| Background and objectiveMore and more interests are attracted in vasculogenesis researches on the stabilization and maturation of nascent vasculature. Proliferation of vascular smooth muscle cells (VSMCs) and recruitment of VSMCs towards endothelial cells (ECs) forming primitive vascular network is a committed step of the stabilization and maturation of nascent vasculature. However, insight in the molecule mechanisms of recruitment and chemotaxis of VSMCs during early embryonic vascular development is currently limited. One of the main reasons is that it is very difficult to separate and purify VSMCs of early stages. During in vitro differentiation, ESCs can spontaneously develop into embryoid bodies (EBs), and can recapitulate many processes of early embryonic development. Embryonic stem cells (ESCs) provide a valuable in vitro model for the investigation of mechanisms in early VSMCs lineage commitment, differentiation, and maturation. Our laboratory had established transgenic ESCs lines expressing the enhanced green fluorescent protein under the transcriptional control of the smooth-muscle-specific SM22αpromoter. In these transgenic ESCs, we can directly observe that ESCs differentiation into VSMCs in living cells without immunocytochemical stainings. By establishing a chemotactic model for VSMCs during early embryonic vasculardevelopment and using platelet derived growth factor-BB (PDGF-BB) as an exogenous chemotatic factor, our aim is to observe initially the chemotactic effect of PDGF-BB on VSMCs during that time.The development and differentiation of VSMCs are extremely complicated and polygene regulating process. PDGF-BB is a potent mitogen. Many researches have shown that treatment of mature VSMCs with PDGF-BB is associated with rapid downregulation of expression of multiple VSMCs differentiation marker genes. However, whether PDGF-BB can repress VSMCs development from pluripotential ESCs remains unkown. Therefore, the goal of the present study is to observe expression regularity of SMα-actin, SM22α, myocardin and SMMHC during early embryonic vascular development, and initially investigate the differentiation effect of PDGF-BB on VSMCs during that period by using AG1296 (a platelet- derived growth factor receptor) as a blocking agent.Methods1. Cell culture Mouse ESCs were cultured on STO feeder cells which were treated by 10μg/mL mitomycin C for 2 hours. ESCs should be passaged when reaching sub-confluent state. Culture medium was replaced everyday to maintain undifferentiated state of ESCs.2. ESCs differentiation and the establishment of EBs model Subconfluent ESCs were dispersed with 0.25% trypsin-0.53 mmol/L ethylenediaminetetraacetic acid and plated onto gelatin-coated dishes for 3 h to allow feeder cells to selectively attach. Nonadherent ESCs aggregates were then dispersed and cultured on bacteriological petri dishes in ESCs medium without leukemia inhibitory factor. ESCs were maintained for 5 days or 6 days in suspension then were allowed to settle onto 0.1% gelatin-coated plates in the presence of DMEM/10% FCS. Medium was changed on a daily basis.3. Preparing of under-agarose model Agarose (2% wt/vol, final) was dissolved in DMEM and then mixed with an equal volume of a solution containing bovine serum albumin (2% wt/vol). The final solution (3 ml per dish) was layered onto cell culture dishes (35 mm diameter) and permitted to solidify at room temperature. Three wells (5 mm diameter) 2 mm apart were cut into the agarose using a hollow metal haustorial tube as a template.A suspended EB at day 6 was placed in the center well under an inverted microscope and allowed to attach in DMEM/10%FCS. Medium was changed on a daily basis.4. Time Lapse Microscopy Embryoid bodies of day 6+20 in the under-agarose gel were kept in a 37℃CO2 supplemented incubation chamber under an Olympus IX-70 microscope with an attached CoolSNAPfx digital camera. Images were taken in 12 h with 10 minutes interval time. Migration velocities were analysed by Image-Pro Plus 5.1 software.5. Immunocytochemical stainings Fixed EBs