| Section A:Developing an efficient differentiaiton model of smooth muscle cells from embryonic stem cellsBackground:As a major cellular component of blood vessels, smooth muscle cells (SMC) play an important role under physiological conditions as well as in a large number of human diseases, including atherosclerosis, hypertension and cancer. Unlike either skeletal or cardiac muscle those are terminally differentiated, SMC possess remarkable plasticity and undergo phenotypic switch at different stages of development, even in adult organisms diseases. An full understanding of the normal regulation of SMC development, maturation and differentiation will not only provide the foundation for elucidating how these processes may be disrupted in vascular disease, accelerating appreciation of pathogenesis and providing novel therapeutic targets, but will also be critical to understanding congenital defects in vascular development. Unfortunately however, the mechanisms underlying SMC development and maturation are poorly understood partly due to the lack of good in vitro and in vivo models. Embryonic stem (ES) cell lines have been previously established from the inner cell mass of blastocysts and have been shown to have the potential to generate all embyonic cell lineages. Developing an innovative culture system tor the efficient differentiaiton of smooth muscle cells from embryonic stem cells will provide a very good platform for studying the molecular mechanism controlling SMC differentiaion.Methods:We plated mouse embryonic stem cells (ES-D3) on collagen Ⅳ-coated dishes/flasks in differentiation medium for2,4,6,8days, and detected a panel of smooth muscle specific markers, including smooth muscle a-actin (SMA), calponin, SM22, mooth muscle myosin heavy chain (SM-MHC) by Quantitative real-time polymerase chain reaction and Western blot. Results:SMC-specific genes were significantly and consistently upregulated at2,4,6,8day in our culture model, and with the maximum expression of both mRNA and protein levels observed at8day. Conclusions:In this part study, we have established an innovative culture system for the efficient differentiaiton of smooth muscle cells from embryonic stem cells. This inducible system in vitro provide a very good platform for studying the molecular mechanism controlling SMC differentiaion.Section B:Angiotensin Ⅱ promote the differentiation of embryonic stem cells to smooth muscle cellsBackground:Angiotensin Ⅱ (Ang Ⅱ), the most important effector peptide of the renin-angiotensin system, plays various roles on different cells from multiform ways. It is now recognized that Ang Ⅱ acts not only as a vasoactive peptide but also as a growth factor that stimulates proliferation, differentiation, apoptosis and hypertrophy. However, the potential impact of Ang Ⅱ on embryonic stem (ES) cells differentiation is still unknown.Methods:The predifferentiated ES cells were cultured on collagen IV and got a60%confluence at day4. For Ang Ⅱ stimulation, the ES cells were cultured in serum-free Ang Ⅱ basal medium for1hour, followed by the addition of different concentrations (10-6-10-14mol/L) of Ang Ⅱ and further incubation for48hours. Then we detected the mRNA and protein expression of smooth muscle specific markers, including smooth muscle a-actin (SMA), calponin, SM22, mooth muscle myosin heavy chain (SM-MHC) by Quantitative real-time polymerase chain reaction and Western blot, and also detected the immunofluorescent staining of SMC-specific markers. Besides, by MTT assay we assessed the viability and the proliferation of cells between Ang Ⅱ groups and control.Results:Ang Ⅱ promote the differentiation of ES cells to SMCs. As we found that Ang Ⅱ dose-dependently increased the mRNA expression level of SMA, SM22, calponin and SM-MHC, with the maximum stimulation observed at10-9mol/L (p<0.01); Ang Ⅱ dose-dependently increased the protein expression level of SMA, SM22and calponin, with the maximum at10-9mol/L (p<0.01); Accompany by the increased immunofluorescent staining of SMC-specific markers (SMA, SM22and calponin) in Ang Ⅱ (10-9mol/L) stimulated cells, comparing with control, but immunofluorescent staining of SM-MHC changed not much. Besides, the MTT assay showed no differences between Ang Ⅱ groups and control (p>0.05). Conclusions:In this part study, we confirmed the promotive effect of Ang Ⅱ on differentiation of ES cells to SMCs from mRNA, protein and morphologic ways. However this effect was not