scholarly journals Production of cartilage link protein by human granulosa-lutein cells

2002 ◽  
Vol 175 (2) ◽  
pp. 505-515 ◽  
Author(s):  
GW Sun ◽  
H Kobayashi ◽  
M Suzuki ◽  
N Kanayama ◽  
T Terao
Keyword(s):  
Time Course ◽  
Matrix Protein ◽  
Cultured Cells ◽  
Extracellular Matrix Protein ◽  
Regulatory Function ◽  
Human Ovary ◽  
Vitro Fertilization ◽  
Link Protein ◽  
Cell Extracts

Link protein (LP), an extracellular matrix protein in cartilage, stabilizes aggregates of hyaluronic acid (HA) and proteoglycans, including aggrecan and inter-alpha-trypsin inhibitor (ITI). We have shown previously that cartilage LP is present in the maturing rat and mouse ovary. In the present study, we have employed immunohistochemistry to examine the anatomical distribution of cartilage LP in the human ovary. The expression of cartilage LP was selectively detected in the cells within the granulosa compartment of the preovulatory dominant follicle. The HA-positive granulosa-lutein cells were found to be a cartilage LP-positive subpopulation. We subsequently studied the in vitro expression of cartilage LP in cultured human granulosa-lutein cells obtained at oocyte retrieval for in vitro fertilization. Analysis of cultured cells by enzyme-linked immunoaffinity assay, Western blotting and immunofluorescence microscopy revealed that gonadotropin stimulates cartilage LP production. Time-course studies indicated that the cartilage LP production was induced as early as with gonadotropin stimulation for 2 h, and the effect was sustained up to 8 h. Western blot analysis further revealed the presence of the macroaggregates composed of HA, ITI and cartilage LP in the gonadotropin-stimulated granulosa-lutein cell extracts. Collectively, the present results raise the possibility that cartilage LP forms extracellular structures that may have a regulatory function in the developing follicle in the human ovary.

Improved in vitro rheological system for studying the effect of fluid shear stress on cultured cells

1993 ◽  
Vol 265 (1) ◽  
pp. C289-C298 ◽  
Author(s):  
H. J. Schnittler ◽  
R. P. Franke ◽  
U. Akbay ◽  
C. Mrowietz ◽  
D. Drenckhahn
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Matrix Protein ◽  
Fluid Shear Stress ◽  
Cultured Cells ◽  
Cellular Adhesion ◽  
Fiber Formation ◽  
Extracellular Matrix Protein ◽  
Fluid Shear

A rheological in vitro system has been developed to study and quantify cellular adhesion under precisely defined external shear forces. The system is similar to a cone-and-plate viscosimeter. A rotating transparent cone produces both steady and pulsatile flow profiles on cultured cells. Direct visualization of cells by phase-contrast or fluorescence optics and connection of the optical system to a computer-controlled x/y-linear stage allows automatic recording of any point of the cell cultures. With the use of up to 12 individual rheological units, this setup allows the quantitative analysis of cell substrate adhesion by determination of cell detachment kinetics. Two examples of application of this rheological system have been studied. First, we show that the extracellular matrix protein laminin strongly increases endothelial cell adhesion under fluid shear stress. In a second approach, we obtained further support for the concept that shear stress-induced formation of actin filament stress fibers is important for endothelial cells to resist the fluid shear stress; inhibition of stress fiber formation by doxorubicin resulted in significant detachment of endothelial cells exposed to medium levels of fluid shear stress (5 dyn/cm2). No detachment was seen under resting conditions.

scholarly journals Low-Intensity Pulsed Ultrasound Promotes Autophagy-Mediated Migration of Mesenchymal Stem Cells and Cartilage Repair

2021 ◽  
Vol 30 ◽  
pp. 096368972098614
Author(s):  
Peng Xia ◽  
Xinwei Wang ◽  
Qi Wang ◽  
Xiaoju Wang ◽  
Qiang Lin ◽  
...  
Keyword(s):  
Cartilage Repair ◽  
Matrix Protein ◽  
Extracellular Matrix Protein ◽  
Histopathological Analysis ◽  
Pulsed Ultrasound ◽  
Low Intensity Pulsed Ultrasound ◽  
Low Intensity ◽  
Autophagy Inhibitor

