scholarly journals Differential Regulation of the Extracellular Cysteine/Cystine Redox State (EhCySS) by Lung Fibroblasts from Young and Old Mice

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Walter H. Watson ◽  
Tom J. Burke ◽  
Igor N. Zelko ◽  
Edilson Torres-González ◽  
Jeffrey D. Ritzenthaler ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Redox State ◽  
Redox Regulation ◽  
Cultured Cells ◽  
Glutamate Transporter ◽  
Lung Fibroblasts ◽  
Extracellular Matrix Components ◽  
Before And After ◽  
Oxidative Challenge

Aging is associated with progressive oxidation of plasma cysteine (Cys)/cystine (CySS) redox state, expressed asEhCySS. Cultured cells condition their media to reproduce physiologicalEhCySS, but it is unknown whether aged cells produce a more oxidized extracellular environment reflective of that seen in vivo. In the current study, we isolated primary lung fibroblasts from young and old female mice and measured the mediaEhCySSbefore and after challenge with Cys or CySS. We also measured expression of genes related to redox regulation and fibroblast function. These studies revealed that old fibroblasts produced a more oxidizing extracellularEhCySSthan young fibroblasts and that old fibroblasts had a decreased capacity to recover from an oxidative challenge due to a slower rate of reduction of CySS to Cys. These defects were associated with 10-fold lower expression of the Slc7a11 subunit of the xCT cystine-glutamate transporter. Extracellular superoxide dismutase (Sod3) was the only antioxidant or thiol-disulfide regulating enzyme among 36 examined that was downregulated in old fibroblasts by more than 2-fold, but there were numerous changes in extracellular matrix components. Thus, aging fibroblasts not only contribute to remodeling of the extracellular matrix but also have a profound effect on the extracellular redox environment.

Cell-extracellular matrix interactions under in vivo conditions during interstitial cell migration in Hydra vulgaris

Development ◽  
1994 ◽  
Vol 120 (2) ◽  
pp. 425-432 ◽  
Author(s):  
X. Zhang ◽  
M.P. Sarras
Keyword(s):  
Extracellular Matrix ◽  
Cell Migration ◽  
Synthetic Peptide ◽  
Interstitial Cell ◽  
Extracellular Matrix Components ◽  
Matrix Interactions ◽  
Apical Half ◽  
Matrix Components ◽  
Extracellular Matrix Interactions

Interstitial cell (I-cell) migration in hydra is essential for establishment of the regional cell differentiation pattern in the organism. All previous in vivo studies have indicated that cell migration in hydra is a result of cell-cell interactions and chemotaxic gradients. Recently, in vitro cell adhesion studies indicated that isolated nematocytes could bind to substrata coated with isolated hydra mesoglea, fibronectin and type IV collagen. Under these conditions, nematocytes could be observed to migrate on some of these extracellular matrix components. By modifying previously described hydra grafting techniques, two procedures were developed to test specifically the role of extracellular matrix components during in vivo I-cell migration in hydra. In one approach, the extracellular matrix structure of the apical half of the hydra graft was perturbed using beta-aminopropionitrile and beta-xyloside. In the second approach, grafts were treated with fibronectin, RGDS synthetic peptide and antibody to fibronectin after grafting was performed. In both cases, I-cell migration from the basal half to the apical half of the grafts was quantitatively analyzed. Statistical analysis indicated that beta-aminopropionitrile, fibronectin, RGDS synthetic peptide and antibody to fibronectin all were inhibitory to I-cell migration as compared to their respective controls. beta-xyloside treatment had no effect on interstitial cell migration. These results indicate the potential importance of cell-extracellular matrix interactions during in vivo I-cell migration in hydra.

Fibronectin enhances Campylobacter fetus interaction with extracellular matrix components and INT 407 cells

2008 ◽  
Vol 54 (1) ◽  
pp. 37-47 ◽  
Author(s):  
L. L. Graham ◽  
T. Friel ◽  
R. L. Woodman
Keyword(s):  
Extracellular Matrix ◽  
Solid Phase ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
Bacterial Attachment ◽  
Binding Assays ◽  
Rgd Sequence ◽  
Campylobacter Fetus ◽  
Extracellular Matrix Components

