scholarly journals Identification of extracellular matrix proteins secreted by human dermal fibroblasts cultured in 3D electrospun scaffolds

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Atena Malakpour-Permlid ◽  
Irina Buzzi ◽  
Cecilia Hegardt ◽  
Fredrik Johansson ◽  
Stina Oredsson
Keyword(s):  
Transforming Growth Factor ◽  
3D Structure ◽  
Three Dimensional ◽  
3D Cell Culture ◽  
Transforming Growth Factor Β1 ◽  
Dermal Fibroblasts ◽  
Human Dermal Fibroblasts ◽  
3D Network ◽  
Tgf Β1

AbstractThe appreciation that cell interactions in tissues is dependent on their three dimensional (3D) distribution has stimulated the development of 3D cell culture models. We constructed an artificial 3D tumour by culturing human breast cancer JIMT-1 cells and human dermal fibroblasts (HDFs) in a 3D network of electrospun polycaprolactone fibres. Here, we investigate ECM components produced by the cells in the artificial 3D tumour, which is an important step in validating the model. Immunostaining and confocal fluorescence microscopy show that the ECM proteins fibronectin, collagen I, and laminin are deposited throughout the entire 3D structure. Secreted soluble factors including matrix metalloproteinases (MMPs) and interleukine-6 (IL-6) were analysed in collected medium and were found to be mainly derived from the HDFs. Treatment with transforming growth factor-β1 (TGF-β1), a major cytokine found in a tumour, significantly alters the MMP activity and IL-6 concentration. In addition, TGF-β1 treatment, changes the morphology of the HDFs to become more elongated and with increased linearized actin filaments compared to non-treated HDFs. Collectively, these novel findings suggest that the artificial 3D tumour displays a clear cell distribution and ECM deposition that resembles a tumour environment in vivo, suggesting an innovative biological model to study a human tumour.

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.

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:.

scholarly journals Expression of pro-inflammatory markers by human dermal fibroblasts in a three-dimensional culture model is mediated by an autocrine interleukin-1 loop

2004 ◽  
Vol 379 (2) ◽  
pp. 351-358 ◽  
Author(s):  
Daniela KESSLER-BECKER ◽  
Thomas KRIEG ◽  
Beate ECKES
Keyword(s):  
Keratinocyte Growth Factor ◽  
Interleukin 1 ◽  
Three Dimensional ◽  
Dermal Fibroblasts ◽  
Human Dermal Fibroblasts ◽  
Inflammatory Processes ◽  
Cox 2 ◽  
Extracellular Matrix Interactions

In vivo, fibroblasts reside in connective tissues, with which they communicate in a reciprocal way. Such cell–extracellular matrix interactions can be studied in vitro by seeding fibroblasts in collagen lattices. Depending upon the mechanical properties of the system, fibroblasts are activated to assume defined phenotypes. In the present study, we examined a transcriptional profile of primary human dermal fibroblasts cultured in a relaxed collagen environment and found relative induction (>2-fold) of 393 out of approx. 7100 transcripts when compared with the same system under mechanical tension. Despite down-regulated proliferation and matrix synthesis, cells did not become generally quiescent, since they induced transcription of numerous other genes including matrix metalloproteinases (MMPs) and growth factors/cytokines. Of particular interest was the induction of gene transcripts encoding pro-inflammatory mediators, e.g. cyclo-oxygenase-2 (COX-2), and interleukins (ILs)-1 and -6. These are apparently regulated in a hierarchical fashion, since the addition of IL-1 receptor antagonist prevented induction of COX-2, IL-1 and IL-6, but not that of MMP-1 or keratinocyte growth factor (KGF). Our results suggest strongly that skin fibroblasts are versatile cells, which adapt to their extracellular environment by displaying specific phenotypes. One such phenotype, induced by a mechanically relaxed collagen environment, is the ‘pro-inflammatory’ fibroblast. We propose that fibroblasts that are embedded in a matrix environment can actively participate in the regulation of inflammatory processes.