simply related with cells proliferation, further study about the real mechanism needs to be processing.Section C:The mechanism of Angiotensin Ⅱ on differentiation of embryonic stem cells to smooth muscle cellsBackground:In previous study, we have confirmed the promotive effect of Ang Ⅱ on differentiation of ES cells to SMCs. However, Ang Ⅱ, the terminal pressor effector arm of the renin-angiotensin-aldosterone system, can exsert it's regulating effect from multiple ways. It is now recognized that Ang Ⅱ acts not only as a vasoactive peptide but also as a growth factor that stimulates proliferation, differentiation, apoptosis and hypertrophy, and even can modulate the expression and function of some transcription factors. So we emphasize our effort on some signal pathways closely related with smooth muscle growth and differentiation, besides also including the trasncriptional level analyse.Methods:The predifferentiated ES cells were cultured on collagen IV and got a60%confluence at day4. For signal pathways analyse, ES cells were pre-incubated with the inhibitors, such as Ang Ⅱ type1(AT1) receptor antagonist losartan (25umol/L,50umol/L), Ang Ⅱ type2(AT2) receptor antagonist PD123319(1umol/L,10umol/L), NF-κB Inhibitor BAY11-7082(5umol/L) and phosphoinositide-3kinase (PI3K) inhibitor LY294002(10umol/L) for1hour, then gave the Ang Ⅱ stimulation (10-9mol/L). The related expression of smooth muscle specific markers and phosphoAkt were detected between Ang II stimulation group and control by Quantitative real-time polymerase chain reaction and Western blot. For transcriptional level analyse, we detected the SMA and SM22promoter activity on different concentrations of Ang II (10-6-10-10mol/L) by Luciferase reporter assay. And we also analyse some transcription factors expression, such as c-Jun, NF-κB p50, NF-κB p65, especially the expression of NF-κB p50during Ang Ⅱ stimulation (10-6-10-10mol/L). Besides we also detected the protein expression of other two proinflammatory factors, TNF-a and c-fos during Ang Ⅱ stimulation.Results:For signal pathways analyse, losartan dose-dependently decreased the protein expression levels of smooth muscle specific markers (SMA and calponin) comparing with control (p<0.01), while the expression levels of SMA and calponin were not affect by PD123319, an AT2receptor antagonist. Ang Ⅱ dose-dependently increased the phosphoAkt expression, with the maximum stimulation observed at10-9mol/L (p<0.01), which were consistent with the expression of smooth muscle specific markers SMA and calponin. LY294002completely abrogated the increased expression of SMA and calponin both in Ang Ⅱ (10-9mol/L) and control, and also decreased the expression of phosphoAkt. However, the suppressed expression level of smooth muscle specific markers and phosphoAkt on control were even lower than that of Ang Ⅱ stimulation (p<0.01), that implies Ang Ⅱ maybe can promote the differentiation of ES cells through other ways. For transcriptional level analyse, our data revealed that Ang Ⅱ dose-dependently increased SMA reporter activity, with the discernable effect observed at10-8mol/L and10-9mol/L (p<0.05). However the changes in SM22-report were not so obviously (p>0.05). We also found Ang Ⅱ increased NF-κB p50expression, with the maximum stimulation observed at10-9mol/L, which were consistent with the expression of smooth muscle specific markers SMA, calponin and phosphoAkt, and the suppressed expression of NF-κB p50by LY294002both in Ang Ⅱ stimulation (10-9mol/L) and control. And NF-κB Inhibitor BAY11-7082can suppressed the protein expression levels of smooth muscle specific markers (SMA and calponin). Besides, the protein expression of two proinflammatory factors TNF-a and c-fos were not changed much, only with TNF-α slightly increasing at the Ang Ⅱ concentration of10-9mol/L(p<0.05).Conclusions:Our study first described the impact of Angiotensin Ⅱ on embryonic stem cell differentiation. Ang Ⅱ can promote the differentiation of ES cells to SMCs by AT1receptor, and PI3K/Akt signaling pathway plays a key role in this process. Besides, Ang Ⅱ can directly promote the smooth muscle specific markers expression at the transcription level, through stimulating many transcription factors especially transcription factor NF-κB p50, which may has the close relationship with SMC differentiation. However, the physiological effect of Ang Ⅱ was not related with inflammation.
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