scholarly journals SAT0299 PROLIFERATION, MIGRATION AND CONTRACTION ARE DIFFERENT BETWEEN TGFΒ AND PDGF STIMULATED DERMAL FIBROBLASTS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1094.2-1095
Author(s):  
A. S. Siebuhr ◽  
S. F. Madsen ◽  
M. Karsdal ◽  
A. C. Bay-Jensen ◽  
P. Juhl
Keyword(s):  
Gene Expression ◽  
Treatment Initiation ◽  
Human Fibroblasts ◽  
Calf Serum ◽  
Collagen Type I ◽  
Dermal Fibroblasts ◽  
Type I ◽  
Full Time ◽  
Ecm Remodeling ◽  
Full Time Employee

Background:Systemic sclerosis has vascular, inflammatory and fibrotic components, which may be associated with different growth factors and cytokines. Platelet derived growth factor (PDGF) is associated with the vasculature, whereas tumor necrosis factor beta (TGFβ) is associated with inflammation and fibrosis. We have developed a fibroblast model system of dermal fibrosis for anti-fibrotic drugs testing, but the effect of the fibroblasts mechanistic properties are unknown.Objectives:We investigated different mechanical capacities of PDGF and TGFβ treated human healthy dermal fibroblasts in the SiaJ setting.Methods:Primary human healthy dermal fibroblasts were grown in DMEM medium containing 0.4% fetal calf serum, ficoll (to produce a crowded environment) and ascorbic acid for up to 17 days. A wound was induced by scratching the cells at 0, 1, 3 or 7 days after treatment initiation. Wound closure was followed for 3 days. Contraction capacity was tested by creating gels of human fibroblasts produced collagens containing dermal fibroblasts and contraction was assessed at day 2 by calculating the percentage of gel size to total well size. Collagen type I, III and VI formation (PRO-C1, PRO-C3 and PRO-C6) and fibronectin (FBN-C) were evaluated by validated ELISAs (Nordic Bioscience). Gene expression was analyzed after 2 days in culture. Statistical analyses included One-way ANOVA and student’s t-test.Results:Generally, PDGF closed the wound in half the time of w/o and TGFβ, when treatment and cells are added concurrently or scratched one day after treatment initiation. When treatments were added 3 or 7 days prior to scratch, the cells treated with PDGF had proliferated to a higher degree than w/o and TGFβ. A consequence of this, was that when cells were scratch the sheet of cells produced was lifted from the bottom and fold over itself, leaving a much greater scratch than in the other treatments. However, despite this increased gap the PDGF treated cells closed the wound at the same time as w/o and TGFβ, confirming the results of the cells scratched at day 0 and 1.Inhibition of contraction by ML-7 of otherwise untreated cells inhibited contraction significantly compared to untreated cells alone (p=0.0009). Contraction was increased in both TGFβ and PDGF treated cells compared to untreated cells (both p<0.0001). TGFβ+ ML-7 inhibited the contraction to the level of w/o (p=0.0024), which was only 35% of ML-7 alone. In the contraction study the cells were terminated after 2 days of culture, thus prior to when biomarker of ECM remodeling is usually assessed. However, FBN-C was detectable and a significant release of fibronectin by TGFβ and PDGF compared to w/o was found in the supernatant (both p<0.0001). The gene expression of FBN was only increased with TGFβ (p<0.05) and not PDGF. ML-7 alone tended to decrease FBN-C and in combination with TGFβ the FBN level was significantly decreased compared to TGFβ alone (p<0.0001). However, the level of TGFβ+ML-7 was significantly higher than ML-7 alone (p=0.038).TGFβ increased the gene expression of most genes assessed, except Col6a1. PDGF increased the gene expression of Col3a1, Col5a1 and Col6a1 above the critical fold change of 2, but only significantly in Col5a1 and Col6a1 (both p<0.05).Conclusion:This study demonstrates that TGFβ and PDGF have different mechanical capacities in human healthy dermal fibroblasts; TGFβ increased gene expression of ECM related genes, such as collagens and have increased FBN release in the supernatant already 2 days after initial treatment. PDGF has increased contraction, proliferation and migratory capacities and less expression of ECM related genes and proteins.Disclosure of Interests:Anne Sofie Siebuhr Employee of: Nordic Bioscience, Sofie Falkenløve Madsen: None declared, Morten Karsdal Shareholder of: Nordic Bioscience A/S., Employee of: Full time employee at Nordic Bioscience A/S., Anne-Christine Bay-Jensen Shareholder of: Nordic Bioscience A/S, Employee of: Full time employee at Nordic Bioscience A/S., Pernille Juhl Employee of: Nordic Bioscience

scholarly journals SAT0298 IS INTERLEUKIN 6 A FACTOR OF FIBROGENESIS IN DERMAL FIBROBLASTS?