CD28 disruption exacerbates inflammation in Tgf-β1-/- mice: in vivo suppression by CD4+CD25+ regulatory T cells independent of autocrine TGF-β1

Blood ◽  
2004 ◽  
Vol 103 (12) ◽  
pp. 4594-4601 ◽  
Author(s):  
Mizuko Mamura ◽  
WoonKyu Lee ◽  
Timothy J. Sullivan ◽  
Angelina Felici ◽  
Anastasia L. Sowers ◽  
...  
Keyword(s):  
T Cells ◽  
Regulatory T Cells ◽  
Transforming Growth Factor ◽  
Cytotoxic T Lymphocyte ◽  
Tumor Necrosis Factor Receptor ◽  
Transforming Growth Factor Β1 ◽  
Autoimmune Inflammatory Disease ◽  
Lymph Node Cells ◽  
Tgf Β1

Abstract Tgf-β1-/- mice develop a progressive, lethal, inflammatory syndrome, but mechanisms leading to the spontaneous activation of Tgf-β1-/- T cells remain unclear. Here we show the disruption of CD28 gene expression accelerates disease in Tgf-β1-/- mice, and we link this increase in severity to a reduction in the number of CD4+CD25+ regulatory T cells. CD4+CD25+ T cells develop normally in Tgf-β1-/- mice and display characteristic expression of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), glucocorticoid-induced tumor necrosis factor receptor (GITR), αEβ7 integrin, and Foxp3. Adoptive transfer of Tgf-β1-/- splenocytes to Tgf-β1+/+/Rag2-/- mice induced an autoimmune inflammatory disease with features similar to those of the Tgf-β1-/- phenotype, and disease transfer was accelerated by the depletion of Tgf-β1-/- CD4+CD25+ T cells from donor splenocytes. Cotransfer of Tgf- β1-/- CD4+CD25+ T cells clearly attenuated disease in Rag2-/- recipients of CD25+-depleted Tgf-β1-/- spleen and lymph node cells, but suppression was incomplete when compared with Tgf-β1+/+ CD4+CD25+ T cells. These data demonstrate that CD4+CD25+ regulatory T cells develop in complete absence of endogenous transforming growth factor-β1 (TGF-β1) expression and that autocrine TGF-β1 expression is not essential for these cells to suppress inflammation in vivo. (Blood. 2004;103:4594-4601)

Comparison of Release Profiles of Various Growth Factors from Biodegradable Carriers

1998 ◽  
Vol 530 ◽  
Author(s):  
Y. Tabata ◽  
M. Yamamoto ◽  
Y. Ikada
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Transforming Growth Factor Β1 ◽  
The Body ◽  
Bone Morphogenetic Protein 2 ◽  
Biodegradable Hydrogel ◽  
Tgf Β1

AbstractA biodegradable hydrogel was prepared by glutaraldehyde crosslinking of acidic gelatin with an isoelectric point (IEP) of 5.0 as a carrier to release basic growth factors on the basis of polyion complexation. Basic fibroblast growth factor (bFGF), transforming growth factor β1 (TGF-β1), and bone morphogenetic protein-2 (BMP-2) were sorbed from their aqueous solution into the dried gelatin hydrogels to prepare respective growth factor-incorporating hydrogels. Under an in vitro non-degradation condition, approximately 20 % of incorporated bFGF and TGF-β1 was released from the hydrogels within initial 40 min, followed by no further release, whereas a large initial release of BMP-2 was observed. After subcutaneous implantation of the gelatin hydrogels incorporating 125I-labeled growth factor in the mouse back, the remaining radioactivity was measured to estimate the in vivo release profile of growth factors. Incorporation into gelatin hydrogels enabled bFGF and TGF-β1 to retain in the body for about 15 days and the retention period well correlated with that of the gelatin hydrogel. Taken together, it is likely that the growth factors ionically complexed with acidic gelatin were released in vivo as a result of hydrogel biodegradation. On the contrary, basic BMP-2 did not ionically interact with acidic gelatin, resulting in no sustained released by the present biodegradable carrier system.

scholarly journals Growth factor regulation of hyaluronan synthesis and degradation in human dermal fibroblasts: importance of hyaluronan for the mitogenic response of PDGF-BB

