A Mechanism Research About Oct4 Mediated Hasmc Phenotypic Transition During The Development Of Thoracic Aortic Dissection

Background and Objective:Acute aortic dissection is a life-threatening disease with a reported annual incidence of 6/100,000 and a 30-day in-hospital mortality of 47.4%. The pathogenesis of thoracic aortic dissection(TAD) was extensively studied in recent years but still has not been thoroughly elucidated. Since the in-depth knowledge on TAD pathogenesis may improve its clinical treatment strategy in the future, it is essential to further clarify the mechanisms of TAD. As the major cellular components of aortic media and the primary source of extracellular matrix, the integrity on structures and functions of human aortic smooth muscle cells(HASMCs) was critical for maintaining normal biomechanical properties of aortic wall. The dysfunctions of HASMCs, however, have been reported to be related to a variety of aortic diseases. Under specific conditions, the terminally differentiated normal HASMCs would acquire some features of phenotypic plasticity which would lead to biological characteristic changes in HASMCs. With regard to TAD, it was acknowledged that the transition of HASMCs phenotype from the physiological contractile type with abundant expression of contractile proteins including smooth muscle alpha-actin(α-SMA), smooth muscle myosin heavy chain, and smooth muscle 22α(SM22α) as its differentiated markers to the synthetic type with decreased expression of these phenotype markers and elevated capability of migration has been reported to be the pivotal pathologic focus and an early event of thoracic aortic diseases. However, the precise molecular mechanisms within the processes of this HASMCs phenotype transition have still not been thoroughly illuminated.Octamer binding protein-4(Oct4), the product encoded by POU5f1, belongs to the family of transcription factors with POU homeodomain. As a key regulator on self-renewal and pluripotency, Oct4 was generally expressed in pluripotent cells. Besides, transduction of Oct4 together with other three transcription factors into primary fibroblasts would establish induced pluripotent stem cells. With regard to the involvement of Oct4 in human diseases, Oct4 has been detected in tissues of lung adenocarcinoma and has been shown to induce epithelial-mesenchymal transition(EMT), a process that contributed to the switch of tumor cells to a more malignant phenotype with higher migration capability and stemness. However, with regard to the biological functions of Oct4 in vascular diseases, extremely rare documents have been reported. To our knowledge, the biological functions and related mechanisms of Oct4 in the aforementioned phenotype transition of HASMCs and their involvement in the pathogenesis of TAD has never been reported till date. In this study, we aim to investigate the fucntion and mechanism of Oct4 in in the regulation of HASMCs phenotype switch during TAD.Methods:1.Tissue Specimens :Dissected ascending aortic specimens(n = 12) were obtained from acute TAD patients classified as Stanford type A cases. Ascending aortic samples(n = 6) in the control group were acquired from donors without vascular diseases. 2.Isolation and Culture of HASMCs: Primary HASMCs were isolated from 3 patients and 3 normal controls. The intima and adventitia of aorta was carefully removed in a super-clean bench. Subsequently, the media of aortic tissues were cut into 4×4 mm squares. Explants were seeded onto the surface of a 24-well culture plate, with 1-2 explants per well. 300μl DMEM with 20% FBS were added to each well and replaced with fresh media twice per week. 3.Immunohistochemistry: After deparaffinization, antigen retrieval and inhibition of endogenous peroxidase activity, the aorta sections were incubated with primary antibodies followed by incubation with horseradish peroxidase-conjugated secondary antibody. A DAB Horseradish Peroxidase Colour Development kit was used for coloration. 4.Immunofluorescence:Paraformaldehyde-fixed HASMCs were first incubated with primary antibodies, followed by incubation with secondary antibodies. Nuclei was counterstained with 4’,6-diamidino-2-phenylindole(DAPI). 5.Small Interfering RNA and Transfection: HASMCs were transfected with si RNA using Lipofectamine 2000 according to the manufacturer’s instructions. 6.Scratch Wound Assay: A liner wound was gently made in the center of the cell monolayer using a 200 ul pipette tip. Images were captured at 0 and 24 hours after scratch. 