scholarly journals Generation, Characterization, and Application of Inducible Proliferative Adult Human Epicardium-Derived Cells

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 2064
Author(s):  
Yang Ge ◽  
Anke M. Smits ◽  
Jia Liu ◽  
Juan Zhang ◽  
Thomas J. van Brakel ◽  
...  
Keyword(s):  
Transforming Growth Factor ◽  
Quantitative Polymerase Chain Reaction ◽  
Expression System ◽  
Marker Gene ◽  
Experimental Studies ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Mesenchymal Transition ◽  
Adult Human ◽  
Proliferation Capacity

Rationale: In recent decades, the great potential of human epicardium-derived cells (EPDCs) as an endogenous cell source for cardiac regeneration has been recognized. The limited availability and low proliferation capacity of primary human EPDCs and phenotypic differences between EPDCs obtained from different individuals hampers their reproducible use for experimental studies. Aim: To generate and characterize inducible proliferative adult human EPDCs for use in fundamental and applied research. Methods and results: Inducible proliferation of human EPDCs was achieved by doxycycline-controlled expression of simian virus 40 large T antigen (LT) with a repressor-based lentiviral Tet-On system. In the presence of doxycycline, these inducible EPDCs (iEPDCs) displayed high and long-term proliferation capacity. After doxycycline removal, LT expression ceased and the iEPDCs regained their cuboidal epithelial morphology. Similar to primary EPDCs, iEPDCs underwent an epithelial-to-mesenchymal transition (EMT) after stimulation with transforming growth factor β3. This was confirmed by reverse transcription-quantitative polymerase chain reaction analysis of epithelial and mesenchymal marker gene expression and (immuno) cytochemical staining. Collagen gel-based cell invasion assays demonstrated that mesenchymal iEPDCs, like primary EPDCs, possess increased invasion and migration capacities as compared to their epithelial counterparts. Mesenchymal iEPDCs co-cultured with sympathetic ganglia stimulated neurite outgrowth similarly to primary EPDCs. Conclusion: Using an inducible LT expression system, inducible proliferative adult human EPDCs were generated displaying high proliferative capacity in the presence of doxycycline. These iEPDCs maintain essential epicardial characteristics with respect to morphology, EMT ability, and paracrine signaling following doxycycline removal. This renders iEPDCs a highly useful new in vitro model for studying human epicardial properties.

scholarly journals Different macrophages equally induce EMT in endometria of adenomyosis and normal

Reproduction ◽  
2017 ◽  
Vol 154 (1) ◽  
pp. 79-92 ◽  
Author(s):  
Min An ◽  
Dong Li ◽  
Ming Yuan ◽  
Qiuju Li ◽  
Lu Zhang ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Transforming Growth Factor ◽  
Epithelial Mesenchymal Transition ◽  
Quantitative Polymerase Chain Reaction ◽  
Immunohistochemical Analysis ◽  
Secretory Phase ◽  
Normal Endometrium ◽  
Mesenchymal Transition ◽  
Expression Levels ◽  
Endometrial Cells

Endometrial cells and microenvironment are two important factors in the pathogenesis of adenomyosis. Our previous study demonstrated that macrophages can induce eutopic epithelial cells of adenomyosis to suffer from epithelial–mesenchymal transition (EMT). The aim of this study is to detect whether macrophages interacting with epithelial cells equally induce the EMT process in normal and eutopic endometria of healthy and adenomyotic patients; and whether macrophages parallelly polarize to M2. We investigated the expression levels of epithelial cadherin (E-cadherin), neural cadherin (N-cadherin), cytokeratin7 (CK7), vimentin, transforming growth factor-β1 (TGFB1), SMAD3 and pSMAD3 using immunohistochemistry and western blot, and then estimated the genetic levels of CD163, IL10 and MMP12 using real-time quantitative polymerase chain reaction (RT-PCR) in macrophages. Eutopic and normal endometrial tissues were obtained from 20 patients with adenomyosis and 11 control patients without adenomyosis, respectively. The immunohistochemical analysis shows distinct EMT in eutopic endometria in secretory phase; the expression levels of TGFB1, SMAD3 and pSMAD3 that indicate signal pathway of EMT were also higher in secretory phase. Macrophages can induce EMT process in primary endometrial epithelial cells derived from normal and eutopic endometria. After co-culturing, THP-1-derived macrophages polarized to M2. Compared with the eutopic endometrium group, further polarization to M2 was observed in the normal endometrium group. These results indicate that adenomyosis may be promoted by the pathologic EMT of epithelial cells, which is induced by macrophages that incapably polarize to M2.

scholarly journals Control of junB and extracellular matrix protein expression by transforming growth factor-beta 1 is independent of simian virus 40 T antigen-sensitive growth-sensitive growth-inhibitory events.

