Re-establishment of VWF-dependent Weibel-Palade bodies in VWD endothelial cells

Type 3 von Willebrand disease (VWD) is a severe hemorrhagic defect in humans. We now identify the homozygous mutation in the Chapel Hill strain of canine type 3 VWD that results in premature termination of von Willebrand factor (VWF) protein synthesis. We cultured endothelium from VWD and normal dogs to study intracellular VWF trafficking and Weibel-Palade body formation. Weibel-Palade bodies could not be identified in the canine VWD aortic endothelial cells (VWD-AECs) by P-selectin, VWFpp, or VWF immunostaining and confocal microscopy. We demonstrate the reestablishment of Weibel-Palade bodies that recruit endogenous P-selectin by expressing wild-type VWF in VWD-AECs. Expression of mutant VWF proteins confirmed that VWF multimerization is not necessary for Weibel-Palade body creation. Although the VWF propeptide is required for the formation of Weibel-Palade bodies, it cannot independently induce the formation of the granule. These VWF-null endothelial cells provide a unique opportunity to examine the biogenesis of Weibel-Palade bodies in endothelium from a canine model of type 3 VWD.     

von Willebrand factor mediates platelet adhesion to virally infected endothelial cells.

von Willebrand factor is an adhesive glycoprotein critical to normal hemostasis. It is stored in the Weibel-Palade body of endothelial cells and upon release may mediate platelet adhesion. Herpesvirus-infected endothelium is known to be prothrombotic and to support enhanced platelet adherence. We previously identified P-selectin as a monocyte receptor that is translocated from the Weibel-Palade body to the endothelial cell surface in response to the local generation of thrombin on herpesvirus infected cells. In this study, we show that viral injury to vascular endothelial cells induces secretion of von Willebrand factor which mediates enhanced platelet adhesion to these cells.     

Small GTP-binding protein Ral modulates regulated exocytosis of von Willebrand factor by endothelial cells

Weibel-Palade bodies are endothelial cell-specific organelles, which contain von Willebrand factor (vWF), P-selectin, and several other proteins. Recently, we found that the small GTP-binding protein Ral is present in a subcellular fraction containing Weibel-Palade bodies. In the present study, we investigated whether Ral is involved in the regulated exocytosis of Weibel-Palade bodies. Activation of endothelial cells by thrombin resulted in transient cycling of Ral from its inactive GDP-bound to its active GTP-bound state, which coincided with release of vWF. Ral activation and exocytosis of Weibel-Palade bodies were inhibited by incubation with trifluoperazine, an inhibitor of calmodulin, before thrombin stimulation. Functional involvement of Ral in exocytosis was further investigated by the expression of constitutively active and dominant-negative Ral variants in primary endothelial cells. Introduction of active Ral G23V resulted in the disappearance of Weibel-Palade bodies from endothelial cells. In contrast, the expression of the dominant-negative Ral S28N did not affect the amount of Weibel-Palade bodies in transfected cells. These results indicate that Ral is involved in regulated exocytosis of Weibel-Palade bodies by endothelial cells.     

Biosynthesis, processing and secretion of von Willebrand factor: biological implications.

von Willebrand factor is a multimeric plasma glycoprotein that is required for normal haemostasis. von Willebrand factor is synthesized by endothelial cells and megakaryocytes, and originates from its precursor pro-von Willebrand factor. The endoproteolytic processing of pro-von Willebrand factor results in mature von Willebrand factor and von Willebrand factor propeptide (also known as von Willebrand Ag II). In endothelial cells, the propeptide controls the polymerization and subsequent targeting of von Willebrand factor to the storage vesicles, the so-called Weibel-Palade bodies. Upon stimulation of the endothelial cells, the Weibel-Palade bodies are translocated to the plasma membrane of the cell, and mature von Willebrand factor and its propeptide are co-secreted. After release, these polypeptides have divergent fates and serve different biological functions. Mature von Willebrand factor both controls platelet adhesion and aggregation at sites of vascular injury and acts as a chaperone protein for coagulation factor VIII. The von Willebrand factor propeptide may serve a role in modulating inflammatory processes. This still growing body of information indicates that the biological function of the von Willebrand factor gene product is more diverse than was previously thought.     

Isolation and longterm culture of human intestinal microvascular endothelial cells.

