Integrin (alpha) and beta subunit contribution to the kinetic properties of (alpha)2beta1 collagen receptors on human keratinocytes analyzed under hydrodynamic conditions

1999 ◽  
Vol 112 (14) ◽  
pp. 2335-2345 ◽  
Author(s):  
B. Masson-Gadais ◽  
A. Pierres ◽  
A.M. Benoliel ◽  
P. Bongrand ◽  
J.C. Lissitzky
Keyword(s):  
Type I Collagen ◽  
Human Keratinocytes ◽  
Flow Chamber ◽  
Dissociation Rate ◽  
Ligand Recognition ◽  
Kinetic Properties ◽  
Type I ◽  
Functional Region ◽  
Laminin 5 ◽  
Bond Stability

The adhesion of keratinocytes to type I collagen or laminin 5 was studied in a laminar flow chamber. These experiments provided an insight into the binding kinetics of integrins in their natural environment and the effects of monoclonal antibodies specific for (alpha) and beta chains. Cells driven by a force too low to alter the natural lifetime of a single bond displayed multiple arrests. Studying the frequency and duration of these arrests yielded fairly direct information on the rate of bond formation (on-rate) and dissociation (off-rate). Off-rate values obtained on collagen or laminin 5 (0.06 seconds-1) were tenfold lower than values determined on selectins. Bond stability was strongly regulated by anti-beta1 chain antibodies since the off-rate was decreased sixfold by activating antibody TS2/16 and increased fivefold by inhibitory antibodies Lia1/2 or P4C10, whereas neutral antibody K20 had no effect on this parameter. Binding frequencies were not significantly changed by all these antibodies. In contrast, both binding frequency and off-rate were altered by antibodies specific for the (alpha)2 chain, suggesting that these antibodies interfered with ligand recognition and also with the ligand-beta1 chain interactions responsible for bond stabilization. The latter hypothesis was supported by the finding that the partial alteration of (alpha)2 chain function by inhibiting antibodies was corrected by anti-beta1 chain antibody TS2/16. These results could not be ascribed to allosteric changes of the functional region of beta1 integrin subunits regulated by TS2/16 since there was no competition between the binding of TS2/16 and anti-(alpha)2 chain antibodies. Interpreted within the framework of current concepts of integrin-ligand binding topology, these data suggest that ligand-alpha chain interactions may be qualitatively important in ligand recognition and also influence the formation of the ligand-beta1 subunit bonding involved in stabilization of the ligand-integrin complex by regulating its dissociation rate.

Activation of human keratinocyte migration on type I collagen and fibronectin

1990 ◽  
Vol 96 (2) ◽  
pp. 197-205
Author(s):  
M. Guo ◽  
K. Toda ◽  
F. Grinnell
Keyword(s):  
Type I Collagen ◽  
Cultured Cells ◽  
Focal Adhesions ◽  
Human Keratinocytes ◽  
Type I ◽  
Integrin Subunit ◽  
Basal Cells ◽  
Keratinocyte Migration ◽  
Beta 1 Integrin ◽  
Cells Cultured

The purpose of our studies was to learn more about the regulation of keratinocyte migration. Human keratinocytes freshly harvested from skin were relatively immotile cells, whereas keratinocytes harvested from cell culture migrated on type I collagen or fibronectin as measured in a phagokinesis assay. Development of migratory competence by keratinocytes varied depending on the culture substratum. Cells cultured on plastic were activated more quickly and to a greater extent than cells cultured on dermis. The effect of the culture substratum on migratory competence was reversible. That is, cells cultured on plastic showed reduced activity after subculture on dermis. Cells cultured on dermis showed increased activity after subculture on plastic. Freshly isolated as well as cultured keratinocytes contained beta 1 integrin subunits, but only cultured cells were able to organize the subunits into focal adhesions. These adhesion sites also contained vinculin. In epidermal explants, beta 1 integrin subunits were mostly in basal cells, often more prominent between lateral cell borders than at the epidermal-dermal interface. In keratinocytes that migrated out of skin explants, there appeared to be an increase in the intensity of beta 1 integrin subunit immunostaining, possibly because of the change in shape of migrating cells. Also, beta 1 integrin subunits were found around and beneath migrating keratinocytes. These results show that changes in the distribution of beta 1 integrin subunits accompany development of migratory competence.

scholarly journals Platelet-derived microparticles associate with fibrin during thrombosis

