| Objective: Nowadays, the most attractive theory and method of therapy in coronary heart disease (CHD) is therapeutic angiogenesis, which is based on vessel forming of early embryonic development to stimulate the growth of new vessels in myocardial ischemia zone and collateral circulation, representing the new direction of therapy in CHD. However, factors, the receptors and related signaling transduction molecules involved in vasculogenesis remains to be elucidated. Murine embryonic stem cells (ESCs) are pluripotent cell lines of embryonic origin, first isolated from the inner cell mass of the preimplantation blastocyst. When cultured without feeder cells, ESCs can spontaneously develop into 3-dimensional, multicellular aggregates called embryoid bodies (EBs), which contains structures of endoderm, mesoderm and ectoderm and can recapitulate early embryonic developmental process and is an ideal model for vascular development. The cellular repressor of E1A stimulated genes (CREG), as a novel transcriptional regulator, was cloned originally from a HeLa cDNA library. It can antagonize the E2F-dependent transcriptional activation and the oncogenic transformation of primary cells induced by the Adenovirus E1A oncoprotein cooperating with the oncogene renin-angiotensin system (RAS). Opposite to the effects of E1A, the physiological function of CREG can inhibit cell growth and/or promote differentiation. Our previous studies showed that a secreted protein, CREG played a role in the maintenance of the mature phenotype of vascular smooth muscle cells (SMCs). In addition, CREG highly expressed in early differentiation of endothelium cells (ECs) in embryonic development, and maintained to the adult. These results suggest that CREG not only participates in differentiated regulation of tumor cells and SMC, but also may be an important factor during the development of vascular angiogenesis. In this study, retrovirus expression vectors were successfully constructed and infected into ESCs, from which EBs differentiation model were developed and used to observe the effect of CREG on the regulation of vasculogenesis.Methods:1.The murine ESs R1 were infected respectively with retrovirus carrying the pCXN2-FLAG-CREG-IRES-EGFP, pDS-shRNA-CREG and pDS-shRNA-GFP. The cells (R1, R1-wtCREG, R1-GFP and R1-shCREG) were cultured on STO feeder cells with DMEM supplied LIF.2.Mouse R1-ESC were trypsinized and suspended in DMEM without LIF. After the 7th day, R1/EBs, R1-wtCREG/EBs, R1-shCREG/EBs were visible by phase-contrast microscope.3.At the 5.5th day, immunofluorescent staining of suspended EBs'cryosection were performed respectively to observe expression of germ layers. The marker of endoderm isα-fetoprotein (AFP) and the basement membrane were marked by Laminin(LN) staining.4.Day 3 EBs were plated onto cover slips coated by 300μl/mL Matri-gel and continued cultured for another 7 days. DiI-acetylated low-density lipoprotein (DiI-Ac-LDL) labeling of differentiated cells was performed to observe the characteristics of ECs differentiation.5.The expression of VEGF in R1, R1-wtCREG and R1-shCREG, and the expression of CREG,VEGF and Flk-1 from R1/EBs and R1-wtCREG /EBs at 1,3 and 5 day were detected by Western blot analysis. The procedure for induction of differentiation of R1-shCREG/EBs with exogenous CREG protein (10μl/mL) or R1-wtCREG/EBs with anti-VEGF neutralized antibody (15ng/mL) was observed by phase contrast microscope. Meanwhile, the expression of VEGF and Flk-1 were detected at optional timepoint.6.The mRNA and protein expression of CREG were detected by real-time PCR and Western blot analysis. AKP staining and teratoma formation assay inoculated into mouse myocardium detected stemness and differentiation potential of infected ESCs.7.The apoptosis rates of the three kinds of EBs (R1/EB,R1-GFP/EB,R1-shCREG/EB) were analyzed by AnnexinV/PI dual-staining flow cytometry analysis. The cleaved caspase-3 was detected by Western blot and semi-quantitative RT-PCR. Results:1.The stably infected ES cells (R1-wtCREG,R1-shCREG and R1-GFP) were obtained by screening G418-resistant clones. The cells grow very well in DMEM with LIF.2.By phase contrast microscope, most suspended R1-wtCREG/EBs has developed into the yolk sac compared with R1-GFP/EBs after 7 days suspended culture without LIF. It is known that the formation time of normal wild-type murine ESC derived EBs develop into yolk sac is 12~14 days. However, R1-shCREG/EBs showed growth slowly, no differentiation and more apoptosis in culture medium.3.Immunofluorescent staining showed that makers of embryonic germ layers, i.e. AFP and LN, are more positive and more cleared in suspended R1-wtCREG/EBs cryosections than that in R1/EBs at the 5.5th day.4.Differentiated vascular endothelial cells were observed by confocal microscope when suspended EBs plated onto Matri-gel for 7 days, there were more endocytose-positive cells were investigate to locate at the edge of R1-wtCREG-EB compared to R1-EB by DiI-Ac-LDL staining.5.The expression of VEGF was higher in R1-wtCREG ESC compared with R1 cells, but no VEGF expression was detected in R1-shCREG. Moreover, the expression of CREG, VEGF and Flk-1 were achieved maximum in R1-wtCREG/EBs group, and were least in R1-shCREG/EBs group at no matter at 1, 3 and 5 day. When R1-shCREG/EBs were pretreated by recombinant wild-type CREG protein (10μl/mL), three layers can be seen at 7 days and the expression of VEGF and Flk-1 were both increased. Conversely, pretreated with antiVEGF (15ng/mL) neutralized antibody, the differentiation of R1-wtCREG/EBs was inhibited and the expression of Flk-1 was decreased obviously at 5 and 7 day.6.Compared with R1/EBs and R1-GFP/EBs, the mRNA and protein expression of CREG was detected decreased by real-time PCR and Western blot analysis. Although the R1, R1-GFP and R1-shCREG cells showed the similar stemness by AKP staining, it was found that R1-shCREG couldn't format teratomas analogue and exhibit lower ability of differentiation compared with R1 and R1-GFP cells by teratomas formation experiments in mice7.Furthermore, AnnexinV/PI FASC assay indicated that apoptotic rate of R1-shCREG/EB was increased significantly compared with the R1/EBs and R1-GFP/EBs when cells were suspended culture for 7 days. Meanwhile, the mRNA and protein expression of cleaved caspase-3 was also increased obviously.Conclusion:1.Overexpression of CREG improves the differentiation of vascular endothelial cells and formation of yolk sac derived from EBs, which suggests that CREG might be an important regulatory factor of vasculogenesis.2.The silencing expression of CREG inhibits ESC differentiation and promotes EBs spontaneously apoptosis in vitro. |
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