Tumor necrosis factor-induced endothelial tissue factor is associated with subendothelial matrix vesicles but is not expressed on the apical surface.

Cultured endothelial cells can be induced by tumor necrosis factor/cachectin (TNF) and other cytokines to synthesize the procoagulant cofactor tissue factor (TF). Intact monolayers of TNF-treated endothelial cells showed only minimal TF activity. In contrast, after permeabilization of these monolayers with detergent (saponin, 0.02%), there was approximately 10- to 20-fold increase in TF-mediated, factor VIIa-dependent factor Xa formation. Extracellular matrix derived from TNF-treated endothelium, prepared after removing the cells by hypotonic lysis or ammonium hydroxide (0.1 N), also had similarly enhanced TF activity. Incubation with a blocking monoclonal antibody to TF inhibited the procoagulant activity of both TNF-stimulated endothelial cells, whether they were intact or permeabilized, and of their matrices. However, when the apical cell surface was pretreated with anti-TF antibody, washed, and then cells were lysed with water or permeabilized with saponin, similar augmentation of TF activity was still observed, suggesting the presence of a pool of TF to which the antibody did not initially gain access. Consistent with this concept, the presence of TF in the matrix of TNF-treated endothelial cells was shown by immunoblotting and morphologic studies; cultured endothelial monolayers and the native endothelium of aortic segments after exposure to TNF showed TF in extracellular matrix, associated with vesicles. In contrast, TF was virtually undetectable on the apical endothelial surface. Taken together, these findings suggest that endothelial TF can be present in a cryptic pool that only gains access to the blood after alteration in the integrity of the endothelial monolayer.     

Induction of permeability across endothelial cell monolayers by tumor necrosis factor (TNF) occurs via a tissue factor-dependent mechanism: relationship between the procoagulant and permeability effects of TNF

Tumor necrosis factor (TNF) has marked effects on permeability and procoagulant activity on tumor-associated neovasculature when used in isolation perfusion, the latter effect primarily mediated via induction of cell surface expression of tissue factor (TF) on endothelial tissue. However, the cellular events that result in rapid alterations in endothelial cell (EC) permeability after intravascular TNF administration in isolation perfusion are not well characterized. We demonstrate that short exposure intervals to TNF induces TF expression on ECs but has no effect on permeability as assessed by flux of Evans blue-bound albumin across confluent EC monolayers using a 2-compartment model under basal culture conditions. However, a rapid and significant increase in EC permeability occurred with TNF in the presence of factor VIII-deficient plasma. Permeability was induced only with luminal versus abluminal TNF exposure and was blocked by antithrombin III, TF pathway inhibitor, or anti-TF antibody cotreatment. These data indicate that EC surface expression of TF and extrinsic clotting factors are critical in augmenting capillary leak following intravascular TNF administration. Alterations in permeability were associated with intercellular gap formation at sites of down-regulation of vascular endothelial (VE)-cadherin expression, the primary endothelial intercellular adhesion molecule, and intracellular contraction and alignment of F-actin cytoskeletal elements. Rapid induction of TF by TNF may be the primary EC response that results in alterations in permeability and procoagulant activity observed following intravascular TNF administration in isolation perfusion.     

Monocytes maintain tissue factor activity after cytolysis of bacteria-infected endothelial cells in an in vitro model of bacterial endocarditis

Intravascular infection with Staphylococcus aureus, Staphylococcus epidermidis, or Streptococcus sanguis can initiate fibrin formation on endocardial tissue, causing bacterial endocarditis. The ability of these bacteria to injure intact endothelial cells (ECs) and to aggravate tissue factor (TF)-dependent coagulation in the presence of blood leukocytes was investigated. Cytolysis of ECs occurred after infection with S. aureus and, with membrane-bound monocytes or granulocytes present, also after infection with S. sanguis or S. epidermidis. Monocytes that subsequently bound to the resultant bacteria-infected subcellular EC matrix (ECM) elicited TF mRNA, TF antigen, and TF activity (TFA). This was most pronounced in ECM prepared after the cytolysis of ECs by infection with S. aureus or S. epidermidis. We demonstrate that monocytes continue and intensify fibrin formation after lysis of bacteria-infected ECs, which suggests that, during the course of intravascular infection, early fibrin formation shifts from being mediated by EC-derived TFA to being mediated by TFA of monocytes bound to bacteria-infected ECM.     

