scholarly journals Bioreactor-Controlled Physoxia Regulates TGF-β Signaling to Alter Extracellular Matrix Synthesis by Human Chondrocytes

2019 ◽  
Vol 20 (7) ◽  
pp. 1715 ◽  
Author(s):  
Holger Jahr ◽  
Seval Gunes ◽  
Annika-Ricarda Kuhn ◽  
Sven Nebelung ◽  
Thomas Pufe
Keyword(s):  
Extracellular Matrix ◽  
Oxygen Tension ◽  
Molecular Mechanisms ◽  
Matrix Metalloproteases ◽  
Human Chondrocytes ◽  
Marker Genes ◽  
Matrix Synthesis ◽  
Altered Expression ◽  
Positive Effects ◽  
Marker Expression

Culturing articular chondrocytes under physiological oxygen tension exerts positive effects on their extracellular matrix synthesis. The underlying molecular mechanisms which enhance the chondrocytic phenotype are, however, still insufficiently elucidated. The TGF-β superfamily of growth factors, and the prototypic TGF-β isoforms in particular, are crucial in maintaining matrix homeostasis of these cells. We employed a feedback-controlled table-top bioreactor to investigate the role of TGF-β in microtissues of human chondrocytes over a wider range of physiological oxygen tensions (i.e., physoxia). We compared 1%, 2.5%, and 5% of partial oxygen pressure (pO2) to the ‘normoxic’ 20%. We confirmed physoxic conditions through the induction of marker genes (PHD3, VEGF) and oxygen tension-dependent chondrocytic markers (SOX9, COL2A1). We identified 2.5% pO2 as an oxygen tension optimally improving chondrocytic marker expression (ACAN, COL2A1), while suppressing de-differentiation markers (COL1A1, COL3A1). Expression of TGF-β isoform 2 (TGFB2) was, relatively, most responsive to 2.5% pO2, while all three isoforms were induced by physoxia. We found TGF-β receptors ALK1 and ALK5 to be regulated by oxygen tension on the mRNA and protein level. In addition, expression of type III co-receptors betaglycan and endoglin appeared to be regulated by oxygen tension as well. R-Smad signaling confirmed that physoxia divergently regulated phosphorylation of Smad1/5/8 and Smad2/3. Pharmacological inhibition of canonical ALK5-mediated signaling abrogated physoxia-induced COL2A1 and PAI-1 expression. Physoxia altered expression of hypertrophy markers and that of matrix metalloproteases and their activity, as well as expression ratios of specific proteins (Sp)/Krüppel-like transcription factor family members SP1 and SP3, proving a molecular concept of ECM marker regulation. Keeping oxygen levels tightly balanced within a physiological range is important for optimal chondrocytic marker expression. Our study provides novel insights into transcriptional regulations in chondrocytes under physoxic in vitro conditions and may contribute to improving future cell-based articular cartilage repair strategies.

scholarly journals Periodic mild heat stimuli diminish extracellular matrix synthesis in pellet cultured human chondrocytes

2019 ◽  
Vol 12 (1) ◽  
Author(s):  
Akira Ito ◽  
Tomoki Aoyama ◽  
Hirotaka Iijima ◽  
Kohei Nishitani ◽  
Junichi Tajino ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Human Chondrocytes ◽  
Matrix Synthesis ◽  
Mild Heat

scholarly journals qPCR analysis of mesenchymal stem cell marker expression during the long-term culture of canine adipocyte derived stem cells

2020 ◽  
Vol 8 (4) ◽  
pp. 139-145
Author(s):  
Rut Bryl ◽  
Claudia Dompe ◽  
Maurycy Jankowski ◽  
Katarzyna Stefańska ◽  
Afsaneh Golkar Narenji ◽  
...  
Keyword(s):  
Stem Cells ◽  
Molecular Mechanisms ◽  
Cultured Cells ◽  
Stem Cell Marker ◽  
Pcr Analysis ◽  
Marker Genes ◽  
Long Term Culture ◽  
Marker Expression