Microvascular endothelial cells play an important part in inflammation as well as in organ specific leucocyte traffic, and may be functionally different from large vessel endothelium in this respect. This study therefore established a method for isolation and longterm culture of human intestinal microvascular endothelial cells (HIMEC). After dissociation by collagenase/dispase/DNase of mucosal and submucosal tissue obtained from normal adult jejunum, cells were plated and cultured to subconfluence in endothelial serum free medium containing 2.5% fetal calf serum, hydrocortisone, and N6, O2-dibutyryladenosine cyclic monophosphate. Primary cultures were trypsinised and endothelial cells were isolated by paramagnetic beads armed with monoclonal antibody to CD31. Optimal growth conditions for HIMEC cultures were established, allowing up to nine passages (three months in vitro). The cells contained Weibel-Palade bodies, expressed von Willebrand factor, CD31, and VE-cadherin; and bound Ulex Europaeus lectin I. A method to establish longterm cell cultures of HIMEC will facilitate further investigation of the function of intestinal endothelial cells and their participation in physiological and pathological events in the gut.     

Activated platelets induce Weibel-Palade-body secretion and leukocyte rolling in vivo: role of P-selectin

The presence of activated platelets and platelet-leukocyte aggregates in the circulation accompanies major surgical procedures and occurs in several chronic diseases. Recent findings that activated platelets contribute to the inflammatory disease atherosclerosis made us address the question whether activated platelets stimulate normal healthy endothelium. Infusion of activated platelets into young mice led to the formation of transient platelet-leukocyte aggregates and resulted in a several-fold systemic increase in leukocyte rolling 2 to 4 hours after infusion. Rolling returned to baseline levels 7 hours after infusion. Infusion of activated P-selectin-/- platelets did not induce leukocyte rolling, indicating that platelet P-selectin was involved in the endothelial activation. The endothelial activation did not require platelet CD40L. Leukocyte rolling was mediated solely by the interaction of endothelial P-selectin and leukocyte P-selectin glycoprotein ligand 1 (PSGL-1). Endothelial P-selectin is stored with von Willebrand factor (VWF) in Weibel-Palade bodies. The release of Weibel-Palade bodies on infusion of activated platelets was indicated by both elevation of plasma VWF levels and by an increase in the in vivo staining of endothelial P-selectin. We conclude that the presence of activated platelets in circulation promotes acute inflammation by stimulating secretion of Weibel-Palade bodies and P-selectin-mediated leukocyte rolling.     

Ceramide triggers Weibel-Palade body exocytosis

The sphingolipid ceramide mediates a variety of stress responses, including vascular inflammation and thrombosis. Activated endothelial cells release Weibel-Palade bodies, granules containing von Willebrand factor (vWF) and P-selectin, which induce leukocyte rolling and platelet adhesion and aggregation. We hypothesized that ceramide induces vascular inflammation and thrombosis in part by triggering Weibel-Palade body exocytosis. We added ceramide to human aortic endothelial cells and assayed Weibel-Palade body exocytosis by measuring the concentration of vWF released into the media. Exogenous ceramide induces vWF release from endothelial cells in a dose-dependent manner. Activators of endogenous ceramide production, neutral sphingomyelinase, or tumor necrosis factor-alpha also induce Weibel-Palade body exocytosis. We next studied NO effects on ceramide-induced Weibel-Palade body exocytosis because NO can inhibit vascular inflammation. The NO donor S-nitroso-N-acetylpenicillamine decreases ceramide-induced vWF release in a dose-dependent manner, whereas the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester increases ceramide-induced vWF release. In summary, our findings show that endogenous ceramide triggers Weibel-Palade body exocytosis, and that endogenous NO inhibits ceramide-induced exocytosis. These data suggest a novel mechanism by which ceramide induces vascular inflammation and thrombosis.     

Molecular and cellular biology of von Willebrand factor

von Willebrand factor (VWF) is a plasma protein that performs 2 main functions in hemostasis: it mediates platelet adhesion to the injured vessel wall, and it carries and protects coagulation factor VIII. VWF is synthesized through a multistep process in endothelial cells and megakaryocytes as a very large polymer composed of identical disulfide-linked 250-kd subunits. In endothelial cells, VWF not only directs the formation of its own storage granules, the Weibel-Palade bodies, but it also acts as a chaperone molecule to direct other proteins, such as P-selectin, into these granules. Upon stimulation of the endothelium, the Weibel-Palade bodies will be translocated to the plasma membrane, and their contents will be secreted into the plasma milieu. The expression of VWF can be regulated at different levels by a number of genetic and environmental factors, resulting in control of its activity. New roles for VWF, especially in inflammatory processes, have recently been suggested, indicating that some aspects of this well-studied protein remain to be investigated.     

