scholarly journals Vascularised human skin equivalents as a novel in vitro model of skin fibrosis and platform for testing of antifibrotic drugs

2019 ◽  
Vol 78 (12) ◽  
pp. 1686-1692 ◽  
Author(s):  
Alexandru-Emil Matei ◽  
Chih-Wei Chen ◽  
Lisa Kiesewetter ◽  
Andrea-Hermina Györfi ◽  
Yi-Nan Li ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Human Skin ◽  
Transforming Growth Factor ◽  
Quantitative Polymerase Chain Reaction ◽  
Skin Fibrosis ◽  
Skin Equivalents ◽  
Artificial Environment ◽  
Key Features ◽  
Antifibrotic Drugs

ObjectivesFibrosis is a complex pathophysiological process involving interplay between multiple cell types. Experimental modelling of fibrosis is essential for the understanding of its pathogenesis and for testing of putative antifibrotic drugs. However, most current models employ either phylogenetically distant species or rely on human cells cultured in an artificial environment. Here we evaluated the potential of vascularised in vitro human skin equivalents as a novel model of skin fibrosis and a platform for the evaluation of antifibrotic drugs.MethodsSkin equivalents were assembled on a three-dimensional extracellular matrix by sequential seeding of endothelial cells, fibroblasts and keratinocytes. Fibrotic transformation on exposure to transforming growth factor-β (TGFβ) and response to treatment with nintedanib as an established antifibrotic agent were evaluated by quantitative polymerase chain reaction (qPCR), capillary Western immunoassay, immunostaining and histology.ResultsSkin equivalents perfused at a physiological pressure formed a mature, polarised epidermis, a stratified dermis and a functional vessel system. Exposure of these models to TGFβ recapitulated key features of SSc skin with activation of TGFβ pathways, fibroblast to myofibroblast transition, increased release of collagen and excessive deposition of extracellular matrix. Treatment with the antifibrotic agent nintedanib ameliorated this fibrotic transformation.ConclusionOur data provide evidence that vascularised skin equivalents can replicate key features of fibrotic skin and may serve as a platform for evaluation of antifibrotic drugs in a pathophysiologically relevant human setting.

scholarly journals OP0195 VASCULARIZED THREE-DIMENSIONAL MODELS OF HUMAN SKIN FIBROSIS

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 121.2-122
Author(s):  
A. E. Matei ◽  
C. W. Chen ◽  
L. Kiesewetter ◽  
A. H. Györfi ◽  
Y. N. LI ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Human Skin ◽  
Three Dimensional ◽  
Cell Polarization ◽  
Skin Fibrosis ◽  
Matrix Deposition ◽  
Skin Equivalents ◽  
Antifibrotic Drugs

Background:The complex pathophysiological processes that result in fibrotic tissue remodeling in systemic sclerosis involve interplay between multiple cell types (1). Experimental models of fibrosis are essential to provide a conceptual understanding of the pathogenesis of these diseases and to test antifibrotic drugs. Current models of fibrosis have important limitations: thein vivomodels rely on species that are phylogenetically distant, whereas thein vitromodels are oversimplified cultures of a single cell type in an artificial two-dimensional environment of excessive stiffness, which imposes an unphysiological cell polarization (2).Objectives:Here we evaluated the potential use of vascularized, three-dimensionalin vitrohuman skin equivalents as a novel model of skin fibrosis and a platform for the evaluation of antifibrotic drugs.Methods:Skin equivalents were generated by seeding human endothelial cells, fibroblasts and keratinocytes on a decellularized porcine extracellular matrix with perfusable vascular structure. The skin models were cultured for one month in a system that ensured perfusion of the vascular network at physiological pressure. Fibrotic transformation induced by TGFβ and response to nintedanib as an established antifibrotic drug was evaluated by capillary Western immunoassays, qPCR, histology and immunostaining.Results:The vascularized human skin equivalents formed the major skin structures relevant for the pathogenesis of fibrosis: a polarized, fully matured epidermis, a stratified dermis and a perfused vessel system with small capillaries. Exposure to TGFβ led to the fibrotic transformation of the skin equivalents, with activated TGFβ downstream pathways, increased fibroblast-to-myofibroblast transition and excessive deposition of extracellular matrix. Treatment of models exposed to TGFβ with nintedanib (a drug with proven antifibrotic effects) ameliorated the fibrotic transformation of skin equivalents with reduced TGFβ signaling, fibroblast-to-myofibroblast transition and decreased extracellular matrix deposition.Conclusion:Here we describe a novelin vitromodel of skin fibrosis. Our data show that vascularized skin equivalents can reproduce all skin layers affected by fibrosis, that, upon exposure to TGFβ, these models recapitulate key features of fibrotic skin and that these skin models can be used as a platform for evaluation of antifibrotic drugs in a setting with high relevance for human disease.References:[1]Distler JHW, Gyorfi AH, Ramanujam M, Whitfield ML, Konigshoff M, Lafyatis R. Shared and distinct mechanisms of fibrosis. Nature reviews Rheumatology. 2019;15(12):705-30.[2]Garrett SM, Baker Frost D, Feghali-Bostwick C. The mighty fibroblast and its utility in scleroderma research. Journal of scleroderma and related disorders. 2017;2(2):69-134.Disclosure of Interests:Alexandru-Emil Matei: None declared, Chih-Wei Chen: None declared, Lisa Kiesewetter: None declared, Andrea-Hermina Györfi: None declared, Yi-Nan Li: None declared, Thuong Trinh-Minh: None declared, Toin van Kuppevelt: None declared, Jan Hansmann: None declared, Astrid Juengel: None declared, Georg Schett Speakers bureau: AbbVie, BMS, Celgene, Janssen, Eli Lilly, Novartis, Roche and UCB, Florian Groeber-Becker: None declared, Jörg Distler Grant/research support from: Boehringer Ingelheim, Consultant of: Boehringer Ingelheim, Paid instructor for: Boehringer Ingelheim, Speakers bureau: Boehringer Ingelheim