AbstractDue to its availability and accessibility, adipose tissue has been the subject of various studies in many different medical fields and is believed to be a useful source of stem cells. The ability of ASCs to differentiate towards different cell lineages, with possibility of directing this differentiation, increases their possible clinical applications, and they have been widely employed in multiple therapies and treatment of different pathologies. However, a deeper understanding of the molecular mechanisms underlying the ASCs osteoblastic and chondrocyte differentiation may lead to novel applications treating a multitude of different bone-related diseases through techniques more likely meeting worldwide consensus. In this study, the RT-qPCR method was used to determine the changes in expression of ASC specific markers (CD105, CD73, CD14, CD34, CD90 and CD45) before and after long-term (14-day) in vitro cultures. To confirm the identity of the investigated cells, flow cytometry was used to evaluate the presence of positive (CD44, CD90) and negative (CD45, CD34) ASC markers. Overall, the results of the PCR analysis showed a significant change in expression of most of the marker genes, indicating significant changes in the cultured cells caused by their long-term culture, potentially altering their original stem-like characteristics.Running title: ASC marker expression during long-term in vitro culture

scholarly journals Targeting and transport: How microtubules control focal adhesion dynamics

2012 ◽  
Vol 198 (4) ◽  
pp. 481-489 ◽  
Author(s):  
Samantha Stehbens ◽  
Torsten Wittmann
Keyword(s):  
Extracellular Matrix ◽  
Cell Migration ◽  
Focal Adhesion ◽  
Molecular Mechanisms ◽  
Rho Gtpase ◽  
Focal Adhesions ◽  
Matrix Metalloproteases ◽  
Force Generation ◽  
Diverse Group ◽  
Directional Cell Migration

Directional cell migration requires force generation that relies on the coordinated remodeling of interactions with the extracellular matrix (ECM), which is mediated by integrin-based focal adhesions (FAs). Normal FA turnover requires dynamic microtubules, and three members of the diverse group of microtubule plus-end-tracking proteins are principally involved in mediating microtubule interactions with FAs. Microtubules also alter the assembly state of FAs by modulating Rho GTPase signaling, and recent evidence suggests that microtubule-mediated clathrin-dependent and -independent endocytosis regulates FA dynamics. In addition, FA-associated microtubules may provide a polarized microtubule track for localized secretion of matrix metalloproteases (MMPs). Thus, different aspects of the molecular mechanisms by which microtubules control FA turnover in migrating cells are beginning to emerge.

Endometriosis is characterized by an impaired localization of laminin-5 and α3β1 integrin receptor

2007 ◽  
Vol 17 (1) ◽  
pp. 242-247 ◽  
Author(s):  
G. Giannelli ◽  
C. Sgarra ◽  
E. Di Naro ◽  
C. Lavopa ◽  
U. Angelotti ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Uterine Cavity ◽  
Matrix Metalloproteases ◽  
Integrin Receptor ◽  
Altered Expression ◽  
Transmembrane Receptors ◽  
Laminin 5 ◽  
Ecm Proteins ◽  
Proliferative Endometrium ◽  
Cell Invasiveness

Endometriosis is an estrogen-correlated benign disease characterized by a marked ability of endometrial-like cells to invade and proliferate outside uterine cavity, resembling for some invasive aspect the cancer growth. The molecular mechanisms regulating endometrial cell invasiveness are mostly unknown, although interactions between extracellular matrix (ECM) proteins and their transmembrane receptors, integrins, are likely to play a central role. In particular, laminin (Ln)-5 could be closely involved, as it is in cancer. We have investigated the expression of Ln-1, Ln-5, and collagen IV (Coll IV) ECM proteins and their receptors, α3β1 and α6β4 integrins, in atrophic, proliferative, and secretive endometrium and in endometriosis. The results show that Ln-5, but not Ln-I and Coll IV, is altered in secretive endometrium as well as in endometriosis tissues. No alterations are observed in atrophic or proliferative endometrium. Consistently, the polarization of both integrin subunits α3 and β1, but not α6 and β4, is altered in secretive endometrium and endometriosis tissues, but not in atrophic and proliferative endometrium. These results seem to suggest that Ln-5 and α3β1 could be involved in the invasive mechanism of endometriosis. The altered expression of Ln-5, by upregulating matrix metalloproteases activity, suggest an invading process similar to that of many cancer processes

scholarly journals Extracellular Matrix Alterations in Metastatic Processes

2019 ◽  
Vol 20 (19) ◽  
pp. 4947 ◽  
Author(s):  
Paolillo ◽  
Schinelli
Keyword(s):  
Extracellular Matrix ◽  
Cancer Cells ◽  
Cancer Progression ◽  
Molecular Mechanisms ◽  
Epithelial To Mesenchymal Transition ◽  
Intercellular Junctions ◽  
Matrix Metalloproteases ◽  
Target Tissue ◽  
Epithelial Tumor ◽  
Mesenchymal Transition

