scholarly journals flt3 Ligand in Cooperation With Transforming Growth Factor-β1 Potentiates In Vitro Development of Langerhans-Type Dendritic Cells and Allows Single-Cell Dendritic Cell Cluster Formation Under Serum-Free Conditions

Blood ◽  
1997 ◽  
Vol 90 (4) ◽  
pp. 1425-1434 ◽  
Author(s):  
Herbert Strobl ◽  
Concha Bello-Fernandez ◽  
Elisabeth Riedl ◽  
Winfried F. Pickl ◽  
Otto Majdic ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β1 ◽  
Langerhans Cell ◽  
Flt3 Ligand ◽  
Serum Free ◽  
Cd34 Cells ◽  
Serum Free Conditions ◽  
Tgf Β1

Using a recently described serum-free culture system of purified human CD34+ progenitor cells, we show here a critical cooperation of flt3 ligand (FL) with transforming growth factor-β1 (TGF-β1) in the induction of in vitro dendritic cell/Langerhans cell (DC/LC) development. The addition of FL to serum-free cultures of CD34+ cells supplemented with TGF-β1, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor α, and stem cell factor strongly increases both percentages (mean, 36% ± 5% v 64% ± 4%; P = .001) and total numbers (4.4- ± 0.8-fold) of CD1a+ dendritic cells. These in vitro-generated CD1a+ cells molecularly closely resemble a particular type of DC known as an epidermal Langerhans cell. Generation of DC under serum-free conditions was found to strictly require supplementation of culture medium with TGF-β1. Upon omission of TGF-β1, percentages of CD1a+ DC decreased (to mean, 10% ± 8%; P = .001) and, in turn, percentages of granulomonocytic cells (CD1a− cells that are lysozyme [LZ+]; myeloperoxidase [MPO+]; CD14+) increased approximately threefold (P < .05). Furthermore, in the absence of TGF-β1, FL consistently promotes generation of LZ+, MPO+, and CD14+ cells, but not of CD1a+ cells. Serum-free single-cell cultures set up under identical TGF-β1– and FL-supplemented culture conditions showed that high percentages of CD34+ cells (mean, 18% ± 2%; n = 4) give rise to day-10 DC colony formation. The majority of cells in these DC-containing colonies expressed the Langerhans cell/Birbeck granule specific marker molecule Lag. Without TGF-β1 supplementation, Lag+ colony formation is minimal and formation of monocyte/macrophage-containing colonies predominates. Total cloning efficiency in the absence and presence of TGF-β1 is virtually identical (mean, 41% ± 6% v 41% ± 4%). Thus, FL has the potential to strongly stimulate DC/LC generation, but has a strict requirement for TGF-β1 to show this costimulatory effect.

scholarly journals flt3 Ligand in Cooperation With Transforming Growth Factor-β1 Potentiates In Vitro Development of Langerhans-Type Dendritic Cells and Allows Single-Cell Dendritic Cell Cluster Formation Under Serum-Free Conditions

Blood ◽  
1997 ◽  
Vol 90 (4) ◽  
pp. 1425-1434 ◽  
Author(s):  
Herbert Strobl ◽  
Concha Bello-Fernandez ◽  
Elisabeth Riedl ◽  
Winfried F. Pickl ◽  
Otto Majdic ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β1 ◽  
Langerhans Cell ◽  
Flt3 Ligand ◽  
Serum Free ◽  
Cd34 Cells ◽  
Serum Free Conditions ◽  
Tgf Β1

