Extracellular matrix modulation of endothelial cell shape and motility following injury in vitro

1985 ◽  
Vol 73 (1) ◽  
pp. 19-32
Author(s):  
W.C. Young ◽  
I.M. Herman
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Fluorescence Microscopy ◽  
Type Iv Collagen ◽  
Type I ◽  
Post Injury ◽  
Type Iv ◽  
Interstitial Collagens

We utilized fluorescence microscopy and affinity-purified antibodies to probe the form and function of cytoplasmic actin in endothelial cells (EC) recovering from injury and grown on extracellular matrices in vitro. Bovine aortic EC were seeded onto glass microscope coverslips that had been coated with either BSA, fibronectin, type I and III (interstitial) collagens, type IV (basement membrane) collagen or gelatin. After EC that had been grown on glass, glass-BSA or extracellular matrix-coated coverslips reached confluence, a 300–400 micron zone of cells was mechanically removed to stimulate EC migration and proliferation. Post-injury EC movements were monitored with time-lapse, phase-contrast videomicrography before fixation for actin localization with fluorescence microscopy using affinity-purified antibodies. We found that the number of stress fibres within EC was inversely proportional to the rate of movement; and, the rates of movement for EC grown on glass or glass-BSA were approximately eight times faster than EC grown on gelatin or type IV collagen (X velocity = 0.5 micron/min versus 0.06 micron/min). EC movements on fibronectin and interstitial collagens were similar (X velocity = 0.2 micron/min). These results suggest that extracellular matrix molecules modulate EC stress fibre expression, thereby producing alterations in the cytoskeleton and the resultant EC movements that follow injury in vitro. Moreover, the induction of stress fibres in the presence of basement membrane (type IV) collagen may explain the failure of aortic EC to migrate and repopulate wounded regions of intima during atherogenesis in vivo.

scholarly journals Shift in collagen type as an early response to induction of the metanephric mesenchyme.

1981 ◽  
Vol 89 (2) ◽  
pp. 276-283 ◽  
Author(s):  
P Ekblom ◽  
E Lehtonen ◽  
L Saxén ◽  
R Timpl
Keyword(s):  
Basal Lamina ◽  
Type Iv Collagen ◽  
Early Response ◽  
Type I ◽  
Metanephric Mesenchyme ◽  
Type Iv ◽  
Interstitial Collagens ◽  
Membrane Components

Conversion of the nephrogenic mesenchyme into epithelial tubules requires an inductive stimulus from the ureter bud. Here we show with immunofluorescence techniques that the undifferentiated mesenchyme before induction expresses uniformly type I and type III collagens. Induction both in vivo and in vitro leads to a loss of these proteins and to the appearance of basement membrane components including type IV collagen. This change correlates both spatially and temporally with the determination of the mesenchyme and precedes and morphological events. During morphogenesis, type IV collagen concentrates at the borders of the developing tubular structures where, by electron microscopy, a thin, often discontinuous basal lamina was seen to cover the first pretubular cell aggregates. Subsequently, the differentiating tubules were surrounded by a well-developed basal lamina. No loss of the interstitial collagens was seen in the metanephric mesenchyme when brought into contact with noninducing tissues or when cultured alone. Similar observations were made with nonnephrogenic mesenchyme (salivary, lung) when exposed to various heterotypic tissues known to induce tubules in the nephrogenic mesenchyme. The sequential shift in the composition of the extracellular matrix from an interstitial, mesenchymal type to a differentiated, epithelial type is so far the first detectable response of the nephrogenic mesenchyme to the tubule-inducing signal.

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

1981 ◽  
Vol 91 (1) ◽  
pp. 1-10 ◽  
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.

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

1985 ◽  
Vol 101 (4) ◽  
pp. 1175-1181 ◽  
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.

scholarly journals Abnormal Mucosal Extracellular Matrix Deposition Is Associated with Increased TGF-β Receptor-expressing Mesenchymal Cells in a Mouse Model of Colitis

2003 ◽  
Vol 51 (9) ◽  
pp. 1177-1189 ◽  
Author(s):  
Christine V. Whiting ◽  
John F. Tarlton ◽  
Michael Bailey ◽  
Clare L. Morgan ◽  
Paul W. Bland
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Mouse Model ◽  
Type Iv Collagen ◽  
Type I Collagen ◽  
Transforming Growth Factor ◽  
Mesenchymal Cells ◽  
Type I ◽  
Matrix Deposition ◽  
Type Iv