The extracellular matrix (ECM) is a complex network of extracellular-secreted macromolecules, such as collagen, enzymes and glycoproteins, whose main functions deal with structural scaffolding and biochemical support of cells and tissues. ECM homeostasis is essential for organ development and functioning under physiological conditions, while its sustained modification or dysregulation can result in pathological conditions. During cancer progression, epithelial tumor cells may undergo epithelial-to-mesenchymal transition (EMT), a morphological and functional remodeling, that deeply alters tumor cell features, leading to loss of epithelial markers (i.e., E-cadherin), changes in cell polarity and intercellular junctions and increase of mesenchymal markers (i.e., N-cadherin, fibronectin and vimentin). This process enhances cancer cell detachment from the original tumor mass and invasiveness, which are necessary for metastasis onset, thus allowing cancer cells to enter the bloodstream or lymphatic flow and colonize distant sites. The mechanisms that lead to development of metastases in specific sites are still largely obscure but modifications occurring in target tissue ECM are being intensively studied. Matrix metalloproteases and several adhesion receptors, among which integrins play a key role, are involved in metastasis-linked ECM modifications. In addition, cells involved in the metastatic niche formation, like cancer associated fibroblasts (CAF) and tumor associated macrophages (TAM), have been found to play crucial roles in ECM alterations aimed at promoting cancer cells adhesion and growth. In this review we focus on molecular mechanisms of ECM modifications occurring during cancer progression and metastatic dissemination to distant sites, with special attention to lung, liver and bone. Moreover, the functional role of cells forming the tumor niche will also be reviewed in light of the most recent findings.

scholarly journals Null cyp1b1 Activity in Zebrafish Leads to Variable Craniofacial Defects Associated with Altered Expression of Extracellular Matrix and Lipid Metabolism Genes

2021 ◽  
Vol 22 (12) ◽  
pp. 6430
Author(s):  
Susana Alexandre-Moreno ◽  
Juan-Manuel Bonet-Fernández ◽  
Raquel Atienzar-Aroca ◽  
José-Daniel Aroca-Aguilar ◽  
Julio Escribano
Keyword(s):  
Extracellular Matrix ◽  
Lipid Metabolism ◽  
Molecular Mechanisms ◽  
Congenital Glaucoma ◽  
Incomplete Penetrance ◽  
Loss Of Function ◽  
Altered Expression ◽  
Matrix Gene ◽  
Lipid Metabolism Genes ◽  
Craniofacial Defects

CYP1B1 loss of function (LoF) is the main known genetic alteration present in recessive primary congenital glaucoma (PCG), an infrequent disease characterized by delayed embryonic development of the ocular iridocorneal angle; however, the underlying molecular mechanisms are poorly understood. To model CYP1B1 LoF underlying PCG, we developed a cyp1b1 knockout (KO) zebrafish line using CRISPR/Cas9 genome editing. This line carries the c.535_667del frameshift mutation that results in the 72% mRNA reduction with the residual mRNA predicted to produce an inactive truncated protein (p.(His179Glyfs*6)). Microphthalmia and jaw maldevelopment were observed in 23% of F0 somatic mosaic mutant larvae (144 hpf). These early phenotypes were not detected in cyp1b1-KO F3 larvae (144 hpf), but 27% of adult (four months) zebrafish exhibited uni- or bilateral craniofacial alterations, indicating the existence of incomplete penetrance and variable expressivity. These phenotypes increased to 86% in the adult offspring of inbred progenitors with craniofacial defects. No glaucoma-related phenotypes were observed in cyp1b1 mutants. Transcriptomic analyses of the offspring (seven dpf) of cyp1b1-KO progenitors with adult-onset craniofacial defects revealed functionally enriched differentially expressed genes related to extracellular matrix and cell adhesion, cell growth and proliferation, lipid metabolism (retinoids, steroids and fatty acids and oxidation–reduction processes that include several cytochrome P450 genes) and inflammation. In summary, this study shows the complexity of the phenotypes and molecular pathways associated with cyp1b1 LoF, with species dependency, and provides evidence for the dysregulation of extracellular matrix gene expression as one of the mechanisms underlying the pathogenicity associated with cyp1b1 disruption.