Desmopressin induces endothelial P-selectin expression and leukocyte rolling in postcapillary venules

Desmopressin, (DDAVP; 1-desamino-8-D-arginine vasopressin) increases the release and activity of von Willebrand factor (vWF); however, its effects on the other major constituent of endothelial Weibel-Palade bodies, P-selectin, has not been investigated. DDAVP-induced P-selectin expression may explain DDAVP's efficacy in bleeding disorders in which vWF levels are normal. Therefore, the objective of this study is to assess the effect of DDAVP on P-selectin expression on endothelial cells of postcapillary venules in vivo and on human umbilical vein endothelium in vitro, and to determine whether DDAVP has direct effects on leukocyte behavior in postcapillary venules. DDAVP (0.1 and 1.0 microgram/mL) induced a significant but transient increase in P- selectin expression on human umbilical vein endothelial cells as well as on rat and human platelets. Immunohistochemical analysis of rat postcapillary venules showed that in contrast to saline, DDAVP injection (1 microgram/kg, intravenous) induced significant endothelial P-selectin expression. DDAVP administration also induced a rapid and significant increase in leukocyte rolling in rat mesenteric venules in vivo. This response was entirely dependent on P-selectin, as an anti-P- selectin antibody rapidly reversed the DDAVP-induced increase in leukocyte rolling. DDAVP induced leukocyte rolling in medium (20 to 40 microns) and large (> 40 microns), but not small (< 20 microns), postcapillary venules. In animals that were treated with DDAVP, there was a steady and significant increase in leukocyte adhesion. This study shows that DDAVP can directly induce P-selectin expression on endothelium in vitro and in vivo and that the latter response is capable of supporting prolonged leukocyte rolling in rat postcapillary venules.     

Storage and secretion of von Willebrand factor by endothelial cells

Endothelial cells synthesize and store von Willebrand factor. We have studied the storage and secretion of von Willebrand factor in cultured human umbilical vein endothelial cells. In particular, we were interested in the nature of the storage compartment and the effects of perturbation on the storage and secretion processes. The storage compartment for von Willebrand factor was isolated from homogenates of endothelial cells. By an immunostaining technique the isolated vesicles stained for von Willebrand factor. The staining pattern was similar to that of Weibel-Palade bodies in intact endothelial cells. We concluded that the storage compartment containing von Willebrand factor is identical to the Weibel-Palade body. The von Willebrand factor of the isolated storage vesicles is predominantly constructed of polypeptide chains with a Mr of 220 kD. On the other hand, von Willebrand factor continuously secreted by endothelial cells is constructed of both a 220 kD and a larger precursor (apparent Mr of 275 kD) subunit. The storage vesicles contain von Willebrand factor that supports ristocetin-induced platelet aggregation. Thus, endothelial cells store fully processed, biologically active von Willebrand factor within Weibel-Palade bodies. Short-term (less than 1 h) treatment of endothelial cells with the perturbing phorbol ester 4 beta-phorbol-12-myristate-13-acetate (PMA) results in release of cellular stored von Willebrand factor. 24-48 h after exposure to PMA the endothelial cell distribution of von Willebrand factor is changed distinctly. While the contents of the von Willebrand factor storage sites in the cells are gradually restored within 48 h, enhanced amounts of von Willebrand factor are secreted into the medium. The number as well as the size of von Willebrand factor storage granules in the endothelial cells increase after exposure to phorbol ester, as determined by immunofluorescence microscopy. Phorbol ester treated cells release stored von Willebrand factor 48 h after they have been stimulated. PMA decreases the von Willebrand factor contents of the extracellular matrix; the deposition of von Willebrand factor in the subendothelium is blocked by PMA, whereas the degradation of matrix von Willebrand factor is not affected. Thus, perturbation of endothelial cells changes the cellular distribution of von Willebrand factor.     

