scholarly journals A comparison of the biological activities of the cell-adhesive proteins vitronectin and fibronectin

1989 ◽  
Vol 93 (4) ◽  
pp. 641-649
Author(s):  
P.A. Underwood ◽  
F.A. Bennett
Keyword(s):  
Endothelial Cells ◽  
Serum Proteins ◽  
Cell Attachment ◽  
Biological Activities ◽  
Cell Types ◽  
Corneal Endothelial Cells ◽  
Response Curves ◽  
Similar Dose ◽  
Bovine Endothelial Cells ◽  
Adhesive Proteins

The effects of vitronectin and fibronectin upon the attachment and growth of bovine corneal endothelial cells (BCE) and BHK-21 cells were compared. Similar dose-response curves for cell attachment to the substratum were obtained for both molecules and both cell types, although BCE cells exhibited a slight preference for vitronectin, and BHK cells for fibronectin. When, however, cells were plated in medium containing bovine serum stripped of fibronectin, they attached and grew normally, whereas in medium containing serum stripped of vitronectin, cells either failed to attach (BHK-21) or attached but exhibited poor cell spreading and growth. This dependence of cells upon vitronectin, rather than fibronectin, in serum for cell attachment, was shown to be due to a failure of fibronectin to coat the substratum in the presence of other serum proteins. Vitronectin was able to coat the substratum efficiently in the presence of other serum proteins. Although dependent upon vitronectin for adhesion to the substratum, bovine endothelial cells were unable to synthesize endogenous vitronectin.

INTERACTION OF S PROTEIN/VITRONECTIN WITH CULTURED ENDOTHELIAL CELLS: PROMOTION OF ATTACHMENT AND SPECIFIC BINDING

1987 ◽  
Author(s):  
K T Preissner ◽  
E Anders ◽  
G Müller-Berghaus
Keyword(s):  
Endothelial Cells ◽  
Cell Attachment ◽  
Specific Binding ◽  
Umbilical Vein ◽  
Attachment Site ◽  
Cell Types ◽  
S Protein ◽  
Specific Association ◽  
Umbilical Vein Endothelial Cells ◽  
Adhesive Proteins

The interaction of the complement inhibitor S protein, which is identical to the serum spreading factor, vitronectin, with cultured human endothelial cells of macro- and microvas- cular origin was investigated. Purified S protein, coated for 2 h on polystyrene petri dishes, induced concentration- and time-dependent attachment and spreading of human umbilical vein endothelial cells (HUVEC) as well as human omental tissqe microvasular endothelial cells (HOTMEC) at 37°C. With 3 × 105 cells/ml (final concentration) more than 50% of the cells attached within 2 h incubation at 0.3 - 3 μg/ml S protein. The effect of S protein was specific, since only monospecific antibodies against S protein prevented attachment of cells, while antibodies against fibronectin, fibrinogen or von Wille-brand factor were uneffective. The pentapeptide Gly-Arg-Gly-Asp-Ser, which contains the cell-attachment site of these adhesive proteins including S protein, inhibited the activity of S protein to promote attachment of endothelial cells in a concentration-dependent fashion; at 200 μM peptide, less than 10% of the cells became attached. Direct binding of S protein to HUVEC and HOTMEC was studied with cells in suspension at a concentration of 1 × 106 cells/ml in the presence of 1% (w/v) human serum albumin and 1 mM CaCl2 and was maximal after 120 min. Both cell types bound S protein in a concentration-dependent fashion with an estimated dissociation constant KD=0.2pM. More than 80% of bound radiolabelled S protein was displaced by unlabelled S protein, whereas binding was reduced to about 50% by the addition in excess of either fibronectin, fibrinogen, von Willebrand factor or the pentapeptide. These findings provide evidence for the specific association of S protein with endothelial cells, ultimately leading to attachment and spreading of cells. Although the promotion of attachment was highly specific for S protein, other adhesive proteins than S protein, also known to associate with endothelial cells, may in part compete with direct S protein binding.

scholarly journals Cell attachment to thrombospondin: the role of ARG-GLY-ASP, calcium, and integrin receptors.