Abstract Using a recently described serum-free culture system of purified human CD34+ progenitor cells, we show here a critical cooperation of flt3 ligand (FL) with transforming growth factor-β1 (TGF-β1) in the induction of in vitro dendritic cell/Langerhans cell (DC/LC) development. The addition of FL to serum-free cultures of CD34+ cells supplemented with TGF-β1, granulocyte-macrophage colony-stimulating factor, tumor necrosis factor α, and stem cell factor strongly increases both percentages (mean, 36% ± 5% v 64% ± 4%; P = .001) and total numbers (4.4- ± 0.8-fold) of CD1a+ dendritic cells. These in vitro-generated CD1a+ cells molecularly closely resemble a particular type of DC known as an epidermal Langerhans cell. Generation of DC under serum-free conditions was found to strictly require supplementation of culture medium with TGF-β1. Upon omission of TGF-β1, percentages of CD1a+ DC decreased (to mean, 10% ± 8%; P = .001) and, in turn, percentages of granulomonocytic cells (CD1a− cells that are lysozyme [LZ+]; myeloperoxidase [MPO+]; CD14+) increased approximately threefold (P < .05). Furthermore, in the absence of TGF-β1, FL consistently promotes generation of LZ+, MPO+, and CD14+ cells, but not of CD1a+ cells. Serum-free single-cell cultures set up under identical TGF-β1– and FL-supplemented culture conditions showed that high percentages of CD34+ cells (mean, 18% ± 2%; n = 4) give rise to day-10 DC colony formation. The majority of cells in these DC-containing colonies expressed the Langerhans cell/Birbeck granule specific marker molecule Lag. Without TGF-β1 supplementation, Lag+ colony formation is minimal and formation of monocyte/macrophage-containing colonies predominates. Total cloning efficiency in the absence and presence of TGF-β1 is virtually identical (mean, 41% ± 6% v 41% ± 4%). Thus, FL has the potential to strongly stimulate DC/LC generation, but has a strict requirement for TGF-β1 to show this costimulatory effect.

scholarly journals Transforming Growth Factor β1 (TGF-β1) Promotes Endothelial Cell Survival during In Vitro Angiogenesis via an Autocrine Mechanism Implicating TGF-α Signaling

2001 ◽  
Vol 21 (21) ◽  
pp. 7218-7230 ◽  
Author(s):  
Francesc Viñals ◽  
Jacques Pouysségur
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Cell Survival ◽  
Network Formation ◽  
Egf Receptor ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β1 ◽  
In Vitro Angiogenesis ◽  
Tgf Β1

ABSTRACT Mouse capillary endothelial cells (1G11 cell line) embedded in type I collagen gels undergo in vitro angiogenesis. Cells rapidly reorganize and form capillary-like structures when stimulated with serum. Transforming growth factor β1 (TGF-β1) alone can substitute for serum and induce cell survival and tubular network formation. This TGF-β1-mediated angiogenic activity depends on phosphatidylinositol 3-kinase (PI3K) and p42/p44 mitogen-activated protein kinase (MAPK) signaling. We showed that specific inhibitors of either pathway (wortmannin, LY-294002, and PD-98059) all suppressed TGF-β1-induced angiogenesis mainly by compromising cell survival. We established that TGF-β1 stimulated the expression of TGF-α mRNA and protein, the tyrosine phosphorylation of a 170-kDa membrane protein representing the epidermal growth factor (EGF) receptor, and the delayed activation of PI3K/Akt and p42/p44 MAPK. Moreover, we showed that all these TGF-β1-mediated signaling events, including tubular network formation, were suppressed by incubating TGF-β1-stimulated endothelial cells with a soluble form of an EGF receptor (ErbB-1) or tyrphostin AG1478, a specific blocker of EGF receptor tyrosine kinase. Finally, addition of TGF-α alone poorly stimulated angiogenesis; however, by reducing cell death, it strongly potentiated the action of TGF-β1. We therefore propose that TGF-β1 promotes angiogenesis at least in part via the autocrine secretion of TGF-α, a cell survival growth factor, activating PI3K/Akt and p42/p44 MAPK.

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.

Ex Vivo Generation of Dendritic Cells from CD34+ Cells in Gas-Permeable Containers Under Serum-Free Conditions

1998 ◽  
Vol 7 (5) ◽  
pp. 403-411 ◽  
Author(s):  
KENNETH L. KOWALKOWSKI ◽  
MORTIMER T. ALZONA ◽  
FREDERICK M. AONO ◽  
DENNIS E. VAN EPPS ◽  
MONA VACHULA
Keyword(s):  
Dendritic Cells ◽  
Ex Vivo ◽  
Serum Free ◽  
Cd34 Cells ◽  
Serum Free Conditions

Proliferation Effect of Mandibular Condylar Chondrocytes Given The Static Tension-Stress and/or TGF-β1 In Vitro

2007 ◽  
Vol 330-332 ◽  
pp. 1125-1128
Author(s):  
Zhi Qiang Wang ◽  
Zhi He Zhao ◽  
Jin Lin Song ◽  
Yu Bo Fan ◽  
Song Jiao Luo
Keyword(s):  
Transforming Growth Factor ◽  
Mandibular Condyle ◽  
Morphological Characters ◽  
Transforming Growth Factor Β1 ◽  
Mechanical Stimulus ◽  
Tension Stress ◽  
Static Tension ◽  
Mitogenic Effect ◽  
Tgf Β1

