Serum enhancement of human endothelial cell attachment to and spreading on collagens I and IV does not require serum fibronectin or vitronectin

The initial attachment and spreading of endothelial cells from human umbilical artery onto type I collagen, type IV collagen or gelatin substrata was shown to be enhanced by inclusion of serum in the culture medium. To test whether this serum effect was mediated by adsorption of serum fibronectin or vitronectin onto the collagen, these adhesive glycoproteins were selectively removed from the serum prior to addition to the culture medium. The stimulatory effect of serum on human endothelial cell spreading on collagens I and IV was also observed with serum from which either fibronectin or vitronectin, or both, had been selectively removed. The stimulatory effect for cell spreading on gelatin was diminished by selective removal of serum fibronectin, but unaffected by removal of vitronectin. Human endothelial cell attachment and spreading onto tissue culture plastic was abolished by removal of vitronectin from the serum in the culture medium. These results emphasize that the native structure of collagens is required for serum-enhancement of human endothelial cell attachment and spreading on native collagen types I and IV, and show that on these substrata the stimulated adhesion and spreading are not dependent upon adsorption of serum fibronectin or vitronectin onto the collagen substratum.

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The VLA-2 (alpha 2 beta 1) I domain functions as a ligand-specific recognition sequence for endothelial cell attachment and spreading: molecular and functional characterization

Blood ◽

10.1182/blood.v84.11.3734.bloodjournal84113734 ◽

1994 ◽

Vol 84 (11) ◽

pp. 3734-3741 ◽

Cited By ~ 25

Author(s):

WF Bahou ◽

CL Potter ◽

H Mirza

Keyword(s):

Endothelial Cell ◽

Type I Collagen ◽

Cell Attachment ◽

Functional Characterization ◽

Comparative Sequence Analysis ◽

Regulatory Function ◽

Type I ◽

Recognition Sequence ◽

Endothelial Cell Attachment

The integrin VLA-2 (alpha 2 beta 1), generally considered to represent the specific collagen receptor on human endothelial cells, contains an alpha 2-subunit inserted I domain with structural similarity to the type A domains found within the recently described superfamily of receptor-ligand recognition proteins. This region of the cDNA has now been isolated and used for molecular and functional characterization of this heterodimeric receptor complex. Comparative sequence analysis with the porcine homologue revealed 93% amino acid sequence identity, suggestive of a developmentally conserved function. To complete structure/function studies, this region of the human cDNA was expressed as a chimeric protein in Escherichia coli, and a rabbit polyclonal antibody (anti-I domain) was used to study determinants of endothelial cell attachment and spreading in vitro. Quantifiable and visual disruption of endothelial cell attachment to gelatin, type I collagen, and laminin was evident using the specific anti-I domain antibody, with minimal inhibitory effects demonstrable using fibronectin or fibrinogen matrices. Therefore, these data would suggest that the alpha 2 beta 1 I domain confers ligand-binding specificity for both known alpha 2 beta 1 substrates (laminin and collagen), and that this region subserves a regulatory function in the molecular processes controlling endothelial cell attachment and spreading in vitro.

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Alpha v beta 1 is a receptor for vitronectin and fibrinogen, and acts with alpha 5 beta 1 to mediate spreading on fibronectin

Journal of Cell Science ◽

10.1242/jcs.108.3.1227 ◽

1995 ◽

Vol 108 (3) ◽

pp. 1227-1238 ◽

Cited By ~ 1

Author(s):

J.F. Marshall ◽

D.C. Rutherford ◽

A.C. McCartney ◽

F. Mitjans ◽

S.L. Goodman ◽

...

Keyword(s):

Type I Collagen ◽

Collagen Type ◽

Collagen Type I ◽

Collagen Type Iv ◽

Type I ◽

Vitronectin Receptor ◽

Type Iv ◽

Beta 1 Integrin ◽

Alpha V Beta 3 ◽

Blocking Antibodies

We have shown previously that VUP was the only line out of ten human melanoma lines that failed to express the vitronectin receptor alpha v beta 3, but instead expressed alpha v beta 1. Levels of alpha v beta 1 expression were low on parental VUP cells so that iterative sorting by FACS, using an anti-alpha v antibody (13C2), was utilised to derive sublines with 8- to 10-fold higher amounts of cell surface alpha v beta 1. There was little difference between low (V-) and high (V+) alpha v beta 1-expressing sublines with regard to adherence to collagen type I, collagen type IV or laminin substrata. However, adherence to vitronectin and fibrinogen correlated closely with alpha v beta 1 expression (35-42% adhesion for V(+) lines versus 6–8% adhesion for V- lines on vitronectin, for example). Utilising a high alpha v beta 1-expressing subline (V + B2) we have shown that binding to vitronectin and fibrinogen was inhibited specifically by function-blocking antibodies to alpha v (17E6 and 14D9) and beta 1 (A11B2). V(+) sublines spread more compared with V(-) sublines on both vitronectin and fibronectin. However, neither alpha 5- nor alpha v-blocking antibodies had any effect on attachment or spreading of V + B2 on fibronectin whereas the combination of alpha 5 (PID6)- and alpha v(17E6)-blocking antibodies abrogated binding to fibronectin almost completely. This is the first report of an alpha v beta 1 integrin able to recognize vitronectin and fibrinogen, and also cooperate with alpha 5 beta 1 to mediate attachment to and spreading on fibronectin.