1988 ◽  
Vol 107 (6) ◽  
pp. 2351-2361 ◽  
Author(s):  
J Lawler ◽  
R Weinstein ◽  
R O Hynes
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Cell Attachment ◽  
Solid Phase ◽  
Rat Kidney ◽  
Muscle Cells ◽  
Cell Types ◽  
Adhesive Properties ◽  
Adhesive Proteins

Thrombospondin is a 420,000-D glycoprotein that has recently been shown to have several properties in common with the members of a class of adhesive proteins. To characterize further the adhesive properties of thrombospondin, we have studied its ability to support cell attachment. Thrombospondin adsorbed to plastic dishes supports the attachment of human endothelial and smooth muscle cells and the monocyte-like cell line (U937) as well as normal rat kidney cells. The majority of attached cells do not spread on the solid-phase thrombospondin. The attachment of all four cell types to thrombospondin is abolished if the assay is performed in the presence of EGTA, although the cells still attach to fibronectin. If thrombospondin is adsorbed to the dishes in the presence of EGTA and then washed with buffer containing calcium before addition of the cells, attachment is still markedly inhibited, indicating that calcium affects the conformation and function of thrombospondin. Attachment of all four cell types is also markedly inhibited by the synthetic peptides gly-arg-gly-asp-ser-pro (GRG-DSP) and gly-arg-gly-asp-ala-cys (GRGDAC) but not by the control peptide gly-arg-gly-glu-ser-pro (GRG-ESP). Affinity chromatography of n-octylglucoside extracts of surface-labeled endothelial cells or smooth muscle cells on thrombospondin-Sepharose and GRG-DSP-Affigel columns was used to identify an integrin complex related to glycoprotein IIb-IIIa as an RGD-dependent receptor for thrombospondin. In addition, a monoclonal antibody (LM609) that blocks attachment of endothelial cells to vitronectin, fibrinogen, and von Willebrand factor also inhibits attachment of endothelial cells to thrombospondin. These data indicate that the attachment of cells to thrombospondin is mediated by RGD and calcium-dependent mechanisms and is consistent with the hypothesis that the GRGDAC sequence in thrombospondin is a site for interaction with an integrin receptor of the beta 3 subclass.

scholarly journals Soluble CD14 participates in the response of cells to lipopolysaccharide.

1992 ◽  
Vol 176 (6) ◽  
pp. 1665-1671 ◽  
Author(s):  
E A Frey ◽  
D S Miller ◽  
T G Jahr ◽  
A Sundan ◽  
V Bazil ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Leukocyte Adhesion ◽  
Umbilical Vein ◽  
Cell Types ◽  
Mononuclear Phagocytes ◽  
Soluble Cd14 ◽  
Bovine Endothelial Cells ◽  
Endothelial Leukocyte Adhesion Molecule ◽  
Umbilical Vein Endothelial Cells ◽  
Serum Dependent

CD14 is a 55-kD protein found both as a glycosylphosphatidyl inositol-linked protein on the surface of mononuclear phagocytes and as a soluble protein in the blood. CD14 on the cell membrane (mCD14) has been shown to serve as a receptor for complexes of lipopolysaccharide (LPS) with LPS binding protein, but a function for soluble CD14 (sCD14) has not been described. Here we show that sCD14 enables responses to LPS by cells that do not express CD14. We have examined induction of endothelial-leukocyte adhesion molecule 1 expression by human umbilical vein endothelial cells, interleukin 6 secretion by U373 astrocytoma cells, and cytotoxicity of bovine endothelial cells. None of these cell types express mCD14, yet all respond to LPS in a serum-dependent fashion, and all responses are completely blocked by anti-CD14 antibodies. Immunodepletion of sCD14 from serum prevents responses to LPS, and the responses are restored by addition of sCD14. These studies suggest that a surface anchor is not needed for the function of CD14 and further imply that sCD14 must bind to additional proteins on the cell surface to associate with the cell and transduce a signal. They also indicate that sCD14 may have an important role in potentiating responses to LPS in cells lacking mCD14.

scholarly journals An integrin receptor on normal and thrombasthenic platelets that binds thrombospondin [see comments]

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 2022-2027 ◽  
Author(s):  
J Lawler ◽  
RO Hynes
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Cell Types ◽  
Melanoma Cells ◽  
Human Platelets ◽  
Alpha Subunit ◽  
Adhesive Proteins ◽  
Alpha V Beta 3

