| The phenotype of vascular smooth muscle cells(VSMC) has changeability. VSMC in the normal mature vascular wall takes on contractile phenotype and belongs to differentiated VSMC. Differentiated VSMC maintains its function by expressing a series of specific contractile proteins. When the vascular endothelium is injured or VSMC is cultured in vitro, VSMC quickly occurs phenotypic modulation, the characteristics are as follows: the expression of differentiated marker genes is down-regulated, contractile function disappears, and extracellular matrix is increased, migration and proliferation abilities of VSMC are obtained. This state of VSMC is named for synthetic or dedifferentiated VSMC. Vascular remodeling induced by VSMC phenotypic modulation is the basis of vascular diseases (such as the vascular renarrow and atherosclerosis). Therefore, it is important for study molecular mechanism of VSMC phenotypic regulation.CArG[CC(A/T)6GG]-box is discovered in all of regulatory sequences of VSMC marker genes. CArG element is involved in activation of muscle specific gene through binding to serum response factor (SRF). SRF is a member of the MADS (MDM1, agamous, deficient, SRF) box family of transcription factor. It widely distributes in all kinds of cells. SRF participates in regulating cell growth and differentiation. But the mechanism of SRF regulating specific gene expression is still unclear. In order to explore the regulation mechanism of promoting smooth muscle specific gene transcription, the model of VSMC redifferentiating induced by serum starvation was used in our study. Firstly we observed SRF expression and the binding ability between SRF and DNA. Furtherly, changes of SRF level and activity in the VSMC phenotypic modulation were detected at different levels such as nucleartranslocation and post-translational modification. We tried to discuss the molecular mechanism and pathophysiology significance of VSMC redifferentiation.1 Expression and activity of SRF in the process of VSMC redifferentiatingIn order to determine the relation between SRF and VSMC redifferentiating, the model of VSMC redifferentiating induced by the serum starvation was used in our study. Firstly we observed SRF expression and the binding ability between SRF and DNA to confirm the relations among SRF and VSMC marker gene expression and cells morphologic change. These results were as follows:1.1 Because SM22a was the most characteristic marker gene of contractile VSMC, we made SM22a expression activity as an evidence for determining VSMC phenotype. During VSMC was cultured by serum stimulation, mRNA of SM22a was scarcely detected by RT-PCR, suggesting that VSMC was in the state of dedifferentiation. The expression of SM22a was quickly up-regulated following serum starvation and maintained at higher levels. It indicated that serum starvation induce VSMC from synthetic state to the contractile state. In serum-restimulated VSMC after serum starvation for 72 hours, the expression of SM22a was quickly down-regulated again. It implied that VSMC restored dedifferentiated state again. In the process, we detected the expression of SRF gene. Northern blotting showed that mRNA levels of SRF was unchanges in different cultured VSMC. Then, we detected the level of SRF mRNA in primary and passaged VSMC by RT-PCR, the similar results were obtained. Western blotting results showed that SRF protein level of different phenotypes VSMC hadn't obvious change. It implied that the activity that SRF started VSMC specific gene expression was unrelated to its protein level.1.2 EMSA showed that nuclear protein, which was extracted from VSMC before and after the serum starvation, all binded to CArG-box. During the serum starvation, the band of DNA-protein complex slightly deepened. Itexplained the binding ability between nuclear protein and DNA was enhanced. In serum-restimulated VSMC after serum starvation for 72 hours, the binding ability restored to the level before the serum starvation again. Nuclear protein extracted from primary VSMC was analyzed by EMSA, the band of DNA-protein complex were obviously deeper than those of passaged cells. Because only a part of VSMC dedifferentiated to contractile phenotype, higher activity nucleus protein-CArG complex could be detected. It made for activating differentiated phenotype marker gene expression that the ability, which SRF induced by serum starvation binded with CArG-box, was obviously enhanced.1.3 Our studies showed SM22a was the regulation protein which participated in regulating VSMC cytoskeleton structure and cell contraction before. After serum starvation, SM22a transcription activity was quickly heightened, it promoted the formation of cytoskeleton. This study proved the reason that SM22a transcription activity was quickly heightened could relate to the binding ability enhancing after serum starvation. SRF was a kind of widespread transcription factor and its main function was to regulating gene expression. However, we discovered there also were SRF in cytoplasm during experiment. In order to explain the physiology meaning, we observed SRF's effect on cytoskeleton. The results showed that cytoskeleton presented discrete network and light dye and dim structure in VSMC cultured naturally. After SRF expression plasmid was transfected to VSMC, the cytoskeleton structure became clearer and dense network and bundle-distributed along VSMC polarity. Under the same condition, the controlled VSMC, which transfected pCGN empty vector, didn't appear those varieties. It showed that forced expression of SRF might promote remodulation of VSMC cytoskeleton. But western blotting showed forced expression of SRF couldn't promote expression of SM22a, so we speculated SRF might influence cytoskeleton's formation by other mechanisms.These results hinted the mRNA and protein level of SRF has no changes during VSMC phenotypic reverse induced by serum strvation. But comparedwith dedifferentiated VSMC, the binding activity between SRF and DNA is obviously enhanced in differentiated VSMC. The latter is positive relation to transcription activity