Substratum-induced stress fiber assembly in vascular endothelial cells during spreading in vitro

1990 ◽  
Vol 95 (3) ◽  
pp. 507-520
Author(s):  
J.C. Yost ◽  
I.M. Herman
Keyword(s):  
Type Iv Collagen ◽  
Vascular Endothelial Cells ◽  
Subcellular Fractionation ◽  
Stress Fiber ◽  
Cytoskeletal Proteins ◽  
Acid Extraction ◽  
Fiber Assembly ◽  
Type Iv ◽  
Collagen Substratum

We tested whether aortic endothelial cell (EC)-synthesized substrata, which modulate smooth muscle cell proliferation and EC motility following injury, could influence EC actin cytoskeleton and spreading in vitro. A partial characterization of the substrata indicates that the substratum prepared by deoxycholic acid extraction (DOC-derived substratum) is enriched with fibronectin and type IV collagen. Substratum prepared by removal of the intact monolayer with 20 mM EGTA in PBS (EGTA-derived substratum) contains fibronectin and heparan sulfate proteoglycan, but no type IV collagen. Morphometric analyses were performed on fixed and cytoskeletal antibody treated EC in order to quantitate the extent of spreading and stress fiber (SF) assembly. Compared to plastic, the DOC-derived substratum, a collagenase-treated DOC-derived substratum (CT-DOC-derived substratum) and the EGTA-derived substratum promote EC spreading 2.3-, 2.9- and 1.7-fold, respectively. In addition, there are 4.2-, 4.1- and 2.0-fold more SF on DOC-, CT-DOC- and EGTA-derived substrata, respectively, when compared to plastic. Subcellular fractionation and immunoprecipitation of cytoskeletal proteins from metabolically labeled EC were performed prior to electrophoresis and fluorography. The DOC-derived substratum increases immunoprecipitable actin and myosin 3- to 4.5-fold in both fractions compared to the EGTA-derived substratum and plastic. Collagenase treatment of the DOC-derived substratum partially inhibits this increase. Cycloheximide treatment prevents the rise in soluble actin and myosin as well as causing a reduction in SF number by 1/2 on the DOC-derived substratum and 2/3 on CT-DOC-derived substratum. We propose that fibronectin-collagen interactions are, in part, responsible for inducing endothelial synthesis of cytoskeletal proteins required for SF assembly. This substratum-induced actin-cytoskeletal reorganization facilitates EC spreading in vitro.

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 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.

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 Chemistry of collagen cross-links: glucose-mediated covalent cross-linking of type-IV collagen in lens capsules

1993 ◽  
Vol 296 (2) ◽  
pp. 489-496 ◽  
Author(s):  
A J Bailey ◽  
T J Sims ◽  
N C Avery ◽  
C A Miles
Keyword(s):  
Type Iv Collagen ◽  
Cross Linking ◽  
Mechanical And Thermal Properties ◽  
Maximum Strain ◽  
Scanning Calorimetry ◽  
Melting Temperatures ◽  
Type Iv ◽  
Collagen Molecules ◽  
Cross Links

The incubation of lens capsules with glucose in vitro resulted in changes in the mechanical and thermal properties of type-IV collagen consistent with increased cross-linking. Differential scanning calorimetry (d.s.c.) of fresh lens capsules showed two major peaks at melting temperatures Tm 1 and Tm 2 at approx. 54 degrees C and 90 degrees C, which can be attributed to the denaturation of the triple helix and 7S domains respectively. Glycosylation of lens capsules in vitro for 24 weeks caused an increase in Tm 1 from 54 degrees C to 61 degrees C, while non-glycosylated, control incubated capsules increased to a Tm 1 of 57 degrees C. The higher temperature required to denature the type-IV collagen after incubation in vitro suggested increased intermolecular cross-linking. Glycosylated lens capsules were more brittle than fresh samples, breaking at a maximum strain of 36.8 +/- 1.8% compared with 75.6 +/- 6.3% for the fresh samples. The stress at maximum strain (or ‘strength’) was dramatically reduced from 12.0 to 4.7 N.mm.mg-1 after glycosylation in vitro. The increased constraints within the system leading to loss of strength and increased brittleness suggested not only the presence of more cross-links but a difference in the location of these cross-links compared with the natural lysyl-aldehyde-derived cross-links. The chemical nature of the fluorescent glucose-derived cross-link following glycosylation was determined as pentosidine, at a concentration of 1 pentosidine molecule per 600 collagen molecules after 24 weeks incubation. Pentosidine was also determined in the lens capsules obtained from uncontrolled diabetics at a level of about 1 per 100 collagen molecules. The concentration of these pentosidine cross-links is far too small to account for the observed changes in the thermal and mechanical properties following incubation in vitro, clearly indicating that another as yet undefined, but apparently more important cross-linking mechanism mediated by glucose is taking place.

