PDE4 inhibitors roflumilast and rolipram augment PGE2 inhibition of TGF-β1-stimulated fibroblasts

2009 ◽  
Vol 296 (6) ◽  
pp. L959-L969 ◽  
Author(s):  
Shinsaku Togo ◽  
Xiangde Liu ◽  
Xingqi Wang ◽  
Hisatoshi Sugiura ◽  
Koichiro Kamio ◽  
...  
Keyword(s):  
Transforming Growth Factor ◽  
Three Dimensional ◽  
Fetal Lung ◽  
Independent Effect ◽  
Collagen Gels ◽  
Fibrotic Diseases ◽  
Human Fetal Lung ◽  
Human Fetal Lung Fibroblast ◽  
Adult Human Lung ◽  
Tgf Β1

Fibrotic diseases are characterized by the accumulation of extracellular matrix together with distortion and disruption of tissue architecture. Phosphodiesterase (PDE)4 inhibitors, by preventing the breakdown of cAMP, can inhibit fibroblast functions and may be able to mitigate tissue remodeling. Transforming growth factor (TGF)-β1, a mediator of fibrosis, can potentially modulate cAMP by altering PGE2 metabolism. The present study assessed whether PDE4 inhibitors functionally antagonize the profibrotic activity of fibroblasts stimulated by TGF-β1. The PDE4 inhibitors roflumilast and rolipram both inhibited fibroblast-mediated contraction of three-dimensional collagen gels and fibroblast chemotaxis toward fibronectin in the widely studied human fetal lung fibroblast strain HFL-1 and several strains of fibroblasts from adult human lung. Roflumilast was ∼10-fold more potent than rolipram. There was a trend for PDE4 inhibitors to inhibit more in the presence of TGF-β1 (0.05 < P < 0.08). The effect of the PDE4 inhibitors was mediated through cAMP-stimulated protein kinase A (PKA), although a PKA-independent effect on gel contraction was also observed. The effect of PDE4 inhibitors depended on fibroblast production of PGE2 and TGF-β1-induced PGE2 production. PDE4 inhibitors together with TGF-β1 resulted in augmented PGE2 production together with increased expression of COX mRNA and protein. The present study supports the concept that PDE4 inhibitors may attenuate fibroblast activities that can lead to fibrosis and that PDE4 inhibitors may be particularly effective in the presence of TGF-β1-induced fibroblast stimulation.

Selective optogenetic stimulation of fibroblasts enables quantification of hetero-cellular coupling to cardiomyocytes in a three-dimensional model of heart tissue

EP Europace ◽  
2020 ◽  
Vol 22 (10) ◽  
pp. 1590-1599
Author(s):  
Maximilian Funken ◽  
Tobias Bruegmann ◽  
Philipp Sasse
Keyword(s):  
Membrane Potential ◽  
Growth Factor ◽  
Connexin 43 ◽  
Transforming Growth Factor ◽  
Three Dimensional ◽  
Transforming Growth Factor Β1 ◽  
Resting Membrane Potential ◽  
Human Cardiomyocytes ◽  
Tgf Β1

Abstract Aims Besides providing mechanical stability, fibroblasts in the heart could modulate the electrical properties of cardiomyocytes. Here, we aim to develop a three-dimensional hetero-cellular model to analyse the electric interaction between fibroblasts and human cardiomyocytes in vitro using selective optogenetic de- or hyperpolarization of fibroblasts. Methods and results NIH3T3 cell lines expressing the light-sensitive ion channel Channelrhodopsin2 or the light-induced proton pump Archaerhodopsin were generated for optogenetic depolarization or hyperpolarization, respectively, and characterized by patch clamp. Cardiac bodies consisting of 50% fibroblasts and 50% human pluripotent stem cell-derived cardiomyocytes were analysed by video microscopy and membrane potential was measured with sharp electrodes. Myofibroblast activation in cardiac bodies was enhanced by transforming growth factor-β1 (TGF-β1)-stimulation. Connexin-43 expression was analysed by qPCR and fluorescence recovery after photobleaching. Illumination of Channelrhodopsin2 or Archaerhodopsin expressing fibroblasts induced inward currents and depolarization or outward currents and hyperpolarization. Transforming growth factor-β1-stimulation elevated connexin-43 expression and increased cell–cell coupling between fibroblasts as well as increased basal beating frequency and cardiomyocyte resting membrane potential in cardiac bodies. Illumination of cardiac bodies generated with Channelrhodopsin2 fibroblasts accelerated spontaneous beating, especially after TGF-β1-stimulation. Illumination of cardiac bodies prepared with Archaerhodopsin expressing fibroblasts led to hyperpolarization of cardiomyocytes and complete block of spontaneous beating after TGF-β1-stimulation. Effects of light were significantly smaller without TGF-β1-stimulation. Conclusion Transforming growth factor-β1-stimulation leads to increased hetero-cellular coupling and optogenetic hyperpolarization of fibroblasts reduces TGF-β1 induced effects on cardiomyocyte spontaneous activity. Optogenetic membrane potential manipulation selectively in fibroblasts in a new hetero-cellular cardiac body model allows direct quantification of fibroblast–cardiomyocyte coupling in vitro.