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1094.1-1094
Author(s):  
A. S. Siebuhr ◽  
P. Juhl ◽  
M. Karsdal ◽  
A. C. Bay-Jensen
Keyword(s):  
Gene Expression ◽  
Type I Collagen ◽  
Collagen Type ◽  
Collagen Type I ◽  
Dermal Fibroblasts ◽  
Type I ◽  
Dependent Manner ◽  
Full Time ◽  
Collagen Formation ◽  
Type Iii

Background:Interleukin 6 (IL-6) is known to have both pro- and anti-inflammatory properties, depending on the receptor activation. The classical IL-6 signaling via the membrane bound receptor is mainly anti-inflammatory, whereas signaling through the soluble receptor (sIL-6R) is pro-inflammatory/pro-fibrotic. However, the direct fibrotic effect of IL-6 stimulation on dermal fibroblasts is unknown.Objectives:We investigated the fibrotic effect of IL-6 + sIL-6R in a dermal fibroblast model and assessed fibrosis by neo-epitope biomarkers of extracellular matrix proteins.Methods:Primary healthy human dermal fibroblasts were grown for up to 17 days in DMEM medium with 0.4% fetal calf serum, ficoll (to produce a crowded environment) and ascorbic acid. IL-6 [1-90 nM]+sIL-6R [0.1-9 nM] alone or in combination with TGFβ [1 nM] were tested in three different donors. TGFβ [1 nM], PDGF-AB [3 nM] and non-stimulated cells (w/o) were used as controls. Tocilizumab (TCZ) with TGFβ + IL-6 + sIL-6R stimulation was tested in one donor. Collagen type I, III and VI formation (PRO-C1, PRO-C3 and PRO-C6) and fibronectin (FBN-C) were evaluated by validated ELISAs (Nordic Bioscience). Western blot analysis investigated signal cascades. Gene expression of selected ECM proteins was analyzed. Statistical analyses included One-way and 2-way ANOVA and area under the curve analysis.Results:formation by the end of the culture period. The fibronectin and collagen type VI signal were consistent between the three tested donors, whereas the formation of type III collagen was only increased in one donor, but in several trials. Type I collagen formation was unchanged by IL-6 + sIL-6R stimulation. The gene expression of type I collagen was induced by IL-6 + sIL-6R. Western blot analysis validated trans-signaling by the IL-6+sIL-6R stimulation as expected.IL-6 + sIL-6R stimulation in combination with TGFβ decreased fibronectin levels compared to TGFβ alone but did not reach the level of unstimulated fibroblasts. The formation of collagen type IV was generally unchanged with IL-6 + sIL-6R + TGFβ compared to TGFβ alone. Collagen type I and III formation was more scattered in the signals when IL-6 + sIL-6R was in combination with TGFβ, as the biomarker level could be either decreased or increased compared to TGFβ alone. In two studies the type I collagen level was synergistic increased by IL-6 + sIL-6R + TGFβ, whereas another study found the level to be decreased compared to TGFβ alone. The gene expression of fibronectin and type I collagen was increased with TGFβ +IL-6+sIL-6R compared to TGFβ alone.Inhibition of IL-6R by TCZ in combination with IL-6 + sIL-6R did only decrease the fibronectin level with the lowest TCZ concentration (p=0.03). TCZ alone decreased the fibronectin level in a dose-dependent manner (One-way ANOVA p=0.0002).Conclusion:We investigated the fibrotic response of dermal fibroblasts to IL-6 + sIL-6R stimulation. IL-6 modulated the fibronectin level and modulated the collagen type III formation level in a somewhat dose-dependent manner. In combination with TGFβ, IL-6 decreased collagen type I and IV formation and fibronectin. However, in this study inhibition of IL-6R by TCZ did not change the fibrotic response of the dermal fibroblasts. This study indicated that IL-6 did not induce collagen formation in dermal fibroblasts, except type III collagen formation with high IL-6 concentration.Figure:Disclosure of Interests:Anne Sofie Siebuhr Employee of: Nordic Bioscience, Pernille Juhl Employee of: Nordic Bioscience, Morten Karsdal Shareholder of: Nordic Bioscience A/S., Employee of: Full time employee at Nordic Bioscience A/S., Anne-Christine Bay-Jensen Shareholder of: Nordic Bioscience A/S, Employee of: Full time employee at Nordic Bioscience A/S.