1991 ◽  
Vol 11 (2) ◽  
pp. 972-978 ◽  
Author(s):  
M Laiho ◽  
L Rönnstrand ◽  
J Heino ◽  
J A Decaprio ◽  
J W Ludlow ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Extracellular Matrix ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Simian Virus 40 ◽  
Plasminogen Activator Inhibitor ◽  
Simian Virus ◽  
T Antigen ◽  
Tgf Beta ◽  
Plasminogen Activator Inhibitor 1

Treatment of Mv1Lu mink lung epithelial cells with transforming growth factor-beta 1 (TGF-beta 1) prevents phosphorylation of the retinoblastoma susceptibility gene product, RB, in late G1 phase of the cell cycle, which is thought to retain RB in a growth-suppressive state. This effect is paralleled by cell cycle arrest in late G1 (M. Laiho, J. A. DeCapric, J. W. Ludlow, D. M. Livingston, and J. Massagué, Cell 62:175-185, 1990). Arrest can be prevented by expression of simian virus 40 T antigen, which binds to underphosphorylated RB, presumably blocking its growth-suppressive activity. The response of cells to TGF-beta 1, however, is complex and includes changes in the levels of expression of genes encoding nuclear transcription factors and extracellular matrix components. To define the relationships among various components of the TGF-beta 1 response, we have investigated the effect of TGF-beta 1 on cells whose growth-inhibitory response to this factor is prevented by T antigen. TGF-beta 1 addition to exponentially growing Mv1Lu cells increased the levels of junB mRNA and of three extracellular matrix proteins: plasminogen activator inhibitor-1, fibronectin, and thrombospondin. Kinetically, the effects on junB and plasminogen activator inhibitor-1 expression occurred faster (half-maximal at 1 to 2 h) than the effects on fibronectin and thrombospondin expression (half-maximal at 6 to 10 h). These effects either preceded or overlapped, respectively, the withdrawal of Mv1Lu cells from the cell cycle. Expression of a transfected T-antigen gene in Mv1Lu cells, however, did not prevent any of these responses to TGF-beta 1. The results indcate that TGF-B1-stimulated expression of junB and extracellular matrix proteins in Mv1Lu cells can occur independently of the T-antigen-sensitive events that lead to growth arrest.

scholarly journals Interactive Effects of c-myc and Transforming Growth Factor α Transgenes on Liver Tumor Development in Simian Virus 40 T Antigen Transgenic Mice

1998 ◽  
Vol 35 (4) ◽  
pp. 283-291 ◽  
Author(s):  
A. Enomoto ◽  
E. P. Sandgren ◽  
R. R. Maronpot
Keyword(s):  
Cell Proliferation ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Liver Tumors ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Transforming Growth Factor Α ◽  
Neoplastic Lesions ◽  
Factor Α

To analyze the effects of c- myc and transforming growth factor α (TGFα) on hepatocarcinogenesis induced by simian virus 40 T antigen (TAg), livers from single and bitransgenic mice, 3 to 11 mice per line, were examined morphologically 1 to 8 weeks after birth. Mice carrying c- myc or TGFα alone exhibited centrilobular hypertrophy and increased apoptosis (c- myc mice only) of hepatocytes after 3 or 4 weeks of age, but no detectable changes in cell proliferation or proliferative lesions were observed in either line during the 8 weeks. Mice carrying TAg alone exhibited increased cell proliferation, apoptosis, and dysplasia of hepatocytes with notably high mitotic and apoptotic indices as major changes before development of putative preneoplastic lesions after 4 weeks of age and neoplastic lesions after 6 weeks. In bitransgenic mice coexpressing c- myc or TGFα with TAg, nonproliferative lesions and mitotic and apoptotic indices were similar to those in mice carrying TAg alone. In TAg X c- myc bitransgenic mice, however, both preneoplastic and neoplastic lesions developed sooner and grew more rapidly than those in TAg mice, whereas in TAg X TGFα bitransgenic mice, rapid tumor growth was the principle observation. Because of the effects of transgene coexpression, livers from TAg X c- myc and TAg X TGFα mice had multiple tumors as early as 3 and 6 weeks of age, respectively. The results indicate cooperative functions of c- myc and TGFα with TAg during development and/or growth of liver tumors in vivo.

