THE BINDING OF ANTITHROMBIN TO CAPILLARY ENDOTHELIAL CELLS GROWN IN VITRO

1987 ◽  
Author(s):  
A de Agostini ◽  
J Marcum ◽  
R Rosenberg

Cloned endothelial cells from rat epididymal fat pads synthesize anticoagulantly active heparan sulfate proteoglycans containing the disaccharide, GlcA→ AMN-3,6-O-SO3, which is a marker for the antithrombin-binding domain of heparin. To demonstrate that antithrombin (AT) binds to cell surface heparan sulfate, a binding assay employing 125I-AT and cell monolayers has been developed. Post-confluent endothelial cells (7 days) were incubated with radiolabeled AT for 1 h at 4° and washed with PBS. Bound radioactivity was quantitated after solubilizing whole cells. Under these conditions, ∼1% (2174±50 cpm/5x104 cells) of the 125I-AT bound to the endothelial cell monolayer, whereas none of the radiolabeled protein bound to CHO cells or bovine smooth muscle cells. Utilization of unlabeled AT (1 μM) in experiments conducted as described above resulted in a reduction (73%) of the binding of the labeled species to endothelial cells. To assess whether heparan sulfate was responsible for AT binding, cell monolayers were incubated for 1 h at 37° with purified Flavobacterium heparinase (0.2 units). Over 90% of 125I-AT binding to these cellular elements was suppressed with the bacterial enzyme. Internalization of radiolabeled AT by endothelial cells was examined by incubating the protease inhibitor and cells at 4° and 37 . An initial rapid binding was observed at both temperatures. At 4° AT binding plateaued within 15 min, whereas at 37° binding did not plateau until 60 min and was 30% greater than that observed at 4. These data suggest that surface-associated AT can be internalized by endothelial cells. In addition, AT binding was shown to increase with the length of endothelial cell postconfluence, indicating an accumulation of heparan sulfate by these cells during quiescence. In conclusion, our studies support the hypothesis that the vascular endothelium is coated with heparan sulfate-bound AT, which is responsible for the antithrombotic properties of these natural surfaces.

1999 ◽  
Vol 112 (10) ◽  
pp. 1599-1609 ◽  
Author(s):  
B.M. Kraling ◽  
D.G. Wiederschain ◽  
T. Boehm ◽  
M. Rehn ◽  
J.B. Mulliken ◽  
...  

Vessel maturation during angiogenesis (the formation of new blood vessels) is characterized by the deposition of new basement membrane and the downregulation of endothelial cell proliferation in the new vessels. Matrix remodeling plays a crucial, but still poorly understood role, in angiogenesis regulation. We present here a novel assay system with which to study the maturation of human capillary endothelial cells in vitro. When human dermal microvascular endothelial cells (HDMEC) were cultured in the presence of dibutyryl cAMP (Bt2) and hydrocortisone (HC), the deposition of a fibrous lattice of matrix molecules consisting of collagens type IV, type XVIII, laminin and thrombospondin was induced. In basal medium (without Bt2 and HC), HDMEC released active matrix metalloproteinases (MMPs) into the culture medium. However, MMP protein levels were significantly reduced by treatment with Bt2 and HC, while protein levels and activity of endogenous tissue inhibitor of MMPs (TIMP) increased. This shift in the proteolytic balance and matrix deposition was inhibited by the specific protein kinase A inhibitors RpcAMP and KT5720 or by substituting analogues without reported glucocorticoid activity for HC. The addition of MMP inhibitors human recombinant TIMP-1 or 1,10-phenanthroline to cultures under basal conditions induced matrix deposition in a dose-dependent manner, which was not observed with the serine protease inhibitor epsilon-amino-n-caproic acid (ACA). The deposited basement membrane-type of matrix reproducibly suppressed HDMEC proliferation and increased HDMEC adhesion to the substratum. These processes of matrix deposition and downregulation of endothelial cell proliferation, hallmarks of differentiating new capillaries in the end of angiogenesis, were recapitulated in our cell culture system by decreasing the matrix-degrading activity. These data suggest that our cell culture assay provides a simple and feasible model system for the study of capillary endothelial cell differentiation and vessel maturation in vitro.