Abstract The members of the integrin family of membrane glycoprotein heterodimer complexes function as cell surface receptors for adhesive proteins. We report here on the identification of two integrins on the surface of human platelets that bind to thrombospondin. When platelet membrane proteins are radiolabeled with 125I-lactoperoxidase, solubilized in n- octylglucoside, (Boehringer Mannheim Biochemicals, Indianapolis, IN), and applied to a column of thrombospondin-Sepharose, both complexes are bound to the column and specifically eluted with the peptide GRGDSP. One of these integrins, glycoprotein (GP) IIb-IIIa, appears to bind relatively weakly. The second integrin shares the same beta subunit (beta 3 or GPIIIa), but has a distinct alpha subunit that comigrates with the alpha subunit (alpha v) of the vitronectin receptor (VnR) on endothelial cells and reacts with a monoclonal antibody, LM142, which was raised against an integrin from M21 melanoma cells. The alpha v beta 3 integrin is present on a variety of cell types and appears to act as a receptor for thrombospondin on endothelial and smooth muscle cells. On endothelial and M21 melanoma cells this receptor is also involved in adhesion to fibrinogen, vitronectin, and von Willebrand factor (vWF). The alpha v beta 3 integrin is present at approximately equal levels on normal and thrombasthenic platelets, whereas levels of GPIIb-IIIa are greatly reduced on thrombasthenic platelets. The alpha v beta 3 integrin on thrombasthenic platelets also binds to thrombospondin-Sepharose and can be eluted with the peptide GRGDSP. These data indicate that the alpha v beta 3 integrin on platelets, endothelial cells, and smooth muscle cells functions as an Arg-Gly-Asp (RGD)-dependent receptor for thrombospondin.

scholarly journals Attachment of cultured human endothelial cells is promoted by specific association with S protein (vitronectin) as well as with the ternary S protein-thrombin-antithrombin III complex

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1581-1589 ◽  
Author(s):  
KT Preissner ◽  
E Anders ◽  
J Grulich-Henn ◽  
G Muller-Berghaus
Keyword(s):  
Endothelial Cells ◽  
Antithrombin Iii ◽  
Cell Attachment ◽  
Umbilical Vein ◽  
Cell Types ◽  
Human Endothelial Cells ◽  
S Protein ◽  
Major Vessel ◽  
Specific Association ◽  
Dose Dependent

Abstract The interaction of the multifunctional S protein (vitronectin) with cultured human endothelial cells of macrovascular and microvascular origin was investigated. Purified S protein, coated on polystyrene Petri dishes, induced dose-dependent and time-dependent attachment and spreading of human umbilical vein endothelial cells (HUVECs) as well as human omental tissue microvascular endothelial cells (HOTMECs) at 37 degrees C. Not only isolated S protein, but also the ternary S protein- thrombin-antithrombin III (STAT) complex promoted attachment of approximately 90% of the cells within 2 hours at an S protein concentration of 0.13 mumol/L. Inhibition of attachment in these experiments was achieved by the addition of the cell-attachment pentapeptide Gly-Arg-Gly-Asp-Ser and by monospecific antibodies against S protein, whereas nonrelated peptides or antibodies against fibronectin, fibrinogen, or von Willebrand factor (vWF) were ineffective. Direct binding of S protein to HUVECs and HOTMECs was studied with cells in suspension at a density of 1 x 10(6) cells/mL and was maximal after 120 minutes. S protein bound to both cell types in a dose-dependent fashion with an estimated dissociation constant Kd = 0.2 mumol/L. At a 200-fold to 500-fold molar excess of unlabeled S protein, greater than 80% of bound radiolabeled S protein was displaceable, whereas binding was reduced to 30% to 50% by addition of the pentapeptide, the STAT complex, or by physiologic concentrations of fibrinogen or vWF as well as Fab fragments of anti(human S protein)IgG, but not by Fab rabbit IgG. These findings present evidence for the specific association of S protein with endothelial cells ultimately leading to attachment and spreading of cells. Moreover, a novel function for the ternary STAT complex, which induced endothelial cell attachment and spreading virtually identical to free S protein, is described. These data further suggest a possible role for S protein during coagulation as major vessel wall-related adhesive protein at sites of vascular injury.

scholarly journals Characterization of a factor that promotes neurite outgrowth: evidence linking activity to a heparan sulfate proteoglycan.

1982 ◽  
Vol 94 (3) ◽  
pp. 574-585 ◽  
Author(s):  
A D Lander ◽  
D K Fujii ◽  
D Gospodarowicz ◽  
L F Reichardt
Keyword(s):  
Endothelial Cells ◽  
Neurite Outgrowth ◽  
Sympathetic Neurons ◽  
Cesium Chloride ◽  
Cell Types ◽  
Sulfate Proteoglycan ◽  
Corneal Endothelial Cells ◽  
Active Factor ◽  
Heparin Sulfate Proteoglycan ◽  
Heparin Sulfate

