Hypoxia Promotes Human Mesenchymal Stem Cells Tube Formation And Endothelial Differentiation And Its Effect On Neovascularization

Seeking for suitable seeding cells to reconstruct endothelium and vessels has been a hotspot of vascular tissue engineering. Considering the limitation of embryonic stem cells, suchas ethical problems and immune rejection, adult stem cells have gradually becomeinvestigative focus, especially mesenchymal stem cells (MSCs).MSCs are multipotent stem cells derived from mesoderm, possessing potentproliferative capacity and self-renewal and multilineage differentiation. MSCs candifferentiate into osteocytes, chondroncytes, adipocytes, muscle cells, liver cells, neuron-likecells and neuroglial cells under the control of different environment and cytokines. MSCshave some advantages, including drawing the materials easily and slightly injury, no ethicalproblems and immune rejection, multiple amplification easily in vitro, induceddifferentiation easily and inserting and expression of exogenous genes easily. Thus, MSCshave been the main source of tissue engineered vascular seeding cells.MSCs can be differentiated into endothelial cell (EC) by the induction of hormone,cytokines, extracelluar matrix (ECM) and cellulr microenvironment. Presently manyscholars emphasize on the research of cytokines and ECM. In fact, differentiation of MSCsin vivo is much more complicated than in vitro and is synaptically regulated by multipleagents. Cell microenvironment is one of important factors which have influence ondifferentiation. Being the significant component in cell microenvironment, oxygen is thedeterminant factor affecting cell differentiation. Under normal circumstance, oxygen tensionin external of body is obviously higher than that in mammal tissue. For instance, oxygentension in bone marrow is 27~49mmHg, corresponding to 4~7% oxygen concentration.Even though there are some protective agents like glutathion peroxidase in the body, cellsand tissue function will be cut down or damaged while oxygen density is excessively high.On the contrary, if oxygen density is less than 20%, cell function will be preferablymaintained. This will offer a new moment for MSCs differentiation into EC.In the present study, highly homologous human MSCs (hMSCs) were obtained by thecombination with orthodox density gradient fractionation, adherence screening method andmonocloning screens. In vitro hMSCs were cultured under hypoxic conditions (5% O2) bysimulating lower oxygen tension in the bone marrow and would better grow and proliferate.Then tube formation and endothelial differentiation induced by hypoxia were observed andmechanism of action was investigated. By using BALB/C nude mice as animal models andmarking hMSCs with pLEGFP-N1 retroviral vector, endothelial differentiation andneovascularization of hMSCs in hypoxic environment in vivo were also studied. In addition,the role and mechanism of hMSCs in the process of tumor neovascularization in vivo werepreliminarily discussed by setting up tumor models.The results were as follows:1. A standardized technique platform was successfully established for isolation,cultivation, purification, identification and amplification of hMSCs. Highly homologoushMSCs were isolated and obtained and the purity was above 98%. They may be the cellsource for extended experiments, because they maintained stable biological characteristics ateven passage 12 in undifferentiation state.2. In vitro hypoxia can accelerate hMSCs spreading, but it had no effect on completeability of cell spreading. It was supposed that hypoxia promoted high expression of CD44,one of the cell adhesion molecules at the surface of hMSCs, while CD44 can participate incell-cell and cell-matrix interactions by surface antigens binding with ECM.3. MT1-MMP mRNA and protein under hypoxia were also significantly higher thanthose under normoxia, which can enhance the ability of hMSCs degrading ECM.4. Under the same chemokine, the speed of hMSCs migration in hypoxia was strikelyfaster, and the cells number under polycarbonate membrane exceeded that in normoxicgroup. This phenomenon was concerned with hypoxia-induced MT1-MMP overexpressionwhich triggered cell migration not only by facilitating ECM proteolysis, but also by cleavingCD44 to regulate cell migration and cell appearance.5. In 3-D Matrigel cultivation, after 4 hours under hypoxia, most of hMSCs sent outbulges and formed complete connection, so more tube-like structures were visible whichwere composed of cell monolayer similar to capillary-like structures formed by HUVEC.However, hMSCs under normoxia showed very few tube-like structures, and majority of thecells stayed round without bulges or the minority sent out bulges without connectiveintegrity. When keeping on culture for 24 hours, a few hMSCs taking part in tube structuresin hypoxic group expressed CD31, one of specific markers for EC, while no expression innormoxic group. It indicated that hypoxia can rapidly induce hMSCs tube formation and partof hMSCs in tube-structures gradually differentiated into endothelium with endothelialphenotype by extension of culture time.6. Under normoxic conditions, hMSCs hypoxic supernatant as chemokine canconspicuously increase the speed of hMSCs migration and the cells number underpolycarbonate membrane were more than that in normoxic supernatant group. In addition,hMSCs hypoxic supernatant can also promote HUVEC capillary-like formation. It suggestedthat there were certain cytokines or chemokines in hMSCs hypoxic supernatant which canstrengthen the capacity of cell migration and tube formation by paracrine action.7. It was confirmed by flow cytometry, AO/EB and AnnexinⅤ-CY3 staining thathMSCs in hypoxia for 24 hours did not show obvious apoptosis in comparison with those innormoxic group. The reason was that 5% O2 density in the study which closely mimicked invivo low oxygen enabled hMSCs to optimally grow, proliferate and differentiate.8. It was detected by semi-quantity RT-PCR, indirect immunofluorescence and WesternBlot that the expression of HIF-1α mRNA and protein in hypoxia for 24 hours were alsosignificantly