Blood ◽  
1983 ◽  
Vol 62 (3) ◽  
pp. 549-556 ◽  
Author(s):  
M Laposata ◽  
DK Dovnarsky ◽  
HS Shin

Abstract When thrombin is incubated with confluent monolayers of human umbilical vein endothelial cells in vitro, there is a change in the shape of the endothelial cells that results in gaps in the monolayer, disrupting the integrity of the endothelium and exposing the subendothelium. Using a grid assay to measure this phenomenon, we observed that up to 80% of the surface area once covered by cells was uncovered after a 15-min incubation with 10(-2) U/ml (10(-10)M) thrombin. The effect was apparent within 2 min and did not remove cells from the surface of the culture dish. The gaps in the monolayer completely disappeared within 2 hr after exposure to thrombin. The effect of thrombin was inhibited by preincubation of thrombin with hirudin or antithrombin III plus heparin or by preincubation of the monolayers with dibutyryl cyclic adenosine monophosphate (dbcAMP). Histamine also induced gap formation in endothelial cell monolayers. Both pyrilamine and cimetidine prevented the histamine-induced effect, but they had no effect on thrombin- induced gap formation. Intact monolayers were not disrupted by bradykinin, serotonin, C5a, or C3a. Our results suggest that small amounts of thrombin can induce repeated and transient exposure of the subendothelium, a situation believed to be conducive to atherogenesis and thrombosis.


1980 ◽  
Vol 152 (4) ◽  
pp. 931-944 ◽  
Author(s):  
R G Azizkhan ◽  
J C Azizkhan ◽  
B R Zetter ◽  
J Folkman

Migration of capillary endothelial cells is an important component of angiogenesis in vivo. Increased numbers of mast cells have been associated with several types of angiogenesis. We have used a quantitative assay in vitro to demonstrate that mast cells release a factor that significantly increases bovine capillary endothelial cell migration. The factor is present in medium conditioned by mast cells as well as lysates of mast cells. The stimulatory effect of mast cells on migration is specific for capillary endothelial cells. Furthermore, mast cells have no mitogenic activity for capillary endothelial cells. Of all the secretory products of mast cells tested, only heparin stimulated capillary endothelial cell migration in vitro. Heparin preparations from a variety of sources stimulated capillary endothelial cell migration to the same degree but did not stimulate migration of several other cell types. The migration activity of heparin and mast cell conditioned medium was blocked by specific antagonists of heparin (protamine and heparinase), but not by chondroitinase ABC. The migration activity of mast cell conditioned medium was resistant to heat (100 degrees C) and incubation with proteolytic enzymes. These results suggest that the role of mast cells in angiogenesis may be to enhance migration of the endothelial cells of growing capillaries.


1988 ◽  
Vol 60 (02) ◽  
pp. 240-246 ◽  
Author(s):  
Erna G Langeler ◽  
Victor W M van Hinsbergh

SummaryA model has been developed to study the transport of fluid and macromolecules through human arterial umbilical cord endothelial cell monolayers in vitro. Cells were cultured on fibronectin- coated polycarbonate filters and formed within a few days a tight monolayer, with an electrical resistance of 17 ± 4 Ohm · cm2. The cells were connected by close cell contacts with tight junctions. The passáge-rate of horse radish peroxidase (HRP) through these filters was 20-40 fold lower than through filters without an endothelial monolayer. The continuous presence of 10% human serum was needed to maintain the electrical resistance of the monolayer and its barrier function towards macromolecules. Chelation of extracellular calcium resulted in an increased permeability and a decreased electrical resistance of the monolayer. This process was reversible by re-addition of calcium ions to the cells. The permeation rate of dextrans of various molecular weights (9-480 kD) was inversely related to the molecular mass of the molecule. No difference was measured between the passage rate of dextran of 480 kD and dextran of 2,000 kD. Incubation of the endothelial cell monolayer with 2-deoxy-D-glucose resulted in a decreased permeability but it had no effect on electrical resistance. This suggests that the passage-process is energy- dependent.Fluid permeation through the endothelial cell monolayer on filters was measured in a perfusion chamber under 20 mmHg hydrostatic pressure. It was decreased by the presence of serum proteins and responded reversibly on the chelation and readdition of extracellular calcium ions.