2007 ◽  
Vol 404 (2) ◽  
pp. 327-336 ◽  
Author(s):  
Lingli Li ◽  
Trias Asteriou ◽  
Berit Bernert ◽  
Carl-Henrik Heldin ◽  
Paraskevi Heldin
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor ◽  
Fold Increase ◽  
Nuclear Factor Κb ◽  
Transforming Growth Factor Β1 ◽  
Dermal Fibroblasts ◽  
Mitogen Activated Protein Kinase ◽  
Signalling Pathways ◽  
Human Dermal Fibroblasts ◽  
High Level

The glycosaminoglycan hyaluronan is important in many tissuerepair processes. We have investigated the synthesis of hyaluronan in a panel of cell lines of fibroblastic and epithelial origin in response to PDGF (platelet-derived growth factor)-BB and other growth factors. Human dermal fibroblasts exhibited the highest hyaluronan-synthesizing activity in response to PDGF-BB. Analysis of HAS (hyaluronan synthase) and HYAL (hyaluronidase) mRNA expression showed that PDGF-BB treatment induced a 3-fold increase in the already high level of HAS2 mRNA, and increases in HAS1 and HYAL1 mRNA, whereas the levels of HAS3 and HYAL2 mRNA were not affected. Furthermore, PDGF-BB also increased the amount and activity of HAS2 protein, but not of HYAL1 and HYAL2 proteins. Using inhibitors for MEK1/2 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase 1/2] (U0126) and for PI3K (phosphoinositide 3-kinase) (LY294002), as well as the SN50 inhibitor, which prevents translocation of the active NF-κB (nuclear factor κB) to the nucleus, we observed a complete inhibition of both HAS2 transcriptional activity and hyaluronan synthesis, whereas inhibitors of other signalling pathways were without any significant effect. TGF-β1 (transforming growth factor-β1) did not increase the activity of hyaluronan synthesis in dermal fibroblasts, but increased the activity of HYALs. Importantly, inhibition of hyaluronan binding to its receptor CD44 by the monoclonal antibody Hermes-1, inhibited PDGF-BB-stimulated [3H]thymidine incorporation of dermal fibroblasts. We conclude that the ERK MAPK and PI3K signalling pathways are necessary for the regulation of hyaluronan synthesis by PDGF-BB, and that prevention of its binding to CD44 inhibits PDGF-BB-induced cell growth.

scholarly journals Genistein does not Inhibit TGF-β1-Induced Conversion of Human Dermal Fibroblasts to Myofibroblasts

2021 ◽  
pp. 815-820
Author(s):  
M KAŇUCHOVÁ ◽  
L URBAN ◽  
N MELEGOVÁ ◽  
M ČOMA ◽  
B DVOŘÁNKOVÁ ◽  
...  
Keyword(s):  
Transforming Growth Factor Beta ◽  
Wound Repair ◽  
Transforming Growth Factor ◽  
Dermal Fibroblasts ◽  
Point Of View ◽  
Human Dermal Fibroblasts ◽  
Receptor Modulator ◽  
Naturally Occurring ◽  
Growth Factor Beta ◽  
Tgf Β1

Transforming growth factor beta 1 (TGF-β1) is a pro-fibrotic cytokine with a key role in wound repair and regeneration, including induction of fibroblast-to-myofibroblast transition. Genistein is a naturally occurring selective estrogen receptor modulator with promising anti-fibrotic properties. In the present study we aimed to investigate whether genistein modulates TGF-β1 (canonical and non-canonical) signaling in normal dermal fibroblasts at the protein level (Western blot and immunofluo-rescence). We demonstrated that TGF-β1 induces the myofibroblast-like phenotype in the studied fibroblast signaling via canonical (SMAD) and non-canonical (AKT, ERK1/2, ROCK) pathways. Genistein induced only ERK1/2 expression, whereas the combination of TGF-β1 and genistein attenuated the ERK1/2 and ROCK signaling. Of note, the other studied pathways remained almost unaffected. From this point of view, genistein does not impair conversion of normal fibroblasts to myofibroblast-like cells.

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