Heparanase, tissue factor, and cancer

Heparanase is an endo-beta- D-glucuronidase that is capable of cleaving heparan sulfate side chains of heparan sulfate proteoglycans on cell surfaces and the extracellular matrix, activity that is strongly implicated in tumor metastasis and angiogenesis. Evidence was provided that heparanase overexpression in human leukemia, glioma, and breast carcinoma cells results in a marked increase in tissue factor (TF) levels. Likewise, TF was induced by exogenous addition of recombinant heparanase to tumor cells and primary endothelial cells, induction that was mediated by p38 phosphorylation and correlated with enhanced procoagulant activity. TF induction was further confirmed in heparanase-overexpressing transgenic mice and correlated with heparanase expression levels in leukemia patients. Heparanase was also found to be involved in the regulation of tissue factor pathway inhibitor (TFPI). It was shown that heparanase overexpression or exogenous addition induces two- to threefold increase of TFPI expression. Similarly, heparanase stimulated accumulation of TFPI in the cell culture medium. Extracellular accumulation exceeded, however, the observed increase in TFPI at the protein level and appeared to be independent of heparan sulfate and heparanase enzymatic activity. Instead, a physical interaction between heparanase and TFPI was demonstrated, suggesting a mechanism by which secreted heparanase interacts with TFPI on the cell surface, leading to dissociation of TFPI from the cell membrane and increased coagulation activity, thus further supporting the local prothrombotic function of heparanase. As heparins are strong inhibitors of heparanase, in view of the effect of heparanase on TF/TFPI pathway, the role of heparins' anticoagulant activity may potentially be expanded.     

Activated factor X and thrombin formation triggered by tissue factor on endothelial cell matrix in a flow model: effect of the tissue factor pathway inhibitor

The procoagulant subcellular matrix of stimulated endothelial cells that contains tissue factor (TF) was used to investigate the mechanism by which TF pathway inhibitor (TFPI) inhibits thrombin formation initiated by TF/factor VIIa (FVIIa) under flow conditions. Purified coagulation factors VII, X, and V and prothrombin were perfused at a wall shear rate of 100 s-1 through a flow chamber containing a coverslip covered with matrix of cultured human umbilical vein endothelial cells. This resulted in a TF- and FVII-dependent FXa and thrombin generation as measured in the effluent at the outlet of the system. Inhibition of this TF/FVIIa-triggered thrombin formation by TFPI purified from plasma was dependent on the amount of TF present on the endothelial cell matrix. The rate of prothrombinase assembly and steady-state levels of thrombin formation were decreased by TFPI. Because persistent albeit decreased steady-state levels of thrombin formation occurred in the presence of TFPI, we conclude that plasma- TFPI does not inhibit FXa present in the prothrombinase complex. The addition of FIX and FVIII to perfusates containing FVII and FX increased the FXa generation on endothelial matrices, and counteracted the inhibition of thrombin formation on endothelial cell matrices by TFPI. Our data provide further evidence for the hypothesis that the rapid inactivation of TF/FVIIa by TFPI in combination with the absence of either FVIII or FIX causes the bleeding tendency of patients with hemophilia A or B.     

Human mesothelial cells express tissue factor when switched to proliferating state. Pharmacological modulation in vitro.

