Endothelial cells in culture: An experimental model for the study of vascular dysfunctions

Culture of endothelial cells started two decades ago and is now a useful tool in understanding endothelial physiology and the study of the interaction of endothelial cells with blood cells and various mediators. In vitro proliferation can be measured by [³H]thymidine incorporation in defined conditions and gives reproducible results. Endothelial cells can be activated by several stimuli, including cytokines such as tumor necrosis factor- and interleukin-1. Part of endothelial cell activation is defined by expression or overexpression of leukocyte adhesion molecules. Intracellular adhesion molecule (ICAM), E-selectin And vascular adhesion molecule (VCAM) are receptor molecules for leukocyte adhesion. Leukocyte adhesion to endothelium can be measured in static but also rn rheologically defined flow conditions. Normal red blood cells (RBCs) do not adhere to endothelium, while RBC from patients with sickle cell anemia, diabetes mellitus, and malaria have an increased adhesion to endothelium which is mediated by specific VCAM, receptor for advanced glycated end-products (RAGE), and ICAM, respectively. Binding of blood cells or activation by cytokine is followed by a series of reactions in endothelial cells associated with the modulation of prostacyclin, nitric oxide, tissue factor, and cytokine production. Modification of endothelial cell functions in culture is correlated to in vivo alteration of vascular wall properties, further supporting these cells in culture as a relevant experimental model.Abbreviations AGEs advanced glycated end-products - ICAM intracellular adhesion molecule - IL-1 interleukin-1 - IFN- interferon- - MECIF monocyte-derived endothelial cell inhibitory factor - NO nitric oxide - PECAM-1 platelet-endothelial cell adhesion molecule-1 - RAGE receptor for advanced glycated end-products - RBCs red blood cells - TNF- tumor necrosis factor- - VCAM vascular adhesion molecule     

Hydroxycarbamide Decreases Sickle Reticulocyte Adhesion to Resting Endothelium by Inhibiting Endothelial Lutheran/Basal Cell Adhesion Molecule (Lu/BCAM) through Phosphodiesterase 4A Activation

Vaso-occlusive crises are the main acute complication in sickle cell disease. They are initiated by abnormal adhesion of circulating blood cells to vascular endothelium of the microcirculation. Several interactions involving an intricate network of adhesion molecules have been described between sickle red blood cells and the endothelial vascular wall. We have shown previously that young sickle reticulocytes adhere to resting endothelial cells through the interaction of α₄β₁ integrin with endothelial Lutheran/basal cell adhesion molecule (Lu/BCAM). In the present work, we investigated the functional impact of endothelial exposure to hydroxycarbamide (HC) on this interaction using transformed human bone marrow endothelial cells and primary human pulmonary microvascular endothelial cells. Adhesion of sickle reticulocytes to HC-treated endothelial cells was decreased despite the HC-derived increase of Lu/BCAM expression. This was associated with decreased phosphorylation of Lu/BCAM and up-regulation of the cAMP-specific phosphodiesterase 4A expression. Our study reveals a novel mechanism for HC in endothelial cells where it could modulate the function of membrane proteins through the regulation of phosphodiesterase expression and cAMP-dependent signaling pathways.     

Adhesion of human granulocytes and T lymphocytes to vascular endothelial cells after stimulation with Bacteroides fragilis endotoxin and enterotoxin]

