scholarly journals The biosynthesis of extracellular-matrix components by bovine retinal endothelial cells displaying distinctive morphological phenotypes

1986 ◽  
Vol 235 (2) ◽  
pp. 375-383 ◽  
Author(s):  
A E Canfield ◽  
A M Schor ◽  
S L Schor ◽  
M E Grant
Keyword(s):  
Endothelial Cells ◽  
Type I ◽  
Collagen Gels ◽  
Early Passage ◽  
Experimental Conditions ◽  
Retinal Endothelial Cells ◽  
Type Iv ◽  
Extracellular Matrix Components ◽  
Cells Cultured

Previous studies have indicated that the morphology and behaviour of bovine retinal microvessel endothelial cells are influenced by culture conditions in vitro. Data are presented here concerning the biosynthesis of matrix macromolecules by bovine retinal endothelial cells cultured under conditions in which the cells display either the ‘cobblestone’ or the ‘sprouting’ phenotype. Newly synthesized matrix proteins were identified by their characteristic electrophoretic mobilities, immunoprecipitation with specific antibodies, susceptibilities to enzymic digestions and chromatographic behaviour. Type IV procollagen was the major collagenous species synthesized by early-passage cells forming a ‘cobblestone’ monolayer. In contrast, cells displaying the ‘sprouting’ morphology switched to the predominant synthesis of interstitial fibrillar collagens (types I and III). Fibronectin was synthesized by retinal endothelial cells under all the experimental conditions studied. A non-collagenous glycoprotein of Mr approx. 47,000 was also a major biosynthetic product of these cells. The synthesis of thrombospondin was very much dependent on the nature of the substratum on which the cells were cultured. This glycoprotein was synthesized in large amounts by ‘cobblestone’ endothelial cells cultured on gelatin-coated dishes, whereas its synthesis was markedly decreased by culturing the cells on collagen gels, and the protein appeared to be absent when the cells were plated within collagen gels (‘sprouting’ cells). Late-passage retinal cells synthesized predominantly type I procollagen, variable amounts of type III procollagen and only traces of type IV procollagen, irrespective of whether the cells displayed a ‘cobblestone’ or ‘sprouting’ morphology.

Abstract 418: Co-Culture of Endothelial Cells, Smooth Muscle Cells and Monocytes in Collagen Scaffold: A Novel i n vitro Model of Atherosclerosis

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Martin Liu ◽  
Angelos Karagiannis ◽  
Matthew Sis ◽  
Srivatsan Kidambi ◽  
Yiannis Chatzizisis
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Type I Collagen ◽  
Smooth Muscle Actin ◽  
Muscle Cells ◽  
Type I ◽  
Collagen Gels ◽  
Human Coronary Artery

Objectives: To develop and validate a 3D in-vitro model of atherosclerosis that enables direct interaction between various cell types and/or extracellular matrix. Methods and Results: Type I collagen (0.75 mg/mL) was mixed with human artery smooth muscle cells (SMCs; 6x10 5 cells/mL), medium, and water. Human coronary artery endothelial cells (HCAECs; 10 5 /cm 2 ) were plated on top of the collagen gels and activated with oxidized low density lipoprotein cholesterol (LDL-C). Monocytes (THP-1 cells; 10 5 /cm 2 ) were then added on top of the HCAECs. Immunofluorescence showed the expression of VE-cadherin by HCAECs (A, B) and α-smooth muscle actin by SMCs (A). Green-labelled LDL-C particles were accumulated in the subendothelial space, as well as in the cytoplasm of HCAECs and SMCs (C). Activated monocytes were attached to HCAECs and found in the subendothelial area (G-I). Both HCAECs and SMCs released IL-1β, IL-6, IL-8, PDGF-BB, TGF-ß1, and VEGF. Scanning and transmission electron microscopy showed the HCAECs monolayer forming gap junctions and the SMCs (D-F) and transmigrating monocytes within the collagen matrix (G-I). Conclusions: In this work, we presented a novel, easily reproducible and functional in-vitro experimental model of atherosclerosis that has the potential to enable in-vitro sophisticated molecular and drug development studies.

scholarly journals PHAGOCYTOSIS OF BACTERIA IN THE ABSENCE OF ANTIBODY AND THE EFFECT OF PHYSICAL SURFACE

1956 ◽  
Vol 104 (2) ◽  
pp. 233-243 ◽  
Author(s):  
Edwin M. Lerner
Keyword(s):  
Filter Paper ◽  
Chemical Effect ◽  
Type I ◽  
Blood Leukocytes ◽  
Experimental Conditions ◽  
Phagocytic Capacity ◽  
E Coli ◽  
Physical Surface ◽  
Type Iv