Rat sympathetic neurons, plated onto extracellular matrix produced by cultured bovine corneal endothelial cells, rapidly extended neurites in the absence of nerve growth factor (NGF). The response was unaffected by antiserum to NGF. Rapid outgrowth also occurred when sympathetic neurons were plated onto polylysine-coated surfaces that had been exposed to serum-free medium conditioned by corneal endothelial cells (CMSF). A response was seen even when the neurons were cultured without serum. When plated onto a polylysine-coated dish treated with CMSF over half its surface, only the neurons on the treated half extended neurites. The active factor in CMSF was destroyed by trypsin, acid (pH 1.6), base (pH 12.7), or heating to 80 degrees C; it was stable to heating to 60 degrees C, collagenase, deoxyribonuclease, and neuraminidase. The factor elutes just after the void volume of a Sepharose 6B column. In associative cesium chloride gradients, it sediments as a peak centered at a density of 1.36-1.37, corresponding to a peak of material that can be biosynthetically labeled with [35S]sulfate or [3H]leucine. Material from this fraction was inactivated by heparinase, but not chondroitinase ABC, implying that a heparin sulfate proteoglycan is essential for the factor's activity. Inactivation by contaminants in the heparinase preparation was ruled out. Further purification indicated that the active factor may exist as an aggregate containing a heparin sulfate proteoglycan and other molecules. CMSF also promoted neurite outgrowth by other types of neurons. Furthermore, a variety of cell types were shown to produce factors similar to that in CMSF.

Reduction in pO2 decreases the fibrinolytic potential of cultured bovine endothelial cells derived from pulmonary arteries and lung microvasculature

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1703-1706
Author(s):  
J Wojta ◽  
RL Jones ◽  
BR Binder ◽  
RL Hoover
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Pulmonary Arteries ◽  
Plasminogen Activator Inhibitor ◽  
Cell Types ◽  
Bovine Endothelial Cells ◽  
The Media ◽  
Fibrinolytic Potential

The effect of anoxia on the fibrinolytic potential of cultured endothelial cells derived from bovine pulmonary artery and bovine lung microvasculature was studied. Both cell types reacted with an increase in plasminogen activator inhibitor (PAI) activity and a decrease in the plasminogen activator (PA) activity in the media after incubation under anoxic conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fibrin autography and reverse fibrin autography indicated that the change in fibrinolytic potential was due to an impaired release of PA and not an increase in the production of PAI. Although anoxia did not affect the viability of the cells as judged by 51Cr release, their metabolism was influenced, which is reflected by increases in the levels of lactate in cell lysates and media. Furthermore, the effect of short-term anoxia on PA and PAI could not be reversed by reoxygenation for 24 hours. The results are discussed in terms of helping to explain the tendency of reocclusion after successful thrombolytic therapy, the development of pulmonary hypertension, and the thrombotic tendency of areas with an impaired circulatory supply.

scholarly journals Reduction in pO2 decreases the fibrinolytic potential of cultured bovine endothelial cells derived from pulmonary arteries and lung microvasculature

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1703-1706 ◽  
Author(s):  
J Wojta ◽  
RL Jones ◽  
BR Binder ◽  
RL Hoover
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Pulmonary Arteries ◽  
Plasminogen Activator Inhibitor ◽  
Cell Types ◽  
Bovine Endothelial Cells ◽  
The Media ◽  
Fibrinolytic Potential

Abstract The effect of anoxia on the fibrinolytic potential of cultured endothelial cells derived from bovine pulmonary artery and bovine lung microvasculature was studied. Both cell types reacted with an increase in plasminogen activator inhibitor (PAI) activity and a decrease in the plasminogen activator (PA) activity in the media after incubation under anoxic conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fibrin autography and reverse fibrin autography indicated that the change in fibrinolytic potential was due to an impaired release of PA and not an increase in the production of PAI. Although anoxia did not affect the viability of the cells as judged by 51Cr release, their metabolism was influenced, which is reflected by increases in the levels of lactate in cell lysates and media. Furthermore, the effect of short-term anoxia on PA and PAI could not be reversed by reoxygenation for 24 hours. The results are discussed in terms of helping to explain the tendency of reocclusion after successful thrombolytic therapy, the development of pulmonary hypertension, and the thrombotic tendency of areas with an impaired circulatory supply.

scholarly journals An integrin receptor on normal and thrombasthenic platelets that binds thrombospondin [see comments]

Blood ◽  
1989 ◽  
Vol 74 (6) ◽  
pp. 2022-2027 ◽  
Author(s):  
J Lawler ◽  
RO Hynes
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Muscle Cells ◽  
Cell Types ◽  
Melanoma Cells ◽  
Human Platelets ◽  
Alpha Subunit ◽  
Adhesive Proteins ◽  
Alpha V Beta 3

