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.

scholarly journals In vitro study of cartilage tissue engineering using human adipose-derived stem cells induced by platelet-rich plasma and cultured on silk fibroin scaffold

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Imam Rosadi ◽  
Karina Karina ◽  
Iis Rosliana ◽  
Siti Sobariah ◽  
Irsyah Afini ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Stem Cells ◽  
Mrna Expression ◽  
Silk Fibroin ◽  
Platelet Rich Plasma ◽  
Cartilage Tissue Engineering ◽  
Cartilage Tissue ◽  
Silk Fibroin Scaffold

Abstract Background Cartilage tissue engineering is a promising technique for repairing cartilage defect. Due to the limitation of cell number and proliferation, mesenchymal stem cells (MSCs) have been developed as a substitute to chondrocytes as a cartilage cell-source. This study aimed to develop cartilage tissue from human adipose-derived stem cells (ADSCs) cultured on a Bombyx mori silk fibroin scaffold and supplemented with 10% platelet-rich plasma (PRP). Methods Human ADSCs and PRP were characterized. A silk fibroin scaffold with 500 μm pore size was fabricated through salt leaching. ADSCs were then cultured on the scaffold (ADSC-SS) and supplemented with 10% PRP for 21 days to examine cell proliferation, chondrogenesis, osteogenesis, and surface marker expression. The messenger ribonucleic acid (mRNA) expression of type 2 collagen, aggrecan, and type 1 collagen was analysed. The presence of type 2 collagen confirming chondrogenesis was validated using immunocytochemistry. The negative and positive controls were ADSC-SS supplemented with 10% foetal bovine serum (FBS) and ADSC-SS supplemented with commercial chondrogenesis medium, respectively. Results Cells isolated from adipose tissue were characterized as ADSCs. Proliferation of the ADSC-SS PRP was significantly increased (p < 0.05) compared to that of controls. Chondrogenesis was observed in ADSC-SS PRP and was confirmed through the increase in glycosaminoglycans (GAG) and transforming growth factor-β1 (TGF-β1) secretion, the absence of mineral deposition, and increased surface marker proteins on chondrogenic progenitors. The mRNA expression of type 2 collagen in ADSC-SS PRP was significantly increased (p < 0.05) compared to that in the negative control on days 7 and 21; however, aggrecan was significantly increased on day 14 compared to the controls. ADSC-SS PRP showed stable mRNA expression of type 1 collagen up to 14 days and it was significantly decreased on day 21. Confocal analysis showed the presence of type 2 collagen in the ADSC-SS PRP and positive control groups, with high distribution outside the cells forming the extracellular matrix (ECM) on day 21. Conclusion Our study showed that ADSC-SS with supplemented 10% PRP medium can effectively support chondrogenesis of ADSCs in vitro and promising for further development as an alternative for cartilage tissue engineering in vivo.

scholarly journals Monocyte Differentiation into Destructive Macrophages on In Vitro Administration of Gingival Crevicular Fluid from Periodontitis Patients

2021 ◽  
Vol 11 (6) ◽  
pp. 555
Author(s):  
Hammam Ibrahim Fageeh ◽  
Hytham N. Fageeh ◽  
Shankargouda Patil
Keyword(s):  
Stem Cells ◽  
Extracellular Matrix ◽  
Stromal Cells ◽  
Pathogenic Bacteria ◽  
Gingival Crevicular Fluid ◽  
Gingival Tissue ◽  
Synthetic Activity ◽  
Periodontal Destruction ◽  
Crevicular Fluid

Background: Periodontitis is an inflammatory condition of the tooth-supporting structures initiated and perpetuated by pathogenic bacteria present in the dental plaque biofilm. In periodontitis, immune cells infiltrate the periodontium to prevent bacterial insult. Macrophages derived from monocytes play an important role in antigen presentation to lymphocytes. However, they are also implicated in causing periodontal destruction and bystander damage to the host tissues. Objectives: The objective of the present study was to quantify the cytokine profile of gingival crevicular fluid (GCF) samples obtained from patients with periodontitis. The study further aimed to assess if GCF of periodontitis patients could convert CD14+ monocytes into macrophages of destructive phenotype in an in vitro setting. The secondary objectives of the study were to assess if macrophages that resulted from GCF treatment of monocytes could affect the synthetic properties, stemness, expression of extracellular matrix proteins, adhesion molecules expressed by gingival stem cells, gingival mesenchymal stromal cells, and osteoblasts. Methods: GCF, blood, and gingival tissue samples were obtained from periodontitis subjects and healthy individuals based on specific protocols. Cytokine profiles of the GCF samples were analyzed. CD14+ monocytes were isolated from whole blood, cultured, and treated with the GCF of periodontitis patients to observe if they differentiated into macrophages. Further, the macrophages were assessed for a phenotype by surface marker analysis and cytokine assays. These macrophages were co-cultured with gingival stem cells, epithelial, stromal cells, and osteoblasts to assess the effects of the macrophages on the synthetic activity of the cells. Results: The GCF samples of periodontitis patients had significantly higher levels of IFN gamma, M-CSF, and GM-CSF. Administration of the GCF samples to CD14+ monocytes resulted in their conversion to macrophages that tested positive for CD80, CD86, and CD206. These macrophages produced increased levels of IL-1β, TNF-α, and IL-6. Co-culture of the macrophages with gingival stem cells, epithelial cells, and stromal cells resulted in increased cytotoxicity and apoptotic rates to the gingival cells. A reduced expression of markers related to stemness, extracellular matrix, and adhesion namely OCT4, NANOG, KRT5, POSTN, COL3A1, CDH1, and CDH3 were seen. The macrophages profoundly affected the production of mineralized nodules by osteoblasts and significantly reduced the expression of COL1A1, OSX, and OCN genes. Conclusion: In periodontitis patients, blood-derived monocytes transform into macrophages of a destructive phenotype due to the characteristic cytokine environment of their GCF. Further, the macrophages affect the genotype and phenotype of the resident cells of the periodontium, aggravate periodontal destruction, as well as jeopardize periodontal healing and resolution of inflammation.