Covering the inner surface of small-diameter arterial prostheses with endothelial cells (ECs) has been proposed as a means of improving biocompatibility and thrombosis resistance. Because the availability of autologous ECs is limited, autologous human mesothelial cells (HMCs) have been suggested as a substitute for ECs. However, HMCs express tissue factor (TF) in vitro, a deleterious characteristic in vivo. We investigated the distribution of TF antigen and of its inhibitor, tissue factor pathway inhibitor, on HMCs and the effect of pharmacological agents on TF expression. TF antigen was measured by enzyme-linked immunosorbent assay and localized by confocal microscopy. Three distinct pools of TF antigen were demonstrated: within the cells, at the cell surface, and in the extracellular matrix. The effects of ilomedin (10 microg/ml) and heparin (500 U/ml), known to affect procoagulant activity, were evaluated by incubating HMCs for 24 h with or without these agents. Ilomedin, but not heparin, decreased TF antigen expression by 30% (P < 0.05). Despite the theoretical potential of HMCs as a vascular prosthesis lining, TF expression by HMCs remains a major drawback. A technique capable of blocking TF expression until the HMCs return to their resting state is needed. Genetic manipulation of HMCs may hold promise for such a technique.     

Priming effect of adrenic acid (22:4(n-6)) on tissue factor activity expressed by thrombin-stimulated endothelial cells.

Tissue factor (TF) which initiates clotting process can be expressed by stimulated endothelial cells (EC). TF is an apolipoprotein requiring an association with phospholipids (PL) in order to become active. Also PL constitute an important storage pool of polyunsaturated fatty acids (PUFAs) in EC which can be modulated by diet or cell medium supplementation. In order to test the effect of such manipulation upon TF activity, we have pre-enriched human EC cultures with different fatty acids of nutritional interest. TF was evaluated after 4 h of thrombin stimulation by using a chromogenic method. Without additional stimulating agents, these acids have no effect on the basal level of TF. Eicosapentaenoic and docosapentaenoic acids appeared to be ineffective at the stimulated TF level. Only adrenic acid (22:4(n-6)) has been found to significantly enhance TF activity of thrombin-stimulated endothelial cells. Other TF inducers were also tested after 22:4(n-6) enrichment. An increase tendency of TF expression was found only with tumor necrosis factor, whereas interleukin-1 beta, lipopolysaccharide and especially phorbol myristate acetate stimulations were not significantly modified. The priming effect of adrenic acid on thrombin stimulated TF expression might involve alterations of signal transduction pathways rather than modifications of apolipoprotein III environment. Adrenic acid, which is a prostacyclin inhibitor, appears to be potential prothrombotic agent.     

Cooperation between VEGF and TNF-alpha is necessary for exposure of active tissue factor on the surface of human endothelial cells

This study was undertaken to characterize tissue factor (TF) induction, localization, and functional activity in cultured human umbilical vein endothelial cells (HUVECs) exposed to recombinant vascular endothelial growth factor (rVEGF) and recombinant tumor necrosis factor-alpha (rTNF-alpha). rVEGF (1 nmol/L) and rTNF-alpha (500 U/mL) synergistically increased TF mRNA, protein, and total activity, as measured in cell lysates. To examine surface TF expression, living cells were treated with antibody to TF and examined microscopically. Almost no staining was seen in control cells or cells treated with a single agent. In contrast, cells treated with both agonists showed intense membrane staining with surface patches, appearing as buds by confocal microscopy. To determine surface TF activity, studies were performed using a parallel-plate flow chamber, which allows detection of factor Xa generation on living cells. rVEGF and rTNF-alpha induced little surface TF activity (0.032+/-0.008 and 0.014+/-0.008 fmol/cm2, respectively). In combination, they significantly increased TF expression on the cell surface (0.429+/-0.094 fmol/cm2, P<0.05). These data indicate that the synergistic effect of rVEGF and rTNF-alpha is necessary to generate functional TF on the surface of endothelial cells. The requirement for multiple agonists to expose active TF may serve to protect endothelial cells from acting as a procoagulant surface, even under conditions of cell perturbation.     