scholarly journals Myofibroblast contraction activates latent TGF-β1 from the extracellular matrix

2007 ◽  
Vol 179 (6) ◽  
pp. 1311-1323 ◽  
Author(s):  
Pierre-Jean Wipff ◽  
Daniel B. Rifkin ◽  
Jean-Jacques Meister ◽  
Boris Hinz
Keyword(s):  
Extracellular Matrix ◽  
Growth Factor ◽  
Mechanical Stress ◽  
Protease Activity ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β1 ◽  
Stress Fibers ◽  
Fibrotic Diseases ◽  
Triton X ◽  
Tgf Β1

The conjunctive presence of mechanical stress and active transforming growth factor β1 (TGF-β1) is essential to convert fibroblasts into contractile myofibroblasts, which cause tissue contractures in fibrotic diseases. Using cultured myofibroblasts and conditions that permit tension modulation on the extracellular matrix (ECM), we establish that myofibroblast contraction functions as a mechanism to directly activate TGF-β1 from self-generated stores in the ECM. Contraction of myofibroblasts and myofibroblast cytoskeletons prepared with Triton X-100 releases active TGF-β1 from the ECM. This process is inhibited either by antagonizing integrins or reducing ECM compliance and is independent from protease activity. Stretching myofibroblast-derived ECM in the presence of mechanically apposing stress fibers immediately activates latent TGF-β1. In myofibroblast-populated wounds, activation of the downstream targets of TGF-β1 signaling Smad2/3 is higher in stressed compared to relaxed tissues despite similar levels of total TGF-β1 and its receptor. We propose activation of TGF-β1 via integrin-mediated myofibroblast contraction as a potential checkpoint in the progression of fibrosis, restricting autocrine generation of myofibroblasts to a stiffened ECM.

Expression of Transforming Growth Factor-αin Mid-gestation Human Fetal Lung

1993 ◽  
Vol 8 (3) ◽  
pp. 266-272 ◽  
Author(s):  
Thomas P. Strandjord ◽  
Joan G. Clark ◽  
W. Alan Hodson ◽  
Rodney A. Schmidt ◽  
David K. Madtes
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Fetal Lung ◽  
Human Fetal Lung

scholarly journals Antenatal glucocorticoids counteract LPS changes in TGF-β pathway and caveolin-1 in ovine fetal lung

2013 ◽  
Vol 304 (6) ◽  
pp. L438-L444 ◽  
Author(s):  
Jennifer J. P. Collins ◽  
Steffen Kunzmann ◽  
Elke Kuypers ◽  
Matthew W. Kemp ◽  
Christian P. Speer ◽  
...  
Keyword(s):  
Growth Factor ◽  
Western Blot ◽  
Transforming Growth Factor ◽  
Fetal Lung ◽  
Tissue Growth ◽  
Caveolin 1 ◽  
Protein Levels ◽  
Smad2 Phosphorylation ◽  
Ovine Fetal ◽  
Tgf Β1