scholarly journals Activated Endothelial TGFβ1 Signaling Promotes Venous Thrombus Nonresolution in Mice Via Endothelin-1

2020 ◽  
Vol 126 (2) ◽  
pp. 162-181 ◽  
Author(s):  
Magdalena L. Bochenek ◽  
Christiane Leidinger ◽  
Nico S. Rosinus ◽  
Rajinikanth Gogiraju ◽  
Stefan Guth ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Transforming Growth Factor ◽  
Quantitative Polymerase Chain Reaction ◽  
Vena Cava ◽  
Chronic Thromboembolic Pulmonary Hypertension ◽  
Type I ◽  
Tgfβ Signaling ◽  
Mesenchymal Transition ◽  
Endothelin 1 ◽  
Venous Thrombus

Rationale: Chronic thromboembolic pulmonary hypertension (CTEPH) is characterized by defective thrombus resolution, pulmonary artery obstruction, and vasculopathy. TGFβ (transforming growth factor-β) signaling mutations have been implicated in pulmonary arterial hypertension, whereas the role of TGFβ in the pathophysiology of CTEPH is unknown. Objective: To determine whether defective TGFβ signaling in endothelial cells contributes to thrombus nonresolution and fibrosis. Methods and Results: Venous thrombosis was induced by inferior vena cava ligation in mice with genetic deletion of TGFβ1 in platelets (Plt.TGFβ-KO) or TGFβ type II receptors in endothelial cells (End.TGFβRII-KO). Pulmonary endarterectomy specimens from CTEPH patients were analyzed using immunohistochemistry. Primary human and mouse endothelial cells were studied using confocal microscopy, quantitative polymerase chain reaction, and Western blot. Absence of TGFβ1 in platelets did not alter platelet number or function but was associated with faster venous thrombus resolution, whereas endothelial TGFβRII deletion resulted in larger, more fibrotic and higher vascularized venous thrombi. Increased circulating active TGFβ1 levels, endothelial TGFβRI/ALK1 (activin receptor-like kinase), and TGFβRI/ALK5 expression were detected in End.TGFβRII-KO mice, and activated TGFβ signaling was present in vessel-rich areas of CTEPH specimens. CTEPH-endothelial cells and murine endothelial cells lacking TGFβRII simultaneously expressed endothelial and mesenchymal markers and transcription factors regulating endothelial-to-mesenchymal transition, similar to TGFβ1-stimulated endothelial cells. Mechanistically, increased endothelin-1 levels were detected in TGFβRII-KO endothelial cells, murine venous thrombi, or endarterectomy specimens and plasma of CTEPH patients, and endothelin-1 overexpression was prevented by inhibition of ALK5, and to a lesser extent of ALK1. ALK5 inhibition and endothelin receptor antagonization inhibited mesenchymal lineage conversion in TGFβ1-exposed human and murine endothelial cells and improved venous thrombus resolution and pulmonary vaso-occlusions in End.TGFβRII-KO mice. Conclusions: Endothelial TGFβ1 signaling via type I receptors and endothelin-1 contribute to mesenchymal lineage transition and thrombofibrosis, which were prevented by blocking endothelin receptors. Our findings may have relevant implications for the prevention and management of CTEPH.

Control of junB and extracellular matrix protein expression by transforming growth factor-beta 1 is independent of simian virus 40 T antigen-sensitive growth-sensitive growth-inhibitory events

1991 ◽  
Vol 11 (2) ◽  
pp. 972-978
Author(s):  
M Laiho ◽  
L Rönnstrand ◽  
J Heino ◽  
J A Decaprio ◽  
J W Ludlow ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Extracellular Matrix ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Simian Virus 40 ◽  
Plasminogen Activator Inhibitor ◽  
Simian Virus ◽  
T Antigen ◽  
Tgf Beta ◽  
Plasminogen Activator Inhibitor 1