Transforming growth factor-β (TGF-β) depresses mucosal inflammation and upregulates extracellular matrix (ECM) deposition. We analyzed TGF-β receptors RI and RII as well as ECM components using the CD4+ T-cell-transplanted SCID mouse model of colitis. The principal change in colitis was an increased proportion of TGF-β RII+ mucosal mesenchymal cells, predominantly α-smooth muscle actin (SMA)+ myofibroblasts, co-expressing vimentin and basement membrane proteins, but not type I collagen. TGF-β RII+ SMA− fibroblasts producing type I collagen were also increased, particularly in areas of infiltration and in ulcers. Type IV collagen and laminin were distributed throughout the gut lamina propria in disease but were restricted to the basement membrane in controls. In areas of severe epithelial damage, type IV collagen was lost and increased type I collagen was observed. To examine ECM production by these cells, mucosal mesenchymal cells were isolated. Cultured cells exhibited a similar phenotype and matrix profile to those of in vivo cells. The data suggested that there were at least two populations of mesenchymal cells responsible for ECM synthesis in the mucosa and that ligation of TGF-β receptors on these cells resulted in the disordered and increased ECM production observed in colitic mucosa.

scholarly journals Extracellular matrix of the human thymus: immunofluorescence studies on frozen sections and cultured epithelial cells.

1985 ◽  
Vol 33 (7) ◽  
pp. 655-664 ◽  
Author(s):  
S Berrih ◽  
W Savino ◽  
S Cohen
Keyword(s):  
Extracellular Matrix ◽  
Epithelial Cells ◽  
Type Iv Collagen ◽  
Type I Collagen ◽  
Basement Membranes ◽  
Thymic Epithelial Cells ◽  
Perivascular Spaces ◽  
Type I ◽  
Type Iv

The immunohistochemical detection of elements of the human thymic extracellular matrix in situ and in vitro is described. In the normal thymus, the intracapsular and intraseptal fibers were strongly labeled by anti-type I collagen antiserum. Basement membranes bordering the capsule, septae, and perivascular spaces were intensely stained by anti-type IV collagen, anti-fibronectin, and anti-laminin sera. In hyperplastic myasthenia gravis thymuses, the major changes consisted of discontinuities of the basement membrane adjacent to clusters of epithelial (keratin-containing) cells, among which an unusual connective framework (densely labeled by all the antisera) was observed. In vitro, most epithelial cells were strongly labeled by antifibronectin serum and to a lesser extent by the anti-type IV collagen and anti-laminin sera. In addition, fibronectin, laminin, and type IV collagen were detected in the intercellular spaces bordering the epithelial cells in culture. Results show that thymic epithelial cells participate in the synthesis of extracellular matrix elements, which as a result of their localization and influence on epithelial cell growth, should be regarded as constitutive components of the thymic microenvironment.

scholarly journals Characterization of the Hemorrhagic Reaction Caused by Vibrio vulnificus Metalloprotease, a Member of the Thermolysin Family

1998 ◽  
Vol 66 (10) ◽  
pp. 4851-4855 ◽  
Author(s):  
Shin-ichi Miyoshi ◽  
Hiromi Nakazawa ◽  
Koji Kawata ◽  
Ken-ichi Tomochika ◽  
Kazuo Tobe ◽  
...  
Keyword(s):  
Basement Membrane ◽  
Type Iv Collagen ◽  
Vibrio Vulnificus ◽  
Vascular Endothelial Cells ◽  
Human Pathogen ◽  
Membrane Breakdown ◽  
Type Iv ◽  
Hemorrhagic Activity

ABSTRACT Vibrio vulnificus is an opportunistic human pathogen causing wound infections and septicemia, characterized by hemorrhagic and edematous damage to the skin. This human pathogen secretes a metalloprotease (V. vulnificus protease [VVP]) as an important virulence determinant. When several bacterial metalloproteases including VVP were injected intradermally into dorsal skin, VVP showed the greatest hemorrhagic activity. The level of the in vivo hemorrhagic activity of the bacterial metalloproteases was significantly correlated with that of the in vitro proteolytic activity for the reconstituted basement membrane gel. Of two major basement membrane components (laminin and type IV collagen), only type IV collagen was easily digested by VVP. Additionally, the immunoglobulin G antibody against type IV collagen, but not against laminin, showed sufficient protection against the hemorrhagic reaction caused by VVP. Capillary vessels are known to be stabilized by binding of the basal surface of vascular endothelial cells to the basement membrane. Therefore, specific degradation of type IV collagen may cause destruction of the basement membrane, breakdown of capillary vessels, and leakage of blood components including erythrocytes.