Inflammation and antenatal glucocorticoids, the latter given to mothers at risk for preterm birth, affect lung development and may contribute to the development of bronchopulmonary dysplasia (BPD). The effects of the combined exposures on inflammation and antenatal glucocorticoids on transforming growth factor (TGF)-β signaling are unknown. TGF-β and its downstream mediators are implicated in the etiology of BPD. Therefore, we asked whether glucocorticoids altered intra-amniotic lipopolysaccharide (LPS) effects on TGF-β expression, its signaling molecule phosphorylated sma and mothers against decapentaplegic homolog 2 (pSmad2), and the downstream mediators connective tissue growth factor (CTGF) and caveolin-1 (Cav-1). Ovine singleton fetuses were randomized to receive either an intra-amniotic injection of LPS and/or maternal betamethasone (BTM) intramuscularly 7 and/or 14 days before delivery at 120 days gestational age (GA; term = 150 days GA). Saline was used for controls. Protein levels of TGF-β1 and -β2 were measured by ELISA. Smad2 phosphorylation was assessed by immunohistochemistry and Western blot. CTGF and Cav-1 mRNA and protein levels were determined by RT-PCR and Western blot. Free TGF-β1 and -β2 and total TGF-β1 levels were unchanged after LPS and/or BTM exposure, although total TGF-β2 increased in animals exposed to BTM 7 days before LPS. pSmad2 immunostaining increased 7 days after LPS exposure although pSmad2 protein expression did not increase. Similarly, CTGF mRNA and protein levels increased 7 days after LPS exposure as Cav-1 mRNA and protein levels decreased. BTM exposure before LPS prevented CTGF induction and Cav-1 downregulation. This study demonstrated that the intrauterine inflammation-induced TGF-β signaling can be inhibited by antenatal glucocorticoids in fetal lungs.

Modulation of bcl-2 and p27 in human primitive proliferating hematopoietic progenitors by autocrine TGF-β1 is a cell cycle–independent effect and influences their hematopoietic potential

Blood ◽  
2000 ◽  
Vol 95 (10) ◽  
pp. 3001-3009 ◽  
Author(s):  
Luca Pierelli ◽  
Maria Marone ◽  
Giuseppina Bonanno ◽  
Simona Mozzetti ◽  
Sergio Rutella ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transforming Growth Factor ◽  
Independent Effect ◽  
Hematopoietic Progenitors ◽  
Cycle Number ◽  
Protein Levels ◽  
Bax Protein ◽  
Relevant Effect ◽  
Tgf Β1 ◽  
Rna Levels

Abstract Primitive, proliferating hematopoietic progenitors (defined as cytokine low-responding primitive progenitors; CLRPP), isolated from human CD34+ cells, expressed endoglin (CD105) and produced transforming growth factor-β1 (TGF-β1). Culture of CLRPP in serum-free conditions with anti-TGF-β1 monoclonal antibody produced a substantial decrease in bcl-2 protein/RNA levels and a significant reduction of cloning and long-term culture-initiating cell (LTC-IC) activities. GATA-1 and PU.1 RNA levels were significantly up-regulated in anti-TGF-β1–treated CLRPP, which generated an increased number of cells expressing CD15/CD11b/glycophorin-A. The described effects of TGF-β1 neutralization were observed in the absence of any relevant effect on cell cycle; number of cell divisions; p53, c-myc, and p21 RNA levels; bcl-xL and bax protein levels; and c-myc/p16/p21/p107/Rb cell cycle–related protein levels. A relevant increase in p27 protein levels was observed in anti-TGF-β1–treated CLRPP, suggesting a role for p27 in the regulation of the hematopoietic potential. The present study on human progenitors and previously reported data on TGF-β1 knockout mice suggest that, at the autocrine level, the cell cycle inhibitor TGF-β1 plays an important role in regulating the survival and differentiation of primitive proliferating hematopoietic progenitors by cell cycle–independent mechanisms.