scholarly journals AB0167 TOFACITINIB AND NINTEDANIB MODULATE COLLAGEN FORMATION IN DERMAL FIBROBLASTS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 1383.1-1384
Author(s):  
A. S. Siebuhr ◽  
M. Karsdal ◽  
P. Juhl ◽  
A. C. Bay-Jensen
Keyword(s):  
Collagen Type ◽  
Dermal Fibroblast ◽  
Collagen Type I ◽  
Dermal Fibroblasts ◽  
Type I ◽  
Full Time ◽  
Collagen Type Iii ◽  
Collagen Formation ◽  
Type Iii ◽  
Full Time Employee

Background:Dermal fibroblasts are responsible for the excessive extracellular matrix (ECM) formation observed in the skin of systemic sclerosis (SSc) patients and fibroblasts are therefore an obvious target for anti-fibrotic treatments. TGFβ, PDGF and IL-6 are known to be central cytokines in systemic sclerosis. Nintedanib, a tyrosine-kinase inhibitor approved for treatment of idiopathic pulmonary fibrosis, did not show effect on dermal fibrosis only on pulmonary fibrosis in SSc patients with interstitial lung disease (ILD). Tofacitinib, as Pan JAK inhibitor, has shown to inhibit dermal fibrosis in mouse models and shown positive indications in patients.Objectives:We investigated the direct effect of Nintedanib and Tofacitinib on ECM production from human dermal fibroblast using translational biomarkers of type I, III and VI collagens and fibronectin.Methods:Primary healthy human dermal fibroblasts were grown in DMEM media containing 0.4% fetal calf serum, Ficoll (to produce a crowded environment) and ascorbic acid for up to 17 days. The cells were stimulated with PDGF [3 nM] and/or TGFβ [1 nM] in combination with Nintedanib [1 nM-10 μM] treatment initiated at day 0 or 7 or Tofacitinib [3-100 nM] treatment initiated at culture start together. Media and treatments were changed twice a week. Non-activated cells (w/o) were used as control. Type I, III and VI collagen formation (PRO-C1, PRO-C3 and PRO-C6, respectively) and fibronectin (FBN-C) were evaluated by validated ELISAs (Nordic Bioscience). Statistical analysis included 1-way and 2-way ANOVA, AUC and Mann-Whitney U-test.Results:PDGF significantly increased collagen type III and VI formation and collagen type I formation minimally. PDGF did not induce changes in fibronectin levels. TGFβ increased collagen type I and VI formation but did not induce formation of collagen type III. TGFβ increased fibronectin levels, where PDGF did not.Nintedanib (≥100 nM) added either from day 0 or 7 reduced PDGF induced collagen type III and VI formation to the levels of w/o throughout the remainder of the study. In TGFβ treated fibroblasts, Nintedanib added either from day 0 or 7 reduced collagen type I and VI formation. The fibronectin levels were dose-dependently reduced by Nintedanib. The biomarker levels were at study end at the level of w/o. Nintedanib at a concentration of 1 uM and higher significantly decreased the biomarker levels. Nintedanib (≥100 nM) in fibroblasts stimulated with both TGFβ and PDGF significantly reduced collagen type I, III and VI collagen and fibronectin.A Tofacitinib concentration of 100 nM was toxic to the dermal fibroblasts as the cell viability was minimal at culture end. However, the viability of Tofacitinib (100 nM) in combination with TGFβ was decreased at study end, but only to half the viability of untreated cells. Tofacitinib dose-dependently decreased the TGFβ induced type I and III collagen formation and fibronectin in the dermal fibroblasts. Tofacitinib (100 nM) decreased the level of collagen type I and III formation to the level of w/o, where as the level of fibronectin was lowered by 80 % of TGFβ. Tofacitinib as low as 12.5 nM significantly lowered the collagen type I formation and fibronectin (both p<0.05) and Tofacitinib of 25 nM decreased collagen type III formation significantly (p<0.0001).Conclusion:Tofacitinib decreased the formation of the collagens and fibronectin. Nintedanib inhibited ECM production differently in PDGF and TGFβ induced dermal fibroblast, but in the combination of TGFβ and PDGF Nintedanib significantly decreased the ongoing fibrosis. In PDGF induced fibrosis, Nintedanib acted as an on-off switch, whereas the inhibition was dose-dependent in TGFβ induced fibrosis. This cell study indicates that Nintedanib and Tofacitinib inhibits collagen production in dermal fibroblasts.Figure:Disclosure of Interests:Anne Sofie Siebuhr Employee of: Nordic Bioscience, Morten Karsdal Shareholder of: Nordic Bioscience A/S., Employee of: Full time employee at Nordic Bioscience A/S., Pernille Juhl Employee of: Nordic Bioscience, Anne-Christine Bay-Jensen Shareholder of: Nordic Bioscience A/S, Employee of: Full time employee at Nordic Bioscience A/S.

