calcified inducersenvironment, the osteoblast-like differentiation of MSC did not appear. Thisphenomenon may be related to the Ror2expression of artery. Part II Vascular smooth muscle cells in vitro regulate the differentiation of bonemarrow mesenchymal stem cellObjective: To study the differentiation of MSC when were direct co-cultured withnormal smooth muscle cells or calcification of smooth muscle cells.Method: Direct co-cultures were established by seeding SMCs or calcified SMCs andMSCs together at ratios of SMC15×104or calcified SMC15×104; SMC5×104orcalcified SMC5×104to MSC10×104, SMC10×104or calcified SMC10×104toMSC5×104. Cells were cultured for9days. Then osteoblastic differentiation wasevaluated by cell morphology and the activity of alkaline phosphatase (ALP) in celllysates. And investigating the inhibit signaling of WNT-signaling, noncanonicalsignaling pathway, wnt5a/Ror2express in each group.Results: We found that MSC cultured in culture media with OS did not differentiate to an osteoblast phenotype when direct contact with noncalcified SMC, independenton the number of MSC. However, an osteoblast phenotype could be seen when MSCcultured in media without OS differentiated towards direct contact with calcifiedSMC, and the level of osteoblastic markers had a direct corelation with the number ofMSC. Interestingly, we found that Wnt5a protein was related to the level ofcalcification, while rarely presents in non-calcification, Ror2mRNA in noncalcifiedgroups are obviously higher than that in calcified groups.Conclusions: MSC can differentiate to different types of cells when directlyco-cultured with normal smooth muscle cells, or calcificed smooth muscle cells, andis proportional to the number of MSC. In addition, Wnt5a/Ror2signaling pathwaymay play an important role in the MSC differentiation.