Human surfactant proteins A1 and A2 are differentially regulated during development and by soluble factors

1998 ◽  
Vol 275 (4) ◽  
pp. L653-L669 ◽  
Author(s):  
Louis M. Scavo ◽  
Robert Ertsey ◽  
Bi Qi Gao
Keyword(s):  
Transforming Growth Factor ◽  
Fetal Lung ◽  
Surfactant Protein ◽  
Soluble Factors ◽  
Adult Lung ◽  
Relative Quantitation ◽  
Ifn Γ ◽  
Factor Α ◽  
Human Fetal Lung

An RT-PCR method for the relative quantitation of the mRNAs for human surfactant protein (SP) A1 and SP-A2 was developed, verified, and then utilized to determine the relative levels of these mRNAs in fetal and adult lung samples in vivo, as well as in cultured human fetal lung explants and H441 cells. For the cultured tissue and cells, we assessed the effects of a variety of soluble factors known to modulate total SP-A. Comprehensive analysis revealed many significant findings, including the following: both mRNAs were expressed as early as 15 wk of gestation; throughout midgestation, SP-A1 was present at higher levels than SP-A2, with an average ratio of 30:1. In the adult lung, SP-A1 mRNA was present at lower levels than SP-A2, with a ratio of 0.4:1, whereas in H441 cells, the ratio was 0.85:1. In fetal lung cultured for 4 days, both mRNAs increased, with a greater increase in SP-A2 (97-fold) than in SP-A1 (15-fold), resulting in a final ratio of 4:1. Differential regulation was demonstrated for 8-(4-chlorophenylthio)-cAMP, interferon (IFN)-γ, tumor necrosis factor-α, and transforming growth factor (TGF)-β in the human fetal lung explant system, with SP-A2 being more affected, and for IFN-γ and TGF-β in the H441 cells, where SP-A1 showed greater regulation. Of the soluble factors tested, IFN-γ and TGF-β had the most potent and consistent effects in both systems.

Role of platelet-derived growth factor-B, vascular endothelial growth factor, insulin-like growth factor-II, mitogen-activated protein kinase and transforming growth factor-β1 in expansion-induced lung growth in fetal sheep

2006 ◽  
Vol 18 (6) ◽  
pp. 655 ◽  
Author(s):  
Megan J. Wallace ◽  
Alison M. Thiel ◽  
Andrea M. Lines ◽  
Graeme R. Polglase ◽  
Foula Sozo ◽  
...  
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Mapk Pathway ◽  
Fetal Lung ◽  
Mitogen Activated Protein Kinase ◽  
Insulin Like Growth Factor ◽  
Lung Expansion ◽  
Mitogen Activated Protein ◽  
Extracellular Matrix Remodelling ◽  
Tgf Β1

Increased fetal lung expansion induces lung growth, cell differentiation and extracellular matrix remodelling, although the mechanisms involved are unknown. Platelet-derived growth factor (PDGF)-B, vascular endothelial growth factor (VEGF) and insulin-like growth factor (IGF)-II are mitogens activating the mitogen-activated protein kinase (MAPK) pathway, whereas transforming growth factor (TGF)-β1 induces differentiation and extracellular matrix remodelling. In the present study, we investigated the mRNA levels of PDGF-B, VEGF, IGF-II and TGF-β1, as well as active MAPK levels, during increased fetal lung expansion induced by tracheal obstruction (TO) in sheep for 0 (controls), 36 h or 2, 4, or 10 days (n = 5 in each group). The 3.7-kb VEGF transcript increased by 30% (P < 0.05) at 36 h TO. The expression of PDGF-B decreased by approximately 25% (P < 0.01) at 2–10 days TO. In contrast, TGF-β1 mRNA increased by 96% (P < 0.05) at 10 days TO, when bioactive TGF-β1 decreased by 55% (P < 0.05). Insulin-like growth factor-II mRNA tended to increase at 10 days TO (37% above controls; P = 0.07), whereas mRNA for its receptor, IGF1R, was reduced by TO. There was no change in active MAPK levels preceding or at the time of a TO-induced 800% increase in cell proliferation. We conclude that VEGF is likely to promote expansion-induced endothelial cell proliferation, but the mechanisms underlying expansion-induced proliferation of fibroblasts and alveolar epithelial cells are unlikely to be mediated by increases in PDGF-B or IGF-II expression or activation of the MAPK pathway.

scholarly journals Decorin-transforming Growth Factor-β Interaction Regulates Matrix Organization and Mechanical Characteristics of Three-dimensional Collagen Matrices