The aim of presented study was to estimates the number of human granulocytes and T lymphocytes adhering to 1 mm2 of vascular endothelial cell culture stimulated by Bacteroides fragilis endotoxins (LPS) and enterotoxin (BFT). HMEC-1 cells were activated with bacterial preparations at the concentration of 10 (micrograms/ml for 4 and 24 hours. Granulocytes and T lymphocytes were isolated from peripheral blood of healthy blood donors. The adhesion tests of granulocytes and adhesion tests of resting and activated with PMA (at the concentration of 10 ng/ml) T lymphocytes to the non-stimulated and stimulated by B. fragilis compounds (LPS and BFT) vascular endothelium were performed. The number of viable leukocytes, which adhered to the endothelium, was determined using inverted microscope (magnification 200x). The results were presented as the number of viable cells adhering to 1 mm2 of the endothelial cell culture. The results of experiments indicate that granulocytes and T lymphocytes (resting and after activation with PMA even in greater number) adhere to the endothelial cells stimulated by B. fragilis endotoxins and enterotoxin. B. fragilis toxins are weaker stimulants of human leukocyte adhesion to the HMEC-1 cells than E. coli O55:B5 LPS. B. fragilis LPS and BFT preparations stimulate endothelial cells to the adhesion of granulocytes in similar manner, whereas the activation of vascular endothelium to the adhesion of T lymphocytes is differentiated.     

Adhesion of human T lymphocytes and granulocytes to endothelial cells stimulated by cell-surface antigens of Bacteroides thetaiotaomicron

The aim of this study was to assay the degree of human T lymphocyte and granulocyte adhesion to the vascular endothelial cells stimulated by Bacteroides thetaiotaomicron lipopolysaccharides, components of LPS and capsular polysaccharide. HMEC-1 cells were activated with bacterial preparations in concentration 10 micrograms/ml for 4 and 24 hours. T lymphocytes and granulocytes were isolated from peripheral blood of healthy blood donors. Thereafter, the adhesion tests of granulocytes and adhesion tests of non-activated and activated with PMA (in concentration 10 ng/ml) T lymphocytes to the resting and stimulated vascular endothelium were performed. The number of viable cells, which adhered to the endothelium, was determined using inverted microscope (magnification 200x). The results were presented as the number of viable cells adhering to 1 mm2 of the endothelial cell culture. The obtained results indicate that granulocytes and T lymphocytes (resting and activated with PMA) adhere to the endothelial cells stimulated by B. thetaiotaomicron cell-surface antigens. B. thetaiotaomicron lipopolysaccharides and capsular polysaccharide are weaker stimulants of human leukocyte adhesion to the HMEC-1 cells than E. coli O55:B5 LPS.     

Inhibition of sickle red cell adhesion and vasoocclusion in the microcirculation by antioxidants

In sickle cell anemia (SCA), inflammatory (i.e., intravascular sickling and transient vasoocclusive) events result in chronic endothelial activation. In addition to sickling behavior, sickle (SS) red blood cells exhibit abnormal interaction with the vascular endothelium, which is considered to have an important role in initiation of vasoocclusion. Upregulation of endothelial adhesion molecules caused by oxidants (and cytokines) may lead to increased SS red cell adhesion. We hypothesize that endothelial activation is indispensable in SS red cell adhesion to the endothelium and that antioxidants will have an inhibitory effect on this interaction. We examined the effect of selected antioxidants in ex vivo mesocecum vasculature, a well-established model that allows measurement of hemodynamic parameters and, by intravital microscopy, can allow quantification of adhesion. We tested antioxidant enzymes (SOD and catalase) and an intravascular SOD mimetic, polynitroxyl albumin (PNA), in the presence of platelet-activating factor (PAF); the latter causes endothelial oxidant generation and endothelial activation, which characterize SCA. In ex vivo preparations, PAF not only induced marked endothelial oxidant generation, it also enhanced SS red cell adhesion, resulting in frequent blockage of small-diameter venules. The adhesion, inversely related to venular diameter, and vasoocclusion were markedly inhibited by antioxidants, resulting in improved hemodynamics. PNA, the most effective antioxidant, also abolished SS red cell adhesion in non-PAF-activated preparations. Thus SS red cell adhesion and related vasoocclusion may be ameliorated by antioxidant therapy with a stable and long-acting molecule (e.g., PNA).     