The purpose of this study is to study the proliferous effect of mandibular condylar chondrocytes given static tension-stress and/or transforming growth factor-β1 (TGF-β1) in vitro. The fourth-passage condylar chondrocytes were harvested for this study, and a pulsatile cellular mechanical system was used to apply stress on cells. The proliferous effect of condylar chondrocytes given continuous static tension-stress and/or TGF-β1 were examined by using flow cytometry. The experiment was divided into two parts. The first part was divided into 20 groups according to different TGF-β1 dosage (0ng/ml, 0.1ng/ml, 1ng/ml and 10ng/ml) for 0, 6, 12, 18 and 24 hours respectively. The second part was divided into eight groups under continuous static tension-stress (0 or 5kPa) and different TGF-β1 dosage (0ng/ml, 0.1ng/ml, 1ng/ml, 10ng/ml) for 12 hours. Experimental data was analyzed with repeated interclass analysis of variance The results showed that chondrocytes which were cultured under different TGF-β1dose combined with 5kPa static tension-stress had multi-horn morphological characters, including a great quantity of chondrocytes with division growth.TGF-β1 had a mitogenic effect on rat mandibular condyle chondrocytes at the concentrations of 0.1 , 1 and 10ng/ml , and the mitogenic effect of TGF-β1 to condylar chondrocytes were demonstrated after 12 to 18 hours, and the peak of mitogenic effects appeared at the 18th hour (P <0.05) . The most active mitogenesis happened in the group whose chondrocytes was under continuous static tension-stress (5kPa) combined with TGF-β1. These results proved that mechanical stimulus and TGF-β1 in vitro could influence and regulate the growth of condylar chondrocytes.

scholarly journals Obesity attenuates the effect of sleep apnea on active TGF-ß1 levels and tumor aggressiveness in patients with melanoma

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Carolina Cubillos-Zapata ◽  
Miguel Ángel Martínez-García ◽  
Elena Díaz-García ◽  
Ana Jaureguizar ◽  
Francisco Campos-Rodríguez ◽  
...  
Keyword(s):  
Sleep Apnea ◽  
Transforming Growth Factor ◽  
Serum Levels ◽  
Transforming Growth Factor Β1 ◽  
Multicenter Observational Study ◽  
Obstructive Sleep ◽  
Obese Subjects ◽  
Vitro Model ◽  
Tgf Β1

Abstract Active transforming growth factor-β1 (TGF-β1), a cytokine partially regulated by hypoxia and obesity, has been related with poor prognosis in several tumors. We determine whether obstructive sleep apnea (OSA) increases serum levels of active TGF-β1 in patients with cutaneous melanoma (CM), assess their relationship with melanoma aggressiveness and analyze the factors related to TGF-β1 levels in obese and non-obese OSA patients. In a multicenter observational study, 290 patients with CM were underwent sleep studies. TGF-β1 was increased in moderate-severe OSA patients vs. non-OSA or mild OSA patients with CM. In OSA patients, TGF-β1 levels correlated with mitotic index, Breslow index and melanoma growth rate, and were increased in presence of ulceration or higher Clark levels. In CM patients, OSA was associated with higher TGF-β1 levels and greater melanoma aggressiveness only in non-obese subjects. An in vitro model showed that IH-induced increases of TGF-β1 expression in melanoma cells is attenuated in the presence of high leptin levels. In conclusion, TGF-β1 levels are associated with melanoma aggressiveness in CM patients and increased in moderate-severe OSA. Moreover, in non-obese patients with OSA, TGF-β1 levels correlate with OSA severity and leptin levels, whereas only associate with leptin levels in obese OSA patients.

scholarly journals Microcarriers Based on Glycosaminoglycan-Like Marine Exopolysaccharide for TGF-β1 Long-Term Protection

Marine Drugs ◽  
2019 ◽  
Vol 17 (1) ◽  
pp. 65 ◽  
Author(s):  
Agata Zykwinska ◽  
Mélanie Marquis ◽  
Mathilde Godin ◽  
Laëtitia Marchand ◽  
Corinne Sinquin ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Target Location ◽  
Cartilage Regeneration ◽  
Transforming Growth Factor Β1 ◽  
Hydrogel Matrix ◽  
Vitro Model ◽  
Tgf Β1