2007 ◽  
Vol 282 (49) ◽  
pp. 35887-35898 ◽  
Author(s):  
Zannatul Ferdous ◽  
Victoria Mariko Wei ◽  
Renato Iozzo ◽  
Magnus Höök ◽  
Kathryn Jane Grande-Allen
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Three Dimensional ◽  
Transforming Growth Factor Β ◽  
Material Behavior ◽  
Wild Type ◽  
Collagen Matrices ◽  
Matrix Organization ◽  
Tgf Β1

The small leucine-rich proteoglycan decorin has been demonstrated to be a key regulator of collagen fibrillogenesis; decorin deficiencies lead to irregularly shaped collagen fibrils and weakened material behavior in postnatal murine connective tissues. In an in vitro investigation of the contributions of decorin to tissue organization and material behavior, model tissues were engineered by seeding embryonic fibroblasts, harvested from 12.5–13.5 days gestational aged decorin null (Dcn-/-) or wild-type mice, within type I collagen gels. The resulting three-dimensional collagen matrices were cultured for 4 weeks under static tension. The collagen matrices seeded with Dcn-/- cells exhibited greater contraction, cell density, ultimate tensile strength, and elastic modulus than those seeded with wild-type cells. Ultrastructurally, the matrices seeded with Dcn-/- cells contained a greater density of collagen. The decorin-null tissues contained more biglycan than control tissues, suggesting that this related proteoglycan compensated for the absence of decorin. The effect of transforming growth factor-β (TGF-β), which is normally sequestered by decorin, was also investigated in this study. The addition of TGF-β1 to the matrices seeded with wild-type cells improved their contraction and mechanical strength, whereas blocking TGF-β1 in the Dcn-/- cell-seeded matrices significantly reduced the collagen gel contraction. These results indicate that the inhibitory interaction between decorin and TGF-β1 significantly influenced the matrix organization and material behavior of these in vitro model tissues.

Role of endogenous TGF-β in glucocorticoid-induced lung type II cell differentiation

2007 ◽  
Vol 292 (1) ◽  
pp. L249-L257 ◽  
Author(s):  
Theresa M. McDevitt ◽  
Linda W. Gonzales ◽  
Rashmin C. Savani ◽  
Philip L. Ballard
Keyword(s):  
Cell Differentiation ◽  
Transforming Growth Factor ◽  
Fetal Lung ◽  
Hormone Treatment ◽  
Type Ii ◽  
Gene Suppression ◽  
Surfactant Production ◽  
Type Ii Cell ◽  
Human Fetal Lung

In the fetal lung, endogenous transforming growth factor (TGF)-β inhibits early morphogenesis and blocks hormone-induced type II cell differentiation. We hypothesized that endogenous TGF-β inhibits type II cell differentiation and that the stimulatory effects of glucocorticoids result in part from suppression of TGF-β. Epithelial cells were isolated from human fetal lung and cultured under defined conditions with and without dexamethasone plus cAMP to promote type II cell differentiation. Control cells produced TGF-β, which was activated in part by αVβ6-integrin. Treatment with dexamethasone, but not cAMP, reduced TGF-β1 and -β2 transcripts and TGF-β bioactivity in culture medium. To examine the effects of decreased TGF-β in the absence of glucocorticoid, cells were treated with antibodies to TGF-β and its receptors. By real-time RT-PCR, antibody blockade of TGF-β reduced serpine1, a TGF-β-inducible gene, and increased gene expression for sftpa, sftpb, sftpc, and titf1, mimicking the response to hormone treatment. By microarray analysis, 29 additional genes were induced by both TGF-β antibody and hormone treatment, and 20 other genes were repressed by both treatments. For some genes, the fold response was comparable for antibody and hormone treatment. We conclude that endogenous TGF-β suppresses expression of surfactant proteins and selected other type II cell genes in fetal lung, in part secondary to increased expression of titf1, and we propose that the mechanism of glucocorticoid-induced type II cell differentiation includes antagonism of TGF-β gene suppression. Surfactant production during fetal development is likely influenced by relative levels of TGF-β and glucocorticoids.

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