scholarly journals Effect of dexamethasone as osteogenic supplementation in in vitro osteogenic differentiation of stem cells from human exfoliated deciduous teeth

2021 ◽  
Vol 32 (1) ◽  
Author(s):  
Mariane Beatriz Sordi ◽  
Raissa Borges Curtarelli ◽  
Izabella Thaís da Silva ◽  
Gislaine Fongaro ◽  
Cesar Augusto Magalhães Benfatti ◽  
...  
Keyword(s):  
Stem Cells ◽  
Extracellular Matrix ◽  
Osteogenic Differentiation ◽  
Culture Media ◽  
Deciduous Teeth ◽  
Free Calcium ◽  
Alizarin Red ◽  
Matrix Mineralization ◽  
Media Supplementation

AbstractIn in vitro culture systems, dexamethasone (DEX) has been applied with ascorbic acid (ASC) and β-glycerophosphate (βGLY) as culture media supplementation to induce osteogenic differentiation of mesenchymal stem cells. However, there are some inconsistencies regarding the role of DEX as osteogenic media supplementation. Therefore, this study verified the influence of DEX culture media supplementation on the osteogenic differentiation, especially the capacity to mineralize the extracellular matrix of stem cells from human exfoliated deciduous teeth (SHED). Five groups were established: G1—SHED + Dulbecco’s Modified Eagles’ Medium (DMEM) + fetal bovine serum (FBS); G2—SHED + DMEM + FBS + DEX; G3—SHED + DMEM + FBS + ASC + βGLY; G4—SHED + DMEM + FBS + ASC + βGLY + DEX; G5—MC3T3-E1 + α Minimal Essential Medium (MEM) + FBS + ASC + βGLY. DNA content, alkaline phosphatase (ALP) activity, free calcium quantification in the extracellular medium, and extracellular matrix mineralization quantification through staining with von Kossa, alizarin red, and tetracycline were performed on days 7 and 21. Osteogenic media supplemented with ASC and β-GLY demonstrated similar effects on SHED in the presence or absence of DEX for DNA content (day 21) and capacity to mineralize the extracellular matrix according to alizarin red and tetracycline quantifications (day 21). In addition, the presence of DEX in the osteogenic medium promoted less ALP activity (day 7) and extracellular matrix mineralization according to the von Kossa assay (day 21), and more free calcium quantification at extracellular medium (day 21). In summary, the presence of DEX in the osteogenic media supplementation did not interfere with SHED commitment into mineral matrix depositor cells. We suggest that DEX may be omitted from culture media supplementation for SHED osteogenic differentiation in vitro studies.

Abstract 292: TRPA1 Promoting Myofibroblast Differentiation Mediate Pressure Overload-Induced Cardiac Fibrosis

Circulation ◽  
2018 ◽  
Vol 138 (Suppl_2) ◽  
Author(s):  
Shuang Li ◽  
Dong Han ◽  
Dachun Yang
Keyword(s):  
Knockout Mice ◽  
Molecular Mechanisms ◽  
Cardiac Fibrosis ◽  
Transient Receptor Potential ◽  
Transforming Growth Factor ◽  
Ventricular Remodeling ◽  
Cardiac Fibroblasts ◽  
Gene Transfection ◽  
Pressure Overload