MicroRNA-223 inhibits tissue factor expression in vascular endothelial cells

Objective: Atherosclerosis is a chronic inflammatory process, in which vascular endothelial cells (ECs) become dysfunctional owing to the effects of chemical substances, such as inflammatory factor and growth factors. Tissue factor (TF) expression is induced by the above chemical substances in activated ECs. TF initiates thrombosis on disrupted atherosclerotic plaques which plays an essential role during the onset of acute coronary syndromes (ACS). Increasing evidences suggest the important role of microRNAs as epigenetic regulators of atherosclerotic disease. The aim of our study is to identify if microRNA-223 (miR-223) targets TF in ECs. Methods and results: Bioinformatic analysis showed that TF is a target candidate of miR-223. Western blotting analysis revealed that tumor necrosis factor α (TNF-α) increased TF expression in aorta of C57BL/6J mice and cultured ECs (EA.hy926 cells and HUVEC) after 4 h treatment. In TNF-α treated ECs, TF mRNA was also increased measured by real-time PCR. Real-time PCR results showed that miR-223 levels were downregulated in TNF-α-treated aorta of C57BL/6J mice and cultured ECs. Transfection of ECs with miR-223 mimic or miR-223 inhibitor modified TF expression both in mRNA and protein levels. Luciferase assays confirmed that miR-223 suppressed TF expression by binding to the sequence of TF 3′-untranslated regions (3′UTR). TF procoagulant activity was inhibited by overexpressing miR-223 with or without TNF-α stimulation. Conclusions: MiR-223-mediated suppression of TF expression provides a novel molecular mechanism for the regulation of coagulation cascade, and suggests a clue against thrombogenesis during the process of atherosclerotic plaque rupture.     

A novel, quantitative model for study of endothelial cell migration and sprout formation within three-dimensional collagen matrices

Interactions between migratory endothelial cells (ECs) and surrounding extracellular matrix (ECM) are of central importance to vascular growth. Here, we present a new model of EC migration and morphogenesis within three-dimensional ECM termed "radial invasion of matrix by aggregated cells" (RIMAC). In the RIMAC model, single aggregates of defined numbers of bovine aortic ECs were embedded within small, lenticular gels of type I collagen supported by annuli of nylon mesh. Culture of the gels in nutrient media resulted in quantifiable, reproducible, radial migration of ECs into the collagen. The angiogenic proteins basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) each stimulated migration of ECs in a concentration-dependent manner. In combination, bFGF and VEGF stimulated migration synergistically. In contrast, transforming growth factor-beta1 inhibited migration of ECs. Low concentrations (0.1-0.5 ng/ml) of VEGF induced ECs to form multicellular sprouts, some of which possessed lumen-like spaces. Mitomycin C, an inhibitor of cell proliferation, did not affect the migration of ECs into collagen induced by 0.5 ng/ml VEGF but moderately inhibited migration induced by 5 ng/ml VEGF. Increasing the density (concentration) of the collagen gel inhibited the migration of single ECs and increased the branching and anastomosis of multicellular sprouts. We conclude that the RIMAC model is a highly efficacious assay for the screening of potentially angiogenic and angiostatic compounds and, moreover, is advantageous for mechanistic studies of vascular morphogenesis.     

Stimulation of tissue factor expression in human microvascular and macrovascular endothelial cells by cultured vascular smooth muscle cells in vitro

The effect of conditioned media obtained from different smooth muscle cells (SMC) on tissue factor (TF) expression in endothelial cells (EC) in vitro was investigated. We could show that conditioned media from cultured human aortic SMC, human umbilical artery SMC or human umbilical vein SMC all resembling the synthetic phenotype of SMC induced TF activity in human umbilical vein EC and human skin microvascular EC in a dose- and time-dependent fashion. This induction was also seen at the level of specific TF mRNA as evidenced by Northern blotting. The TF inducing activity was heat-labile and acid-stable and had an approximate molecular mass of 38 kD. This activity was found to be distinct from known inducers of TF expression in EC such as interleukin-1, tumor necrosis factor-alpha, bacterial lipopolysaccharide or vascular endothelial growth factor. Such as factor, if released by SMC in vivo, could contribute to the activation of EC under conditions such as when EC are in close contact with SMC of the synthetic (nondifferentiated) phenotype seen in processes like vessel development or neo-intima formation.     