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 Inhibition of laminin self-assembly and interaction with type IV collagen by antibodies to the terminal domain of the long arm.

1986 ◽  
Vol 103 (5) ◽  
pp. 1689-1697 ◽  
Author(s):  
A S Charonis ◽  
E C Tsilibary ◽  
T Saku ◽  
H Furthmayr
Keyword(s):  
Self Assembly ◽  
Binding Sites ◽  
Type Iv Collagen ◽  
Specific Interaction ◽  
Basement Membranes ◽  
Complex Domain ◽  
Peptide Fragment ◽  
Type Iv ◽  
Collagen Molecules

Laminin is a major glycoprotein of the basement membrane. Although its precise localization and orientation within this structure is unknown, it is presumably anchored to other macromolecules such as type IV collagen or proteoheparan sulfate. In vitro, laminin has the ability to self-assemble and to bind to type IV collagen molecules at distinct sites. To identify more precisely the domains of the complex, cross-shaped laminin molecule that are involved in these interactions, images of laminin-laminin dimers and laminin-type IV collagen complexes obtained by the rotary shadowing method were analyzed. We observed that the complex domain at the end of the long arm of laminin is predominantly involved in these interactions. By using Fab fragments of antibodies specific for a peptide fragment derived from this complex domain, it is shown that laminin self-assembly is inhibited in their presence, as measured by turbidity and by electron microscopy. In addition, these antibodies inhibit the specific interaction of laminin with type IV collagen. These data suggest that the complex domain at the end of the long arm of laminin contains binding sites of potential importance for the assembly of basement membranes.

In vitro and post-transplantation differentiation of human keratinocytes grown on the human type IV collagen film of a bilayered dermal substitute

1991 ◽  
Vol 193 (2) ◽  
pp. 310-319 ◽  
Author(s):  
Estelle Tinois ◽  
Jerome Tiollier ◽  
Martine Gaucherand ◽  
Henri Dumas ◽  
Michel Tardy ◽  
...  
Keyword(s):  
Type Iv Collagen ◽  
Human Keratinocytes ◽  
Dermal Substitute ◽  
Post Transplantation ◽  
Collagen Film ◽  
Human Type ◽  
Type Iv

Regulation of the RhoA pathway in human endothelial cell spreading on type IV collagen: role of calcium influx

1999 ◽  
Vol 112 (19) ◽  
pp. 3205-3213 ◽  
Author(s):  
L. Masiero ◽  
K.A. Lapidos ◽  
I. Ambudkar ◽  
E.C. Kohn
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Focal Adhesion ◽  
Type Iv Collagen ◽  
Cell Spreading ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Stress Fibers ◽  
Type Iv ◽  
Stress Fiber Formation