Background: Hypertensive ventricular remodeling is a common cause of heart failure. Activation and accumulation of cardiac fibroblasts is the key contributors to this progression. Our previous studies indicate that transient receptor potential ankyrin 1 (TRPA1), a Ca 2+ channel necessary and sufficient, play a prominent role in ventricular remodeling. However, the molecular mechanisms regulating remain poorly understood. Methods: We used TRPA1 agonists cinnamaldehyde (CA) pretreatment and TRPA1 knockout mice to understand the role of TRPA1 in ventricular remodeling of hypertensive heart. We also examine the mechanisms through gene transfection and in vitro experiments. Results: TRPA1 overexpression fully activated myofibroblast transformation, while fibroblasts lacking TRPA1 were refractory to transforming growth factor β (TGF-β) -induced transdifferentiation. TRPA1 knockout mice showed hypertensive ventricular remodeling reversal following pressure overload. We found that the TGF-β induced TRPA1 expression through calcineurin-NFAT-Dyrk1A signaling pathway via the TRPA1 promoter. Once induced, TRPA1 activates the Ca 2+ -responsive protein phosphatase calcineurin, which itself induced myofibroblast transdifferentiation. Moreover, inhibition of calcineurin prevented TRPA1-dependent transdifferentiation. Conclusion: Our study provides the first evidence that TRPA1 regulation in cardiac fibroblasts transformation in response to hypertensive stimulation. The results suggesting a comprehensive pathway for myofibroblast formation in conjunction with TGF-β, Calcineurin, NFAT and Dyrk1A. Furthermore, these data indicate that negative modulation of cardiac fibroblast TRPA1 may represent a therapeutic strategy against hypertensive cardiac remodeling.

Abstract 782: Fra2 Mediates Oxygen-induced TGFbeta-1 mRNA Expression In Adult Cardiac Fibroblasts: Significance In Myocardial Fibrosis Following Ischemia-reperfusion Injury

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Sashwati Roy ◽  
Savita Khanna ◽  
Chandan K Sen
Keyword(s):  
Mrna Expression ◽  
Transforming Growth Factor Beta ◽  
Cardiac Fibrosis ◽  
Transforming Growth Factor ◽  
Ischemia Reperfusion Injury ◽  
Cardiac Fibroblasts ◽  
Ischemia Reperfusion ◽  
Luciferase Reporter ◽  
Mrna Levels

Background . Transforming growth factor beta-1 (TGFbeta-1) is a key cytokine implicated in the development of cardiac fibrosis following ischemia-reperfusion (IR) injury. The profibrotic effects of TGFbeta-1 are primarily attributable to the differentiation of cardiac fibroblasts (CF) to myofibroblasts. Previously, we have reported perceived hyperoxia (Circ Res 92:264 –71), sub-lethal reoxygenation shock during IR, induces differentiation of CF to myofibroblasts at the infarct site. The mechanisms underlying oxygen-sensitive induction of TGFbeta-1 mRNA remain to be characterized. Hypothesis . Fra2 mediates oxygen-induced TGFbeta-1 mRNA expression in adult cardiac fibroblasts. Methods. TGFbeta-1 mRNA expression in infarct tissue was investigated in an IR injury model. The left anterior descending coronary artery of mice was transiently occluded for 60 minutes followed by reperfusion to induce IR injury. Spatially resolved infarct and non-infarct tissues were collected at 0, 1, 3, 5, and 7 days post-IR using laser capture microdissection. TGFbeta-1 mRNA levels were measured using real-time PCR. To investigate the role of oxygen in the regulation of TGFbeta-1, we used our previously reported model of perceived hyperoxia where CF (from 5wks old mice) after isolation were cultured at 5%O 2 (physiological pO 2 ) followed by transferring them to 20%O 2 to induce hyperoxic insult. Results & Conclusions. In vivo, a significant increase (p<0.01; n=5) in TGFbeta-1 mRNA was observed at the infarct site already at day 1 post-IR. The levels continued to increase until day 7 post-IR. In vitro, exposure of CF to 20%O 2 hyperoxic insult induced TGFbeta-1 mRNA (p<0.001; n=4) and protein (p<0.01; n=4) expression. Using a TGFbeta-1 promoter-luciferase reporter and DNA binding assays, we collected first evidence that AP-1 and its component Fra2 as major mediators of oxygen-induced TGFbeta-1 expression. Exposure to 20%O 2 resulted in increased localization of Fra2 in nucleus. siRNA-dependent Fra-2 knock-down completely abrogated oxygen-induced TGFbeta1 expression. In conclusion, this study presents first evidence that Fra-2 is involved in inducible TGFbeta1 expression in CF. Fra2 was noted as being central in regulating oxygen-induced TGFbeta-1 expression.s

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 Trypanosoma cruzi activates mouse cardiac fibroblasts in vitro leading to fibroblast-myofibroblast transition and increase in expression of extracellular matrix proteins

2018 ◽  
Vol 11 (1) ◽  
Author(s):  
Laura Lacerda Coelho ◽  
Isabela Resende Pereira ◽  
Mirian Claudia de Souza Pereira ◽  
Liliane Mesquita ◽  
Joseli Lannes-Vieira ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Trypanosoma Cruzi ◽  
Cardiac Fibroblasts ◽  
Extracellular Matrix Proteins ◽  
Matrix Proteins
Sign in / Sign up

Export Citation Format

Share Document