All-trans-retinoic acid counteracts endothelial cell procoagulant activity induced by a human promyelocytic leukemia-derived cell line (NB4)

Therapy with all-trans-retinoic acid (ATRA) can rapidly improve the coagulopathy of acute promyelocytic leukemia (APL). This study was designed to evaluate whether the APL cell line NB4 induces the procoagulant activity (PCA) of human endothelial cells (ECs) in vitro, and whether this property is modified after ATRA-induced NB4 maturation. EC monolayers were incubated for 4 hours at 37 degrees C with the conditioned media (CM) of NB4 treated with 1 mumol/L ATRA (ATRA-NB4-CM) or the vehicle (control-NB4-CM). EC lysates were tested for PCA. ATRA-NB4-CM induced significantly more PCA:tissue factor (TF) than control-NB4-CM (P < .01). To identify the cause of TF induction, interleukin (IL)-1 beta antigen levels were measured in CM samples. ATRA-NB4-CM contained significantly more IL-1 beta than control-NB4-CM. EC PCA was significantly inhibited by an anti-IL-1 beta antibody. The addition to the media of 10 mumol/L ATRA counteracted the EC TF expression induced by NB4-CM. These data indicate that ATRA increases the promyelocyte-induced EC TF, partly through increased IL-1 beta production. However, ATRA can protect the endothelium from the procoagulant stimulus of leukemic cells.     

Probing antiphospholipid-mediated thrombosis: the interplay between anticardiolipin antibodies and endothelial cells

The association of antiphospholipid (aPL) antibodies with thrombosis in patients with antiphospholipid syndrome (APS) is well documented in humans and in animal studies. However, the mechanisms by which aPL antibodies induce thrombosis are the subject of much current study. It has been suggested that aPL may activate endothelial cells (ECs), thus creating a hypercoagulable state that precedes and contributes to thrombosis in patients with APS. Several studies have shown that aPL upregulate ECs' adhesion molecules (CAMs): intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and E-selectin (E-sel) or induce tissue factor (TF) in monocytes in vitro. Similarly, the incubation of EC with antibodies reacting with beta2glycoprotein I (beta2GPI) has been shown to induce EC activation with concomitant upregulation of CAMs, IL-6 production and alteration of prostaglandin metabolism. Our group has shown that aPL-mediated upregulation of adhesion molecules on ECs correlates with an increased adhesion of leukocytes to endothelium in the microcirculation of mouse cremaster muscle, a n indication of EC activation in vivo, andwith enhanced thrombosis in vivo. In another series of studies, investigators have shown that upregulation of expression of adhesion molecules by some murine monoclonal anti-beta2glycoprotein I (anti-beta2GPI) antibodies correlated with fetal resorption in mice in vivo. More recently, one study showed that the anti-hypercholesterolaemic drug fluvastatin inhibited the aPL-mediated enhanced adhesion of monocytes to ECs in vitro. Data from our laboratories indicate that fluvastatin also reverses thrombus formation and activation of EC induced by aPL in an in vivo mouse model. As additional support for the hypothesis that aPL antibodies activate ECs and may create an hypercoagulable state in APS patients, two recent studies indicated that levels of soluble ICAM-1 and VCAM-1 were significantly increased in the plasma of patients with APS and recurrent thrombosis. Furthermore, studies utilizing knockout mice and specific monoclonal anti-VCAM-1 antibodies have demonstrated that expression of ICAM-1, P-selectin, E-selectin and VCAM-1 are important in in vivo aPL-mediated thrombosis and EC activation in mice. Recent data suggests that aPL antibodies also induce expression of TF not only in monocytes but in ECs. Hence, the interference of aPL with the TF mechanism may be another important mechanism by which these antibodies create a hypercoagulable state and prone patients to thrombosis. Specifically, how aPL alters EC activation state and the molecular and intracellular mechanisms involved have not yet been defined. APL may interact with specific cell surface receptors (proteins and/or lipids) induce signals that have consequences downstream, and that ultimately will result in upregulation of cell surface proteins (i.e., CAMs and TF) and subsequently induce EC activation. In that regard, our group recently showed that aPL-mediated upregulation of adhesion molecules in ECs is preceded by activation of the nuclear factor kappa B (NFkappaB). Other intracellular mechanisms triggered by aPL are not completely understood and are the subject of current investigation. In conclusion, studies suggest that activation of ECs by aPL is an important mechanism that may precede thrombus formation in patients with APS. Hence, the interplay between aPL antibodies and ECs is important inthe pathogenesis of thrombosis in APS.     