Treatment of Mv1Lu mink lung epithelial cells with transforming growth factor-beta 1 (TGF-beta 1) prevents phosphorylation of the retinoblastoma susceptibility gene product, RB, in late G1 phase of the cell cycle, which is thought to retain RB in a growth-suppressive state. This effect is paralleled by cell cycle arrest in late G1 (M. Laiho, J. A. DeCapric, J. W. Ludlow, D. M. Livingston, and J. Massagué, Cell 62:175-185, 1990). Arrest can be prevented by expression of simian virus 40 T antigen, which binds to underphosphorylated RB, presumably blocking its growth-suppressive activity. The response of cells to TGF-beta 1, however, is complex and includes changes in the levels of expression of genes encoding nuclear transcription factors and extracellular matrix components. To define the relationships among various components of the TGF-beta 1 response, we have investigated the effect of TGF-beta 1 on cells whose growth-inhibitory response to this factor is prevented by T antigen. TGF-beta 1 addition to exponentially growing Mv1Lu cells increased the levels of junB mRNA and of three extracellular matrix proteins: plasminogen activator inhibitor-1, fibronectin, and thrombospondin. Kinetically, the effects on junB and plasminogen activator inhibitor-1 expression occurred faster (half-maximal at 1 to 2 h) than the effects on fibronectin and thrombospondin expression (half-maximal at 6 to 10 h). These effects either preceded or overlapped, respectively, the withdrawal of Mv1Lu cells from the cell cycle. Expression of a transfected T-antigen gene in Mv1Lu cells, however, did not prevent any of these responses to TGF-beta 1. The results indcate that TGF-B1-stimulated expression of junB and extracellular matrix proteins in Mv1Lu cells can occur independently of the T-antigen-sensitive events that lead to growth arrest.

scholarly journals Production of simian virus 40 large tumor antigen in bacteria: altered DNA-binding specificity and dna-replication activity of underphosphorylated large tumor antigen

1989 ◽  
Vol 86 (17) ◽  
pp. 6479-6483 ◽  
Author(s):  
I J Mohr ◽  
Y Gluzman ◽  
M P Fairman ◽  
M Strauss ◽  
D McVey ◽  
...  
Keyword(s):  
Tumor Antigen ◽  
Mammalian Cells ◽  
Expression System ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Origin Of Replication ◽  
Wild Type ◽  
Large Tumor ◽  
Large Tumor Antigen

A bacterial expression system was used to produce simian virus 40 large tumor antigen (T antigen) in the absence of the extensive posttranslational modifications that occur in mammalian cells. Wild-type T antigen produced in bacteria retained a specific subset of the biochemical activities displayed by its mammalian counterpart. Escherichia coli T antigen functioned as a helicase and bound to DNA fragments containing either site I or the wild-type origin of replication in a manner identical to mammalian T antigen. However, T antigen purified from E. coli did not efficiently bind to site II, an essential cis element within the simian virus 40 origin of replication. It therefore could not unwind origin-containing plasmids or efficiently replicate simian virus 40 DNA in vitro. The ability of protein phosphorylation to modulate the intrinsic preference of full-length T antigen for either site I or site II is discussed.

scholarly journals trans activation of type 1 interferon promoters by simian virus 40 T antigen.

1988 ◽  
Vol 8 (8) ◽  
pp. 3397-3405 ◽  
Author(s):  
J Hiscott ◽  
A Wong ◽  
D Alper ◽  
S Xanthoudakis
Keyword(s):  
Hela Cells ◽  
Sendai Virus ◽  
Expression System ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Relative Contribution ◽  
Ifn Alpha ◽  
293 Cells

A human transient expression system was used to measure the influence of simian virus 40 T antigen and adenovirus E1a proteins on the activation of alpha interferon subtype 1 (IFN-alpha 1) and IFN-beta promoters linked to the reporter chloramphenicol acetyltransferase gene. Large T-antigen production, amplified by expression plasmid replication in transfected 293 cells, was able to trans activate the IFN-beta promoter 5- to 10-fold, increasing both the constitutive and Sendai virus-induced levels of expression. Surprisingly, the previously quiescent transfected IFN-alpha 1 promoter in T-antigen-expressing cells displayed a level of inducibility similar to IFN-beta. The endogenous IFN-alpha 1 gene was also inducible to a limited extent in cells expressing T antigen. A truncated IFN-beta promoter deleted to position -37 relative to the CAP site was neither inducible nor trans activated by T antigen, suggesting that sequences required for efficient induction were also needed for trans activation. Since 293 cells express adenoviral E1a proteins, experiments were also performed in HeLa cells to assess the relative contribution of T antigen and E1a proteins to IFN trans activation. In HeLa cells, T-antigen coexpression increased the constitutive level of IFN-beta and IFN-alpha 1 promoter activity without augmenting relative inducibility. Coexpression of T antigen and E1a proteins did not have a cooperative effect on type 1 IFN expression.

Sign in / Sign up

Export Citation Format

Share Document