scholarly journals Adapting the Scar-in-a-Jar to Skin Fibrosis and Screening Traditional and Contemporary Anti-Fibrotic Therapies

2021 ◽  
Vol 9 ◽  
Author(s):  
João Q. Coentro ◽  
Ulrike May ◽  
Stuart Prince ◽  
John Zwaagstra ◽  
Olli Ritvos ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Histone Deacetylases ◽  
Transforming Growth Factor ◽  
Dermal Fibroblast ◽  
Trichostatin A ◽  
Macromolecular Crowding ◽  
Skin Fibrosis ◽  
Type I ◽  
Matrix Deposition

Skin fibrosis still constitutes an unmet clinical need. Although pharmacological strategies are at the forefront of scientific and technological research and innovation, their clinical translation is hindered by the poor predictive capacity of the currently available in vitro fibrosis models. Indeed, customarily utilised in vitro scarring models are conducted in a low extracellular matrix milieu, which constitutes an oxymoron for the in-hand pathophysiology. Herein, we coupled macromolecular crowding (enhances and accelerates extracellular matrix deposition) with transforming growth factor β1 (TGFβ1; induces trans-differentiation of fibroblasts to myofibroblasts) in human dermal fibroblast cultures to develop a skin fibrosis in vitro model and to screen a range of anti-fibrotic families (corticosteroids, inhibitors of histone deacetylases, inhibitors of collagen crosslinking, inhibitors of TGFβ1 and pleiotropic inhibitors of fibrotic activation). Data obtained demonstrated that macromolecular crowding combined with TGFβ1 significantly enhanced collagen deposition and myofibroblast transformation. Among the anti-fibrotic compounds assessed, trichostatin A (inhibitors of histone deacetylases); serelaxin and pirfenidone (pleiotropic inhibitors of fibrotic activation); and soluble TGFβ receptor trap (inhibitor of TGFβ signalling) resulted in the highest decrease of collagen type I deposition (even higher than triamcinolone acetonide, the gold standard in clinical practice). This study further advocates the potential of macromolecular crowding in the development of in vitro pathophysiology models.

Targeting ER protein TXNDC5 in hepatic stellate cell mitigates liver fibrosis by repressing non-canonical TGFβ signalling

Gut ◽  
2021 ◽  
pp. gutjnl-2021-325065
Author(s):  
Chen-Ting Hung ◽  
Tung-Hung Su ◽  
Yen-Ting Chen ◽  
Yueh-Feng Wu ◽  
You-Tzung Chen ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Liver Fibrosis ◽  
Liver Injury ◽  
Transforming Growth Factor ◽  
Stellate Cell ◽  
Transforming Growth Factor Β1 ◽  
Duct Ligation ◽  
Extracellular Matrix Production ◽  
Matrix Production