Articular cartilage is an avascular, non-innervated connective tissue with limited ability to regenerate. Articular degenerative processes arising from trauma, inflammation or due to aging are thus irreversible and may induce the loss of the joint function. To repair cartilaginous defects, tissue engineering approaches are under intense development. Association of cells and signalling proteins, such as growth factors, with biocompatible hydrogel matrix may lead to the regeneration of the healthy tissue. One current strategy to enhance both growth factor bioactivity and bioavailability is based on the delivery of these signalling proteins in microcarriers. In this context, the aim of the present study was to develop microcarriers by encapsulating Transforming Growth Factor-β1 (TGF-β1) into microparticles based on marine exopolysaccharide (EPS), namely GY785 EPS, for further applications in cartilage engineering. Using a capillary microfluidic approach, two microcarriers were prepared. The growth factor was either encapsulated directly within the microparticles based on slightly sulphated derivative or complexed firstly with the highly sulphated derivative before being incorporated within the microparticles. TGF-β1 release, studied under in vitro model conditions, revealed that the majority of the growth factor was retained inside the microparticles. Bioactivity of released TGF-β1 was particularly enhanced in the presence of highly sulphated derivative. It comes out from this study that GY785 EPS based microcarriers may constitute TGF-β1 reservoirs spatially retaining the growth factor for a variety of tissue engineering applications and in particular cartilage regeneration, where the growth factor needs to remain in the target location long enough to induce robust regenerative responses.

Identification of Platelet Releasate Proteins that Bind to Mastoparan, a Peptide that Inhibits Shear-Induced TGF-β1 Activation,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3271-3271
Author(s):  
Teresa M Brophy ◽  
Jasimuddin Ahamed ◽  
Barry S. Coller
Keyword(s):  
Transforming Growth Factor ◽  
Coagulation Factor ◽  
Transforming Growth Factor Β1 ◽  
Affinity Column ◽  
Factor V ◽  
Disulfide Exchange ◽  
Control Peptide ◽  
Tgf Β1

Abstract Abstract 3271 Transforming growth factor β1 (TGF-β1) is a disulfide-bonded, 25 kD homodimeric protein produced by most cell types, including platelets, that functions as a cytokine in many physiologic and pathologic processes. Platelets contain 40–100 times more TGF-β1 than other cells and release it as an inactive large latent complex (LLC) comprised of TGF-β1 non-covalently associated with its latency-associated peptide (LAP), which is, in turn, disulfide-bonded to latent TGF-β binding protein 1 (LTBP-1). Thrombospondin-1 (TSP1), proteases, and reactive oxygen species have all been shown to activate TGF-β1 in vitro and a role for integrins in vivo has been inferred from studies of transgenic mice. Recently, we discovered that shear force can activate latent TGF-β1 released from platelets in vitro and that thiol-disulfide exchange contributes to shear-dependent TGF-β1 activation. A number of thiol isomerase enzymes that can catalyze thiol-disulfide exchange have been identified in platelets, including protein disulfide isomerase (PDI), ERp5, ERp57, ERp72, ERp44, ERp29, and TMX3. As shear-induced activation of TGF-β1 is partially thiol-dependent, we investigated if thiol isomerases can affect this process. Mastoparan is a non-thiol-containing wasp venom peptide known to inhibit the chaperone activity of PDI, ERp5, and perhaps other thiol isomerases. We recently showed that mastoparan, (INLKALAALAKKIL), inhibits stirring-induced TGF-β1 activation by more than 90% (100 μM; n=3, p=0.03), whereas no inhibition was observed with an inactive mastoparan-like control peptide (INLKAKAALAKKLL) at 100 μM (n=3, p=0.66). To identify the proteins that bind to mastoparan, either directly or indirectly, platelet releasates were chromatographed on a mastoparan affinity column prepared from N-hydroxysuccinimide Sepharose. Two control columns were employed: 1. unconjugated Sepharose, and 2. Sepharose conjugated with the mastoparan-like control peptide. Elution of bound proteins was achieved by increasing the NaCl concentration. Proteins identified by mass spectrometry as specifically binding to the mastoparan peptide column included LTBP-1, TGF-β1 precursor, clusterin, coagulation Factor V, multimerin-1, 14-3-3 protein zeta/delta, and α-actinin 4. These results were confirmed by immunoblotting. Furthermore, the thiol isomerases PDI, ERp5, ERp57, and ERp72 were all found to bind specifically to mastoparan as confirmed by immunoblotting. We conclude that mastoparan affinity chromatography identified a number of proteins in platelet releasates that may contribute to shear-induced TGF-β1 activation. Disclosures: Coller: Centocor/Accumetrics/Rockefeller University:.

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