Blood ◽  
1983 ◽  
Vol 62 (3) ◽  
pp. 549-556 ◽  
Author(s):  
M Laposata ◽  
DK Dovnarsky ◽  
HS Shin

When thrombin is incubated with confluent monolayers of human umbilical vein endothelial cells in vitro, there is a change in the shape of the endothelial cells that results in gaps in the monolayer, disrupting the integrity of the endothelium and exposing the subendothelium. Using a grid assay to measure this phenomenon, we observed that up to 80% of the surface area once covered by cells was uncovered after a 15-min incubation with 10(-2) U/ml (10(-10)M) thrombin. The effect was apparent within 2 min and did not remove cells from the surface of the culture dish. The gaps in the monolayer completely disappeared within 2 hr after exposure to thrombin. The effect of thrombin was inhibited by preincubation of thrombin with hirudin or antithrombin III plus heparin or by preincubation of the monolayers with dibutyryl cyclic adenosine monophosphate (dbcAMP). Histamine also induced gap formation in endothelial cell monolayers. Both pyrilamine and cimetidine prevented the histamine-induced effect, but they had no effect on thrombin- induced gap formation. Intact monolayers were not disrupted by bradykinin, serotonin, C5a, or C3a. Our results suggest that small amounts of thrombin can induce repeated and transient exposure of the subendothelium, a situation believed to be conducive to atherogenesis and thrombosis.


2002 ◽  
Vol 115 (9) ◽  
pp. 1951-1959
Author(s):  
Jian Li ◽  
Nicholas W. Shworak ◽  
Michael Simons

Binding of basic fibroblast growth factor (FGF2) to its high affinity receptors requires the presence of specific heparan sulfate (HS) moieties on the cell surface that act as coreceptors. To determine the contribution of cell-surface HS to modulation of FGF2-dependent cell growth, we studied the changes in the cell mass and FGF2 binding of endothelial cell HS under normoxic and hypoxic conditions in vitro. Both large vein and cardiac microvascular endothelial cells cultured under hypoxic conditions demonstrated an increase in the ratio of cell-surface HS to chondroitin sulfate (CS), as well as an increase in the number of low affinity (HS-associated) binding sites for FGF2 with no change in the apparent Kd. This increase in the number of HS-FGF2 binding sites, in the absence of a significant change in FGF receptor expression, resulted in enhanced responsiveness of hypoxic,compared with normoxic, endothelial cells to FGF2 stimulation. Gene expression studies demonstrated increased expression of the key regulatory enzyme responsible for HS chain synthesis, 1,4 GlcNAc transferase(GlcNAcT-I), as well as increased expression of 2-O sulfotransferase (HS2ST),the enzyme responsible for sulfation of IdoA, a crucial part of the HS-FGF2 binding site. Transduction of cells with an adenovirus encoding a HIF-1αexpression construct resulted in a similar increase in GlcNAcT-I and HS2ST expression. We conclude that hypoxia increases endothelial cell responsiveness to FGF2 by promoting preferential synthesis of HS rather than CS chains and increasing the number of FGF2-binding sites on HS chains. Both of these events are mediated by a HIF-1α-dependent increase in expression of the enzymes GlnNAcT-I and HS2ST. This shift in cell-surface HS composition results in enhanced cell sensitivity to FGF2-induced growth stimulation.


1992 ◽  
Vol 119 (3) ◽  
pp. 629-641 ◽  
Author(s):  
F Bussolino ◽  
M F Di Renzo ◽  
M Ziche ◽  
E Bocchietto ◽  
M Olivero ◽  
...  

Hepatocyte Growth Factor (HGF, also known as Scatter Factor) is a powerful mitogen or motility factor in different cells, acting through the tyrosine kinase receptor encoded by the MET protooncogene. Endothelial cells express the MET gene and expose at the cell surface the mature protein (p190MET) made of a 50 kD (alpha) subunit disulfide linked to a 145-kD (beta) subunit. HGF binding to endothelial cells identifies two sites with different affinities. The higher affinity binding site (Kd = 0.35 nM) corresponds to the p190MET receptor. Sub-nanomolar concentrations of HGF, but not of a recombinant inactive precursor, stimulate the receptor kinase activity, cell proliferation and motility. HGF induces repairs of a wound in endothelial cell monolayer. HGF stimulates the scatter of endothelial cells grown on three-dimensional collagen gels, inducing an elongated phenotype. In the rabbit cornea, highly purified HGF promotes neovascularization at sub-nanomolar concentrations. HGF lacks activities related to hemostasis-thrombosis, inflammation and endothelial cells accessory functions. These data show that HGF is an in vivo potent angiogenic factor and in vitro induces endothelial cells to proliferate and migrate.


