Adhesion, growth and morphology of human mesothelial cells on extracellular matrix

Human mesothelial cells (HMC) cover a variety of serosal surfaces and have been shown to rest upon an underlying subcellular basement membrane in vivo. Bovine corneal endothelial cells produce an extracellular matrix (ECM) in vitro that mimics HMC subcellular basement membrane and was found to modulate HMC adhesion, morphology and proliferation in vitro. Our results indicated that within minutes after plating, a high percentage (greater than 80%) of HMC firmly attached to ECM. Active cellular migration and subsequent proliferation were observed leading to the formation of a well-organized closely apposed cell monolayer. However, when cells were plated on plastic, the rate of cell attachment was much lower and the proliferative rate of HMC grown on plastic also was strikingly lower (exponential doubling time 4.3 days) than that of cells grown on ECM (exponential doubling time 2.4 days). Cells upon reaching confluency on plastic were markedly enlarged as compared to confluent cells grown on ECM. These observations corroborated differences in final cell density where it was noted that HMC cultured on ECM demonstrated a 10-fold greater final cell density as compared to cells grown on plastic. Results from these studies illustrate the fact that phenotypic expression as well as proliferative responsiveness of HMC can be modulated by adhesive interactions with preformed ECM.

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Cytokeratin expression in rat mesothelial cells in vitro is controlled by the extracellular matrix

Journal of Cell Science ◽

10.1242/jcs.95.1.97 ◽

1990 ◽

Vol 95 (1) ◽

pp. 97-107

Author(s):

A.M. Mackay ◽

R.P. Tracy ◽

J.E. Craighead

Keyword(s):

Extracellular Matrix ◽

Basement Membrane ◽

Intermediate Filament ◽

Culture Conditions ◽

Mesothelial Cells ◽

Intermediate Filament Protein ◽

Cellular Contact ◽

Filament Protein

Rat mesothelial cells co-express vimentin and the simple epithelial cytokeratins. While cytokeratins predominate in situ, under most culture conditions vimentin is the major intermediate filament protein of the cells. This loss of cytokeratin production upon culture can be partly prevented by growing mesothelial cells on a basement membrane matrix. However, the basement membrane-promoted persistence of cytokeratin synthesis is not accompanied by expression of cytokeratin G (no. 19), the major acidic cytokeratin of mesothelium in vivo. While cells grown on plastic establish a prominent juxtanuclear assemblage of tonofilaments, those cultured on basement membrane exhibit cytokeratin filaments which are distributed throughout the cytoplasm and attach to neighboring cells at the plasma membrane. This latter pattern resembles that seen in the intact mesothelium. Intermediate filaments are markers of cellular differentiation, but their roles are obscure. The response of cultured mesothelial cells to different growth substrata supports the hypothesis that intermediate filament synthesis is influenced by cellular contact with the extracellular matrix.

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The influence of the epithelium on palate shelf reorientation

Development ◽

10.1242/dev.88.1.265 ◽

1985 ◽

Vol 88 (1) ◽

pp. 265-279

Author(s):

Robert F. Bulleit ◽

Ernest F. Zimmerman

Keyword(s):

Extracellular Matrix ◽

Wound Healing ◽

Basement Membrane ◽

Soft Palate ◽

Hard Palate ◽

Significant Inhibition ◽

Oral Epithelium ◽

Secondary Palate ◽

Intrinsic Forces

The intrinsic forces necessary for directing the reorientation of the secondary palate appear to reside in the anterior two thirds of the palate or presumptive hard palate. The hard palate could reorient regardless of whether it was intact or separated from the posterior third or presumptive soft palate. The soft palate could only reorient if the palate shelves are left intact. These intrinsic forces, within the hard palate, may be mediated by the mesenchymal cells, their extracellular matrix, or the epithelium surrounding the shelves. This latter possibly was tested by removing the epithelium, from either the presumptive oral or nasal surface followed by measurement of reorientation in vitro. Only after removal of the oral epithelium was a significant inhibition in reorientation observed. The treatment used to remove the epithelium, EDTA and scraping, was shown to remove 41 % of the oral epithelium leaving the majority of the basement membrane intact. The observed inhibition of reorientation did not appear to be a consequence of wound healing. Creation of wounds twice the area that was observed after treatment with EDTA and scraping inhibited reorientation minimally. These results suggest that the epithelium and particularly the anterior oral epithelium plays a major role in the reorientation of the murine secondary palate.