Mesenchymal stem cell (MSC) migration is promoted by low-intensity pulsed ultrasound (LIPUS), but its mechanism is unclear. Since autophagy is known to regulate cell migration, our study aimed to investigate if LIPUS promotes the migration of MSCs via autophagy regulation. We also aimed to investigate the effects of intra-articular injection of MSCs following LIPUS stimulation on osteoarthritis (OA) cartilage. For the in vitro study, rat bone marrow-derived MSCs were treated with an autophagy inhibitor or agonist, and then they were stimulated by LIPUS. Migration of MSCs was detected by transwell migration assays, and stromal cell-derived factor-1 (SDF-1) and C-X-C chemokine receptor type 4 (CXCR4) protein levels were quantified. For the in vivo study, a rat knee OA model was generated and treated with LIPUS after an intra-articular injection of MSCs with autophagy inhibitor added. The cartilage repair was assessed by histopathological analysis and extracellular matrix protein expression. The in vitro results suggest that LIPUS increased the expression of SDF-1 and CXCR4, and it promoted MSC migration. These effects were inhibited and enhanced by autophagy inhibitor and agonist, respectively. The in vivo results demonstrate that LIPUS significantly enhanced the cartilage repair effects of MSCs on OA, but these effects were blocked by autophagy inhibitor. Our results suggest that the migration of MSCs was enhanced by LIPUS through the activation autophagy, and LIPUS improved the protective effect of MSCs on OA cartilage via autophagy regulation.

Contrasting migratory response of astrocytoma cells to tenascin mediated by different integrins

1996 ◽  
Vol 109 (8) ◽  
pp. 2161-2168 ◽  
Author(s):  
A. Giese ◽  
M.A. Loo ◽  
S.A. Norman ◽  
S. Treasurywala ◽  
M.E. Berens
Keyword(s):  
Cell Adhesion ◽  
Matrix Protein ◽  
Glioma Cells ◽  
Extracellular Matrix Protein ◽  
Integrin Receptors ◽  
Migration Assay ◽  
Dose Dependent Fashion ◽  
Beta 1 Integrin

Tenascin, an extracellular matrix protein, is expressed in human gliomas in vitro and in vivo. The distribution of tenascin at the invasive edge of these tumors, even surrounding solitary invading cells, suggests a role for this protein as a regulator of glioma cell migration. We tested whether purified tenascin, passively deposited on surfaces, influenced the adhesion or migration of a human gliomaderived cell line, SF-767. Adhesion of glioma cells to tenascin increased in a dose-dependent fashion up to a coating concentration of 10 micrograms/ml. Higher coating concentrations resulted in progressively fewer cells attaching. Cell adhesion could be blocked to basal levels using anti-beta 1 integrin antibodies. In contrast, when anti-alpha v antibodies were added to the medium of cells on tenascin, cell adhesion was enhanced slightly. Using a microliter scale migration assay, we found that cell motility on tenascin was dose dependently stimulated at coating concentrations of 1 and 3 micrograms/ml, but migration was inhibited below levels of non-specific motility when tested at coating concentrations of 30 and 100 micrograms/ml. Migration on permissive concentrations of tenascin could be reversibly inhibited with anti-beta 1, while treatment with anti-alpha v antibodies increased migration rates. We conclude that SF-767 glioma cells express two separate integrin receptors that mediate contrasting adhesive and migratory responses to tenascin.

scholarly journals FOS, a Critical Downstream Mediator of PGR and EGF Signaling Necessary for Ovulatory Prostaglandins in the Human Ovary

2018 ◽  
Vol 103 (11) ◽  
pp. 4241-4252 ◽  
Author(s):  
Yohan Choi ◽  
Katherine L Rosewell ◽  
Mats Brännström ◽  
James W Akin ◽  
Thomas E Curry ◽  
...  
Keyword(s):  
Target Genes ◽  
Activator Protein 1 ◽  
Egf Receptors ◽  
Human Ovary ◽  
Promoter Regions ◽  
Vitro Fertilization ◽  
Egf Signaling ◽  
Periovulatory Follicles