Defect in regulated secretion of P-selectin affects leukocyte recruitment in von Willebrand factor-deficient mice

Stimulation of endothelial cells by various inflammatory mediators leads to release of Weibel--Palade bodies and therefore to exocytosis of both P-selectin (adhesion receptor for leukocytes) and von Willebrand factor (vWf) (platelet ligand). The potential role of vWf in leukocyte recruitment was investigated with the use of vWf-deficient mice. We report a strong reduction of leukocyte rolling in venules of vWf-deficient mice. Similarly, vWf deficiency led to a decrease in neutrophil recruitment in a cytokine-induced meningitis model as well as in early skin wounds. In all instances with an antibody that preferentially recognizes plasma membrane P-selectin, we observed a dramatic reduction in P-selectin expression at the cell surface of vWf-deficient endothelium. With confocal microscopy, we found that the typical rodlike shape of the Weibel--Palade body is missing in vWf -/- endothelial cells and that part of the P-selectin content in the vWf -/- cells colocalized with LAMP-1, a lysosomal marker. However, intracellular P-selectin levels were similar in tumor necrosis factor alpha- and lipopolysaccharide-activated cells of both genotypes. We conclude that the absence of vWf, as found in severe von Willebrand disease, leads to a defect in Weibel--Palade body formation. This defect results in decreased P-selectin translocation to the cell surface and reduced leukocyte recruitment in early phases of inflammation.     

von Willebrand factor synthesized by endothelial cells from a patient with type IIB von Willebrand disease supports platelet adhesion normally but has an increased affinity for platelets.

Endothelial cells were isolated from the umbilical vein of a patient with subtype IIB von Willebrand disease, and the biosynthesis and function of von Willebrand factor (vWF) synthesized by these cells were compared with those of vWF synthesized by endothelial cells from normal individuals. The patient's endothelial cells synthesized, stored, and secreted vWF indistinguishably from normal endothelial cells: it was synthesized as a prepolypeptide of Mr 270,000 and had a mature form of Mr 220,000; the full spectrum of multimers was found both inside the cells and in the culture medium; it was stored normally, in the Weibel-Palade bodies; and similar amounts of vWF were secreted into the medium and deposited in the extracellular matrix. In a perfusion set-up, the extracellular matrix from IIB cells supported platelet adhesion similarly to the matrix from normal cells. vWF secreted constitutively by IIB cells into the culture medium bound to platelets at concentrations of ristocetin lower than those necessary for vWF from normal cells. vWF stored in the Weibel-Palade bodies of type IIB cells was released upon stimulation with phorbol ester and bound almost completely to platelets even in the absence of ristocetin. Moreover, spontaneous platelet aggregation was induced by vWF synthesized by type IIB cells. These data support the hypothesis that the absence of highly multimeric forms of vWF in plasma of type IIB von Willebrand disease patients is due to specific removal of these multimers by platelets.     

CD63 is a component of Weibel-Palade bodies of human endothelial cells

Weibel-Palade bodies are secretory granules of vascular endothelial cells specialized in the storage of von Willebrand factor (vWF) and P- selectin, two adhesion proteins that can be rapidly mobilized to the cell surface by exocytosis in response to thrombin or other agonists. In this study, we attempted to identify additional components of Weibel- Palade bodies by raising monoclonal antibodies to these granules, purified by cell fractionation. One antibody, 2C6, was found to be specific for CD63, a membrane glycoprotein previously described in the lysosomes of platelets and other cell types. The immunopurified 2C6 antigen was recognized by an anti-CD63 reference antibody, 2.28, by Western blotting. Also, the biosynthetic profile of the 2C6 antigen in endothelial cells showed a nascent molecular mass and a glycosylation pattern identical to that of CD63. Immunofluorescence staining with 2C6 showed the lysosomes, and also elongated structures identified as Weibel-Palade bodies by their shape, distribution, and positive staining with anti-vWF antibodies, CD63 was also found by Western blotting of subcellular fractions highly enriched in Weibel-Palade bodies. Our results indicate that CD63 colocalizes with vWF and P- selectin in the Weibel-Palade bodies of endothelial cells, and together with these adhesion proteins it could be rapidly expressed on the cell surface in areas of vascular injury and inflammation.     