Background and objectivesLiver fibrosis (LF) occurs following chronic liver injuries. Currently, there is no effective therapy for LF. Recently, we identified thioredoxin domain containing 5 (TXNDC5), an ER protein disulfide isomerase (PDI), as a critical mediator of cardiac and lung fibrosis. We aimed to determine if TXNDC5 also contributes to LF and its potential as a therapeutic target for LF.DesignHistological and transcriptome analyses on human cirrhotic livers were performed. Col1a1-GFPTg, Alb-Cre;Rosa26-tdTomato and Tie2-Cre/ERT2;Rosa26-tdTomato mice were used to determine the cell type(s) where TXNDC5 was induced following liver injury. In vitro investigations were conducted in human hepatic stellate cells (HSCs). Col1a2-Cre/ERT2;Txndc5fl/fl (Txndc5cKO) and Alb-Cre;Txndc5fl/fl (Txndc5Hep-cKO) mice were generated to delete TXNDC5 in HSCs and hepatocytes, respectively. Carbon tetrachloride treatment and bile duct ligation surgery were employed to induce liver injury/fibrosis in mice. The extent of LF was quantified using histological, imaging and biochemical analyses.ResultsTXNDC5 was upregulated markedly in human and mouse fibrotic livers, particularly in activated HSC at the fibrotic foci. TXNDC5 was induced by transforming growth factor β1 (TGFβ1) in HSCs and it was both required and sufficient for the activation, proliferation, survival and extracellular matrix production of HSC. Mechanistically, TGFβ1 induces TXNDC5 expression through increased ER stress and ATF6-mediated transcriptional regulation. In addition, TXNDC5 promotes LF by redox-dependent JNK and signal transducer and activator of transcription 3 activation in HSCs through its PDI activity, activating HSCs and making them resistant to apoptosis. HSC-specific deletion of Txndc5 reverted established LF in mice.ConclusionsER protein TXNDC5 promotes LF through redox-dependent HSC activation, proliferation and excessive extracellular matrix production. Targeting TXNDC5, therefore, could be a potential novel therapeutic strategy to ameliorate LF.

Abstract 3030: Differential Regulation of Valvular Interstitial Cell Dysfunction by Extracellular Matrix Components

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Karien J Rodriguez ◽  
Kristyn S Masters
Keyword(s):  
Extracellular Matrix ◽  
Transforming Growth Factor ◽  
Neutralizing Antibody ◽  
Lower Amount ◽  
Valvular Interstitial Cells ◽  
Cell Dysfunction ◽  
Endogenous Production ◽  
Extracellular Matrix Components ◽  
Culture Surface

Calcification is the leading cause of bioprosthetic and native aortic valve failure, but relatively little is known regarding the factors that contribute to the progression of valvular calcification. Because extracellular matrix (ECM) disarray is often observed in explanted diseased valves, we have investigated the role of individual ECM components in the in vitro calcification of valvular interstitial cells (VICs). The transformation of VICs to an osteoblast-like phenotype was quantified in VICs cultured on different types of ECM coatings. The results show that the number and size of calcific nodules formed in VIC cultures, as well as the expression of mineralization markers alkaline phosphatase (ALP) and CBFa1, were highly dependent upon the composition of the culture surface. In fact, VICs cultured on certain ECM components, namely collagen (Coll) and fibronectin (FN), were resistant to calcification, even upon treatment with potent mineralization-inducing growth factors, such as transforming growth factor beta1 (TGFb1). Meanwhile, VIC cultures on fibrin (FB), laminin, and heparin not only had a high number of calcified nodules (p<0.001 vs. Coll, FN), but also elevated levels of ALP and CBFa1 (p<0.02), and the number of nodules on these ‘pro-calcific’ coatings significantly increased upon treatment with exogenous TGFb1 (p<0.05). To explain the ECM-dependence of calcification, the endogenous production of a pro-mineralization factor (TGFb1) was assessed in VICs on anti-calcific (Coll) and pro-calcific (FB) substrates. Quantification of TGFb1 mRNA revealed that VICs on Coll surfaces expressed a significantly lower amount of TGFb1 mRNA than VICs on FB (p<0.01). Furthermore, treatment with a neutralizing antibody to TGFb1 decreased TGFb1 mRNA expression by VICs on Coll in comparison to VICs on FB or polystyrene controls (p<0.02). Thus, we have discovered a strong correlation between VIC calcification and ECM composition. Our findings show that the ECM plays an important role in controlling TGFb1 expression and subsequent calcification of VICs, which may significantly impact the design of biomaterials for valve tissue engineering, understanding of valvular disease, and the development of preventative treatments for valve calcification.