Campylobacter fetus is a recognized pathogen of cattle and sheep that can also infect humans. No adhesins specific for C. fetus have to date been identified; however, bacterial attachment is essential to establish an infecting population. Scanning electron microscopy revealed C. fetus attachment to the serosal surface of human colonic biopsy explants, a location consistent with the presence of the extracellular matrix (ECM). To determine whether the ECM mediated C. fetus adherence, 7 C. fetus strains were assessed in a solid-phase binding assay for their ability to bind to immobilized ECM components. Of the ECM components assayed, adherence to fibronectin was noted for all strains. Attachment to ECM components was neither correlated with S-layer expression nor with cell-surface hydrophobicity. Ligand immunoblots, however, identified the S-layer protein as a major site of fibronectin binding, and modified ECM binding assays revealed that soluble fibronectin significantly enhanced the attachment of S-layer-expressing C. fetus strains to other ECM components. Soluble fibronectin also increased C. fetus adherence to INT 407 cells. This adherence was inhibited when INT 407 cells were incubated with synthetic peptides containing an RGD sequence, indicating that integrin receptors were involved in fibronectin-mediated attachment. Together, this data suggests that C. fetus can bind to immobilized fibronectin and use soluble fibronectin to enhance attachment to other ECM components and intestinal epithelial cells. In vivo, fibronectin would promote bacterial adherence, thereby, contributing to the initial interaction of C. fetus with mucosal and submucosal surfaces.

scholarly journals The Lack of Mitochondrial Thioredoxin TRXo1 Affects In Vivo Alternative Oxidase Activity and Carbon Metabolism under Different Light Conditions

2019 ◽  
Vol 60 (11) ◽  
pp. 2369-2381 ◽  
Author(s):  
Igor Florez-Sarasa ◽  
Toshihiro Obata ◽  
N�stor Fern�ndez Del-Saz ◽  
Jean-Philippe Reichheld ◽  
Etienne H Meyer ◽  
...  
Keyword(s):  
Carbon Metabolism ◽  
Redox State ◽  
Redox Regulation ◽  
Alternative Oxidase ◽  
Tricarboxylic Acid Cycle ◽  
Reduced Form ◽  
Photosynthetic Parameters ◽  
Primary Metabolism ◽  
Redox Systems

Abstract The alternative oxidase (AOX) constitutes a nonphosphorylating pathway of electron transport in the mitochondrial respiratory chain that provides flexibility to energy and carbon primary metabolism. Its activity is regulated in vitro by the mitochondrial thioredoxin (TRX) system which reduces conserved cysteines residues of AOX. However, in vivo evidence for redox regulation of the AOX activity is still scarce. In the present study, the redox state, protein levels and in vivo activity of the AOX in parallel to photosynthetic parameters were determined in Arabidopsis knockout mutants lacking mitochondrial trxo1 under moderate (ML) and high light (HL) conditions, known to induce in vivo AOX activity. In addition, 13C- and 14C-labeling experiments together with metabolite profiling were performed to better understand the metabolic coordination between energy and carbon metabolism in the trxo1 mutants. Our results show that the in vivo AOX activity is higher in the trxo1 mutants at ML while the AOX redox state is apparently unaltered. These results suggest that mitochondrial thiol redox systems are responsible for maintaining AOX in its reduced form rather than regulating its activity in vivo. Moreover, the negative regulation of the tricarboxylic acid cycle by the TRX system is coordinated with the increased input of electrons into the AOX pathway. Under HL conditions, while AOX and photosynthesis displayed similar patterns in the mutants, photorespiration is restricted at the level of glycine decarboxylation most likely as a consequence of redox imbalance.

scholarly journals Agrin-induced reorganization of extracellular matrix components on cultured myotubes: relationship to AChR aggregation.

1990 ◽  
Vol 111 (3) ◽  
pp. 1161-1170 ◽  
Author(s):  
R M Nitkin ◽  
T C Rothschild
Keyword(s):  
Extracellular Matrix ◽  
Type Iv Collagen ◽  
Neuromuscular Junctions ◽  
Synaptic Organization ◽  
Type Iv ◽  
Extracellular Matrix Components ◽  
Cultured Myotubes ◽  
Matrix Components ◽  
Induced Aggregation

Agrin, an extracellular matrix-associated protein extracted from synapse-rich tissues, induces the accumulation of acetylcholine receptors (AChRs) and other synaptic components into discrete patches on cultured myotubes. The appearance of agrin-like molecules at neuromuscular junctions suggests that it may direct synaptic organization in vivo. In the present study we examined the role of extracellular matrix components in agrin-induced differentiation. We used immunohistochemical techniques to visualize the spatial and temporal distribution of laminin, a heparan sulfate proteoglycan (HSPG), fibronectin, and type IV collagen on cultured chick myotubes during agrin-induced aggregation of AChRs. Myotubes displayed significant amounts of laminin and HSPG, lesser amounts of type IV collagen, and little, if any, fibronectin. Agrin treatment caused cell surface laminin and HSPG to patch, while collagen and fibronectin distributions were generally unaffected. Many of the agrin-induced laminin and HSPG patches colocalized with AChR patches, raising the possibility of a causal relationship between matrix patching and AChR accumulations. However, patching of AChRs (complete within a few hours) preceded that of laminin or HSPG (not complete until 15-20 h), making it unlikely that matrix accumulations initiate AChR patching at agrin-induced sites. Conversely, when AChR patching was blocked by treatment with anti-AChR antibody mAb 35, agrin was still able to effect patching of laminin and HSPG. Taken together, these findings suggest that agrin-induced accumulations of AChR and laminin/HSPG are not mechanistically linked.

scholarly journals Laminin promotes neuritic regeneration from cultured peripheral and central neurons.