Tissue factor on the loose

The enzymatic complex of tissue factor (TF) and the blood coagulation factor VIIa is generally considered to be the initiator of coagulation. Coagulation that occurs at the site of luminal injury to an artery is, along with platelet deposition, the cause of arterial thrombosis, which is the leading cause of death in Western society. Under pathological conditions the intima, the neointima and the atherosclerotic plaque contain active TF. Therefore the initiation of thrombosis is believed to be due to TF present in the wall of the pathologically changed artery. This classical view of thrombosis has been challenged. In this article we review the evidence for the presence of TF activity in various tissues outside the vessel wall, in extracellular form, in encrypted form, and even in plasma. We found TF expression in a variety of cells in culture after growth factor or cytokine stimulation. This TF was often also present in the extracellular matrix, and in addition we found latent TF on the outside of unbroken smooth muscle cells. Freeze-thawing the cells or detergent lysis could activate this TF. We also found TF activity in native whole blood and in plasma. Inhibition of this circulating TF prevented formation of thrombi on collagen-coated glass slides in an ex vivo perfusion system. Furthermore, in a thrombosis model in which rat aorta was injured, TF was found on the intimal surface of the injured aorta. TF activity was measured in a flow chamber, and it was shown that all measurable activity was extracellular. We conclude that blood-borne TF plays a major role in thrombosis. Encryption of TF present in circulation could be a mechanism that prevents thrombosis. Alternatively, circulating TF may be active but below the threshold required for the initiation of blood coagulation.     

Induction of transporter associated with antigen processing by interferon γ confers endothelial cell cytoprotection against natural killer-mediated lysis

T lymphocytes react minimally with nonactivated endothelial cells (ECs). However, natural killer (NK) lymphocyte interactions with resting ECs are rapid, avid, and result in endothelial activation and/or cytotoxicity. The molecular basis for these interactions and EC sensitivity to NK-mediated lysis is unclear. To address the EC-specific nature of NK sensitivity, we used syngeneic human umbilical vein ECs, dermal microvascular ECs, dermal fibroblasts, and B lymphoblastoid cell lines in calcein-AM retention NK assays with allogeneic NK effector cells and found the EC lines consistently more NK-sensitive. Because NK inhibitory receptors are engaged by membrane major histocompatibility complex (MHC) I molecules and MHC I-deficient targets are NK-sensitive, we investigated the quantitative levels of membrane MHC I on the panel of syngeneic lines. Highly sensitive ECs expressed similar (or higher) levels of membrane MHC I than their syngeneic NK-resistant counterparts. Pretreatment of ECs with γ interferon (IFN-γ) conferred protection against NK-mediated lysis, with much more rapid kinetics (2–6 hr) than those required for membrane MHC I hyperinduction (>8 hr). These kinetics are consistent with induction of transporter associated with antigen processing (TAP) expression and function. As opposed to NK-resistant cell lines, TAP-1 was undetectable in resting ECs. Recombinant expression of the TAP inactivator ICP47 by adenoviral-mediated transduction was used to selectively inhibit IFN-γ-mediated EC TAP function. ICP47 expression abrogated EC cytoprotection conferred by IFN-γ. We demonstrate a relationship between both basal and induced TAP-1 expression/function and EC sensitivity to NK-mediated cytotoxicity. We discuss the influence of an induced MHC I-associated peptide repertoire on vascular vulnerability to cytotoxic lymphocytes.     