Abstract Context Fos null mice failed to ovulate and form a corpus luteum (CL) even when given exogenous gonadotropins, suggesting that ovarian Fos expression is critical for successful ovulation and CL formation. However, little is known about FOS in the human ovary. Objectives To determine the expression, regulation, and function of FOS in human periovulatory follicles. Design/Participants Timed periovulatory follicles were obtained from normally cycling women. Granulosa/lutein cells were collected from in vitro fertilization patients. Main Outcome Measures The in vivo expression after human chorionic gonadotropin (hCG) administration and in vitro regulation of FOS, JUN, JUNB, and JUND was evaluated at the mRNA and protein level. Binding of progesterone receptor (PGR) and FOS to their target genes was assessed by chromatin immunoprecipitation analyses. Prostaglandin E2 (PGE2) and progesterone were measured. Results The expression of FOS, JUNB, and JUND drastically increased in ovulatory follicles after hCG administration. In human granulosa/lutein cell cultures, hCG increased the expression of FOS and JUN proteins. Inhibitors of PGR and epidermal growth factor (EGF) receptors reduced hCG-induced increases in the expression and phosphorylation of FOS. PGR bound to the FOS gene. A selective FOS inhibitor blocked hCG-induced increases in PGE2 and the expression of prostaglandin (PG) synthases and transporters (PTGES, SLCO2A1, and ABCC1). FOS bound to the promoter regions of these genes. Conclusions The increase of FOS/activator protein 1 in human periovulatory follicles after hCG administration is mediated by collaborative actions of PGR and EGF signaling and critical for the upregulated expression of key ovulatory genes required for the rise in ovulatory PG in human granulosa cells.

scholarly journals LTBP1 promotes fibrillin incorporation into the extracellular matrix

2021 ◽  
Author(s):  
Matthias Przyklenk ◽  
Veronika Georgieva ◽  
Fabian Metzen ◽  
Sebastian Mostert ◽  
Birgit Kobbe ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Matrix Protein ◽  
Wide Spectrum ◽  
Extracellular Matrix Protein ◽  
Connective Tissues ◽  
Pathogenic Variants ◽  
Human Pathology ◽  
Fibrillin 1

LTBP1 is a large extracellular matrix protein and an associated ligand of fibrillin-microfibrils. Knowledge of LTBP1 functions is largely limited to its role in targeting and sequestering TGFβ growth factors within the extracellular matrix, thereby regulating their bioavailability. However, the recent description of a wide spectrum of phenotypes in multiple tissues in patients harboring LTBP1 pathogenic variants suggests a multifaceted role of the protein in the homeostasis of connective tissues. To better understand the human pathology caused by LTBP1 deficiency it is important to investigate its functional role in extracellular matrix formation. In this study, we show that LTBP1 coordinates the incorporation of fibrillin-1 and -2 into the extracellular matrix in vitro. We also demonstrate that this function is differentially exerted by the two isoforms, the short and long forms of LTBP1. Thereby our findings uncover a novel TGFβ-independent LTBP1 function potentially contributing to the development of connective tissue disorders.

Effect of microenvironment and cell-line type on carbohydrate-binding proteins of macrophage-like cells

1988 ◽  
Vol 66 (11) ◽  
pp. 1169-1176 ◽  
Author(s):  
Hans-Joachim Gabius ◽  
Katalin Vehmeyer
Keyword(s):  
Binding Proteins ◽  
Cultured Cells ◽  
Sodium Dodecyl ◽  
Carbohydrate Binding ◽  
Intercellular Interaction ◽  
Cell Extracts ◽  
Carbohydrate Binding Proteins ◽  
Sugar Receptors

The pattern of sugar inhibition of rosette formation, a model for intercellular interaction between cultured cells and glutaraldehyde-fixed, trypsinated rabbit erythrocytes, served to infer the presence of carbohydrate-binding proteins. This profile from cell extracts for the two murine macrophage-like cell lines, P388D1 and J774A.1, was comparatively analyzed by affinity chromatography on supports with immobilized carbohydrates (lactose, L-fucose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, and maltose) or with the immobilized mannose-rich yeast glycoprotein mannan or fetuin-derived glycopeptides containing sialic acid residues. After elution with specific sugar in the absence of Ca2+ ions, the proteins were separated by sodium dodecyl sulfate – polyacrylamide slab gel electrophoresis. The composition of carbohydrate-binding proteins of the two lines clearly exhibited quantitative and qualitative differences. Moreover, the pattern of P388D1 cells was also demonstrated to change significantly in response to alterations in the conditions of the physiological environment. These alterations were imposed by in vitro growth, by subsequent in vivo growth in nude mice, and by re-adaptation of cells to culture after in vivo passage. Collectively, our observations and other physiological and biochemical reports on macrophage lectins indicate that the presence of sugar receptors with different specificities may be an indicator of macrophage differentiation, being reversibly modulated to a considerable extent by external factors, e.g., microenvironment. Extensive but selective alterations in this respect could play an important role in the control of recognition and effector mechanisms within diverse functions of macrophage subpopulations.

scholarly journals Xenopus embryonic cell adhesion to fibronectin: position-specific activation of RGD/synergy site-dependent migratory behavior at gastrulation.