1983 ◽  
Vol 97 (6) ◽  
pp. 1882-1890 ◽  
Author(s):  
M Manthorpe ◽  
E Engvall ◽  
E Ruoslahti ◽  
F M Longo ◽  
G E Davis ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Neurite Growth ◽  
Sensory Ganglia ◽  
Culture Methods ◽  
Fetal Rat ◽  
Extracellular Matrix Components ◽  
Ciliary Ganglion Neurons ◽  
Ganglion Neurons

The ability of axons to grow through tissue in vivo during development or regeneration may be regulated by the availability of specific neurite-promoting macromolecules located within the extracellular matrix. We have used tissue culture methods to examine the relative ability of various extracellular matrix components to elicit neurite outgrowth from dissociated chick embryo parasympathetic (ciliary ganglion) neurons in serum-free monolayer culture. Purified laminin from both mouse and rat sources, as well as a partially purified polyornithine-binding neurite promoting factor (PNPF-1) from rat Schwannoma cells all stimulate neurite production from these neurons. Laminin and PNPF-1 are also potent stimulators of neurite growth from cultured neurons obtained from other peripheral as well as central neural tissues, specifically avian sympathetic and sensory ganglia and spinal cord, optic tectum, neural retina, and telencephalon, as well as from sensory ganglia of the neonatal mouse and hippocampal, septal, and striatal tissues of the fetal rat. A quantitative in vitro bioassay method using ciliary neurons was used to (a) measure and compare the specific neurite-promoting activities of these agents, (b) confirm that during the purification of laminin, the neurite-promoting activity co-purifies with the laminin protein, and (c) compare the influences of antilaminin antibodies on the neurite-promoting activity of laminin and PNPF-1. We conclude that laminin and PNPF-1 are distinct macromolecules capable of expressing their neurite-promoting activities even when presented in nanogram amounts. This neurite-promoting bioassay currently represents the most sensitive test for the biological activity of laminin.

scholarly journals In vivo engineered extracellular matrix scaffolds with instructive niches for oriented tissue regeneration

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Meifeng Zhu ◽  
Wen Li ◽  
Xianhao Dong ◽  
Xingyu Yuan ◽  
Adam C. Midgley ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Tissue Regeneration ◽  
Spatial Organization ◽  
Cultured Cells ◽  
Functional Integration ◽  
Host Cells ◽  
Hierarchical Porous ◽  
Parallel Microchannels

Abstract Implanted scaffolds with inductive niches can facilitate the recruitment and differentiation of host cells, thereby enhancing endogenous tissue regeneration. Extracellular matrix (ECM) scaffolds derived from cultured cells or natural tissues exhibit superior biocompatibility and trigger favourable immune responses. However, the lack of hierarchical porous structure fails to provide cells with guidance cues for directional migration and spatial organization, and consequently limit the morpho-functional integration for oriented tissues. Here, we engineer ECM scaffolds with parallel microchannels (ECM-C) by subcutaneous implantation of sacrificial templates, followed by template removal and decellularization. The advantages of such ECM-C scaffolds are evidenced by close regulation of in vitro cell activities, and enhanced cell infiltration and vascularization upon in vivo implantation. We demonstrate the versatility and flexibility of these scaffolds by regenerating vascularized and innervated neo-muscle, vascularized neo-nerve and pulsatile neo-artery with functional integration. This strategy has potential to yield inducible biomaterials with applications across tissue engineering and regenerative medicine.

scholarly journals Lysyl oxidase directly contributes to extracellular matrix production and fibrosis in systemic sclerosis

2021 ◽  
Vol 320 (1) ◽  
pp. L29-L40
Author(s):  
Xinh-Xinh Nguyen ◽  
Tetsuya Nishimoto ◽  
Takahisa Takihara ◽  
Logan Mlakar ◽  
Amy D. Bradshaw ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Systemic Sclerosis ◽  
Pulmonary Fibrosis ◽  
Nuclear Localization ◽  
Amine Oxidase ◽  
Ex Vivo ◽  
Lysyl Oxidase ◽  
Lung Fibroblasts ◽  
Transcriptional Level