of VSMC marker gene SM22a.2 Phosphorylation and nuclear translocation change of SRF during VSMC phenotypic reversedOn the basis of part one, we discussed the molecular mechanism that led to binding activity change between SRF and DNA. The chemical modification of protein was one of the most common cell signal transmission forms. Nuclear translocation of SRF was the key point which transmitted upstream signal to target gene. There were many kinds of kinases which all might make SRF to occur phosphorylation modification, but the change characteristic of SRF phosphorylation modification was still unclear during VSMC phenotypic modulation. Therefore, we studied SRF's phosphorylation and cell orientation during VSMC phenotype reversed in molecular and cell levels. These results were as followed:2.1 SRF threonine phosphorylation level of VSMC before and after serum starvation was detected by immunity blotting. The result showed SRF phosphorylation level was lower in dedifferentiated VSMC, obviously enhanced after serum starvation for 6 hours. In serum-restimulated VSMC after serum starvation for 72 hours, the level was obviously lowered again. The result implied that serum starvation could promote SRF phosphorylation. It could be one of mechanisms that serum starvation induced VSMC redifferentiation.2.2 For the sake of discussing physiology meaning that serum starvation induced SRF phosphorylation, we analyzed SRF phosphorylation's effect on its distribution in VSMC nuclear /cytoplasmic. Western blotting showed that the protein level of SRF was high in the nucleus of VSMC cultured naturally, then lowered after serum starvation. In serum-restimulated VSMC after serum starvation for 72 hours, SRF protein level ascended again. But under the same conditions, SRF protein level in cytoplasmic occurred reverse trend. This change was confirmed by the cell immunohistochemistry. In VSMC culturednaturally, brown dark-dye particles were found in cytonucleus and cytoplasm was light dyed, indicated that SRF mainly distributed in cytonucleus. When serum starvation for 6 hours, cytoplasm was dark-dyed and cytonucleus was light-dyed. It implied that SRF had shifted from cytonucleus to cytoplasm. In serum-restimulated VSMC after serum starvation for 72 hours, subcell distribution of SRF restored the state before serum was withdrawn. The actual meaning of this phenomena, which nuclear translocation of SRF occurred reverse change with its phosphorylation level, was still unclear. Perhaps low-concentrated SRF in the cytonucleus was in favor of transcription stimulation of differentiation genes.These results showed serum starvation can induce phosphorylation level of SRF to increase. Phosphorylation of SRF and suitable SRF level in the nucleus are necessary environment conditions that SRF binding to CArG element.3 Expression change of serum response factor during neointimal formationIn this part, we built the rat model of endometrial hyperplasia after de-endodermis in arteria carotis communis-thorax and abdominal aorta, observed changes of SRF expression in the process of endometrial hyperplasia and orientation characteristic in the neoinaimal. We tried to explore the relation among SRF expression level and endometrial hyperplasia degree and VSMC phenotypic modulation. These results were as follows:3.1 The carotid artery sections by denuding the endothelium in different times stained with HE were observed under microscope, we found that compared with orderly arranged medial VSMC of artery in the normal rats, pathological changes of injured areas were first found in sections on day 3 after de-endothelialization. On day 7-21, widespread intima thickening and lumen stenosis were observed. These results demonstrated that the rat intimal hyperplasia model was successful.Immunohistochemistry showed that positive dye particles of SRF could be observed in intima and media of the normal carotid artery, but its amount was lower. On day 3 after de-endothelialization, expression of SRF began toincrease. On day 7, it was markedly increased and mainly distributed in cytoplasm of neoinaimal; On day 14-21, expression of SRF was gradually decreased; On day 21, it reached the lowest, but it still didn't restore to the normal level. The similar results were found by western blotting. It implied that the increased SRF level possibly participated in VSMC phenotypic modulation and proliferation's startup.3.2 Northern blotting showed expression activity of SRF gene was weaker in the normal vascular wall, then increased after de-endodermis, reached peak on day 7 and began to decrease on day 14. It didn't still restore to the normal level until day 21. The change was similar with SRF protein's. Expression activity of SRF was consistent in the trend of intima progressive incrassation, implied that SRF level increase induced by de-endodermis was the result of gene transcription increase. The latter had direct relation to the vascular endometrial hyperplasia.3.3 In order to explore the relation between the increased SRF level and VSMC phenotype during neointimal formation, we observed the expression of SM22a after de-endothelialization. Western blotting showed the expression of SM22a gradually decreased after de-endothelialization and reached the lowest level on day7-14 and began to increase on day 21. It demonstrated that VSMC experienced phenotype remodeling during neointimal formation. The result implied that high level SRF wasn't necessary for marker genes expression of contractile VSMC.These result showed that the increase in SRF after de-endodermis might have direct relation to dedifferentiation of VSMC and neointimal formation. Phenotypic modulation and proliferation of VSMC had intimate correlation with expression amount of SRF.Conclusions1 The mRNA and protein level of SRF has no changes during VSMC phenotypic reverse induced by serum starvation .But compared with dedifferentiated VSMC, the binding activity between SRF and DNA is obviously enhanced in differentiated VSMC. The latter is positive relation to...
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