scholarly journals Cell-derived extracellular matrix-coated silk fibroin scaffold for cardiogenesis of brown adipose stem cells through modulation of TGF-β pathway

2020 ◽  
Vol 7 (4) ◽  
pp. 403-412
Author(s):  
Wei Liu ◽  
Yanfeng Sun ◽  
Xiaohui Dong ◽  
Qi Yin ◽  
Huimin Zhu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Extracellular Matrix ◽  
Silk Fibroin ◽  
Transforming Growth Factor ◽  
Troponin T ◽  
Cardiac Fibroblasts ◽  
Adipose Stem Cells ◽  
Silk Fibroin Scaffold ◽  
Brown Adipose

Abstract The cell-derived extracellular matrix (ECM)-modified scaffolds have advantages of mimic tissue specificity and been thought to better mimic the native cellular microenvironment in vitro. ECM derived from cardiac fibroblasts (CFs) are considered as key elements that provide a natural cell growth microenvironment and change the fate of cardiomyocytes (CMs). Here, a new hybrid scaffold is designed based on silk fibroin (SF) scaffold and CFs-derived ECM. CFs were seeded on the SF scaffold for 10 days culturing and decellularized to produce CFs-derived ECM-coated SF scaffold. The results showed that the cell-derived ECM-modified silk fibroin scaffold material contained collagen, laminin, fibronectin and other ECM components with myocardial-like properties. Further to explore its effects on brown adipose stem cells (BASCs) differentiation into CMs. We found that the CF-derived ECM-coated scaffold also increased the expression of CM-specific proteins (e.g. cardiac troponin T and α-actinin) of BASCs. Notably, the β1-integrin-dependent transforming growth factor-β1 signaling pathway was also involved in the regulation of CF-derived ECM by promoting the differentiation of BASCs into CMs. Overall, these findings provide insights into the bionic manufacturing of engineered cardiac tissues (ECTs) and establish a theoretical basis for the construction of ECTs.

Interrelation of permeation and penetration parameters obtained from in vitro experiments with human skin and skin equivalents

2001 ◽  
Vol 75 (3) ◽  
pp. 283-295 ◽  
Author(s):  
Heike Wagner ◽  
Karl-Heinz Kostka ◽  
Claus-Michael Lehr ◽  
Ulrich F Schaefer
Keyword(s):  
Human Skin ◽  
In Vitro Experiments ◽  
Skin Equivalents

scholarly journals Contribution of Palmitic Acid to Epidermal Morphogenesis and Lipid Barrier Formation in Human Skin Equivalents

2019 ◽  
Vol 20 (23) ◽  
pp. 6069 ◽  
Author(s):  
Arnout Mieremet ◽  
Richard Helder ◽  
Andreea Nadaban ◽  
Gert Gooris ◽  
Walter Boiten ◽  
...  
Keyword(s):  
Palmitic Acid ◽  
Human Skin ◽  
De Novo ◽  
Skin Equivalents ◽  
Barrier Formation ◽  
Lipid Organization ◽  
Epidermal Morphogenesis ◽  
Lateral Organization

The outermost barrier layer of the skin is the stratum corneum (SC), which consists of corneocytes embedded in a lipid matrix. Biosynthesis of barrier lipids occurs de novo in the epidermis or is performed with externally derived lipids. Hence, in vitro developed human skin equivalents (HSEs) are developed with culture medium that is supplemented with free fatty acids (FFAs). Nevertheless, the lipid barrier formation in HSEs remains altered compared to native human skin (NHS). The aim of this study is to decipher the role of medium supplemented saturated FFA palmitic acid (PA) on morphogenesis and lipid barrier formation in HSEs. Therefore, HSEs were developed with 100% (25 μM), 10%, or 1% PA. In HSEs supplemented with reduced PA level, the early differentiation was delayed and epidermal activation was increased. Nevertheless, a similar SC lipid composition in all HSEs was detected. Additionally, the lipid organization was comparable for lamellar and lateral organization, irrespective of PA concentration. As compared to NHS, the level of monounsaturated lipids was increased and the FFA to ceramide ratio was drastically reduced in HSEs. This study describes the crucial role of PA in epidermal morphogenesis and elucidates the role of PA in lipid barrier formation of HSEs.

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