We have shown that nonvoltage-operated Ca(2+) entry regulates human umbilical vein endothelial cell adhesion, migration, and proliferation on type IV collagen. We now demonstrate a requirement for Ca(2+) influx for activation of the RhoA pathway during endothelial cell spreading on type IV collagen. Reorganization of actin into stress fibers was complete when the cells where fully spread at 90 minutes. No actin organization into stress fibers was seen in endothelial cells plated on type I collagen, indicating a permissive effect of type IV collagen. CAI, a blocker of nonvoltage-operated Ca(2+) channels, prevented development of stress fiber formation in endothelial cells on type IV collagen. This permissive effect was augmented by Ca(2+) influx, as stimulated by 0. 5 microM thapsigargin or 0.1 microM ionomycin, yielding faster development of actin stress fibers. Ca(2+) influx and actin rearrangement in response to thapsigargin and ionomycin were abrogated by CAI. Activated, membrane-bound RhoA is a substrate for C3 exoenzyme which ADP-ribosylates and inactivates RhoA, preventing actin stress fiber formation. Pretreatment of endothelial cells with C3 exoenzyme prevented basal and thapsigargin-augmented stress fiber formation. While regulation of Ca(2+) influx did not alter RhoA translocation, it reduced in vitro ADP-ribosylation of RhoA (P(2)<0. 05), suggesting Ca(2+) influx is needed for RhoA activation during spreading on type IV collagen; no Ca(2+) regulated change in RhoA was seen in HUVECs spreading on type I collagen matrix. Blockade of Ca(2+) influx of HUVEC spread on type IV collagen also reduced tyrosine phosphorylation of p190Rho-GAP and blocked thapsigargin-enhanced binding of p190Rho-GAP to focal adhesion kinase. Thus, Ca(2+) influx is necessary for RhoA activation and for linkage of the RhoA/stress fiber cascade to the focal adhesion/focal adhesion kinase pathway during human umbilical vein endothelial cell spreading on type IV collagen.

scholarly journals Multiple mechanisms of dissociated epidermal cell spreading.

1983 ◽  
Vol 96 (1) ◽  
pp. 63-67 ◽  
Author(s):  
K S Stenn ◽  
J A Madri ◽  
T Tinghitella ◽  
V P Terranova
Keyword(s):  
Serum Albumin ◽  
Epidermal Cell ◽  
Type Iv Collagen ◽  
Cell Spreading ◽  
Specific Protein ◽  
Epidermal Cells ◽  
Type Iv ◽  
Basement Membrane Protein ◽  
Specific Proteins

To test the possibility that epidermal cells use a common basement membrane protein whenever they spread, in vitro experiments were conducted using trypsin-dissociated guinea pig epidermal cells and the following proteins: human serum, bovine serum albumin, serum fibronectin, Type IV collagen, laminin, and epibolin (a recently described serum glycoprotein which supports epidermal cell spreading; Stenn, K.S., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:6907.). When the cells were added to media containing the specific proteins, all the tested proteins, except for serum albumin, supported cell spreading. Added to protein-coated substrates in defined media, the cells spread on fibronectin, epibolin, and laminin-Type IV collagen, but not on albumin or whole serum. In none of these experiments were the results qualitatively affected by the presence of cycloheximide. Antibodies to a specific protein blocked cell spreading on that protein but not on the other active proteins, e.g. whereas antibodies to epibolin blocked cell spreading on epibolin, they did not affect spreading on fibronectin, collagen, or laminin. In a second assay in which the cells were allowed to adhere to tissue culture plastic before the protein-containing medium was added, the cells spread only if the medium contained epibolin. Moreover, under these conditions the spreading activity of whole serum and plasma was neutralized by antiepibolin antibodies. These results support the conclusion that dissociated epidermal cells possess multiple spreading modes which depend, in part, on the proteins of the substrate, proteins of the medium, and the sequence of cell adhesion and protein exposure.

scholarly journals Retraction Note: Type IV collagen α1-chain noncollagenous domain blocks MMP-2 activation both in-vitro and in-vivo

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Yakkanti Akul Sudhakar ◽  
Raj Kumar Verma ◽  
Smita C. Pawar
Keyword(s):  
Type Iv Collagen ◽  
Link Type ◽  
Type Iv

Editor's Note: this Article has been retracted; the Retraction Note is available at https://www.nature.com/articles/s41598-020-76500-9

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