Isolation and culture of endothelial cells from human bone marrow

Summary : Adhesive interactions between haemopoietic progenitor cells and bone marrow sinusoidal endothelium are potentially important in the homing of these cells back to re-establish haemopoiesis following stem cell transplantation. A simple method for the isolation and culture of human bone marrow endothelial cells is described using bone marrow aspirates obtained from patients undergoing bone marrow harvests for autologous or syngeneic bone marrow transplantation. The method is based on the selective binding of the lectin Ulex europaeus agglutinin-1 (UEA-1) to endothelial cells. Magnetic Dynabeads coupled with UEA-1 were incubated with single cell suspensions of bone marrow following red cell lysis, and bound cells were isolated with a magnet. The isolated cells demonstrated positive immunofluorescence staining for von Willebrand factor. Cells were plated onto tissue culture flasks coated with extracellular matrix derived from human umbilical vein endothelial cells in an endothelial serum-free medium together with 5% fetal calf serum for 24h. Cells were then cultured in endothelial serum-free growth medium supplemented with 5% fetal calf serum, endothelial cell growth supplemented with 5% fetal calf serum, endothlial cell growth supplement and heparin. After 2–4 weeks in culture, two morphologically different cell populations can be identified. One has a polygonal spindle-shaped morphology with a rapid growth rat, the other a rounded morphology and a slow growth rate. Both population have a vasiculated cytoplasm. Positive immunostaining of the cells was demonstrated with a number of endothelial cell markers including von Willebrand factor, and antibodies to ICAM-1, VCAM-1, E-wlectin, CD31 and BMA120. Weibel-Palade bodies were observed by electron microscopy.
Culture of these cells will allow detailed in vitro studies of adhesion mechanisms in the homing of haemopoietic progenitor cells.     

Isolation and characterization of human bone marrow microvascular endothelial cells: hematopoietic progenitor cell adhesion

To examine potential mechanisms by which hematopoiesis may be regulated by endothelial cells within the bone marrow (BM) microenvironment, we have devised a technique for the in vitro study of the interaction of human BM microvascular endothelial cells (BMEC) with hematopoietic cells. Microvessels isolated by collagenase digestion of spicules obtained from filtered BM aspirate were plated on gelatin-coated plastic dishes, and colonies of endothelial cells grown from microvessel explants were further purified by Ulex europaeus lectin affinity separation. BMEC monolayers isolated by this technique grew in typical cobblestone fashion, stained positively with antibody to factor VIII/von Willebrand factor, and incorporated acetylated LDL. Immunohistochemical studies showed that BM microvessels and BMEC monolayers express CD34, PECAM, and thrombospondin. Incubation of resting BMEC with BM mononuclear hematopoietic cells resulted in the selective adhesion of relatively large numbers of CD34+ progenitor cells and megakaryocytes. The binding of purified BM-derived CD34+ progenitor cells to BMEC was dependent on divalent cations and was partially blocked by antibodies to CD34. IL-1 beta treatment of BMEC monolayers resulted in an increase of CD34+ progenitor cell adhesion by mechanisms independent of CD34 or divalent cations. BMEC exhibit specific affinity for CD34+ progenitor cells and megakaryocytes, suggesting that the BM microvasculature may play a role in regulating the trafficking, proliferation, and differentiation of lineage specific hematopoietic elements, and possibly of pluripotent stem cells within the CD34+ population.     

Pathophysiological levels of soluble P-selectin mediate adhesion of leukocytes to the endothelium through Mac-1 activation

Plasma soluble P-selectin (sP-selectin) levels are increased in pathologies associated with atherosclerosis, including peripheral arterial occlusive disease (PAOD). However, the role of sP-selectin in regulating leukocyte-endothelial adhesion is unclear. The aim of this study was to assess the ability of exogenous and endogenous sP-selectin to induce leukocyte responses that promote their adhesion to various forms of endothelium. In flow chamber assays, sP-selectin dose-dependently increased neutrophil adhesion to resting human iliac artery endothelial cells. Similarly, sP-selectin induced neutrophil adhesion to the endothelial surface of murine aortae and human radial venous segments in ex vivo flow chamber experiments. Using intravital microscopy to examine postcapillary venules in the mouse cremaster muscle, in vivo administration of sP-selectin was also found to significantly increase leukocyte rolling and adhesion in unstimulated postcapillary venules. Using a Mac-1-specific antibody and P-selectin knockout mouse, it was demonstrated that this finding was dependent on a contribution of Mac-1 to leukocyte rolling and endothelial P-selectin expression. This was confirmed in an ex vivo perfusion model using viable mouse aorta and human radial vessels. In contrast, with tumor necrosis factor-alpha-activated endothelial cells and intact endothelium, where neutrophil adhesion was already elevated, sP-selectin failed to further increase adhesion. Plasma samples from PAOD patients containing pathologically elevated concentrations of sP-selectin also increased neutrophil adhesion to the endothelium in a sP-selectin-dependent manner, as demonstrated by immunodepletion of sP-selectin. These studies demonstrate that raised plasma sP-selectin may influence the early progression of vascular disease by promoting leukocyte adhesion to the endothelium in PAOD, through Mac-1-mediated rolling and dependent on endothelial P-selectin expression.     