scholarly journals Acute Exhaustive Exercise under Normoxic and Normobaric Hypoxic Conditions Differentially Regulates Angiogenic Biomarkers in Humans

Medicina ◽  
2021 ◽  
Vol 57 (7) ◽  
pp. 727
Author(s):  
Frank Suhr ◽  
Sarah Knuth ◽  
Silvia Achtzehn ◽  
Joachim Mester ◽  
Markus de Marees
Keyword(s):  
Extracellular Matrix ◽  
Matrix Metalloproteases ◽  
Short Term ◽  
Post Exercise ◽  
Exercise Interventions ◽  
Positive Effects ◽  
Hypoxic Conditions ◽  
Ecm Extracellular Matrix ◽  
And Performance ◽  
Angiogenic Biomarkers

Background and Objectives: Angiogenesis describes the outgrowth of new capillaries from already existing ones. Different biomarkers regulate this process. Physical exercise and hypoxia are key stimuli for the activation of different angiogenic molecules, such as the vascular endothelial growth factor (VEGF). matrix metalloproteases (MMPs)-2 and -9 or the extracellular matrix cleavage fragment endostatin. The present study aimed to investigate influences of short-term, intensive cycling exercise under both normoxic and normobaric hypoxic conditions on the mentioned parameters. Materials and Methods: Twelve male subjects (age: 23.3 ± 2.0 years) participated in the study. All subjects conducted four intensive cycling tests until individual exhaustion in a randomized order under the following conditions: normoxia, 2000 m, 3000 m and 4000 m above sea level. Blood samples were taken before (pre) and 10 min, 30 min, 60 min and 240 min post exercise and were analyzed by ELISA. Results: VEGF showed a significantly reduced concentration compared to the pre-value solely under 4000 m at 10 min post exercise. MMP-2 showed significantly reduced concentrations at 240 min post exercise under 4000 m. MMP-9 increased at 240 min post exercise under both 2000 m and 4000 m conditions. Endostatin was significantly increased at 10 min post exercise independently of the applied stimulus. Conclusions: The presented data show that intensive short-term exercise bouts facilitate the bioavailability of angiogenic, ECM (extracellular matrix)-related biomarkers. This finding is interesting for both health- and performance-related research as it demonstrates the positive effects of intensive short exercise interventions.

scholarly journals TGFβ1, MMPs and cytokines profiles in ocular surface: Possible tear biomarkers for pseudoexfoliation

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0249759
Author(s):  
Prity Sahay ◽  
Shweta Reddy ◽  
Birendra Kumar Prusty ◽  
Rahul Modak ◽  
Aparna Rao
Keyword(s):  
Growth Factor ◽  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Gelatin Zymography ◽  
Matrix Metalloproteases ◽  
Enzyme Linked Immunosorbent Assay ◽  
Altered Expression ◽  
Gm Csf ◽  
Tenon’S Capsule ◽  
Tenon's Capsule