The members of the integrin family of membrane glycoprotein heterodimer complexes function as cell surface receptors for adhesive proteins. We report here on the identification of two integrins on the surface of human platelets that bind to thrombospondin. When platelet membrane proteins are radiolabeled with 125I-lactoperoxidase, solubilized in n- octylglucoside, (Boehringer Mannheim Biochemicals, Indianapolis, IN), and applied to a column of thrombospondin-Sepharose, both complexes are bound to the column and specifically eluted with the peptide GRGDSP. One of these integrins, glycoprotein (GP) IIb-IIIa, appears to bind relatively weakly. The second integrin shares the same beta subunit (beta 3 or GPIIIa), but has a distinct alpha subunit that comigrates with the alpha subunit (alpha v) of the vitronectin receptor (VnR) on endothelial cells and reacts with a monoclonal antibody, LM142, which was raised against an integrin from M21 melanoma cells. The alpha v beta 3 integrin is present on a variety of cell types and appears to act as a receptor for thrombospondin on endothelial and smooth muscle cells. On endothelial and M21 melanoma cells this receptor is also involved in adhesion to fibrinogen, vitronectin, and von Willebrand factor (vWF). The alpha v beta 3 integrin is present at approximately equal levels on normal and thrombasthenic platelets, whereas levels of GPIIb-IIIa are greatly reduced on thrombasthenic platelets. The alpha v beta 3 integrin on thrombasthenic platelets also binds to thrombospondin-Sepharose and can be eluted with the peptide GRGDSP. These data indicate that the alpha v beta 3 integrin on platelets, endothelial cells, and smooth muscle cells functions as an Arg-Gly-Asp (RGD)-dependent receptor for thrombospondin.

Hemostatic Factors and Inflammatory Disease

1999 ◽  
Vol 82 (08) ◽  
pp. 858-864 ◽  
Author(s):  
Jay Degen
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Biological Activities ◽  
Bacterial Species ◽  
Physiological Role ◽  
Muscle Cells ◽  
Cell Types ◽  
Hemostatic Factors

IntroductionVascular integrity is preserved by a sophisticated system of circulating and cell-associated hemostatic factors that control local thrombin generation, platelet deposition, and the conversion of soluble fibrinogen to an insoluble fibrin matrix.1,2 However, there is considerable evidence that hemostatic factors play both a wider physiological role than simply controlling blood loss, and a wider pathological role than simply triggering inopportune thrombotic events, such as myocardial infarction and stroke. In tissue repair, a crucial physiological process, fibrin(ogen) is thought to provide a critical provisional matrix on which cells can proliferate, organize, and carry out specialized functions. A variety of cell types specifically bind to and migrate on fibrin(ogen) matrices. These include endothelial cells, macrophages, neutrophils, smooth muscle cells, fibroblasts, and keratinocytes.3-8 Direct binding to fibrin(ogen) through both integrin [e.g., αvβ3, α1β5, αMβ2 (CD11b/CD18, Mac-1)] and non-integrin receptors (e.g., intercellular adhesion molecule (ICAM-1)) appears to contribute to these cell-fibrin interactions.8-11 Fibrin(ogen) degradation products have also been reported to have an impressive array of biological activities, including mitogenic, angiogenic, chemotactic, and immunosuppressive activities.12-14 There are now substantial data indicating that fibrin(ogen) may plays an important role in the inflammatory response15,16 and that it may, in fact, direct leukocyte transendothelial cell migration.11 Similarly, through several G-protein coupled protease-activated receptors on fibroblasts, endothelial cells, leukocytes, smooth muscle cells, and other cell types, thrombin is thought to play an important role in inflammatory and fibroproliferative responses.17 Fibrinolytic factors, such as plasmin(ogen), also appear to be important modulators of inflammation.18 Finally, host fibrinogen, prothrombin, plasminogen, plasminogen activator, and other hemostatic factors appear to be crucial to the pathogenesis and virulence of many bacterial species.19-21 Unfortunately, despite a myriad of provocative observations made using in vitro systems, there is little direct in vivo evidence supporting an important role of fibrin(ogen) or other hemostatic factors in either the inflammatory response or disease progression. Direct and definitive analyses have been hampered by the lack of an experimental means to specifically manipulate the level or structure of selected hemostatic factors in vivo. Fortunately, this experimental roadblock has been effectively removed by the development of gene-targeting and gene transfer technologies in mice (see below).

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