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 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.

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 Synthesis and physiological activity of heterodimers comprising different splice forms of vascular endothelial growth factor and placenta growth factor

1996 ◽  
Vol 316 (3) ◽  
pp. 703-707 ◽  
Author(s):  
Ralf BIRKENHÄGER ◽  
Bernard SCHNEPPE ◽  
Wolfgang RÖCKL ◽  
Jörg WILTING ◽  
Herbert A. WEICH ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Vascular Endothelial Cells ◽  
Physiological Activity ◽  
Expression System ◽  
Vascular Endothelial ◽  
Placenta Growth Factor ◽  
Splice Forms

Vascular endothilial growth factor (VEGF) and placenta growth factor (PIGF) are members of a dimeric-growth-factor family with angiogenic properties. VEGF is a highly potent and specific mitogen for endothelial cells, playing a vital role in angiogenesis in vivo. The role of PIGF is less clear. We expressed the monomeric splice forms VEGF-165, VEGF-121, PIGF-1 and PlGF-2 as unfused genes in Escherichia coli using the pCYTEXP expression system. In vitro dimerization experiments revealed that both homo- and hetero-dimers can be formed from these monomeric proteins. The dimers were tested for their ability to promote capillary growth in vivo and stimulate DNA synthesis in cultured human vascular endothelial cells. Heterodimers comprising different VEGF splice forms, or combinations of VEGF/PlGF splice forms, showed mitogenic activity. The results demonstrate that four different heterodimeric growth factors are likely to have as yet uncharacterized functions 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 Identification of a multifunctional, cell-binding peptide sequence from the a1(NC1) of type IV collagen.

1990 ◽  
Vol 111 (4) ◽  
pp. 1583-1591 ◽  
Author(s):  
E C Tsilibary ◽  
L A Reger ◽  
A M Vogel ◽  
G G Koliakos ◽  
S S Anderson ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Basement Membrane ◽  
Type Iv Collagen ◽  
Solid Phase ◽  
Peptide Sequence ◽  
Cell Binding ◽  
Type Iv ◽  
Specific Manner ◽  
Dose Dependent ◽  
Nc1 Domain

We have previously identified three distinctive amino acid sequences from type IV collagen which specifically bound to heparin and also inhibited the binding of heparin to intact type IV collagen. One of these chemically synthesized domains, peptide Hep-I, has the sequence TAGSCLRKFSTM and originates from the a1(noncollagenous [NC1]) chain of type IV collagen (Koliakos, G. G., K. K. Koliakos, L. T. Furcht, L. A. Reger, and E. C. Tsilibary. 1989. J. Biol. Chem. 264:2313-2323). We describe in this report that this same peptide also bound to intact type IV collagen in solid-phase assays, in a dose-dependent and specific manner. Interactions between peptide Hep-I and type IV collagen in solution resulted in inhibition of the assembly process of this basement membrane glycoprotein. Therefore, peptide Hep-I should represent a major recognition site in type IV collagen when this protein polymerizes to form a network. In addition, solid phase-immobilized peptide Hep-I was able to promote the adhesion and spreading of bovine aortic endothelial cells. When present in solution, peptide Hep-I competed for the binding of these cells to type IV collagen- and NC1 domain-coated substrata in a dose-dependent manner. Furthermore, radiolabeled peptide Hep-I in solution also bound to endothelial cells in a dose-dependent and specific manner. The binding of radiolabeled Hep-I to endothelial cells could be inhibited by an excess of unlabeled peptide. Finally, in the presence of heparin or chondroitin/dermatan sulfate glycosaminoglycan side chains, the binding of endothelial cells to peptide Hep-I and NC1 domain-coated substrates was also inhibited. We conclude that peptide Hep-I should have a number of functions. The role of this type IV collagen-derived sequence in such diverse phenomena as self-association, heparin binding and cell binding and adhesion makes Hep-I a crucial domain involved in the determination of basement membrane ultrastructure and cellular interactions with type IV collagen-containing matrices.

scholarly journals A genome-wide view of the de-differentiation of central nervous system endothelial cells in culture

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Mark F Sabbagh ◽  
Jeremy Nathans
Keyword(s):  
Central Nervous System ◽  
Endothelial Cells ◽  
Nervous System ◽  
In Vitro Culture ◽  
Vascular Endothelial Cells ◽  
Beta Catenin ◽  
A Genome ◽  
Accessible Chromatin

Vascular endothelial cells (ECs) derived from the central nervous system (CNS) variably lose their unique barrier properties during in vitro culture, hindering the development of robust assays for blood-brain barrier (BBB) function, including drug permeability and extrusion assays. In previous work (Sabbagh et al., 2018) we characterized transcriptional and accessible chromatin landscapes of acutely isolated mouse CNS ECs. In this report, we compare transcriptional and accessible chromatin landscapes of acutely isolated mouse CNS ECs versus mouse CNS ECs in short-term in vitro culture. We observe that standard culture conditions are associated with a rapid and selective loss of BBB transcripts and chromatin features, as well as a greatly reduced level of beta-catenin signaling. Interestingly, forced expression of a stabilized derivative of beta-catenin, which in vivo leads to a partial conversion of non-BBB CNS ECs to a BBB-like state, has little or no effect on gene expression or chromatin accessibility in vitro.