1996 ◽  
Vol 134 (1) ◽  
pp. 227-240 ◽  
Author(s):  
J W Ramos ◽  
D W DeSimone
Keyword(s):  
Cell Adhesion ◽  
Matrix Protein ◽  
Marginal Zone ◽  
Cell Attachment ◽  
Extracellular Matrix Protein ◽  
Mesoderm Induction ◽  
Type Iii ◽  
Basic Body ◽  
And Migration

During Xenopus laevis gastrulation, the basic body plan of the embryo is generated by movement of the marginal zone cells of the blastula into the blastocoel cavity. This morphogenetic process involves cell adhesion to the extracellular matrix protein fibronectin (FN). Regions of FN required for the attachment and migration of involuting marginal zone (IMZ) cells were analyzed in vitro using FN fusion protein substrates. IMZ cell attachment to FN is mediated by the Arg-Gly-Asp (RGD) sequence located in the type III-10 repeat and by the Pro-Pro-Arg-Arg-Ala-Arg (PPRRAR) sequence in the type III-13 repeat of the Hep II domain. IMZ cells spread and migrate persistently on fusion proteins containing both the RGD and synergy site sequence Pro-Pro-Ser-Arg-Asn (PPSRN) located in the type III-9 repeat. Cell recognition of the synergy site is positionally regulated in the early embryo. During gastrulation, IMZ cells will spread and migrate on FN whereas presumptive pre-involuting mesoderm, vegetal pole endoderm, and animal cap ectoderm will not. However, animal cap ectoderm cells acquire the ability to spread and migrate on the RGD/synergy region when treated with the mesoderm inducing factor activin-A. These data suggest that mesoderm induction activates the position-specific recognition of the synergy site of FN in vivo. Moreover, we demonstrate the functional importance of this site using a monoclonal antibody that blocks synergy region-dependent cell spreading and migration on FN. Normal IMZ movement is perturbed when this antibody is injected into the blastocoel cavity indicating that IMZ cell interaction with the synergy region is required for normal gastrulation.

Asporin competes with decorin for collagen binding, binds calcium and promotes osteoblast collagen mineralization

2009 ◽  
Vol 423 (1) ◽  
pp. 53-59 ◽  
Author(s):  
Sebastian Kalamajski ◽  
Anders Aspberg ◽  
Karin Lindblom ◽  
Dick Heinegård ◽  
Åke Oldberg
Keyword(s):  
Matrix Protein ◽  
Collagen Type I ◽  
Full Length ◽  
Extracellular Matrix Protein ◽  
Type I ◽  
Similar System ◽  
Collagen Binding ◽  
Osteoblastic Activity ◽  
Collagen Mineralization

The interactions of the ECM (extracellular matrix) protein asporin with ECM components have previously not been investigated. Here, we show that asporin binds collagen type I. This binding is inhibited by recombinant asporin fragment LRR (leucine-rich repeat) 10–12 and by full-length decorin, but not by biglycan. We demonstrate that the polyaspartate domain binds calcium and regulates hydroxyapatite formation in vitro. In the presence of asporin, the number of collagen nodules, and mRNA of osteoblastic markers Osterix and Runx2, were increased. Moreover, decorin or the collagen-binding asporin fragment LRR 10–12 inhibited the pro-osteoblastic activity of full-length asporin. Our results suggest that asporin and decorin compete for binding to collagen and that the polyaspartate in asporin directly regulates collagen mineralization. Therefore asporin has a role in osteoblast-driven collagen biomineralization activity. We also show that asporin can be expressed in Escherichia coli (Rosetta-gami™) with correctly positioned cysteine bridges, and a similar system can possibly be used for the expression of other SLRPs (small LRR proteoglycans/proteins).

