Tongcun Zhang

The role of endothelial–mesenchymal transition in development and pathological process

Epithelial–mesenchymal transition is an important developmental process, participates in tumors formation, invasion, and metastasis and has been extensively studied. Recently, endothelial–mesenchymal transition (EndMT), a newly recognized type of cellular transdifferentiation, has been demonstrated to participate in a number of diseases by causing morphology changes and pathological processes. Previous studies showed that EndMT was a critical process of embryonic cardiac development. Not only that recent advances also suggested that EndMT occurred postnatally in cancer and cardiac fibrosis and emerged as a possible source of cancer‐associated fibroblasts (CAFs). CAFs were found to acquire properties that promoted tumor development and metastasis formation. Resident endothelial cells undergoing EndMT lose their endothelial markers, acquire a mesenchymal or myofibroblastic phenotype, express mesenchymal cell products such as α‐smooth muscle actin and type I collagen and develop invasive and migratory abilities. EndMT‐derived cells are believed to function as fibroblasts in damaged tissue and may therefore have an important role in pathological process. However, little is known about the signaling mechanisms that cause endothelial cells to transform into mesenchymal cells. Transforming growth factor‐β, Notch, or other signaling pathways could direct or interact to mediate EndMT. Therefore, to explore the signaling mechanisms of EndMT may provide novel therapeutic strategies for treating cancer.

STAT3 Protein Regulates Vascular Smooth Muscle Cell Phenotypic Switch by Interaction with Myocardin

Background: The Hippo-Yap signaling pathway is one of the critical pathways regulating cell proliferation, differentiation, and apoptosis. Results: Knockdown of endogenous Yap1 impairs VSMC proliferation and enhances VSMC contractile phenotype by promoting the association of the myocardin-SRF-CArG complex. Conclusion: The Yap1 signaling pathway is a central regulator of the phenotypic switch of VSMCs. Significance: The phenotypic switch of VSMCs and vessel injury response can be controlled by modulation of Hippo-Yap signaling. The Hippo-Yap (Yes-associated protein) signaling pathway has emerged as one of the critical pathways regulating cell proliferation, differentiation, and apoptosis in response to environmental and developmental cues. However, Yap1 roles in vascular smooth muscle cell (VSMC) biology have not been investigated. VSMCs undergo phenotypic switch, a process characterized by decreased gene expression of VSMC contractile markers and increased proliferation, migration, and matrix synthesis. The goals of the present studies were to investigate the relationship between Yap1 and VSMC phenotypic switch and to determine the molecular mechanisms by which Yap1 affects this essential process in VSMC biology. Results demonstrated that the expression of Yap1 was rapidly up-regulated by stimulation with PDGF-BB (a known inducer of phenotypic switch in VSMCs) and in the injured vessel wall. Knockdown of Yap1 impaired VSMC proliferation in vitro and enhanced the expression of VSMC contractile genes as well by increasing serum response factor binding to CArG-containing regions of VSMC-specific contractile genes within intact chromatin. Conversely, the interaction between serum response factor and its co-activator myocardin was reduced by overexpression of Yap1 in a dose-dependent manner. Taken together, these results indicate that down-regulation of Yap1 promotes VSMC contractile phenotype by both up-regulating myocardin expression and promoting the association of the serum response factor-myocardin complex with VSMC contractile gene promoters and suggest that the Yap1 signaling pathway is a central regulator of phenotypic switch of VSMCs.

Myocardin‐related transcription factor‐A induces cardiomyocyte hypertrophy

Myocardin is a remarkably potent transcriptional coactivator expressed specifically in cardiac muscle lineages and smooth muscle cells during postnatal development. Myocardin shares homology with myocardin‐related transcription factor‐A (MRTF‐A), which are expressed in a broad range of embryonic and adult tissues. Our previous results show that myocardin induces cardiac hypertrophy. However, the effects of MRTF‐A in cardiac hypertrophy remain poorly understood. Our present work further demonstrates that myocardin plays an important role in inducing hypertrophy. At the same time, we find that overexpression of MRTF‐A in neonatal rat cardiomyocytes might induce cardiomyocyte hypertrophy. Furthermore, MRTF‐A expression is induced in phenylephrine, angiotensin‐II, and transforming growth factor‐β‐stimulated cardiac hypertrophy, whereas a dominant‐negative form of MRTF‐A or MRTF‐A siRNA strongly inhibited upregulation of hypertrophy genes in response to hypertrophic agonists in neonatal rat cardiomyocytes. Our studies indicate that besides myocardin, MRTF‐A might play an important role in cardiac hypertrophy. Our findings provide novel evidence for the future studies to explore the roles of MRTFs in cardiac hypertrophy.

Cooperation of myocardin and smad2 in inducing differentiation of mesenchymal stem cells into smooth muscle cells

Several reports demonstrated that mesenchymal stem cells (MSCs) might differentiate into smooth muscle cells (SMCs) in vitro and in vivo. It has been shown that myocardin protein is a strong inducer of smooth muscle genes and MSCs can differentiate into SMCs in response to transforming growth factor‐β (TGF‐β). However, the relationship or link between myocardin and TGF‐β3‐induced MSC differentiation has not been fully elucidated. Here, we demonstrated that both myocardin and TGF‐β3 were able to induce differentiation of rat bone marrow‐derived MSCs toward smooth‐muscle‐like cell types, as evidenced by increasing expression of SMC‐specific genes. Of note, myocardin cooperated with Smad2 to synergistically activate SM22α promoter and significantly enhance the expression of SM22α. Report assays with site‐direct mutation analysis of SM22α promoter demonstrated that myocardin and Smad2 coactivated SM22α promoter mainly depending on CArG box and less on smad binding elements (SBE) sites as well. These findings reveal the cooperation of myocardin and Smad2 in process of MSC differentiation into SMCs.