Prevalence of diabetes mellitus in individuals heterozygous and homozygous for sickle cell anemia

Colorimetric determinations of glycosylated Hb were carried out in a sample (n = 97) of sickle cell anemia patients, and in an age- and sex-matched group of individuals (n = 45) heterozygous for sickle cell anemia, from the Eastern Province of Saudi Arabia. A statistically significant increase in the value of glycosylated Hb was found in sickle cell trait (HbAS) group, when compared with those of sickle cell anemia (HbSS) and normal (HbAA) groups. Since glycosylated Hb is considered a valid indicator of long-term blood glucose, and assuming normal red blood cell survival in HbAS carriers, the increased value of glycosylated Hb may suggest that there exists a higher incidence of undiagnosed diabetes mellitus in individuals with heterozygous inheritance for sickle cell hemoglobin than homozygous sickle cell patients and normal individuals. The mechanism underlying this observation remains to be defined.     

Critical role of endothelial cell activation in hypoxia-induced vasoocclusion in transgenic sickle mice

Activation of vascular endothelium plays an essential role in vasoocclusion in sickle cell disease. The anti-inflammatory agents dexamethasone and adhesion molecule-blocking antibodies were used to inhibit endothelial cell activation and hypoxia-induced vasoocclusion. Transgenic sickle mice, expressing human alpha-, beta(S)-, and beta(S-Antilles)-globins, had an activated vascular endothelium in their liver, lungs, and skin, as exhibited by increased activation of NF-kappaB compared with normal mice. NF-kappaB activation increased further in the liver and skin after sickle mice were exposed to hypoxia. Sickle mice had decreases in red blood cell (RBC) velocities and developed vasoocclusions in subcutaneous venules in response to hypoxia. Dexamethasone pretreatment prevented decreases in RBC velocities and inhibited vasoocclusions and leukocyte-endothelium interactions in venules after hypoxia. Dexamethasone treatment inhibited NF-kappaB, VCAM-1, and ICAM-1 expression in the liver, lungs, and skin of sickle mice after hypoxia-reoxygenation. VCAM-1 or ICAM-1 blockade with monoclonal antibodies mimicked dexamethasone by inhibiting vasoocclusion and leukocyte adhesion in sickle mice, demonstrating that endothelial cell activation and VCAM-1 and ICAM-1 expression are necessary for hypoxia-induced vasoocclusion in sickle mice. VCAM-1, ICAM-1, and vasoocclusion increased significantly 3 days after dexamethasone discontinuation, possibly explaining rebounds in vasoocclusive crises observed after withdrawal of glucocorticosteroids in sickle patients. We conclude that anti-inflammatory treatments that inhibit endothelial cell activation and adhesion molecule expression can inhibit vasoocclusion in sickle cell disease. Rebounds in vasoocclusive crises after dexamethasone withdrawal are caused by rebounds in endothelial cell activation.     

Co-localization of the immunoreactivities of corticotropin-releasing factor and arginine vasotocin in the brain and pituitary system of the teleost Catostomus commersoni

Summary Using the label-fracture technique, an ultrastructural comparison was made of the number and distribution of wheat germ agglutinin (WGA)-binding sites between human normal and sickle red blood cells. The WGA was adsorbed to colloidal gold, and quantitative analysis of the electron micrographs revealed that more binding sites were present on the sickle erythrocytes than on the normal erythrocytes. Moreover, the sites were more clustered on the sickle red cells than on the normal red cells. Use of another lectin, Bandieraea simplicifolia-II, revealed that it did not bind to normal or sickle red cells. Because of the affinity of the WGA for sialic acid residues, it is probable that the WGA is binding to a transmembrane sialoglycoprotein, glycophorin A. The conformation and/or distribution of the glycophorin A molecules may be altered by the sickle hemoglobin that binds to the red cell membrane. Hence, as detected by WGA, new surface receptors, which could play a role in the adhesion of sickle cells to endothelium may be exposed.     