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4651-4663 ◽  
Author(s):  
P Siljander ◽  
O Carpen ◽  
R Lassila
Keyword(s):  
Type I Collagen ◽  
Thrombus Formation ◽  
Flow Chamber ◽  
Type I ◽  
Binding Studies ◽  
Vitro Binding ◽  
Large Platelet ◽  
Platelet Aggregates ◽  
Thrombin Antithrombin Iii Complex

Platelet-derived microparticles (MP) are reported to express both pro- and anticoagulant activities. Nevertheless, their functional significance has remained unresolved. The present study monitored the generation and fate of MP in an experimental model of thrombosis with costimulation of platelets by collagen and thrombin. When minimally anticoagulated (0.5 micromol/L PPACK) blood was perfused over immobilized fibrillar type I collagen in a flow chamber at a low shear rate (300 s(-1)), endogenous thrombin was generated, as evidenced by thrombin-antithrombin III complex. In contrast to full anticoagulation 150 micromol/L PPACK) and the absence of collagen, large platelet aggregates and fibrin ensued during perfusions over collagen in the presence of thrombin. In these thrombi, MP, defined as GPIIbIIIa- and P- selectin-positive vesicles (<1 micron), were found to align fibrin in immunofluorescence and scanning immunoelectron microscopy. Moreover, in sections of embolectomized thromboemboli from patients GPIIbIIIa- and P- selectin-positive material compatible with MP was detected in a fibrin strand-like pattern. In vitro binding studies showed that MP bound to fibrin and acted there as procoagulants. In summary, we show that MP generated during thrombus formation associate with local fibrin. This adhesive function fibrin could imply a sustained modulatory role for MP in evolving thrombi.

Induction of interstitial collagenase in human keratinocytes during wound repair requires contact with native type I collagen

1994 ◽  
Vol 14 (5) ◽  
pp. 379
Author(s):  
W.C. Parks ◽  
B.D. Sudbeck ◽  
U.S. Saariahlo-Kere ◽  
A.P. Pentland ◽  
H.G. Welgus
Keyword(s):  
Wound Repair ◽  
Type I Collagen ◽  
Human Keratinocytes ◽  
Type I ◽  
Interstitial Collagenase

scholarly journals The Activity of Collagenase-1 Is Required for Keratinocyte Migration on a Type I Collagen Matrix

1997 ◽  
Vol 137 (6) ◽  
pp. 1445-1457 ◽  
Author(s):  
Brian K. Pilcher ◽  
Jo Ann Dumin ◽  
Barry D. Sudbeck ◽  
Stephen M. Krane ◽  
Howard G. Welgus ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Type I Collagen ◽  
Cell Movement ◽  
Collagen Matrix ◽  
Human Keratinocytes ◽  
Type I ◽  
Integrin Subunit ◽  
Primary Keratinocytes ◽  
Keratinocyte Migration ◽  
Dermal Collagen

We have shown in a variety of human wounds that collagenase-1 (MMP-1), a matrix metalloproteinase that cleaves fibrillar type I collagen, is invariably expressed by basal keratinocytes migrating across the dermal matrix. Furthermore, we have demonstrated that MMP-1 expression is induced in primary keratinocytes by contact with native type I collagen and not by basement membrane proteins or by other components of the dermal or provisional (wound) matrix. Based on these observations, we hypothesized that the catalytic activity of MMP-1 is necessary for keratinocyte migration on type I collagen. To test this idea, we assessed keratinocyte motility on type I collagen using colony dispersion and colloidal gold migration assays. In both assays, primary human keratinocytes migrated efficiently on collagen. The specificity of MMP-1 in promoting cell movement was demonstrated in four distinct experiments. One, keratinocyte migration was completely blocked by peptide hydroxymates, which are potent inhibitors of the catalytic activity of MMPs. Two, HaCaTs, a line of human keratinocytes that do not express MMP-1 in response to collagen, did not migrate on a type I collagen matrix but moved efficiently on denatured type I collagen (gelatin). EGF, which induces MMP-I production by HaCaT cells, resulted in the ability of these cells to migrate across a type I collagen matrix. Three, keratinocytes did not migrate on mutant type I collagen lacking the collagenase cleavage site, even though this substrate induced MMP-1 expression. Four, cell migration on collagen was completely blocked by recombinant tissue inhibitor of metalloproteinase-1 (TIMP-1) and by affinity-purified anti–MMP-1 antiserum. In addition, the collagen-mediated induction of collagenase-1 and migration of primary keratinocytes on collagen was blocked by antibodies against the α2 integrin subunit but not by antibodies against the α1 or α3 subunits. We propose that interaction of the α2β1 integrin with dermal collagen mediates induction of collagenase-1 in keratinocytes at the onset of healing and that the activity of collagenase-1 is needed to initiate cell movement. Furthermore, we propose that cleavage of dermal collagen provides keratinocytes with a mechanism to maintain their directionality during reepithelialization.