were washed with 0.05 mol/L TBS and permeabilized with 0.25% Triton X-100 and 0.5 mmol/L NH4Cl in 0.05 mol/L TBS. Before incubation with antibodies, EBs were blocked with 5% bovine serum albumin in TBS. Primary antibodies were SM-α-actin, Laminin, myocardin, SMMHC and PECAM-1(CD31) used at 1:100 dilution. Negative controls were performed without primary antibodies. Detection was performed with a TRITC or FITC-labeled secondary antibody.6. RT-PCR RNA was extracted from EBs at a variety of time points with Trizol. Reverse transcription was carried out on 1μg of total RNA: PECAM-1, SM22α, myocardin, SMMHC and GAPDH.7. Western blot analysis Total cell protein was extracted from EBs at a variety of time points more solito, then was separated by SDS/PAGE, transferred onto a poly membrane, combined antibody and colored. Immunoblot analyses were carried out using SMα-actin, myocardin, SMMHC andβ-actin antibodies. Specific proteins were detected with the enhanced chemiluminescence (ECL) system.Results1. Morphological characteristics of ESCs ESCs can maintain undifferentiated on STO feeder cells, which formed compact cell colony with smooth margin and undistinguished cell boundary.2. Morphological characteristics of EBs at different developmental stage EBs underwent rapid differentiation when cultured in suspension, typical simple embryoid bodies (SEBs) were formed 1 to 2 days later after suspension. With culture time going on, SEBs differentiated further to form mature cystic embryoid bodies (CEBs). CEBs were composed of structuresof primitive endoderm, base membrane, columnar primitive ectoderm and cystic cavity, which were similar to those in early embryo.3. Differentiated characteristics of EBs in under-agarose model In the under-agarose gel, EBs could spontaneously differentiate into myocardial cells, endothelial cells and VSMCs in the absence of exogenous growth factors, indicating that EBs in under-agarose gel have full potential of differentiation.4. Expression phases of VSMCs and ECs specfic marker genes SMα-actin, myocardin, SM22αand SMMHC were found to be expressed in EBs from day 0 (ESCs), 8, 11, 13 respectively during early embryonic vascular development. Maximal increases in SMα-actin, myocardin, SM22αand SMMHC expression occured about day 10, 20, 25, 25 respectively. CD31 was present in undifferentiated ESCs, downregulated as differentiation proceeded, but then upregulated by day 5 as ECs developed. CD31-positive EBs at day 14 exhibited many cord-like structure or vessel-like structure. The greatest increase in CD31 occurred at about day 8.5. The chemotactic effect of exogenous PDGF-BB on VSMCs The mean migration velocities of VSMCs at day 20 after embryoid bodies attachment in control group and 5 ng/ml, 10 ng/ml, 20 ng/ml, 50 ng/ml PDGF-BB groups were (94.07±23.80)μm/h, (118.08±31.63)μm/h, (173.53±24.58)μm/h, (380.74±39.56)μm/h and (335.62±32.16)μm/h, respectively, with 20 ng/ml as the peak concentration. VSMCs migrated randomly in control and 5 ng/ml PDGF-BB groups, but migrated toward PDGF-BB in 10ng/ml to 50 ng/ml PDGF-BB groups.6. The differentiated effect of exogenous PDGF-BB on VSMCs There were no clear differences among the three groups of different concentrations (0μmol/L, 10μmol/L, 50μmol/L) of AG1296 in the expression of SMα-actin, SM22α, myocardin and SMMHC protein and of SM22α, myocardin and SMMHC mRNA.Conclusion1. The under-agarose model can imitate the whole procedure of early embryonic vascular development and can be used to investigate chemotactic influences of different growth factors on VSMCs. Certain concentrations ofexogenous PDGF-BB can induce the migration of VSMCs in a concentration-dependent manner.2. A spontaneous VSMCs differentiation occurs during EBs development, SMα-actin is the first to be detected, the following are myocardin, SM22αand SMMHC; the expression of VSMCs in EBs is much later than that of ECs; PDGF-BB may not be indispensable for the regulation of expression of VSMCs markers during early EBs differentiation. |
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