Identification and function of transmembrane glycoproteins--the red cell model

Transmembrane glycoproteins in the red cell membrane traverse the plasma membrane, have their carbohydrate moieties on the extracellular surface, are sialyated (except for band 3) and are tethered to the membrane cytoskeleton proteins on the cytoplasmic surface. This linkage between the transmembrane proteins and the skeletal membrane proteins provides a two-way communication between the extracellular surface and the interior of the red cell; i.e., a transmembrane effect can be initiated from either side. These interactions are discussed in this review, including the example of sickle cell anemia in which the membrane bound hemoglobin may exert a transmembrane effect to change the conformation or distribution of transmembrane glycoproteins and and hence the extracellular surface receptors. This, in turn, may explain why sickle cells adhere to endothelium in vitro. Although the RBC transmembrane sialoglycoproteins may function in communication, regulation of cell shape, and adhesion, uncertainties exist regarding many of their functions. To study these sialoglycoproteins, we have developed a double staining technique (Dzandu et al., 1984) that differentially stains human RBC membrane sialoglycoproteins and asialoproteins in SDS-polyacrylamide gels. This should aid in elucidating the conformational structure and function of transmembrane glycoproteins.     

Heterotypic and homotypic cell-cell adhesion molecules in endothelial cells

Sickle red blood cells display an abnormal propensity to adhere to cultured bovine aortic endothelial cells when compared to normal red blood cells. The adherence was potentiated three-fold by endothelial cell derived conditioned medium, enriched in multimers of von Willebrand factor. Such adherence was ablated by 80% by either the synthetic peptide (RGDS) or antibody to GPIIb/IIIa, indicating the presence of RGD peptide recognition domain/receptor in either endothelial cells or sickle cells or both. The adherence was also inhibited by 70% by phosphatidylserine, but not by other phospholipids, indicating the presence of putative receptors for this phospholipid in endothelial cells. The labeling of cultured bovine aortic endothelial cells with monoclonal antibodies revealed the localization of MAB D2 to regions of cell-cell contact. The antigen on endothelial cells which cross-reacts with this antibody has a Mr of 130,000. The addition of such an antibody during the plating of endothelial cells disrupted monolayer formation. It appears that a 130-kDa polypeptide antigen in endothelial cells which is recognized by MAB D2, may be a cell-cell adhesion molecule.     

Capillary plugging by granulocytes and the no-reflow phenomenon in the microcirculation

Granulocytes are large, stiff viscoelastic cells that adhere naturally to the vascular endothelium. On their passage through the capillary network they have to be deformed, and recent evidence indicates that they may impose a significant hemodynamic resistance. The entry time of granulocytes into capillaries is about three orders of magnitude longer than that for red cells. Inside the capillary the granulocytes move with a lower velocity than red cells. Under conditions when the capillary perfusion pressure is reduced and/or elevated levels of inflammatory products are present that increase the adhesion stress to the endothelium, granulocytes may become stuck in the capillary. In such a situation, the granulocytes form a large contact area with the capillary endothelium, they obstruct the lumen, and they may initiate tissue injury. After the restoration of the perfusion pressure the granulocytes may not be removed from the capillary owing to the adhesion to the endothelium. Capillary plugging by granulocytes appears to be the mechanism responsible for the no-reflow phenomenon, and together with oxygen free radical formation and lysosomal enzyme activity may constitute the origin for ischemic injury as well as other microvascular occlusive diseases.     

Cytoadherence and sequestration in Plasmodium falciparum: defining the ties that bind

Infected erythrocytes containing the more mature stages of the human malaria Plasmodium falciparum may adhere to endothelial cells and uninfected red cells. These phenomena, called sequestration and rosetting, respectively, are involved in both host pathogenesis and parasite survival. This review provides a critical summary of recent advances in the characterization of the molecules of the infected red blood cell involved in adhesion, i.e. parasite-encoded molecules (PfEMP1, MESA, rifins, stevor, clag 9, histidine-rich protein), a modified host membrane protein (band 3) and exofacial exposure of phosphatidylserine, as well as receptors on the endothelium, i.e. thrombospondin, CD36, ICAM-1 (intercellular adhesion molecule), and chondroitin sulfate.     