scholarly journals Laminin-5 and type I collagen promote adhesion and osteogenic differentiation of animal serum-free expanded human mesenchymal stromal cells

2012 ◽  
Vol 4 (4) ◽  
pp. 36 ◽  
Author(s):  
Falk Mittag ◽  
Eva-Maria Falkenberg ◽  
Alexandra Janczyk ◽  
Marco Götze ◽  
Tino Felka ◽  
...  
Keyword(s):  
Osteogenic Differentiation ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Type I Collagen ◽  
Cell Attachment ◽  
Expression Patterns ◽  
Marker Genes ◽  
Type I ◽  
Laminin 5 ◽  
Laminin 1

Mesenchymal stromal cells (MSC) are differentiation competent cells and may generate, among others, mature osteoblasts or chondrocytes<em> in vitro</em> and <em>in vivo</em>. Laminin-5 and type I collagen are important components of the extracellular matrix. They are involved in a variety of cellular and extracellular activities including cell attachment and osteogenic differentiation of MSC. MSC were isolated and expanded using media conforming good medical practice (GMP)-regulations for medical products. Cells were characterized according to the defined minimal criteria for multipotent MSC. MTT- and BrdU-assays were performed to evaluate protein-dependent (laminin-5, laminin-1, type I collagen) metabolic activity and proliferation of MSC. MSC-attachment assays were performed using protein-coated culture plates. Osteogenic differentiation of MSC was measured by protein-dependant mineralization and expression of osteogenic marker genes (osteopontin, alkaline phophatase, Runx2) after three, seven and 28 days of differentiation. Marker genes were identified using quantitative reverse-transcription polymerase chain reaction. Expansion of MSC in GMP-conforming media yielded vital cells meeting all minimal criteria for MSC. Attachment assay revealed a favorable binding of MSC to laminin-5 and type I collagen at a protein concentration of 1-5 fmol/mL. Compared to plastic, osteogenic differentiation was significantly increased by laminin-5 after 28 days of culture (P&lt;0.04). No significant differences in gene expression patterns were observed. We conclude that laminin-5 and type I collagen promote attachment, but laminin-1 and laminin-5 promote osteogenic differentiation of MSC. This may influence future clinical applications.

Cultured Human Keratinocytes on Type I Collagen Membranes to Reconstitute the Epidermis

2000 ◽  
Vol 6 (1) ◽  
pp. 53-67 ◽  
Author(s):  
Raymund E. Horch ◽  
Markus Debus ◽  
Gilbert Wagner ◽  
G. Bjoern Stark
Keyword(s):  
Type I Collagen ◽  
Human Keratinocytes ◽  
Type I ◽  
Collagen Membranes

Collagen fibrillogenesis in vitro: interaction of types I and V collagen

1986 ◽  
Vol 44 ◽  
pp. 210-211
Author(s):  
Arthur J. Wasserman ◽  
Kathy C. Kloos ◽  
David E. Birk
Keyword(s):  
Type I Collagen ◽  
Corneal Stroma ◽  
Minor Component ◽  
Homogeneous Distribution ◽  
Type I ◽  
Type V Collagen ◽  
Fibril Morphology ◽  
Type V ◽  
In Vitro Interaction

Type I collagen is the predominant collagen in the cornea with type V collagen being a quantitatively minor component. However, the content of type V collagen (10-20%) in the cornea is high when compared to other tissues containing predominantly type I collagen. The corneal stroma has a homogeneous distribution of these two collagens, however, immunochemical localization of type V collagen requires the disruption of type I collagen structure. This indicates that these collagens may be arranged as heterpolymeric fibrils. This arrangement may be responsible for the control of fibril diameter necessary for corneal transparency. The purpose of this work is to study the in vitro assembly of collagen type V and to determine whether the interactions of these collagens influence fibril morphology.

948: Clinical Safety, Histologic Findings and Surgical Methods in Complex Phalloplasty Using Type I Collagen

2007 ◽  
Vol 177 (4S) ◽  
pp. 314-314 ◽  
Author(s):  
Joon-Yang Kim ◽  
Hoon Seog Jean ◽  
Beom Joon Kim ◽  
Kye Yong Song
Keyword(s):  
Type I Collagen ◽  
Type I ◽  
Clinical Safety ◽  
Surgical Methods
Sign in / Sign up

Export Citation Format

Share Document