Interaction between vascular endothelial cells and vascular intimal spindle-shaped cells in vitro

1983 ◽  
Vol 60 (1) ◽  
pp. 89-102
Author(s):  
D de Bono ◽  
C. Green
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Three Dimensional ◽  
Vascular Endothelial ◽  
Pure Cultures ◽  
Species Specific ◽  
Endothelial Growth Factor

The interactions between human or bovine vascular endothelial cells and fibroblast-like vascular intimal spindle-shaped cells have been studied in vitro, using species-specific antibodies to identify the different components in mixed cultures. Pure cultures of endothelial cells grow as uniform, nonoverlapping monolayers, but this growth pattern is lost after the addition of spindle cells, probably because the extracellular matrix secreted by the latter causes the endothelial cells to modify the way they are attached to the substrate. The result is a network of tubular aggregates of endothelial cells in a three-dimensional ‘polylayer’ of spindle-shaped cells. On the other hand, endothelial cells added to growth-inhibited cultures of spindle-shaped cells will grow in sheets over the surface of the culture. Human endothelial cells grown in contact with spindle-shaped cells have a reduced requirement for a brain-derived endothelial growth factor. The interactions of endothelial cells and other connective tissue cells in vitro may be relevant to the mechanisms of endothelial growth and blood vessel formation in vivo, and emphasize the potential importance of extracellular matrix in controlling endothelial cell behaviour.

scholarly journals Control of Angiogenesis via a VHL/miR-212/132 Axis

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 1017 ◽  
Author(s):  
Zhiyong Lei ◽  
Timothy D. Klasson ◽  
Maarten M. Brandt ◽  
Glenn van de Hoek ◽  
Ive Logister ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Vascular Endothelial Cells ◽  
Suppressor Gene ◽  
Protein Product ◽  
Cell Renal Cell Carcinoma ◽  
Von Hippel Lindau ◽  
Clinical Interest ◽  
Biallelic Mutations

A common feature of tumorigenesis is the upregulation of angiogenesis pathways in order to supply nutrients via the blood for the growing tumor. Understanding how cells promote angiogenesis and how to control these processes pharmaceutically are of great clinical interest. Clear cell renal cell carcinoma (ccRCC) is the most common form of sporadic and inherited kidney cancer which is associated with excess neovascularization. ccRCC is highly associated with biallelic mutations in the von Hippel–Lindau (VHL) tumor suppressor gene. Although upregulation of the miR-212/132 family and disturbed VHL signaling have both been linked with angiogenesis, no evidence of a possible connection between the two has yet been made. We show that miRNA-212/132 levels are increased after loss of functional pVHL, the protein product of the VHL gene, in vivo and in vitro. Furthermore, we show that blocking miRNA-212/132 with anti-miRs can significantly alleviate the excessive vascular branching phenotype characteristic of vhl−/− mutant zebrafish. Moreover, using human umbilical vascular endothelial cells (HUVECs) and an endothelial cell/pericyte coculture system, we observed that VHL knockdown promotes endothelial cells neovascularization capacity in vitro, an effect which can be inhibited by anti-miR-212/132 treatment. Taken together, our results demonstrate an important role for miRNA-212/132 in angiogenesis induced by loss of VHL. Intriguingly, this also presents a possibility for the pharmaceutical manipulation of angiogenesis by modulating levels of MiR212/132.

scholarly journals Perturbations in the fibrinolytic pathway abolish cyst formation but not capillary-like organization of cultured murine endothelial cells

Blood ◽  
1994 ◽  
Vol 83 (11) ◽  
pp. 3206-3217 ◽  
Author(s):  
N Dubois-Stringfellow ◽  
A Jonczyk ◽  
VL Bautch
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Basement Membrane ◽  
Organizational Behavior ◽  
Cell Lines ◽  
Primary Cultures ◽  
Cyst Formation ◽  
Fibrin Gels

Abstract Fibrinolytic activity and its relation to morphogenesis was investigated in several transformed murine endothelial cell lines and primary cultures of endothelial cells. Two in vitro systems, fibrin gels and Matrigel (Collaborative Research, Bedford, MA), were used. Fibrin gels model a fibrin-rich extracellular matrix that frequently supports neovascularization in vivo, and Matrigel models the basement membrane surrounding quiescent endothelial cells in vivo. The transformed endothelial cell lines have higher levels of plasminogen activator (PA) mRNA than primary cultures of endothelial cells, and an increased PA-mediated proteolytic activity was correlated with formation of cysts in fibrin gels. Addition of neutralizing anti- urokinase antibodies, plasminogen depletion, or addition of a plasmin inhibitor prevented cyst formation. Addition of plasminogen restored the ability to form cysts in the plasminogen-depleted system. Normal endothelial cells organized into capillary-like structures in fibrin gels regardless of manipulations affecting the fibrinolytic pathway. In Matrigel, both transformed and primary cultures of endothelial cells rapidly formed a capillary-like network that was not affected by plasminogen depletion or addition of plasmin inhibitors. Thus, elements of the fibrinolytic pathway necessary for cyst formation are not critical in capillary-like structure formation on a reconstituted basement membrane. These results suggest that plasmin is essential for hemangioma formation but is not critical to the organizational behavior of normal endothelial cells.

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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