Blood ◽  
2004 ◽  
Vol 104 (1) ◽  
pp. 92-99 ◽  
Author(s):  
Marco Rusnati ◽  
Maura Camozzi ◽  
Emanuela Moroni ◽  
Barbara Bottazzi ◽  
Giuseppe Peri ◽  
...  

Abstract The long pentraxin PTX3 is a soluble pattern recognition receptor produced by monocytes and endothelial cells that plays a nonredundant role in inflammation. Several pathologic conditions are characterized by local production of both PTX3 and the angiogenic fibroblast growth factor-2 (FGF2). Here, solid-phase binding assays demonstrated that PTX3 binds with high affinity to FGF2 but not to a panel of cytokines and growth factors, including FGF1, FGF4, and FGF8. Accordingly, PTX3 prevented 125I-FGF2 binding to endothelial cell receptors, leading to specific inhibition of FGF2-induced proliferation. PTX3 hampered also the motogenic activity exerted by endogenous FGF2 on a wounded endothelial cell monolayer. Moreover, PTX3 cDNA transduction in FGF2-transformed endothelial cells inhibited their autocrine FGF2-dependent proliferation and morphogenesis in vitro and their capacity to generate vascular lesions when injected in nude mice. Finally, PTX3 suppressed neovascularization triggered by FGF2 in the chick embryo chorioallantoic membrane with no effect on physiologic angiogenesis. In contrast, the short pentraxin C-reactive protein was a poor FGF2 ligand/antagonist. These results establish the selective binding of a member of the pentraxin superfamily to a growth factor. PTX3/FGF2 interaction may modulate angiogenesis in various physiopathologic conditions driven by inflammation, innate immunity, and/or neoplastic transformation.


1988 ◽  
Vol 64 (1) ◽  
pp. 308-322 ◽  
Author(s):  
S. M. Albelda ◽  
P. M. Sampson ◽  
F. R. Haselton ◽  
J. M. McNiff ◽  
S. N. Mueller ◽  
...  

The purpose of this study was to characterize the permeability characteristics of an in vitro endothelial cell monolayer system and relate this information to available in vivo data. We cultured bovine fetal aortic endothelial cells on fibronectin-coated polycarbonate filters and confirmed that our system was similar to others in the literature with regard to morphological appearance, transendothelial electrical resistance, and the permeability coefficient for albumin. We then compared our system with in vivo endothelium by studying the movement of neutral and negatively charged radiolabeled dextran tracers across the monolayer and by using electron microscopy to follow the pathways taken by native ferritin. There were a number of differences. The permeability of our monolayer was 10-100 times greater than seen in intact endothelium, there was no evidence of "restricted" diffusion or charge selectivity, and ferritin was able to move freely into the subendothelial space. The reason for these differences appeared to be small (0.5-2.0 micron) gaps between 5 and 10% of the endothelial cells. Although the current use of cultured endothelial cells on porous supports may provide useful information about the interaction of macromolecules with the endothelium, there appear to be differences in the transendothelial permeability characteristics of these models and in vivo blood vessels.


1988 ◽  
Vol 89 (3) ◽  
pp. 441-447 ◽  
Author(s):  
R. Montesano ◽  
L. Orci

The endothelium of visceral capillaries is characterized by the occurrence of numerous fenestrae, which are usually bridged by a thin, single-layered diaphragm. Both in vivo and in vitro, diaphragmed fenestrae perforate the endothelial cell cytoplasm in the most attenuated regions of the cell. We report here that in capillary endothelial cells grown under experimental conditions promoting the development of intracellular lumina (for example, suspension within a three-dimensional collagen matrix), diaphragmed fenestrae can form in a unique, previously undescribed intracellular location - that is, within thin cytoplasmic septa separating contiguous luminal compartments.


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