Hypoxia – as a Possible Regulator of the Activity of Epicardial Mesothelial Cells After Myocardial Infarction

Kardiologiia ◽  
2021 ◽  
Vol 61 (6) ◽  
pp. 59-68
Author(s):  
K. V. Dergilev ◽  
Z. I. Tsokolaeva ◽  
Yu. D. Vasilets ◽  
I. B. Beloglazova ◽  
B. N. Kulbitsky ◽  
...  
Keyword(s):  
Myocardial Infarction ◽  
Extracellular Matrix ◽  
Matrix Protein ◽  
Mesothelial Cells ◽  
Immunofluorescent Staining ◽  
Extracellular Matrix Protein ◽  
Hypoxic Exposure ◽  
Mesenchymal Transition ◽  
Epicardial Cells

Aim      To study the effect of hypoxia on the activity of epithelial-mesenchymal transition (EMT) in epicardial cells, which provides formation of a specialized microenvironment.Material and methods   This study used a model of experimental myocardial infarction created by ligation of the anterior descendent coronary artery. The activity of epicardial cells after a hypoxic exposure was studied with the hypoxia marker, pimonidazole, bromodeoxyuridine, immunofluorescent staining of heart cryosections, and in vitro mesothelial cell culture.Results The undamaged heart maintained the quiescent condition of mesothelial cells and low levels of their proliferation, extracellular matrix protein production, and of the EMT activity. Acute ischemic injury induced moderate hypoxia in the epicardial/subepicardial region. This caused a global rearrangement of this region due to the initiation of EMT in cells, changes in the cell composition, and accumulation of extracellular matrix proteins. We found that the initiation of EMT in mesothelial cells may result in the formation of smooth muscle cell precursors, fibroblasts, and a population of Sca-1+ cardiac progenitor cells, which may both participate in construction of new blood vessels and serve as a mesenchymal link for the paracrine support of microenvironmental cells. In in vitro experiments, we showed that 72‑h hypoxia facilitated activation of EMT regulatory genes, induced dissembling of intercellular contacts, cell uncoupling, and increased cell plasticity.Conclusion      The epicardium of an adult heart serves as a “reparative reserve” that can be reactivated by a hypoxic exposure. This creates a basis for an approach to influence the epicardium to modulate its activity for regulating reparative processes.

Abstract TMP118: Potential Therapeutic Benefits of Perlecan Domain V in Preclinical Vascular Dementia and Cerebral Angiopathy

Stroke ◽  
2017 ◽  
Vol 48 (suppl_1) ◽  
Author(s):  
Amanda L Trout ◽  
Zuohui Zhang ◽  
Jill Roberts ◽  
Gillian Grohs ◽  
Ela Patel ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Parietal Cortex ◽  
Matrix Protein ◽  
Extracellular Matrix Protein ◽  
Amyloid Angiopathy ◽  
Cognitive Changes ◽  
Cerebral Microvessels ◽  
Therapeutic Benefits ◽  
Domain V

Vascular contributions to cognitive impairment and dementia (VCID), the second leading cause of dementia behind Alzheimer’s disease (AD), is a broad term that encompasses a spectrum of initial asymptomatic cerebrovascular changes (seen in small vessel disease and cerebral amyloid angiopathy where pathologic Aβ1-42 protein accumulates around brain blood vessels) to the profound symptomatic damage following acute stroke(s). Cerebrovascular remodeling and new blood vessel growth (angiogenesis) may represent early compensatory changes to reduced cerebral blood flow that can initiate VCID. Angiogenesis, in turn, is supported by various growth factors and the proteolytic turnover of surrounding extracellular matrix. We have demonstrated that one such extracellular matrix protein, perlecan (a heparan sulfate proteoglycan), possesses a C terminal domain V (DV) protein that upon cleavage greatly enhances brain angiogenesis. We characterized VCID induced changes in DV expression in the human parietal cortex and a distinct mouse model (diabetic APP/PS1 knock in (db/AD)), that has a gradual cognitive decline by 9 months with microangiopathy, Aβ1-42 deposition, aneurysms, and microhemorrhages. We also utilized an in vitro model of the blood-brain barrier (BBB) to assess the transport of human Aβ1-42 in the presence of DV. In the human parietal cortex, dementia patients had increased expression of DV despite having fewer cells. In db/AD animals (3-6 months), we observed a decrease in BBB proteins (i.e. claudin-5), indicating that altered function correlated with an increase in brain DV expression during the asymptomatic angiogenic stage, which precedes cognitive changes (9-12 months). In vitro, DV doubled the transport of Aβ1-42 into the lumen of cerebral microvessels over 24 hours with increased activity and total protein expression of P-glycoprotein (P-gp), one of Aβ’s known transport proteins. Collectively, these data indicate that early cerebrovascular changes induce angiogenic-remodeling that correlates with increased expression of DV. DV, in turn, may further enhance angiogenesis and increase brain Aβ clearance into the vascular compartment through P-gp, suggesting that DV could represent a novel therapeutic for VCID.

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