higher than those in normoxic group. Likewise, the expression of VEGF mRNAand protein markedly increased. So we speculated that the reason why hypoxia can promotehMSCs endothelial differentiation and tube formation was that hypoxia induced theexpression of HIF-1α, one of transcription factors in turn upregulated expression of VEGF,which contributed to endothelial differentiation and formation of tube-like structures.9. Under hypoxic conditions, hMSCs migration can be effectively inhibited by ablocking antibody directed against MT1-MMP catalytic domain. In 3-D Matrigel culture, thetube-like structures were strongly diminished because of utilizing this antibody. Theseresults showed that overexpression of MT1-MMP under hypoxia was indispensable forhMSCs migration, capillary tube formation and neovascularization.10. Under laser scanning confocal microscope (LSCM), the change of calcium ion(Ca2+)channel was dynamically observed by Fluo-3/AM staining. When hMSCs were in restingconditions, Ca2+ mean fluorescent intensity in hypoxic group was markedly higher than thatin normoxic group. It was shown that hypoxia promoted Ca2+ accumulation. Compared withnormoxic group, Ca2+ elevated extent in hypoxic group after adding exogenous Ca2+ wassignificantly higher, which manifested that the patency of Ca2+ channel increased in hypoxia.It may be another cause to induce hMSCs differentiation.11. In comparison with physiological saline, the quantity of vessels branch on thechicken chorioallantoic membrane (CAM) induced by hypoxic or normoxic supernatant ofhMSCs markedly increased. Furthermore, the quantity of vessels branch in hypoxicsupernatant group was much higher than that in normoxic supernatant group. These dataindicated that hypoxic supernatant of hMSCs can induce a clearly angiogenesis on CAM. Itwas further confirmed by ex-vivo experiment that hypoxia can promote hMSCs secretingangiogenic factors.12. After hMSCs were transduced with pLEGFP-N1 retroviral vector, hMSCsEGFP+retained their own morphology, immunophenotype, cell cycle, growth characteristics andosteogenic or adipogenic potential. During cell transplantation and gene therapy, EGFP as areporter in vivo is a simple, safe and reliable method to monitor the changes of implantedhMSCs. It is useful for clinical application.13. In Matrigel assay in vivo, at 2 weeks or 4 weeks after subcutaneous implantationinto nude mice, it could be seen from the gross that blood vessels grew into Matrigel plugsin hMSCsEGFP+ or VEGF group, but there was avascularity or less vessels in plain Matrigel.The mean vascular density (MVD) in Matrigel plugs in hMSCsEGFP+ group was strikelyhigher than that in VEGF or plain Matrigel group. There was an increase in MVD inhMSCsEGFP+ group from 2 weeks to 4 weeks. In contrast, the MVD in VEGF group showeda reduction in the same time period. These results showed that hMSCsEGFP+ could contributeto neovascularization, and the angiogenic effects were time-dependent, namely angiogeniccapacity correspondingly raised with the extension of time. Taking account of the finitehalf-time of VEGF, the biological effect of VEGF would be a decline with continuousconsumption. However, hMSCs possessed potent capacity of self-renewal. When they wereimplanted into the body, hMSCs under the physiological regulation could continuouslyproduce growth factors. Thereby, hMSCs could maintain the long-lasting effect ofneovascularization. In addition, there was a dose-dependence between the neovascularizedeffect of hMSCs and cells number, namely angiogenic capacity accordingly enhanced withthe multiplication of implanted cells.14. In Matrigel assay in vivo, CD31-CY3 immunofluorescent staining showed most ofhMSCsEGFP+ in Matrigel plugs were dispersed and presented a fibroblast-like shape. But veryfew hMSCsEGFP+ appeared in the endothelium of vessel wall which were also positive forCD31. It indicated that the majority of vessels were host-derived angiogenesis mediated byhMSCs and very few new vessels were formed due to hMSCs differentiation into EC(vasculogenesis).15. In Matrigel assay in vivo, at 2 weeks after subcutaneous implantation, the quantityof vessels in Matrigel plugs reduced obviously when utilizing anti-VEGF antibody or VEGFantagonist. But there was no significant change while using nonspecific anti-IgG antibody. Itwas concluded that hMSCs contributed to neovascularization by secreting VEGF which maybe associated with low oxygen tension of Matrigel implant.16. Boyden Chamber assay showed that tumor cells or EC promoted hMSCs migrationbecause growth factors, such as VEGF, secreted by tumor cells or EC were more thanthose secreted by normal cells.17. In tumor neovascularization experiment, at 4 weeks after subcutaneous inoculationwith MCF-7 alone or in combination with hMSCsEGFP+ (1:1) into nude mice, the size andweight of tumor mass in hMSCsEGFP+/MCF-7 group were markedly increased and MVD wasalso significantly higher than that in MCF-7 group. CD31-CY3 immunofluorescent stainingshowed that very few hMSCsEGFP+ in hMSCsEGFP+/MCF-7 group localized in theendothelium of vessels, which expressed CD31, but most hMSCsEGFP+ were in sporadicdistribution. The results not only suggested that hMSCs contributed to tumorneovascularization, but also that most of vessels were host-derived angiogenesis mediatedby hMSCs. Only very few vessles were attributed to hMSCs transdifferentiation into EC.The role of hMSCs in support of tumor neovascularization was owing to low oxygentension caused by tumor mass.In a word, this experiment from in vivo to in vitro systemically explained that hypoxiacan promote hMSCs endothelial differentiation, tube formation and its contribution toneovascularization, as well as the mechanisms were profoundly investigated. It will providenot only a new technological route and substantial method for MSCs differentiation into EC,but also important theoretic and experimental basis for better utilizing MSCs to treatischemic and vaso-dependent diseases. In addition, it will establish a novel route for tumortherapy to utilize engineered MSCs to secrete anti-tumor agents and to antagonize tumorprogression.