scholarly journals Platelet-derived growth factors-AA and -BB regulate collagen and collagenase gene expression differentially in human fibroblasts

1995 ◽  
Vol 310 (2) ◽  
pp. 585-588 ◽  
Author(s):  
E M L Tan ◽  
H Qin ◽  
S H Kennedy ◽  
S Rouda ◽  
J W Fox ◽  
...  
Keyword(s):  
Gene Expression ◽  
Time Course ◽  
Type I Collagen ◽  
Biological Activities ◽  
Human Fibroblasts ◽  
Dermal Fibroblasts ◽  
Type I ◽  
Mrna Levels ◽  
Interstitial Collagenase ◽  
Collagenase Gene

Platelet-derived growth factor (PDGF) is a mitogen associated with tissue repair, a process involving collagen synthesis and remodelling by interstitial collagenase. This study examines and compares the regulation of interstitial collagenase and collagen gene expression by PDGF-AA and -BB in human fibroblasts. Time-course analysis showed that neither PDGF-AA or -BB had a consistent effect on the expression of pro-alpha 1(I) or pro-alpha 2(I) type-I collagen genes. In contrast, interstitial collagenase gene expression was found to be consistently up-regulated severalfold by PDGF-BB. Enhanced expression of the collagenase gene was not apparently due to up-regulation of its promoter activity in human dermal fibroblasts, as indicated by transient and stable transfection experiments. Unlike PDGF-BB, PDGF-AA did not alter collagenase mRNA levels under low-serum culture conditions. Thus, the biological activities of the PDGF homodimers are different, with PDGF-BB being clearly more potent than PDGF-AA in its regulation of collagenase gene expression.

Potential Wound Healing Activities of Galla Rhois in Human Fibroblasts and Keratinocytes

2015 ◽  
Vol 43 (08) ◽  
pp. 1625-1636 ◽  
Author(s):  
Hyo-Hyun Park ◽  
Na-Young Park ◽  
Sun-Gun Kim ◽  
Kyu-Tae Jeong ◽  
Eu-Jin Lee ◽  
...  
Keyword(s):  
Wound Healing ◽  
Tissue Injury ◽  
Human Fibroblasts ◽  
Ethanol Extract ◽  
Collagen Type I ◽  
Dermal Fibroblasts ◽  
Type I ◽  
Collagen Deposition ◽  
Soluble Collagen ◽  
Ldh Release

Wound healing is a complex process orchestrated by the regeneration of the epithelium and the remodeling of the extracellular matrix through processes like collagen deposition. Galla Rhois has been widely used in traditional Korean medicine for its various pharmacological effects, including an anticoccidial effect, however, little is known about its healing activity. The purpose of this study was to determine the effects of Galla Rhois ethanol extract (GRE) on wound healing activities, including H2O2-induced oxidative stress, cell migration, and lactate dehydrogenase (LDH) release assays using human keratinocyte (HaCaT) and dermal fibroblasts (CCD-986SK). In addition, total soluble collagen deposition and collagen gene expression for Type I and III collagen were evaluated in CCD-986SK. Total tannin and flavonoid contents for GRE were measured. GRE induced a significant increase in the number and migration of cells, along with a decrease in cell death and LDH release. In addition, it also induced the over-expression of collagen Type I and III mRNA and caused increased synthesis of total soluble collagen. The contents of total tannin and flavonoid for GRE were 55.7% ([Formula: see text][Formula: see text]mg/g) and 62.9% ([Formula: see text][Formula: see text]mg/g), respectively. The results suggest that GRE can cause accelerated wound healing by increasing cell survival, proliferation, migration, and collagen synthesis along with a potential anti-oxidant property. This evidence provides novel insight into natural therapy for tissue injury.

scholarly journals Endothelial cell growth factor and heparin regulate collagen gene expression in keloid fibroblasts