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Interactions between mesoderm cells and the extracellular matrix following gastrulation in the chick embryo

Journal of Cell Science ◽

10.1242/jcs.99.2.431 ◽

1991 ◽

Vol 99 (2) ◽

pp. 431-441

Author(s):

A.J. Brown ◽

E.J. Sanders

Keyword(s):

Extracellular Matrix ◽

Chick Embryo ◽

Basement Membrane ◽

Primitive Streak ◽

Cell Binding ◽

Fab Fragment ◽

Fibronectin Receptor ◽

In Vitro Methods

In the gastrulating chick embryo, the mesoderm cells arise from the epiblast layer by ingression through the linear accumulation of cells called the primitive streak. The mesoderm cells emerge from the streak with a fibroblastic morphology and proceed to move away from the mid-line of the embryo using, as a substratum, the basement membrane of the overlying epiblast and the extracellular matrix. We have investigated the roles of fibronectin and laminin as putative substrata for mesoderm cells using complementary in vivo and in vitro methods. We have microinjected agents into the tissue space adjacent to the primitive streak of living embryos and, after further incubation, we have examined the embryos for perturbation of the mesoderm tissue. These agents were: cell-binding regions from fibronectin (RGDS) and laminin (YIGSR), antibodies to these glycoproteins, and a Fab' fragment of the antibody to fibronectin. We find that RGDS, antibody to fibronectin, and the Fab' fragment cause a decrease in the number of mesoderm cells spread on the basement membrane, and a perturbation of cell shape suggesting locomotory impairment. No such influence was seen with YIGSR or antibodies to laminin. These results were extended using in vitro methods in which mesoderm cells were cultured in fibronectin-free medium on fibronectin or laminin in the presence of various agents. These agents were: RGDS; YIGSR; antibodies to fibronectin, fibronectin receptor, laminin and vitronectin; and a Fab' fragment of the fibronectin antiserum. We find that cell attachment and spreading on fibronectin is impaired by RGDS, antiserum to fibronectin, the Fab' fragment of fibronectin antiserum, and antiserum to fibronectin receptor. The results suggest that although the RGDS site in fibronectin is important, it is probably not the only fibronectin cell-binding site involved in mediating the behaviour of the mesoderm cells. Cells growing on laminin were perturbed by YIGSR, RGDS and antibodies to laminin, suggesting that mesoderm cells are able to recognise at least two sites in the laminin molecule. We conclude that the in vivo dependence of mesoderm cells on fibronectin is confirmed, but that although these cells have the ability to recognise sites in laminin as mediators of attachment and spreading, the in vivo role of this molecule in mesoderm morphogenesis is not yet certain.

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

Kardiologiia ◽

10.18087/cardio.2021.6.n1476 ◽

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.

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Capillary endothelial cell cultures: phenotypic modulation by matrix components.

The Journal of Cell Biology ◽

10.1083/jcb.97.1.153 ◽

1983 ◽

Vol 97 (1) ◽

pp. 153-165 ◽

Cited By ~ 417

Author(s):

J A Madri ◽

S K Williams

Keyword(s):

Endothelial Cells ◽

Basement Membrane ◽

Membrane Surface ◽

Phenotypic Expression ◽

Aortic Endothelial Cells ◽

Capillary Endothelial Cells ◽

Endothelial Cell Cultures ◽

Cell Densities

Capillary endothelial cells of rat epididymal fat pad were isolated and cultured in media conditioned by bovine aortic endothelial cells and substrata consisting of interstitial or basement membrane collagens. When these cells were grown on interstitial collagens they underwent proliferation, formed a continuous cell layer and, if cultured for long periods of time, formed occasional tubelike structures. In contrast, when these cells were grown on basement membrane collagens, they did not proliferate but did aggregate and form tubelike structures at early culture times. In addition, cells grown on basement membrane substrata expressed more basement membrane constituents as compared with cells grown on interstitial matrices when assayed by immunoperoxidase methods and quantitated by enzyme-linked immunosorbent inhibition assays. Furthermore, when cells were grown on either side of washed, acellular amnionic membranes their phenotypes were markedly different. On the basement membrane surface they adhered, spread, and formed tubelike structures but did not migrate through the basement membrane. In contrast, when seeded on the stromal surface, these cells were observed to proliferate and migrate into the stromal aspect of the amnion and ultimately formed tubelike structures at high cell densities at longer culture periods (21 d). Thus, connective tissue components play important roles in regulating the phenotypic expression of capillary endothelial cells in vitro, and similar roles of the collagenous components of the extracellular matrix may exist in vivo following injury and during angiogenesis. Furthermore, the culture systems outlined here may be of use in the further study of differentiated, organized capillary endothelial cells in culture.