7.Western Blot and Quantitative Real-time Polymerase Chain Reaction Analysis were used to detect the expresison levels of protein and m RNA respectively. 8.Luciferase Reporter Assay: The PCR products of wild type and mutant type promotor sequence of KLF5 were respectively cloned into p GL3-basic vector upstream of the luciferase gene to construct p GL3-Pro-WT-KLF5 and p GL3-Pro-MT-KLF5. Overexpression plasmid pc DNA-Oct4 was constructed by PCR cloning human genomic DNA. Luciferase activities were measured using the Dual Luciferase Reporter Assay system. Relative promoter activities are expressed as luminescence relative units normalized to renilla luciferase activity expression in the cell extracts. 9. The chromatin immunoprecipitation(Ch IP) assay was performed using the EZ-Ch IP Chromatin Immunoprecipitation Kit according the manufacturer’s instruction. 10.Statistical Analysis: Data were presented as mean ± standard deviation. Statistical analysis was performed by unpaired Student’s test using SPSS software. A p value less than 0.05 is considered to be statistially significant.Results: 1.Oct4 expression is increased in aortic tissues and HASMCs from TAD patients.Western blot and q RT-PCR showed that the protein and m RNA level of Oct4 was significantly increased in aortic tissues of TAD group, as compared with normal aortic samples. Immunohistochemistry demonstrated that there was a significant upregulation in Oct4 expression and remarkable nuclear localization of Oct4 in HASMCs within the aortic media of TAD patients. Immunofluorescence revealed that HASMCs from TAD patients presented more prominent Oct4 positive stains which were specifically localized in nucleus of HASMCs. On the contrary, Oct4 was mildly expressed and diffusely distributed in both the cytoplasm and nucleus of HASMCs from normal controls. These results suggested that Oct4 might play an important role in the transcriptional regulation of some genes in HASMCs which contributed to the pathogenesis of TAD.2.Oct4 induced the dedifferentiated phenotype of HASMCs and up-regulated its migration and proliferation capability.HASMCs transfected with Ad.Oct4 showed significantly reduced mRNA and protein levels of α-SMA and MYH11. Scratch wound healing assay demonstrated that the increased Oct4 expression significantly up-regulated the migration capability of HASMCs. CCK8 assay showed that overexpression of Oct4 can increase the proliferation of HASMCs. Futhermore, our results showed that downregulation of Oct4 can inhibit the TNF-α-induced reduction of the expression of α-SMA and MYH11 and the enhancement of HASMC migration and proliferation.3.KLF5 expression is elevated in aortic tissues from TAD patients and Oct4 induced the phenotype transition of HASMCs via directly up-regulating KLF5 expression.It was demonstrated that as compared with the Ad.GFP group, overexpression of Oct4 in HASMCs significantly increased the m RNA levels of Nanog, Sox2, BMP4, Etv5 and KLF5, while the m RNA levels of TDGF1, Nodal and Sfrp1 markedly decreased.Western blot showed that the protein level of KLF5 in the TAD group was significantly higher than that in the normal controls. Immunohistochemistry revealed that KLF5 was highly expressed in the HASMCs of aortic media in TAD cases, but was rarely expressed in the normal aorta. Ch IP assay suggested that Oct4 could directly bind to the promoter of KLF5. Furthermore, we constructed a plasmid p GL3-Pro-WT-KLF5 containing wild type KLF5 promoter and a motif-mutant plasmid p GL3-Pro-MT-KLF5 in which the sequence of the predicted binding site of Oct4 was changed. These two plasmids were respectively co-transfected with an Oct4 overexpression plasmid into 293 cells. As compared with the motif-mutant plasmid, co-transfection of Oct4 significantly transactivated the luciferase reporter plasmid of p GL3-Pro-WT-KLF5 containing the wild type KLF5 promoter. These results indicated that Oct4 upregulated the HASMCs KLF5 expression by directly binding to its promoter sequence and therefore increasing the transcription of KLF5. At last, we performed rescue experiment and confirmed that the effects of Oct4 on inducing the phenotypic switch of HASMCs from the contractile type to the synthetic type were, at least partially, mediated by its up-regulation of KLF5 expression.ConclusionsOct4 expression was significantly upregulated in aortic tissues and primary HASMCs of TAD patients. The increased Oct4 induced phenotype transition of HASMCs from the contractile type to the synthetic type by directly up-regulating KLF5.