Single Cell and Spheroid Collagen Type I Invasion Assay

2013 ◽  
pp. 13-35 ◽  
Author(s):  
Olivier De Wever ◽  
An Hendrix ◽  
Astrid De Boeck ◽  
Frank Eertmans ◽  
Wendy Westbroek ◽  
...  
Keyword(s):  
Single Cell ◽  
Collagen Type ◽  
Collagen Type I ◽  
Type I ◽  
Invasion Assay

scholarly journals Revealing Early Steps of α2β1 Integrin-mediated Adhesion to Collagen Type I by Using Single-Cell Force Spectroscopy

2007 ◽  
Vol 18 (5) ◽  
pp. 1634-1644 ◽  
Author(s):  
Anna Taubenberger ◽  
David A. Cisneros ◽  
Jens Friedrichs ◽  
Pierre-Henri Puech ◽  
Daniel J. Muller ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Single Cell ◽  
Collagen Type ◽  
Chinese Hamster ◽  
Force Spectroscopy ◽  
Collagen Type I ◽  
Time Range ◽  
Type I ◽  
Actomyosin Contractility ◽  
Cell Force

We have characterized early steps of α2β1 integrin-mediated cell adhesion to a collagen type I matrix by using single-cell force spectroscopy. In agreement with the role of α2β1 as a collagen type I receptor, α2β1-expressing Chinese hamster ovary (CHO)-A2 cells spread rapidly on the matrix, whereas α2β1-negative CHO wild-type cells adhered poorly. Probing CHO-A2 cell detachment forces over a contact time range of 600 s revealed a nonlinear adhesion response. During the first 60 s, cell adhesion increased slowly, and forces associated with the smallest rupture events were consistent with the breakage of individual integrin–collagen bonds. Above 60 s, a fraction of cells rapidly switched into an activated adhesion state marked by up to 10-fold increased detachment forces. Elevated overall cell adhesion coincided with a rise of the smallest rupture forces above the value required to break a single-integrin–collagen bond, suggesting a change from single to cooperative receptor binding. Transition into the activated adhesion mode and the increase of the smallest rupture forces were both blocked by inhibitors of actomyosin contractility. We therefore propose a two-step mechanism for the establishment of α2β1-mediated adhesion as weak initial, single-integrin–mediated binding events are superseded by strong adhesive interactions involving receptor cooperativity and actomyosin contractility.

scholarly journals Relative rates of biosynthesis of collagen type I, type V and type VI in calf cornea

1991 ◽  
Vol 274 (2) ◽  
pp. 615-617 ◽  
Author(s):  
P Kern ◽  
M Menasche ◽  
L Robert
Keyword(s):  
Type I Collagen ◽  
Collagen Type ◽  
Corneal Stroma ◽  
Collagen Type I ◽  
Type I ◽  
Type Vi Collagen ◽  
Sds Page ◽  
Proline Incorporation ◽  
Type V

The biosynthesis of type I, type V and type VI collagens was studied by incubation of calf corneas in vitro with [3H]proline as a marker. Pepsin-solubilized collagen types were isolated by salt fractionation and quantified by SDS/PAGE. Expressed as proportions of the total hydroxyproline solubilized, corneal stroma comprised 75% type I, 8% type V and 17% type VI collagen. The rates of [3H]proline incorporation, linear up to 24 h for each collagen type, were highest for type VI collagen and lowest for type I collagen. From pulse-chase experiments, the calculated apparent half-lives for types I, V and VI collagens were 36 h, 10 h and 6 h respectively.

scholarly journals Confocal Blood Flow Videomicroscopy of Thrombus Formation over Human Arteries and Local Targeting of P2X7

2021 ◽  
Vol 22 (8) ◽  
pp. 4066
Author(s):  
Patrizia Marchese ◽  
Maria Lombardi ◽  
Maria Elena Mantione ◽  
Domenico Baccellieri ◽  
David Ferrara ◽  
...  
Keyword(s):  
Blood Flow ◽  
Vascular Function ◽  
Collagen Type ◽  
Thrombus Formation ◽  
Collagen Type I ◽  
Type I ◽  
Prevention Therapy ◽  
Healthy Donors ◽  
Internal Mammary ◽  
Human Arteries

Atherothrombosis exposes vascular components to blood. Currently, new antithrombotic therapies are emerging. Herein we investigated thrombogenesis of human arteries with/without atherosclerosis, and the interaction of coagulation and vascular components, we and explored the anti-thrombogenic efficacy of blockade of the P2X purinoceptor 7 (P2X7). A confocal blood flow videomicroscopy system was performed on cryosections of internal mammary artery (IMA) or carotid plaque (CPL) determining/localizing platelets and fibrin. Blood from healthy donors elicited thrombi over arterial layers. Confocal microscopy associated thrombus with tissue presence of collagen type I, laminin, fibrin(ogen) and tissue factor (TF). The addition of antibodies blocking TF (aTF) or factor XI (aFXI) to blood significantly reduced fibrin deposition, variable platelet aggregation and aTF + aFXI almost abolished thrombus formation, showing synergy between coagulation pathways. A scarce effect of aTF over sub-endothelial regions, more abundant in tissue TF and bundles of laminin and collagen type I than deep intima, may suggest tissue thrombogenicity as molecular structure-related. Consistently with TF-related vascular function and expression of P2X7, the sections from CPL but not IMA tissue cultures pre-treated with the P2X7 antagonist A740003 demonstrated poor thrombogenesis in flow experiments. These data hint to local targeting studies on P2X7 modulation for atherothrombosis prevention/therapy.