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Formation of basement membranes in the embryonic kidney: an immunohistological study

The Journal of Cell Biology ◽

10.1083/jcb.91.1.1 ◽

1981 ◽

Vol 91 (1) ◽

pp. 1-10 ◽

Cited By ~ 180

Author(s):

P Ekblom

Keyword(s):

Extracellular Matrix ◽

Basement Membrane ◽

Type Iv Collagen ◽

Basement Membranes ◽

Glomerular Endothelial Cell ◽

Type Iv ◽

Endothelial Cell Differentiation ◽

Inductive Interaction ◽

Immunohistological Study

Specific antibodies to laminin, type IV collagen, basement-membrane proteoglycan, and fibronectin have been used in immunofluorescence microscopy to study the development of basement membranes of the embryonic kidney. Kidney tubules are known to form from the nephrogenic mesenchyme as a result of an inductive tissue interaction. This involves a change in the composition of the extracellular matrix. The undifferentiated mesenchyme expresses in the composition of the extracellular matrix. The undifferentiated mesenchyme expresses fibronectin but no detectable laminin, type IV collagen, or basement-membrane proteoglycan. During the inductive interaction, basement-membrane specific components (laminin, type IV collagen, basement membrane proteoglycan) become detectable in the induced area, whereas fibronectin is lost. While the differentiation to epithelial cells of the kidney requires an inductive interaction, the development of the vasculature seems to involve an ingrowth of cells which throughout development deposits basement-membrane specific components, as well as fibronectin. These cells form the endothelium and possibly also the mesangium of the glomerulus, and contribute to the formation of the glomerular basement membrane. An analysis of differentiation of the kidney mesenchyme in vitro in the absence of circulation supports these conclusions. Because a continuity with vasculature is required for glomerular endothelial cell differentiation, it is possible that these cells are derived from outside vasculature.

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Cells that emerge from embryonic explants produce fibers of type IV collagen.

The Journal of Cell Biology ◽

10.1083/jcb.101.4.1175 ◽

1985 ◽

Vol 101 (4) ◽

pp. 1175-1181 ◽

Cited By ~ 16

Author(s):

J M Chen ◽

C D Little

Keyword(s):

Extracellular Matrix ◽

Epithelial Cells ◽

Basement Membrane ◽

Type Iv Collagen ◽

Polyclonal Antibodies ◽

Mouse Lung ◽

Embryonic Mouse ◽

Type Iv ◽

Collagen Substratum

Double immunofluorescence staining experiments designed to examine the synthesis and deposition of collagen types I and IV in cultured explants of embryonic mouse lung revealed the presence of connective tissue-like fibers that were immunoreactive with anti-type IV collagen antibodies. This observation is contrary to the widely accepted belief that type IV collagen is found only in sheet-like arrangements beneath epithelia or as a sheath-like layer enveloping bundles of nerve or muscle cells. The extracellular matrix produced by cells that migrate from embryonic mouse lung rudiments in vitro was examined by double indirect immunofluorescence microscopy. Affinity-purified monospecific polyclonal antibodies were used to examine cells after growth on glass or native collagen substrata. The data show that embryonic mesenchymal cells can produce organized fibers of type IV collagen that are not contained within a basement membrane, and that embryonic epithelial cells deposit fibers and strands of type IV collagen beneath their basal surface when grown on glass; however, when grown on a rat tail collagen substratum the epithelial cells produce a fine meshwork. To our knowledge this work represents the first report that type IV collagen can be organized by cells into a fibrous extracellular matrix that is not a basement membrane.

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Comparison of the ability of basement membranes produced by corneal endothelial and mouse-derived Endodermal PF-HR-9 cells to support the proliferation and differentiation of bovine kidney tubule epithelial cells in vitro.

The Journal of Cell Biology ◽

10.1083/jcb.99.3.947 ◽

1984 ◽

Vol 99 (3) ◽

pp. 947-961 ◽

Cited By ~ 64

Author(s):

D Gospodarowicz ◽

J Lepine ◽

S Massoglia ◽

I Wood

Keyword(s):