Massive sequestration of human sickle cells after transfusion to a baboon

A baboon was exchange-transfused with sickle cell anemia patients' blood. The animal died suddenly, and postmortem examination showed widespread red cell sequestration, particularly in the spleen and liver. The clinical and pathological findings were similar to those in children with sickle cell anemia who die of acute splenic sequestration syndrome. A control animal, exchange-transfused with normal human blood, tolerated the procedure without difficulties for a period of 4 days, when a delayed transfusion reaction occurred. Thus the baboon can be used as a model for the abnormal circulatory behavior of sickle cells and for the sickle cell sequestration syndrome.     

Leukocyte kinetics in the microcirculation

The transport of leukocytes in the microcirculation is specific for the type, size, and the rheological and adhesive properties, the microanatomy of the host organ, and the hemodynamics. The adhesion to the endothelium is determined largely by the degree of activation via chemotactic factors. Leukocyte motion differs from that of red cells or platelets in several respects. When granulocytes enter into capillaries, they are deformed just like red cells. Under normal flow conditions, the time to deform at the entry to capillaries is typically 1,000 times larger than for the red cell, leading to temporary obstruction of the capillaries. After entry, granulocytes move with lower velocity than red cells which causes a cell train formation inside the capillary. At the venular side, the granulocyte is displaced from the center stream toward the endothelium by faster moving red cells. This process leads to systematic attachment of the granulocytes to the endothelium. At a reduced perfusion pressure or in the presence of locally elevated levels of chemotactic factors, the granulocytes may not be able to pass through the capillary network, which leads to microvascular obstruction. Organs with a narrow capillary network may thereby become filters for circulating granulocytes. This event is accompanied in many situations with damage to the host organ.     

Non-adsorbing macromolecules promote endothelial adhesion of erythrocytes with reduced sialic acids

Background

Abnormal adhesion of red blood cells (RBCs) to vascular endothelium is often associated with reduced levels of sialic acids on RBC membranes and with elevated levels of pro-adhesive plasma proteins. However, the synergistic effects of these two factors on the adhesion are not clear. In this work, we tested the hypothesis that macromolecular depletion interaction originating from non-adsorbing macromolecules can promote the adhesion of RBCs with reduced sialic acid content to the endothelium.

Methods

RBCs are treated with neuraminidase to specifically remove sialic acids from their surface followed by the evaluation of their deformability, zeta potential and membrane proteins. The adhesion of these enzyme-treated RBCs to cultured human umbilical vein endothelial cells (ECs) is studied in the presence of 70 or 500 kDa dextran with a flow chamber assay.

Results

Our results demonstrate that removal of sialic acids from RBC surface can induce erythrocyte adhesion to endothelial cells and that such adhesion is significantly enhanced in the presence of high-molecular weight dextran. The adhesion-promoting effect of dextran exhibits a strong dependence on dextran concentration and molecular mass, and it is concluded to originate from macromolecular depletion interaction.

Conclusion

These results suggest that elevated levels of non-adsorbing macromolecules in plasma might play a significant role in promoting endothelial adhesion of erythrocytes with reduced sialic acids.

General significance

Our findings should therefore be of great value in understanding abnormal RBC–EC interactions in pathophysiological conditions (e.g., sickle cell disease and diabetes) and after blood transfusions.     