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Transforming Growth Factor-Beta Binds Reversibly in Vitro to Guglielmi Detachable Coils

Interventional Neuroradiology ◽

10.1177/159101999800400102 ◽

1998 ◽

Vol 4 (1) ◽

pp. 21-26 ◽

Cited By ~ 2

Author(s):

D.F. Kallmes ◽

G.R. Hankins ◽

M.K. Borland ◽

H.J. Cloft ◽

M.E. Jensen ◽

...

Keyword(s):

Growth Factor ◽

Type Iv Collagen ◽

Type I Collagen ◽

Transforming Growth Factor ◽

Absolute Amount ◽

Collagen Type Iv ◽

Type I ◽

Guglielmi Detachable Coils ◽

Type Iv ◽

Detachable Coils

We determined the propensity for and reversibility of transforming growth factor-ß (TGFß) binding to uncoated Guglielmi Detachable Coils (GDC) and to GDC coated with extracellular matrix (ECM) proteins. Three 1.0 centimetre samples each of uncoated GDC-18 and of GDC-18 coated with either poly-L-lysine, laminin, type I collagen, type IV collagen, fibronectin, or poly-L-lysine and laminin were prepared. These samples were immersed briefly in a solution containing I125-labelled TGFß at a concentration of 0.225 μg/ml with initial specific activity of 123.3 mCi/mg (DuPont-NEN, Billerica, MA), and were counted using a scintillation counter. Each sample was then placed in a vial containing saline, shaken for 60 seconds, and counted again. Selected samples were immersed for varying periods within the TGFß solution and counted before and after saline rinse. Samples were rinsed one week after initial rinsing and counted again. The amount of binding between coil types was compared using the Student t test. For all samples initial binding of TGFß was in the order of 60–120 pg/cm. For the pre-rinse data there were no statistically significant differences between the amount bound to any single coil coating type relative to other coatings. Compared to the initial accumulations, the amount remaining after rinsing ranged from 40% (poly-L-lysine) to 63% (poly-L-lysine with laminin), with a mean of 55% among the seven coil types. After rinsing there was more growth factor remaining on uncoated coils than on poly-L-lysine-coated coils (p=0.05), fibronectin-coated coils (p=0.01), and type IV collagen-coated coils (p=0.04). There was a trend toward greater residual growth factor on coils coated with poly-L-lysine and laminin compared to coils coated with poly-L-lysine alone (p=0.10). Delayed, second rinsing of the samples one week after initial testing demonstrated only minor incremental loss of TGFß from the coil surfaces. After five minutes of immersion, accumulation was approximately 200% greater than that noted with brief submersion, but immersions lasting over five minutes did not yield increasing levels of TGFß binding. TGFß binds to GDC coils. Binding is not improved with ECM protein-coated coils compared to uncoated coils. The absolute amount of TGFß bound to the coil will likely result in local concentrations of growth factor in the order of those required for biological activity in vivo.

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Dualistic nature of adhesive protein function: fibronectin and its biologically active peptide fragments can autoinhibit fibronectin function.

The Journal of Cell Biology ◽

10.1083/jcb.99.1.29 ◽

1984 ◽

Vol 99 (1) ◽

pp. 29-36 ◽

Cited By ~ 297

Author(s):

K M Yamada ◽

D W Kennedy

Keyword(s):

Type I Collagen ◽

Cell Attachment ◽

Cell Spreading ◽

Biologically Active ◽

Cell Surface Receptor ◽

Type I ◽

Peptide Fragments ◽

Adhesive Protein ◽

Active Peptide ◽

Biologically Active Peptide

Fibronectin and certain polypeptide regions of this adhesive glycoprotein mediate cell attachment and spreading on various substrates. We explored the theoretical prediction that this adhesive protein could become a competitive inhibitor of fibronectin-mediated processes if present in solution at appropriately high concentrations. Fibronectin function was inhibited by purified plasma fibronectin at 5-10 mg/ml, by a 75,000-dalton cell-interaction fragment of the protein at 0.5-1 mg/ml, and even by two synthetic peptides containing a conserved, hydrophilic amino acid sequence at 0.1-0.5 mg/ml. Inhibition of fibronectin-dependent cell spreading was dose dependent, noncytotoxic, and reversible. It was competitive in nature, since increased quantities of substrate-adsorbed fibronectin or longer incubation periods decreased the inhibition. A peptide inhibitory for fibronectin-mediated cell spreading also inhibited fibronectin-mediated attachment of cells to type I collagen, but it did not affect concanavalin A-mediated spreading. These results demonstrate the potential of a cell adhesion molecule and its biologically active peptide fragments to act as competitive inhibitors, and they suggest that fibronectin may act by binding to a saturable cell surface receptor.

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