The synergistic effects of cytomegalovirus IE2 and myocardin on cardiomyocyte hypertrophy

Human cytomegalovirus immediate early proteins (CMV IEs) are involved in transcriptional activities of both host and virus gene expression. This study shows that the transcriptional activity of myocardin in regulating cardiomyocyte hypertrophy is enhanced by co‐expressing CMV IE2. Forced expression of IE2 increases the augmented cell size of neonatal rat cardiac myocytes induced by myocardin, as well as the mRNA and protein levels of hypertrophic genes, whereas deletion of CArG boxes in the atrial natriuretic factor (ANF) promoter attenuates the effect of CMV IE2 with myocardin. In conclusion, CMV IE2 synergistically stimulates myocardin transactivity in the hypertrophic marker gene ANF in a CArG box‐dependent manner. Our study indicates that the association of CMV IEs with myocardin‐induced transcription may be involved in myocardin‐mediated cardiac hypertrophy.

Human cytomegalovirus immediate early protein 2 enhances myocardin-mediated survival of rat aortic smooth muscle cells

Human cytomegalovirus (HCMV) may increase the incidence of restenosis and predispose to atherosclerosis. The lesions of restenosis and atherosclerosis often contain smooth muscle cells (SMCs) with high rates of proliferation and apoptosis. One of the immediate early (IE) gene products of HCMV-IE2 affects transcriptional activities of some cellular factors in SMCs, including myocardin. In this study, we studied the effects of IE2 and myocardin on PI3K pathway inducer wortmannin induced apoptosis in rat aortic SMCs. We show that the transcriptional activity of myocardin on Mcl-1 promoter is enhanced by co-expression of HCMV IE2 in rat aortic SMCs; and the expressions of mRNA and protein of antiapoptotic genes-Mcl-1 and Bcl-2 are upregulated by IE2 alone and co-transfection of myocardin and IE2, but decreased by myocardin-specific shRNA in rat aortic SMCs. We further demonstrate that co-expression of myocardin and HCMV IE2 declines apoptotic cell numbers and caspase-3 activities induced by serum starvation plus wortmannin in rat aortic SMCs. The results suggest that HCMV IE2 enhances myocardin-mediated survival of rat aortic SMCs under serum deprivation and PI3-kinase inhibition, partly via activation of Mcl-1s antiapoptosis effect. Our study connects HCMV IE2 to myocardin-induced transcriptional program for rat aortic SMCs survival and proliferation, involving in HCMV related restenosis and atherosclerosis.

Cytotoxicity effects of three-dimensional graphene in NIH-3T3 fibroblasts

The 3D configuration of graphene materials has been intensively investigated due to their novel properties in biomedical, electrical and optical applications. But few reports have been intentionally carried out to understand the biological influence of 3D graphene materials so far. Herein, we presented an evaluation of the in vitro cytotoxicity of 3D graphene sheets fabricated by carbonization of polydopamine (PDA) films on a template of aligned nanopore arrays (NPAs) on a stainless steel surface. The prepared 3D graphene sheets with a thickness of ∼20 nm displayed a nanoporous architecture that can be readily tuned by the NPA template to control the morphology of the 3D configuration. The in vitro toxicity of the nanoporous 3D graphene sheets with pore sizes of ∼50 nm and ∼240 nm was evaluated using NIH-3T3 fibroblasts as the representative mammal fibroblast cell type. The NPA structure exhibits enhanced properties in cell attachment, spreading, proliferation, and the assembly of focal adhesions and actin filament associated proteins. Morphology-dependent cytotoxicity aroused by the 3D graphene configuration should be attributed to the engulfment of the carbon nanospheres embedded in the 3D configuration and the lack of both focal adhesions and actin filament associated proteins assembling at the nanoscale.

Construction and Expression of Human Coagulation Factor IX in the Pichia Pastoris

Human Coagulation Factor IX (hFIX) plays a crucial role in the process of human endogenous blood clotting. Patients with deficiency of hFIX will suffer from hemophilia B. In this study, we use Pichia Pastoris to produce recombinant hFIX to cure hemophilia B Human liver hFIX cDNA were amplified by RT-PCR and inserted into pPIC9k vector, to construct pPIC9k-hFIX. The pPIC9k-hFIX were integrated into Pichia Pastoris SMD1168 chromosome by electro-transformation. Then we screened recombinant by PCR analysis and G418 selection to obtain high-copy recombinants. SDS-PAGE and Western Blot analysis show that protein expression volume is up to 100 mg/L. The protein was purifed and concentrated by anion exchange membrane diffusion dialysis. Then we assay activity by using lack coagulation factor IX. The activity calculation by standard curve show that the coagulation factor IX expressed by Pichia Pastoris has similar clotting activity (5.5%) compared to normal human plasma. Thus, expression of hFIX in Pichia Pastoris is of feasibility.