scholarly journals 14 Characterization of adipose tissue extracellular matrix component mRNA expression during porcine adipogenesis using real-time PCR

2020 ◽  
Vol 98 (Supplement_2) ◽  
pp. 35-35
Author(s):  
Maegan A Reeves ◽  
Courtney E Charlton ◽  
Terry D Brandebourg
Keyword(s):  
Extracellular Matrix ◽  
Adipose Tissue ◽  
Mrna Expression ◽  
Real Time ◽  
Real Time Pcr ◽  
Collagen Type ◽  
Primary Cultures ◽  
Collagen Type I ◽  
Type I ◽  
Matrix Metallopeptidase

Abstract Given adipose tissue is histologically classified as connective tissue, we hypothesized expression of extracellular matrix (ECM) components are significantly altered during adipogenesis. However, little is known about the regulation of the ECM during adipose tissue development in the pig. Therefore, the objective of this study was to characterize expression of ECM components during porcine adipogenesis. Primary cultures of adipose tissue stromal-vascular cells were harvested from 3-day-old neonatal pigs (n=6) and preadipocytes induced to differentiate in vitro for 8 days in the presence of insulin, hydrocortisone, and rosiglitazone. Total RNA was extracted from these cultures on days 0 and 8 post-induction. Real-time PCR was then utilized to determine changes in mRNA expression for collagen type I alpha 1 chain (COL1A), collagen type I alpha 2 chain (COL2A), collagen type I alpha 3 chain (COL3A), collagen type I alpha 4 chain (COL4A), collagen type I alpha 6 chain (COL6A), biglycan, fibronectin, laminin, nitogen-1 (NID1), matrix metallopeptidase 2 (MMP2), matrix metallopeptidase 9 (MMP9), metallopeptidase inhibitor 3 (TIMP3). The mRNA abundances of COL1A, COL3A and MMP2 were significantly downregulated 2.86-fold (P < 0.05), 16.7-fold (P < 0.01) and 3.1-fold (P < 0.05) respectively in day 8 (differentiated) compared to day 0 (undifferentiated) cultures. Meanwhile, mRNA abundances were significantly upregulated during adipogenesis for the COL2A (2.82-fold; P < 0.05), COL4A (2.01-fold; P < 0.05), COL6A (2.8-fold; P < 0.05), biglycan (49.9- fold; P < 0.001), fibronectin (452-fold; P < 0.001), laminin (6.1-fold; P < 0.05), NID1(47.4-fold; P < 0.01), MMP9 (76.8- fold; P < 0.01), and TIMP3(3.04-fold; P < 0.05) genes. These data support the hypothesis that significant changes in ECM components occur during porcine adipogenesis. Modulating adipose tissue ECM remodeling might be a novel strategy to manipulate adiposity in the pig.

scholarly journals Non-Syndromic Dentinogenesis Imperfecta Caused by Mild Mutations in COL1A2

2021 ◽  
Vol 11 (6) ◽  
pp. 526
Author(s):  
Yejin Lee ◽  
Youn Jung Kim ◽  
Hong-Keun Hyun ◽  
Jae-Cheoun Lee ◽  
Zang Hee Lee ◽  
...  
Keyword(s):  
Collagen Type ◽  
Molecular Genetic ◽  
Collagen Type I ◽  
Type I ◽  
Dentinogenesis Imperfecta ◽  
Gene Encoding ◽  
Korean Families ◽  
Whole Exome ◽  
Genes Encoding ◽  
Candidate Gene Sequencing

Hereditary dentin defects can be categorized as a syndromic form predominantly related to osteogenesis imperfecta (OI) or isolated forms without other non-oral phenotypes. Mutations in the gene encoding dentin sialophosphoprotein (DSPP) have been identified to cause dentinogenesis imperfecta (DGI) Types II and III and dentin dysplasia (DD) Type II. While DGI Type I is an OI-related syndromic phenotype caused mostly by monoallelic mutations in the genes encoding collagen type I alpha 1 chain (COL1A1) and collagen type I alpha 2 chain (COL1A2). In this study, we recruited families with non-syndromic dentin defects and performed candidate gene sequencing for DSPP exons and exon/intron boundaries. Three unrelated Korean families were further analyzed by whole-exome sequencing due to the lack of the DSPP mutation, and heterozygous COL1A2 mutations were identified: c.3233G>A, p.(Gly1078Asp) in Family 1 and c.1171G>A, p.(Gly391Ser) in Family 2 and 3. Haplotype analysis revealed different disease alleles in Families 2 and 3, suggesting a mutational hotspot. We suggest expanding the molecular genetic etiology to include COL1A2 for isolated dentin defects in addition to DSPP.

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