The present experiments have shown that phagocytosis occurs in the absence of specific antibody and in the absence of a "suitable physical surface", as further that the presence of a rough surface does not increase the in vitro phagocytosis of pneumococci by polymorphonuclear leukocytes. This held true during repetition of Wood's experiments, as well as when more controlled quantitative techniques were employed, when conditions were made optimal for phagocytosis by increasing bacterial concentrations, and when blood leukocytes were substituted for exudate leukocytes. Evidence has been presented previously that the stimulation of phagocytosis of E. coli, B. abortus, and Type IV Pneumococcus, after contact with filter paper or an active compound present in filter paper, is a chemical effect rather than a physical effect. This type of stimulation did not occur with the Type I A5 Pneumococcus. The leukocyte of the circulating blood was found to be definitely superior to the exudate leukocyte in phagocytic capacity, under all the experimental conditions tested.

scholarly journals Thrombospondin gene expression by endothelial cells in culture is modulated by cell proliferation, cell shape and the substratum

1990 ◽  
Vol 268 (1) ◽  
pp. 225-230 ◽  
Author(s):  
A E Canfield ◽  
R P Boot-Handford ◽  
A M Schor
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
Endothelial Cells ◽  
Cell Shape ◽  
Type I Collagen ◽  
Three Dimensional ◽  
Type I ◽  
Mrna Levels ◽  
Collagen Gels ◽  
Cells Cultured

Endothelial cells plated on the surface of a two-dimensional substratum (gelatin-coated dishes, dishes coated with native type I collagen or collagen gels) form a cobblestone monolayer at confluence, whereas cells plated within a three-dimensional gel matrix elongate into a sprouting morphology and self-associate into tube-like structures. In this study, we have compared the synthesis of thrombospondin by quiescent endothelial cells displaying (a) the same morphological phenotype (cobblestone) on different substrata (gelatin and collagen) and (b) different morphological phenotypes (cobblestone and sprouting) on the same substratum (collagen). We demonstrate that thrombospondin is a major biosynthetic product of confluent, quiescent cells cultured on dishes coated with either gelatin or collagen, and that the synthesis of this protein is markedly decreased when cells are plated on or in three-dimensional collagen gels. Moreover, we demonstrate that cells plated in gel (sprouting) secrete less thrombospondin than do cells plated on the gel surface (cobblestone). The regulation of thrombospondin synthesis is reversible and occurs at the level of transcription, as steady-state mRNA levels for thrombospondin decrease in a manner comparable with the levels of protein secreted by these cells. We also show that mRNA levels for laminin B2 chains are increased when cells are cultured on and in collagen gels compared with on gelatin-coated dishes, suggesting that the syntheses of thrombospondin and laminin are regulated by different mechanisms. When cells are cultured on gelatin- or collagen-coated dishes, thrombospondin gene expression is directly proportional to the proliferative state of the cultures. By contrast, the synthesis of thrombospondin by cells cultured on collagen gels remains at equally low levels whether they are labelled when they are sparse and rapidly proliferating or when they are confluent and quiescent. Fibronectin synthesis was found to increase with increasing confluency of the cells plated on all three substrata. These results demonstrate that thrombospondin gene expression is modulated by cell shape, cell proliferation and the nature of the substratum used for cell culture.

scholarly journals Expression of extracellular matrix components is regulated by substratum.

1990 ◽  
Vol 110 (4) ◽  
pp. 1405-1415 ◽  
Author(s):  
C H Streuli ◽  
M J Bissell
Keyword(s):  
Extracellular Matrix ◽  
Epithelial Cells ◽  
Basement Membrane ◽  
Type I Collagen ◽  
Basement Membranes ◽  
Type I ◽  
Collagen Gels ◽  
Extracellular Matrix Components ◽  
Matrix Components ◽  
Cells Cultured

Reconstituted basement membranes and extracellular matrices have been demonstrated to affect, positively and dramatically, the production of milk proteins in cultured mammary epithelial cells. Here we show that both the expression and the deposition of extracellular matrix components themselves are regulated by substratum. The steady-state levels of the laminin, type IV collagen, and fibronectin mRNAs in mammary epithelial cells cultured on plastic dishes and on type I collagen gels have been examined, as has the ability of these cells to synthesize, secrete, and deposit laminin and other, extracellular matrix proteins. We demonstrate de novo synthesis of a basement membrane by cells cultured on type I collagen gels which have been floated into the medium. Expression of the mRNA and proteins of basement membranes, however, are quite low in these cultures. In contrast, the levels of laminin, type IV collagen, and fibronectin mRNAs are highest in cells cultured on plastic surfaces, where no basement membrane is deposited. It is suggested that the interaction between epithelial cells and both basement membrane and stromally derived matrices exerts a negative influence on the expression of mRNA for extracellular matrix components. In addition, we show that the capacity for lactational differentiation correlates with conditions that favor the deposition of a continuous basement membrane, and argue that the interaction between specialized epithelial cells and stroma enables them to create their own microenvironment for accurate signal transduction and phenotypic function.