Epithelial Cells ◽

Basement Membrane ◽

Cell Monolayer ◽

Basement Membranes ◽

Low Density ◽

Kidney Tubule ◽

Confluent Monolayer ◽

Bovine Kidney ◽

Fibroblastic Cells

The proliferation and morphological differentiation of bovine kidney collecting-tubule epithelial cells has been examined as a function of substrata and plasma factors. Collecting kidney tubule explant maintained in vitro gave rise to two distinct cell populations; one was composed mostly of fibroblastic cells whereas the other was epithelioid (EP cells). The proliferation of fibroblastic cells when exposed to serum-supplemented medium was best expressed when cells were maintained on a basement membrane produced by bovine corneal endothelial cells. This basement membrane has a composition, which in previous studies has been shown to favor the proliferation of mesenchymal cells. In contrast, the proliferation of EP cells was best expressed when cells were maintained on a basement membrane produced by the mouse-derived endodermal cell line PF-HR-9 (HR-9-BM). This basement membrane has a biochemical composition very similar to the basement membrane underlying the kidney tubules. Although the fibroblast confluent monolayer maintained on bovine corneal endothelial cell extracellular matrix did not undergo morphogenesis, the confluent monolayer of EP cells maintained on HR-9-BM shows hemicyst formation, suggesting that they were capable of vectorial fluid transport. They also built a complex three-dimensional kidney tubulelike network. Some tubules became grossly visible and floated into the tissue culture medium, remaining tethered to the cell monolayer at either end of the tubule. On an ultrastructural level, the tubules consisted of cells held together with junctional complexes arranged so as to form a lumen. The smallest lumen were bordered by 2-3 cells, and the largest ones by 8-15 cells. The lumens of the larger tubules did contain granular fibrillar and amorphous debris. Low-density EP cell cultures maintained on HR-9-BM could be induced to proliferate at a rate approaching that of cultures exposed to serum when they were exposed to medium supplemented with high-density lipoprotein (HDL, 750 micrograms protein/ml) and transferrin (50 micrograms/ml). When exposed to HDL concentrations equal or lower than 250 micrograms protein/ml, low-density cultures proliferated at a slow rate and readily formed tubulelike structures. This observation indicates that EP cells do not need to reach confluence to undergo morphogenesis, and that HDL, which in the presence of transferrin supports the cell proliferation, can favor their differentiation into tubulelike structures once its concentration becomes limiting for mitogenesis.

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Elastin synthesis by ligamentum nuchae fibroblasts: effects of culture conditions and extracellular matrix on elastin production.

The Journal of Cell Biology ◽

10.1083/jcb.90.2.332 ◽

1981 ◽

Vol 90 (2) ◽

pp. 332-338 ◽

Cited By ~ 84

Author(s):

R P Mecham ◽

G Lange ◽

J Madaras ◽

B Starcher

Keyword(s):

Extracellular Matrix ◽

Cultured Cells ◽

Elastic Fiber ◽

Calf Serum ◽

Phenotypic Expression ◽

Fetal Tissue ◽

Culture Conditions ◽

Population Doubling

Fetal bovine ligamentum nuchae fibroblasts maintained in culture synthesized soluble elastin but were unable to form the insoluble elastic fiber. Secreted elastin precursors accumulated in culture medium and were measured using a radioimmunoassay for elastin. When elastin production was examined in ligament tissue from fetal calves of various gestational ages, cells from tissue taken during the last trimester of development produced significantly more elastin than did cells from younger fetal tissue, with maximal elastin synthesis occurring shortly before birth. Soluble elastin was detected in ligament cells plated at low density until proliferation began to be density inhibited and the cells became quiescent. Also, soluble elastin production per cell declined with increasing population doubling or with age in culture. Cells grown in the presence of 5% fetal calf serum produced approximately four times as much soluble elastin as cells grown in serum-free medium. The addition of dexamethasone (0.1 microM) and bleomycin (1 microgram/ml) increased soluble elastin production by cultured cells 180% and 50%, respectively, whereas theophylline (5 micrograms/ml) depressed production 50% and antagonized stimulation by dexamethasone. Ascorbate (50 micrograms/ml), soybean trypsin inhibitor (1 mg/ml), insulin (100 microunits/ml), and aminoacetonitrile (50 micrograms/ml) had no effect, but cycloheximide at 10(-4) M completely inhibited soluble elastin production. In contrast to cells in culture, ligament tissue minces (ligament cells surrounded by in vivo extracellular matrix) efficiently incorporated soluble elastin precursors into insoluble, cross-linked elastin. In addition, soluble elastin production per cell (per microgram of DNA) was higher in tissue minces than elastin production by cells maintained on plastic. These results suggest a role for extracellular matrix in formation of the elastic fiber and in stabilizing elastin phenotypic expression by ligament fibroblasts. Fibroblasts from the bovine ligamentum nuchae present an excellent model for in vitro studies of elastin biosynthesis.

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