MEK Inhibitors,Novel Anti-Adhesive Molecules,Reduce Sickle Red Blood Cell Adhesion In Vitro and In Vivo,and Vasoocclusion In Vivo

In sickle cell disease, sickle erythrocyte (SSRBC) interacts with endothelial cells, leukocytes, and platelets, and activates coagulation and inflammation, promoting vessel obstruction, which leads to serious life-threatening complications, including acute painful crises and irreversible damage to multiple organs. The mitogen-activated protein kinase, ERK1/2, is abnormally activated in SSRBCs. However, the therapeutic potential of SSRBC ERK1/2 inactivation has never been investigated. I tested four different inhibitors of MEK1/2 (MEK), the kinase that activates ERK1/2, in a model of human SSRBC adhesion to TNFα-activated endothelial cells (ECs). SSRBC MEK inhibition abrogated adhesion to non-activated and TNFα-activated ECs to levels below baseline SSRBC adhesion to non-activated ECs in vitro. SSRBC MEK inhibition also prevented SSRBCs from activating naïve neutrophils to adhere to endothelium. To determine the effect of MEK inhibitors on SSRBC adherence in vivo, sham-treated or MEK inhibitor-treated SSRBCs were infused to nude mice previously treated with TNFα. Sham-treated SSRBCs displayed marked adhesion and occlusion of enflamed vessels, both small and large. However, SSRBC treatment with MEK inhibitors ex vivo showed poor SSRBC adhesion to enflamed vessels with no visible vasoocclusion in vivo. In addition, MEK inhibitor treatment of SSRBCs reduced SSRBC organ trapping and increased the number of SSRBCs circulating in bloodstream. Thus, these data suggest that SSRBC ERK1/2 plays potentially a critical role in sickle pathogenesis, and that MEK inhibitors may represent a valuable intervention for acute sickle cell crises.     

Small-molecule cyclic alpha V beta 3 antagonists inhibit sickle red cell adhesion to vascular endothelium and vasoocclusion

Abnormal adhesion of sickle red blood cells (SS RBCs) to vascular endothelium may play an important role in vasoocclusion in sickle cell disease. Accruing evidence shows that endothelial alpha V beta 3-integrin has an important role in SS RBC adhesion because of its ability to bind several adhesive proteins implicated in this interaction. In the present studies, we tested therapeutic efficacy of small-molecule cyclic pentapeptides for their ability to block alpha V beta 3-mediated SS RBC adhesion by using two well-established assay systems, i.e., cultured human umbilical vein endothelial cells (HUVEC) and artificially perfused mesocecum vasculature of the rat under flow conditions. We tested the efficacy of two RGD-containing cyclic pentapeptides, i.e., cRGDFV (EMD 66203) and cRGDF-ACHA (alpha-amino cyclohexyl carboxylic acid) (EMD 270179), based on their known ability to bind alpha V beta 3. An inactive peptide, EMD 135981 (cR beta-ADFV) was used as control. Cyclization and the introduction of D-Phe (F) results in a marked increase in the ability of cyclic peptides to selectively bind alpha V beta 3 receptors. In the mesocecum vasculature, both EMD 66203 and EMD 270179 ameliorated platelet-activating factor-induced enhanced SS RBC adhesion, postcapillary blockage, and significantly improved hemodynamic behavior. Infusion of a fluorescent derivative of EMD 66203 resulted in colocalization of the antagonist with vascular endothelium. Also, pretreatment of HUVEC with either alpha V beta 3 antagonist resulted in a significant decrease in SS RBC adhesion. Because of their metabolic stability, the use of these cyclic alpha V beta 3 antagonists may constitute a novel therapeutic strategy to block SS RBC adhesion and associated vasoocclusion under flow conditions.     

Oxidative status of valinomycin-resistant normal, beta-thalassemia and sickle red blood cells