Megakaryocytes Contribute To The Establishment Of The Bone Marrow Environment By Expressing Extracellular Matrix proteins

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3695-3695
Author(s):  
Alessandro Malara ◽  
Cristian Gruppi ◽  
Manuela Currao ◽  
Alessandra Balduini
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Type Iv Collagen ◽  
Extracellular Matrix Component ◽  
Matrix Component ◽  
Type Iv ◽  
Extracellular Matrix Components ◽  
Matrix Components

Abstract Introduction the bone marrow microenvironment consists of various types of cells and their secreted extracellular matrix components that surround capillary-venous sinusoids, and plays a key role in the regulation of hematopoiesis. In general, extracellular matrix components interact with each other to form a structural framework that supports tissue organization and positional cues that regulate cellular processes. Megakaryocytes are rare cells in the bone marrow and, besides platelet release, growing evidences attribute new functions to these cells in the generation and maintenance of the bone marrow cell niche. Recent evidences, by our group, demonstrated that megakaryocytes are involved in matrix deposition and remodeling, as demonstrated by their role in fibronectin fibrillogenesis and the expression of matrix cross-linking enzymes, such as factor XIIIa, essential in the dynamic of megakaryocyte-matrix component interactions. Interestingly, individual extracellular matrix components were demonstrated to play a role in the regulation of megakaryocytes development in vitro. Fibronectin was shown to regulate megakaryocyte maturation and proplatelet extension, while type III and type IV collagens were demonstrated to support proplatelet formation in vitro. In contrast, type I collagen is an important physiological inhibitor of platelet release in vitro. However, little is known about the exact localization as well as function of these matrix components in vivo. Results in this work we have analyzed the spatial distribution of megakaryocytes and extracellular matrix components by immunofluorescence in murine femur sections. We found that megakaryocytes were predominantly located in the femur diaphysis with only 20% of megakaryocytes within 50μm from the endosteal surface and more than 80% of megakaryocytes located less than 50 μm from a sinusoid. Correlation between megakaryocyte distance from sinusoids and dimension suggested a gradient of maturing megakaryocytes towards the vascular niche. Next, we deciphered bone marrow extracellular matrix component composition by western blotting and mapped the location in situ of different collagens (I, III, IV, VI) and glycoproteins (fibronectin, laminin). We found that all these proteins were differently located in the endosteal and sinusoidal districts supporting the concept that regulation of hemopoiesis, in the bone marrow, may also depend from matrix distribution. Further, we showed, for the first time, that megakaryocytes were surrounded by a pericellular matrix mainly composed of fibronectin, laminin and type IV collagen. Interestingly, these three proteins were also demonstrated to promote thrombopoietin-dependent megakaryocyte differentiation in in vitro cultures of bone marrow hemopoietic progenitor cells. Finally, fibronectin, laminin and type IV collagen were also demonstrated to be expressed and synthesized by differentiated megakaryocytes in vitro as demonstrated by PCR and western blotting analysis. Most importantly, megakaryocyte expression of these extracellular matrix components was up-regulated in vivo during bone marrow reconstitution upon drug induced myelosuppression and, at a lesser extent, thrombocytopenia. Conclusions all together these results suggested that megakaryocytes are important extracellular matrix component-producing bone marrow cells and that released extracellular matrix components support megakaryopoiesis and concur to the generation of bone marrow niches. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Basement membrane-like structures containing NTH α1(IV) are formed around the endothelial cell network in a novel in vitro angiogenesis model

2019 ◽  
Vol 317 (2) ◽  
pp. C314-C325
Author(s):  
Yongchol Shin ◽  
Akane Moriya ◽  
Yuta Tohnishi ◽  
Takafumi Watanabe ◽  
Yasutada Imamura
Keyword(s):  
Endothelial Cells ◽  
Basement Membrane ◽  
Blood Vessels ◽  
Type Iv Collagen ◽  
Vascular Endothelial Cells ◽  
Culture Dish ◽  
Cell Network ◽  
Type Iv