Platelets adhere to and translocate on von Willebrand factor presented by endothelium in stimulated veins

With the use of intravital microscopy, a new type of platelet-endothelial interaction in mouse mesenteric venules at low shear (80-100 seconds(-1)) is described. Stimulation of these vessels with calcium ionophore A23187, a known secretagogue of Weibel-Palade bodies, induced immediate platelet adhesion (within 15 seconds) and translocation without the formation of aggregates. This stop-and-go process reached a maximum in approximately 1 minute, when approximately 25 000 platelets adhered/mm(2).s, and then adhesion progressively decreased. This adhesion process was dependent on von Willebrand factor (vWF) and independent of P-selectin. Immunohistologic analysis showed that the venules were not denuded with A23187 treatment, suggesting that platelets adhered to vWF secreted on the luminal face of the endothelial cells. Histamine treatment induced a similar adhesion phenomenon. Platelet adhesion was not abolished in beta3-deficient mice or when the platelets were treated with inhibitory antibodies to PECAM-1 or PSGL-1, indicating that these molecules are not required for platelet-endothelium interaction at low shear. The adhesion was mediated by platelet glycoprotein Ibalpha (GPIbalpha) because the adhesion of murine platelets expressing exclusively the human GPIbalpha could be prevented by a pretreatment with mocarhagin, a snake venom protease that cleaves human GPIbalpha. The results indicate that vWF released from Weibel-Palade bodies can dramatically increase the concentration of platelets along the vessel wall through an interaction with GPIbalpha. It is proposed that this process may rapidly recruit platelets to sites of injury or inflammation in veins.     

Divergent fates of von Willebrand factor and its propolypeptide (von Willebrand antigen II) after secretion from endothelial cells.

The intracellular site of cleavage of pro-von Willebrand factor subunit and the subsequent fate of the propolypeptide (von Willebrand antigen II) and of the mature von Willebrand factor (vWf) were investigated. Both the propolypeptide, which was found to be a homodimer of noncovalently linked subunits, and mature vWf were released from Weibel-Palade bodies of endothelial cells following stimulation with secretagogues. The stoichiometry of the two released proteins was essentially equimolar. This indicates that vWf and the propolypeptide were packaged into the Weibel-Palade bodies as one unit, pro-vWf, and that the proteolytic cleavage of pro-vWf is likely to be a post-Golgi event. The association of prosequences into dimers supports their hypothetical role in the multimerization process. After secretion, the two proteins were distributed differently, as based on the following observations. The propolypeptide did not associate with vWf in the culture medium, did not codistribute with vWf in the extracellular "patches of release" on stimulated endothelial cells, and was not detected in the endothelial cell extracellular matrix, which did contain vWf. Additionally, in contrast to vWf, the propolypeptide did not bind to the matrix of human foreskin fibroblasts. Since the propolypeptide does not associate with vWf and does not interact with extracellular matrices in vitro, it is highly unlikely that it would promote platelet adhesion to subendothelium in vivo.     

Soluble intercelluar adhesion molecule-1, E-selectin, vascular cell adhesion molecule-1 and von Willebrand factor in stroke.

We investigated the role of endothelial cell and leukocyte adhesion in the pathophysiology of acute stroke. The immunocytochemical expression of adhesion molecules in brain tissue from six patients who died following acute ischaemic stroke showed weak endothelial expression of intercellular adhesion molecule-1 (ICAM-1), but intense expression of vascular cell adhesion molecule-1 (VCAM-1) by astrocytes and endothelial cells from the infarcted, but not the non-infarcted areas. We also measured soluble adhesion molecules E-selectin, ICAM-1, VCAM-1 and von Willebrand factor (all by enzyme-linked immunosorbent assay) in 21 patients after an acute ischaemic stroke (ictus < 12 h), and again 3 months later. Blood levels in the stroke patients were compared with 82 healthy controls and 22 subjects with carotid atherosclerosis. Compared with healthy controls, both patient groups had raised levels of von Willebrand factor (P < 0.02) but the level of soluble VCAM-1 was raised only in patients with acute stroke (P < 0.02). Levels of von Willebrand factor and soluble VCAM-1 in the stroke patients were still high at 3 months follow-up. We suggest that there might be changes in adhesion molecule expression and release in the acute and chronic stages of ischaemic stroke, where blood levels are related to immunocytochemical findings on infarcted brain. These changes may perhaps be part of the complex pathophysiological responses to infarction and repair of brain tissue following stroke.