Exposure of red blood cells (RBC) to the K+ -ionophore valinomycin (val), causes loss of KCl and water, resulting in cell dehydration, manifested by increased cell density. While almost all normal val-treated RBC dehydrate, in sickle cell anemia (SCA) a portion of the RBC fail to dehydrate and maintain a light density, indicating the existence of val-resistant (val-res) RBC. In thalassemia and sickle cell disease (SCD), although the primary lesion is in the globin genes, damage to the RBC is partly mediated by oxidative stress. We previously showed that such RBC are under oxidative stress, having more reactive oxygen species (ROS) and less reduced glutathione than normal RBC. We now report a relationship between the phenomenon of val-res and the RBC oxidative status: Treatment with oxidants that increase ROS, also increased the frequency of val-res cells. Val-res cells had higher oxidative status than other RBC in the sample. Similar to SCA, thalassemic blood has more val-res cells than does normal blood. Val-res cells in thalassemic and sickle blood showed a higher oxidative status than normal val-res cells. Thus, oxidative stress might be involved in generation of val-res cells. Further studies are required to elucidate the origin and significance of these cells.     

Proinflammatory and procoagulant effects of herpes simplex infection on human endothelium

Atherosclerotic lesions have been reported to contain herpes simplex virus (HSV) genomic material. This and other evidence suggests that latent viral infection may be an atherogenic trigger. Moreover, active HSV lesions manifest histologically marked fibrin deposition in microvessels. Our laboratory tested in vitro whether HSV infection would cause human umbilical vein endothelial cells to become procoagulant and attract inflammatory cells. Early infection of human endothelial cells with HSV-1 alters the surface conformation as detected by merocyanine 540 staining. The efficiency of prothrombinase complex assembly increases, resulting in a two- to threefold accelerated rate of thrombin generation on the cell surface of virally infected endothelium. HSV infection of endothelium results in a marked increase in thrombin-induced platelet adhesion with a concomitant decrease in prostacyclin secretion in response to thrombin. Viral infection enhances coagulation by decreasing endothelial thrombomodulin expression and subsequent activation of protein C. Viral infection also induces tissue factor in human endothelial cells within 4 hours of infection. Not only does the endothelial monolayer become procoagulant when infected with HSV, it also becomes a more adherent surface for granulocytes. Resting and stimulated granulocyte adherence is enhanced twofold on virally infected endothelium. Enhanced adhesion is accompanied by excessive granulocyte-mediated lysis of 51Cr-labeled HSV-infected endothelium and endothelial cell detachment from its substrate. Exaggerated endothelial detachment correlated with poor binding of infected endothelial cells to substratum matrix proteins. Resuspended virus-infected cells bound significantly less well to tissue culture containers coated with fibronectin, laminin, and type IV collagen. HSV-infected endothelium alters the anticoagulant properties of the endothelium causing it to become procoagulant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of interaction of thrombospondin with human endothelium and inhibition of sickle erythrocyte adhesion to human endothelial cells by heparin

Thrombospondin (TSP) mediates sickle erythrocyte adhesion to endothelium, but the mechanism remains unknown. Since TSP is comprised of heterogeneously distinct domains, this adhesion may depend on the interaction of specific regions of TSP with different cell surface receptors. To examine the mechanisms of interaction of TSP with human umbilical vein endothelial cells (HUVEC), we performed binding studies using soluble [¹²⁵I]TSP. Our data showed that (i) monoclonal antibodies (MoAbs) against cell surface heparan sulfate (HS) or the heparin-binding domain of TSP, or cleavage of HS on HUVEC by heparitinase reduced TSP binding by 28–40%, (ii) the RGD peptide or MoAbs against integrin α_vβ₃ or the calcium binding region of TSP inhibited binding by 18–28%, and (iii) a MoAb against the cell-binding domain of TSP inhibited binding by 36%. Unmodified heparin inhibited the binding of TSP to endothelial cells by 70% and did so far more effectively than selectively desulfated heparins, HS or chondroitin sulfate. Heparin inhibited TSP binding to HUVEC at much lower concentrations than were required to inhibit TSP binding to sickle erythrocytes. Unmodified heparin effectively inhibited the TSP-mediated adhesion of sickle erythrocytes to HUVEC. These data imply that cell surface HS-mediated mechanisms play a key role in TSP-mediated sickle erythrocyte adhesion to endothelium, and heparin may be of use for inhibition of this adhesion.