Pulmonary fibrosis is one of the important causes of morbidity and mortality in fibroproliferative disorders such as systemic sclerosis (SSc) and idiopathic pulmonary fibrosis (IPF). Lysyl oxidase (LOX) is a copper-dependent amine oxidase whose primary function is the covalent crosslinking of collagens in the extracellular matrix (ECM). We investigated the role of LOX in the pathophysiology of SSc. LOX mRNA and protein levels were increased in lung fibroblasts of SSc patients compared with healthy controls and IPF patients. In vivo, bleomycin induced LOX mRNA expression in lung tissues, and LOX activity increased in the circulation of mice with pulmonary fibrosis, suggesting that circulating LOX parallels levels in lung tissues. Circulating levels of LOX were reduced upon amelioration of fibrosis with an antifibrotic peptide. LOX induced ECM production at the transcriptional level in lung fibroblasts, human lungs, and human skin maintained in organ culture. In vivo, LOX synergistically exacerbated fibrosis in bleomycin-treated mice. Further, LOX increased the production of interleukin (IL)-6, and the increase was mediated by LOX-induced c-Fos expression, the nuclear localization of c-Fos, and its engagement with the IL-6 promoter region. Our findings demonstrate that LOX expression and activity correlate with fibrosis in vitro, ex vivo, and in vivo. LOX induced ECM production via upregulation of IL-6 and nuclear localization of c-Fos. Thus, LOX has a direct pathogenic role in SSc-associated fibrosis that is independent of its crosslinking function. Our findings also suggest that measuring circulating LOX levels and activity can be used for monitoring response to antifibrotic therapy.

Oxidation of an Adjacent Methionine Residue Inhibits Regulatory Seryl-Phosphorylation of Pyruvate Dehydrogenase

2009 ◽  
Vol 2 ◽  
pp. PRI.S2799 ◽  
Author(s):  
Jan A. Miernyk ◽  
Mark L. Johnston ◽  
Steve C. Huber ◽  
Alejandro Tovar-Méndez ◽  
Elizabeth Hoyos ◽  
...  
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Redox State ◽  
Cultured Cells ◽  
Pyruvate Dehydrogenase Complex ◽  
Phosphorylation Site ◽  
Methionine Sulfoxide ◽  
Tryptic Peptides ◽  
Methionine Residue ◽  
Chemical Agents

A Met residue is located adjacent to phosphorylation site 1 in the sequences of mitochondrial pyruvate dehydrogenase E1α subunits. When synthetic peptides including site 1 were treated with H2O2, the Met residue was oxidized to methionine sulfoxide (MetSO), and the peptides were no longer phosphorylated by E1α-kinase. Isolated mitochondria were incubated under state III or IV conditions, lysed, the pyruvate dehydrogenase complex (PDC) immunoprecipitated, and tryptic peptides analyzed by MALDI-TOF mass spectrometry. In all instances both Met and MetSO site 1 tryptic-peptides were detected. Similar results were obtained when suspension-cultured cells were incubated with chemical agents known to stimulate production of reactive oxygen species within the mitochondria. Treatment with these agents had no effect upon the amount of total PDC, but decreased the proportion of P-PDC. We propose that the redox-state of the Met residue adjacent to phosphorylation site 1 of pyruvate dehydrogenase contributes to overall regulation of PDC activity in vivo.

Effect of matrigel on development of A549 lung epithelial cells

1993 ◽  
Vol 51 ◽  
pp. 358-359
Author(s):  
Yvonne Kress ◽  
Barry R. Bloom ◽  
Kathleen A. McDonough
Keyword(s):  
Extracellular Matrix ◽  
Cultured Cells ◽  
Alveolar Epithelial Cell ◽  
Membrane Filter ◽  
A549 Cells ◽  
Lung Epithelial Cells ◽  
Epithelial Cell Line ◽  
Polycarbonate Membrane ◽  
Extracellular Matrix Components ◽  
Matrix Components

Extracellular matrix components are known to influence the growth and differentiation of cultured cells, often causing them to behave more like their in vivo counterparts than cells grown on plastic. To develop an in vitro model in which to study the interaction of pathogenic microorganisms with the lung epithelium, we have undertaken a morphological study of the effects of extracellular matrix components on the human lung alveolar epithelial cell line A549. A549 cells were grown for varying amounts of time on plastic; Costar polycarbonate membrane filter inserts; or on Matrigel coated polycarbonate filter inserts. Cells were fixed for 1 h in 2% glutaraldehyde in 0.1 M cacodylate buffer, post-fixed in 1 % OsO4 for 45 min, dehydrated in ascending ethanol, and embedded in Spurr’s resin.A549 cells grown on a plastic slide formed an even monolayer with cells connected by wellformed junctional complexes or separated by interdigitating microvilli. The cytoplasm showed many polyribosomes, rough endoplasmic reticulum, small golgi complexes, mitochondria, occasional lysosomes and bundles of microfilaments.

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