Angiogenesis is a process through which new blood vessels are formed by sprouting and elongating from existing blood vessels. Several methods have been used to replicate angiogenesis in vitro, including culturing vascular endothelial cells on Matrigel and coculturing with endothelial cells and fibroblasts. However, the angiogenesis elongation process has not been completely clarified in these models. We therefore propose a new in vitro model of angiogenesis, suitable for observing vascular elongation, by seeding a spheroid cocultured from endothelial cells and fibroblasts into a culture dish. In this model, endothelial cells formed tubular networks elongated from the spheroid with a lumen structure and were connected with tight junctions. A basement membrane (BM)-like structure was observed around the tubular network, similarly to blood vessels in vivo. These results suggested that blood vessel-like structure could be reconstituted in our model. Laminin and type IV collagen, main BM components, were highly localized around the network, along with nontriple helical form of type IV collagen α1-chain [NTH α1(IV)]. In an ascorbic acid-depleted condition, laminin and NTH α1(IV) were observed around the network but not the triple-helical form of type IV collagen and the network was unstable. These results suggest that laminin and NTH α1(IV) are involved in the formation of tubular network and type IV collagen is necessary to stabilize the network.

scholarly journals Extracellular matrix components of the mouse thymus microenvironment: ontogenetic studies and modulation by glucocorticoid hormones.

1991 ◽  
Vol 39 (11) ◽  
pp. 1539-1546 ◽  
Author(s):  
J Lannes-Vieira ◽  
M Dardenne ◽  
W Savino
Keyword(s):  
Extracellular Matrix ◽  
Basement Membrane ◽  
Type I Collagen ◽  
Basement Membranes ◽  
Thymic Epithelial Cell ◽  
Epithelial Cell Line ◽  
Type I ◽  
Extracellular Matrix Components

The present investigation was an ontogenetic study on the distribution of extracellular matrix (ECM) components in the thymic microenvironment of C57BL/6 mice (comprising young and old adults and developing embryos) and NZB mice. In addition, we evaluated the in vivo and in vitro influence of hydrocortisone treatment on basement membrane protein production by a thymic epithelial cell line. In young normal animals, Type I collagen was restricted to the interstitial spaces of the capsule and septa, where Type IV collagen, fibronectin, and laminin could be detected in the basement membranes. In addition, fibronectin-containing fibers were seen within the medulla of the thymic lobules. The ECM distribution pattern in the developing embryos was distinct from that observed in adults, since a fine meshwork of basement membrane-containing proteins was clearly seen throughout the parenchyma. Moreover, aging normal and NZB mice exhibited a denser ECM pattern than young adult normal animals. Treatment with hydrocortisone, both in vivo and in vitro, resulted in enhancement of ECM expression, detected in mice as early as 2 hr post injection and lasting for several days. Considering that the fluctuations of ECM expression parallel important events in thymocyte differentiation, we discuss the possibility that the two phenomena may be associated.

The extracellular matrix of human amniotic epithelium: ultrastructure, composition and deposition

1985 ◽  
Vol 79 (1) ◽  
pp. 119-136
Author(s):  
J.D. Aplin ◽  
S. Campbell ◽  
T.D. Allen
Keyword(s):  
Extracellular Matrix ◽  
Epithelial Cells ◽  
Basal Lamina ◽  
Basal Cell ◽  
Type I ◽  
Type Iv ◽  
Cell Processes ◽  
Complete Matrix

Ultrastructural comparisons have been made between human amnion extracellular matrix in tissue and cell culture. Immunochemical analysis of matrix deposited by monolayers of cultured amnion epithelial cells has also been undertaken. The basal cell surfaces are highly invaginated with an associated basal lamina that is more electron dense at the distal tips of basal cell processes where hemidesmosomes are frequent. Immediately below the lamina densa is a zone rich in collagen bundles. In the underlying stroma two types of fibril predominate, one striated of 50 nm diameter and one of 18 nm diameter. The observations suggest that at gestational term the epithelial cells are still active in the production of matrix. Secretion appears to occur into invaginations in the basal cell surface where a loosely organized mixture of stromal-type and basal laminal-type aggregates is formed. In culture on plastic, cells also deposit a mixture of basal laminal (type IV collagen + laminin) and stromal (collagens type I + III) components as well as fibronectin. However, segregation into a true basal lamina with underlying stroma does not occur in vitro, suggesting the need for an organized subcellular template to complete matrix morphogenesis. The in vitro and in vivo evidence suggest that the epithelium contributes to the subjacent dense collagenous zone as well as to the basal lamina.

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