scholarly journals Galectin-3 expression and effects on cyst enlargement and tubulogenesis in kidney epithelial MDCK cells cultured in three-dimensional matrices in vitro

1995 ◽  
Vol 108 (8) ◽  
pp. 2791-2800 ◽  
Author(s):  
Q. Bao ◽  
R.C. Hughes
Keyword(s):  
Cell Surface ◽  
Mdck Cells ◽  
Three Dimensional ◽  
Immunofluorescent Staining ◽  
Epifluorescence Microscopy ◽  
Type I ◽  
Collagen Gels ◽  
Galectin 3 ◽  
Cells Cultured

Galectin-3 is a member of a closely related family of beta-galactoside-binding soluble proteins found in many vertebrate epithelial and myeloid cell types. The developmentally regulated presence of galectin-3 in tissues, for example kidney, and an affinity for many cell-surface and matrix glycoproteins indicate its importance in extracellular biological processes. Since a polarised expression and secretion of galectin-3 was observed in monolayer-cultured MDCK cells, an understanding of the secretion and distribution of this lectin in a three-dimensional in vitro model would help to uncover its role(s) in the interplay between cell-surface adhesion molecules and extracellular matrix components occurring during cell aggregation and polarisation in tissue formation. In this study, the cellular distribution and secretion of galectin-3 were examined in MDCK cells cultured within a gel matrix. MDCK cells were cultured within type I collagen or Matrigel to obtain multicellular cysts, and tubule formation was induced in collagen gels with hepatocyte growth factor. Immunofluorescent staining of these structures using antibodies against galectin-3 and other cell-surface domain markers was carried out either in situ or on cryosections and was visualised by confocal and conventional epifluorescence microscopy. Our results show that MDCK cells suspended in hydrated collagen gels or Matrigel exhibit differential and polarised galectin-3 expression on the baso-lateral surface domains of cells lining the cysts. The lectin is colocalised with laminin on the basal surface. In tubule-forming cysts, galectin-3 is excluded from the initial spikes and the progressing tips of the tubules although its basolateral expression on the cyst body remains. Galectin-3 added exogenously to cultures, as well as antibodies against laminin subunits and integrin beta 1 subunit, exerted an inhibitory effect on cyst enlargement of MDCK cells in 3-D Matrigel while galectin-3-specific antibodies could promote this process. The results suggest that galectin-3 exerts its effect on MDCK cells in a three-dimensional environment through modulation of both cell-cell and cell-substratum adhesions, and the interplay between these adhesions is important in the growth of multicellular aggregates and extensions occurring during normal kidney tubulogenesis.

Characterization of Human Anulus Fibrosus– and Nucleus Pulposus–Derived Cells During Monolayer Expansion and in Hydrogel Cultures

2014 ◽  
Vol 5 ◽  
pp. BTRI.S13604 ◽  
Author(s):  
Gundula Schulze-Tanzil ◽  
Marion Lemke ◽  
Carola Meier ◽  
Wolfgang Ertel ◽  
Benjamin Kohl ◽  
...  
Keyword(s):  
Nucleus Pulposus ◽  
Cell Types ◽  
Type I ◽  
Collagen Gels ◽  
Anulus Fibrosus ◽  
Tenascin C ◽  
In Situ Conditions ◽  
Extracellular Matrix Components

In vitro-expanded intervertebral disc (IVD) cells could be a source for disc repair. However, IVD cell characterization still remains challenging and is demanded to detect phenotypical shifts. Therefore, the aim of the present study was to determine IVD cell expression profile during two- and three-dimensional culturing in direct comparison to in situ conditions. Human IVD tissue was analyzed immunohistologically and anulus fibrosus (AF) and nucleus pulposus (NP) cells were isolated and characterized for cytoskeletal architecture and expression of typical markers (type I, II, and III collagens, aggrecan, decorin, cartilage oligomeric protein, the chondrogenic transcription factor sox9, the tendon markers scleraxis and tenascin C) during 6 monolayer passages using real-time detection polymerase chain reaction and/or immunolabellings. Cells were introduced in alginate and collagen hydrogels and cell morphology and viablility was determined after 7 days. In addition to typical extracellular matrix components, IVD tissue and isolated cells revealed scleraxis expression. In early passages of cell expansion, genes of sox9, scleraxis, and the small proteoglycan decorin were expressed higher, but type I and III collagen genes were expressed lower in NP cells compared with AF cells. However, in passage 6, actin stress fibers increased and the expression levels of sox9 were nearly similar in NP and AF cells. The immunolabeling indicated that the fibroblast marker tenascin C could only be detected in vitro in both cell types but not in situ. Decorin protein expression decreased in both cell types in vitro in passage 6. IVD cells survived in both hydrogel cultures, and some cells elongated in collagen gels.