Purpose Pseudoexfoliation (PXF) is a unique form of glaucoma characterized by accumulation of exfoliative material in the eyes. Changes in tear profile in disease stages may give us insights into molecular mechanisms involved in causing glaucoma in the eye. Methods All patients were categorized into three main categories; pseudoexfoliation (PXF), pseudoexfoliation glaucoma (PXG) and cataract, which served as control. Cytokines, transforming growth factor β1 (TGFβ1), matrix metalloproteases (MMPs) and fibronectin (FN1) were assessed with multiplex bead assay, enzyme-linked immunosorbent assay (ELISA), gelatin zymography, and immunohistochemistry (IHC) respectively in different ocular tissues such as tears, tenon’s capsule, aqueous humor (AH) and serum samples of patients with PXF stages. Results We found that TGFβ1, MMP-9 and FN1 protein expression were upregulated in tears, tenon’s capsule and AH samples in PXG compared to PXF, though the MMP-9 protein activity was downregulated in PXG compared with control or PXF. We have also found that in PXG tears sample the fold change of TGF-α (Transforming Growth Factor-α), MDC (Macrophage Derived Chemokine), IL-8 (Interleukin-8), VEGF (Vascular Endothelial Growth Factor) were significantly downregulated and the levels of GM-CSF (Granulocyte Macrophage Colony Stimulating Factor), IP-10 (Interferon- γ produced protein-10) were significant upregulated. While in AH; IL-6 (Interleukin-6), IL-8, VEGF, IFN-a2 (Interferon- α2), GRO (Growth regulated alpha protein) levels were found lower and IL1a (Interleukin-1α) level was higher in PXG compared to PXF. And in serum; IFN-a2, Eotaxin, GM-CSF, Fractalkine, IL-10 (Interleukin-10), IL1Ra (Interleukin-1 receptor antagonist), IL-7 (Interleukin-7), IL-8, MIP1β (Macrophage Inflammatory Protein-1β), MCP-1 (Monocyte Chemoattractant Protein-1) levels were significantly upregulated and PDGF-AA (Platelet Derived Growth Factor-AA) level was downregulated in the patients with PXG compared to PXF. Conclusions Altered expression of these molecules in tears may therefore be used as a signal for onset of glaucoma or for identifying eyes at risk of developing glaucoma in PXF.

scholarly journals Null cyp1b1 Activity in Zebrafish Leads to Variable Craniofacial Defects Associated with Altered Expression of Extracellular Matrix and Lipid Metabolism Genes

2021 ◽  
Author(s):  
Susana Alexandre-Moreno ◽  
Juan-Manuel Bonet-Fernández ◽  
Raquel Atienzar-Aroca ◽  
José-Daniel Aroca-Aguilar ◽  
Julio Escribano
Keyword(s):  
Extracellular Matrix ◽  
Lipid Metabolism ◽  
Molecular Mechanisms ◽  
Congenital Glaucoma ◽  
Incomplete Penetrance ◽  
Loss Of Function ◽  
Altered Expression ◽  
Matrix Gene ◽  
Lipid Metabolism Genes ◽  
Craniofacial Defects

CYP1B1 loss-of-function (LoF) is the main known genetic alteration present in recessive primary congenital glaucoma (PCG), an infrequent disease characterized by delayed embryonic development of the ocular iridocorneal angle and caused by poorly understood molecular mechanisms. To model CYP1B1 LoF underlying PCG, we developed a cyp1b1 knockout (KO) zebrafish line using CRISPR/Cas9 genome editing. This line carries the c.535_667del frameshift mutation that results in a 72% mRNA reduction with residual mRNA predicted to produce an inactive truncated protein (p.(His179Glyfs*6)). Craniofacial defects and jaw maldevelopment were observed in 23% of somatic mosaic F0 crispant larvae (144 hpf). These early phenotypes were not detected in KO F3 larvae (144 hpf) but 27% of adult fishes (4 months) showed uni or bilateral craniofacial alterations, indicating the existence of incomplete penetrance and variable expressivity. These phenotypes increased to 86% in the adult offspring of inbred progenitors with craniofacial defects. No glaucoma-related phenotypes were observed in the cyp1b1 mutants. Transcriptomic analyses of the offspring (7dpf) of KO cyp1b1 progenitors with adult-onset craniofacial defects revealed that differentially expressed genes were functionally enriched in groups related with extracellular matrix and cell adhesion, cell growth and proliferation, lipid metabolism (retinoids, steroids, and fatty acids, and oxidation-reduction processes which included several cytochrome P450 genes) and inflammation. In summary, this study shows the complexity of phenotypes and molecular pathways associated with cyp1b1 LoF, with species-dependency, and provides evidence for dysregulation of extracellular matrix gene expression as one of the mechanisms underlaying pathogenicity associated with cyp1b1 disruption.

Sign in / Sign up

Export Citation Format

Share Document