Localization of 13-hydroxyoctadecadienoic acid and the vitronectin receptor in human endothelial cells and endothelial cell/platelet interactions in vitro

Blood/vessel wall cell interactions depend, in part, on the expression of adhesion receptors on cell surfaces, such as expression of the vitronectin receptor (VnR) on the apical surface of endothelial cells (ECs) for platelet/EC adhesion. However, it is unclear how receptor expression is regulated from within cells. In previous studies, we found that ECs metabolize linoleic acid into the lipoxygenase monohydroxide, 13-hydroxyoctadecadienoic acid (13-HODE), and that the intracellular level of 13-HODE correlates inversely with VnR expression and platelet adhesion to the EC apical surface. In this study, we determined the physical associations of 13-HODE and VnR in unstimulated and stimulated ECs, ie, at times when ECs were and were not adhesive for specific ligands and platelets, using double antibody immunofluorescent staining techniques and binding assays. 13-HODE and the VnR were colocalized within unstimulated ECs. When ECs were stimulated, 13-HODE was no longer detectable, either in or outside the ECs, and the VnR was detected on the apical surface of the ECs. These changes were paralleled by increased vitronectin binding and increased platelet adhesion to the ECs. We suggest that colocalization of 13-HODE with VnR reflects a 13-HODE/VnR interaction, confining the VnR in a nonadhesive form inside unstimulated ECs, and, as a result, the ECs are nonadhesive. When the ECs are stimulated, 13-HODE and VnR dissociate, allowing the VnR to relocate on the EC surface, where the VnR undergoes a conformational change resulting in increased EC adhesivity.     

Fibrinogen induces endothelial cell adhesion and spreading via the release of endogenous matrix proteins and the recruitment of more than one integrin receptor.

We have previously shown that fibrinogen (fg) acts as a subendothelial matrix protein in promoting human endothelial cell (EC) adhesion and cytoskeletal organization. In this study we report that EC spreading on fg, at variance with other matrix proteins, requires endogenous matrix protein synthesis and secretion. ECs, upon seeding on fg, promptly released and organized a fibronectin (fn) matrix. Fg was more effective than vitronectin (vn) in promoting the deposition of this protein. ECs treated with monensin to block matrix protein secretion still adhered to fg but did not properly organize their cytoskeleton and adhesion structures. In contrast, monensin did not affect EC spreading either on vn or on fn. Using antibodies to the alpha and beta chains of fn (alpha 5 beta 1) and vn (alpha v beta 3) receptors, it was found that ECs adherent to fg show clustering and organization in adhesion structures of both type of receptors. A faint staining of adhesion structures with alpha 2 but not alpha 3 and alpha 6 antibodies was also observed. Antibodies either to vn or fn receptors were able to disrupt the EC monolayer and to induce EC retraction and detachment, thus indicating that both receptors are important in maintaining a sustained EC adhesion to fg. However, when ECs were treated with monensin only the vn receptor was organized in adhesion structures while the fn receptor was diffusely distributed. This suggests that clustering of the fn receptor is mediated by the release of endogenous matrix proteins induced by the exposure to fg. In conclusion, fg has a peculiar and complex type of interaction with ECs since it requires endogenous matrix protein release and the recruitment of more than one adhesive receptor. This suggests a specific way of response of ECs to each extracellular matrix component.