Type I collagen permits invasive behaviour by retinal pigmented epithelial cells in vitro

1987 ◽  
Vol 87 (3) ◽  
pp. 399-409
Author(s):  
R.J. Docherty ◽  
J.V. Forrester ◽  
J.M. Lackie
Keyword(s):  
Epithelial Cells ◽  
Type I Collagen ◽  
Phenotypic Expression ◽  
Type I ◽  
Collagen Gels ◽  
Rpe Cells ◽  
Dome Formation ◽  
Type Iv

Epithelial cells cultured on type I collagen gels adopt a typical apical—basal polarity and undergo differentiation. We have compared the behaviour of chick embryo retinal pigmented epithelial (RPE) cells on collagen and on plastic with and without gelatin coats. RPE cell proliferation was similar on all three substrata, and post-confluent cultures exhibited multilayering. On plastic and gelatin-coated plastic, dome formation, typical of transporting epithelia, occurred. On type I collagen gels, however, dome formation did not occur, but rather invasion of the gel matrix by cords of epithelial cells took place. In contrast, invasive behaviour of the cells was markedly reduced on type IV coated collagen gels, particularly in the presence of laminin. These results illustrate the prominent role of the extracellular matrix on phenotypic expression by RPE cells and may represent a more general phenomenon.

Desmopressin (DDAVP) Enhances Platelet Adhesion to the Extracellular Matrix of Cultured Human Endothelial Cells through Increased Expression of Tissue Factor

1997 ◽  
Vol 77 (05) ◽  
pp. 0975-0980 ◽  
Author(s):  
Angel Gálvez ◽  
Goretti Gómez-Ortiz ◽  
Maribel Díaz-Ricart ◽  
Ginés Escolar ◽  
Rogelio González-Sarmiento ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Tissue Factor ◽  
Platelet Adhesion ◽  
Umbilical Vein ◽  
Procoagulant Activity ◽  
Experimental Conditions ◽  
Chromogenic Assay

SummaryThe effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DD A VP-treated ECMs were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin®, 20 U/ml). Perfusions were run for 5 min at a shear rate of 800 s1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p <0.05 and p <0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p <0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.

scholarly journals Patterns and Relevance of Langerhans Islet Invasion in Pancreatic Cancer

Cancers ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 249
Author(s):  
Ruediger Goess ◽  
Ayse Ceren Mutgan ◽  
Umut Çalışan ◽  
Yusuf Ceyhun Erdoğan ◽  
Lei Ren ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Pancreatic Cancer ◽  
Cancer Cells ◽  
Tumor Cells ◽  
Islet Cells ◽  
Human Pancreatic Cancer ◽  
Type I ◽  
Type Iv ◽  
Pancreatic Cancer Cells

Background: Pancreatic cancer‐associated diabetes mellitus (PC‐DM) is present in most patients with pancreatic cancer, but its pathogenesis remains poorly understood. Therefore, we aimed to characterize tumor infiltration in Langerhans islets in pancreatic cancer and determine its clinical relevance. Methods: Langerhans islet invasion was systematically analyzed in 68 patientswith pancreatic ductal adenocarcinoma (PDAC) using histopathological examination and 3D in vitro migration assays were performed to assess chemoattraction of pancreatic cancer cells to isletcells. Results: Langerhans islet invasion was present in all patients. We found four different patterns of islet invasion: (Type I) peri‐insular invasion with tumor cells directly touching the boundary, but not penetrating the islet; (Type II) endo‐insular invasion with tumor cells inside the round islet; (Type III) distorted islet structure with complete loss of the round islet morphology; and (Type IV)adjacent cancer and islet cells with solitary islet cells encountered adjacent to cancer cells. Pancreatic cancer cells did not exhibit any chemoattraction to islet cells in 3D assays in vitro. Further, there was no clinical correlation of islet invasion using the novel Islet Invasion Severity Score (IISS), which includes all invasion patterns with the occurrence of diabetes mellitus. However, Type IV islet invasion was related to worsened overall survival in our cohort. Conclusions: We systematically analyzed, for the first time, islet invasion in human pancreatic cancer. Four different main patterns of islet invasion were identified. Diabetes mellitus was not related to islet invasion. However, moreresearch on this prevailing feature of pancreatic cancer is needed to better understand underlying principles.

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