1991 ◽  
Vol 278 (3) ◽  
pp. 863-869 ◽  
Author(s):  
E M L Tan ◽  
J Peltonen
Keyword(s):  
Gene Expression ◽  
Endothelial Cell ◽  
Collagen Synthesis ◽  
Type I Collagen ◽  
Collagen Type ◽  
Collagen Type I ◽  
Type I ◽  
Collagen Gene ◽  
Endothelial Cell Growth Factor ◽  
Endothelial Cell Growth

Keloids are benign cutaneous tumours characterized by excess deposition of collagen, specifically type I collagen. We report here that collagen biosynthesis, as measured by hydroxyproline synthesis, was markedly inhibited by 65-80% by the combination of endothelial cell growth factor (ECGF) supplement and heparin in keloid fibroblast cultures. Fibroblast cultures that were incubated with ECGF alone also demonstrated a measurable decrease of approx. 50% in collagen synthesis compared with control cultures. The inhibition of collagen synthesis was related to the down-regulation of collagen gene expression. Quantitative measurements of mRNA-cDNA hybrids revealed that the gene expression of collagen type I was decreased by more than 80% by heparin and ECGF. Markedly diminished levels of mRNA encoding collagen type I were also observed in cultures incubated with ECGF alone. The results show that ECGF and heparin elicit a negative regulatory effect on collagen production, and that this inhibition is due largely to the down-regulation of the pro-alpha 1(I) of type I collagen gene. Furthermore, ECGF has a potent suppressive effect, and heparin provides an additive effect to this inhibitory phenomenon.

Mechanical Stimulation Increases Collagen Type I and Collagen Type III Gene Expression of Stem Cell–Collagen Sponge Constructs for Patellar Tendon Repair

2007 ◽  
Vol 13 (6) ◽  
pp. 1219-1226 ◽  
Author(s):  
Natalia Juncosa-Melvin ◽  
Karl S. Matlin ◽  
Robert W. Holdcraft ◽  
Victor S. Nirmalanandhan ◽  
David L. Butler
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Patellar Tendon ◽  
Mechanical Stimulation ◽  
Collagen Type ◽  
Tendon Repair ◽  
Collagen Type I ◽  
Collagen Sponge ◽  
Type I ◽  
Collagen Type Iii

scholarly journals Fli-1 Inhibits Collagen Type I Production in Dermal Fibroblasts via an Sp1-dependent Pathway

2001 ◽  
Vol 276 (24) ◽  
pp. 20839-20848 ◽  
Author(s):  
Joanna Czuwara-Ladykowska ◽  
Fumiaki Shirasaki ◽  
Pascale Jackers ◽  
Dennis K. Watson ◽  
Maria Trojanowska
Keyword(s):  
Collagen Type ◽  
Collagen Type I ◽  
Dermal Fibroblasts ◽  
Type I ◽  
Dependent Pathway

scholarly journals Stimulation of the chemotactic migration of human fibroblasts by transforming growth factor beta.

1987 ◽  
Vol 165 (1) ◽  
pp. 251-256 ◽  
Author(s):  
A E Postlethwaite ◽  
J Keski-Oja ◽  
H L Moses ◽  
A H Kang
Keyword(s):  
Growth Factor ◽  
Transforming Growth Factor Beta ◽  
Transforming Growth Factor ◽  
Human Fibroblasts ◽  
Critical Role ◽  
Dermal Fibroblasts ◽  
Type I ◽  
Tgf Beta ◽  
Chemotactic Migration ◽  
Growth Factor Beta

Transforming growth factor beta (TGF-beta) is a potent chemoattractant in vitro for human dermal fibroblasts. Intact disulfide and perhaps the dimeric structure of TGF-beta is essential for its ability to stimulate chemotactic migration of fibroblasts, since reduction with 2-ME results in a marked loss of its potency as a chemoattractant. Although epidermal growth factor (EGF) appears to be capable of modulating some effects of TGF-beta, it does not alter the chemotactic response of fibroblasts to TGF-beta. Specific polyvalent rabbit antibodies to homogeneously pure TGF-beta block its chemotactic activity but has no effect on the other chemoattractants tested (platelet-derived growth factor, fibronectin, and denatured type I collagen). Since TGF-beta is secreted by a variety of neoplastic and normal cells including platelets, monocytes/macrophages, and lymphocytes, it may play a critical role in vivo in embryogenesis, host response to tumors, and the repair response that follows damage to tissues by immune and nonimmune reactions.

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