U937 Adhesion Research Articles

Homocysteine Inhibits TNF-α-Induced Endothelial Adhesion Molecule Expression and Monocyte Adhesion via Nuclear Factor-κB Dependent Pathway

Cellular adhesion molecules play a pivotal role in the pathogenesis of atherosclerosis by mediating the adherence of blood leukocytes. Since hyperhomocysteinemia appears to be an independent risk factor for the development of atherosclerosis, in this study we investigated the effect of homocysteine on basal and TNF-α-induced expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell-adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (E-selectin) on human umbilical-vein endothelial cells. Incubation of endothelial cells with homocysteine resulted in dose-dependent reduction in TNF-α-induced (5 ng/ml) expression of VCAM-1, E-selectin, and ICAM-1 (the latter less pronounced). This effect was found to be specific since other thiol compounds—cysteine and glutathione—did not mimic homocysteine activity. Homocysteine attenuated TNF-α-stimulated U-937 adhesion to the endothelial monolayer and reduced TNF-α-induced activation of the transcription factor NF-κB, indicating that NF-κB inhibition may play a role in inhibiting expression of adhesion molecules in endothelial cells.

Statins prevent endothelial cell activation induced by antiphospholipid (anti-beta2-glycoprotein I) antibodies: effect on the proadhesive and proinflammatory phenotype.

To investigate the ability of statins, the inhibitors of the hydroxymethylglutaryl-coenzyme A reductase enzyme, to affect endothelial cell activation induced by anti-beta2-glycoprotein I (anti-beta2GPI) antibodies in vitro. Human umbilical vein endothelial cell (HUVEC) activation was evaluated as U937 monocyte adhesion, E-selectin, and intercellular adhesion molecule I (ICAM-1) expression by cell enzyme-linked immunosorbent assay and as interleukin-6 (IL-6) messenger RNA (mRNA) expression by RNA protection assay. E-selectin-specific nuclear factor kappaB (NF-kappaB) DNA-binding activity was evaluated by the gel-shift assay. HUVECs were activated by polyclonal affinity-purified IgG, human monoclonal IgM anti-beta2GPI antibodies, human recombinant IL-1beta, tumor necrosis factor alpha, or lipopolysaccharide (LPS). Fluvastatin reduced, in a concentration-dependent manner (1-10 microM), the adhesion of U937 to HUVECs and the expression of E-selectin and ICAM-1 induced by anti-beta2GPI antibodies as well as by cytokines or LPS. Another lipophilic statin, simvastatin, displayed similar effects but to a lesser extent than fluvastatin. The inhibition of E-selectin expression exerted by fluvastatin was related to the impairment of NF-kappaB binding to DNA. Moreover, the drug attenuated the expression of IL-6 mRNA in HUVEC exposed to anti-beta2GPI antibodies or cytokines. Incubation of HUVECs with mevalonate (100 microM), concomitantly with fluvastatin, greatly prevented the inhibitory effect of statin. Endothelial activation mediated by anti-beta2GPI antibody can be inhibited by statins. Because of the suggested role of endothelial cell activation in the pathogenesis of antiphospholipid syndrome (APS), our data provide, for the first time, a rationale for using statins as an additional therapeutic tool in APS.

Annexin 1 binds to U937 monocytic cells and inhibits their adhesion to microvascular endothelium: involvement of the alpha 4 beta 1 integrin.

Annexin 1 (ANX1), a calcium-binding protein, participates in the regulation of early inflammatory responses. Whereas some of its effects depend on intracellular interactions, a growing number of observations indicate that ANX1 may also act via autocrine/paracrine functions following externalization to the outer side of the plasma membrane. We studied the effects of ANX1 on leukocyte adhesion to endothelial cells using as a model system the monocytic cell line U937 and human bone marrow microvascular endothelial cells. Exogenous rANX1, as well as endogenous ANX1 externalized by U937 differentiated in vitro, inhibited monocyte firm adhesion to vascular endothelium. Both binding of ANX1 to U937 cells and ANX1-mediated inhibition of cell adhesion involved the short N-terminal domain of the ANX1 molecule. Under experimental conditions in which ANX1 inhibited U937 adhesion to human bone marrow microvascular endothelial cells, this protein specifically colocalized with the alpha 4 integrin, and a direct interaction between ANX1 and the alpha 4 integrin could be documented by immunoprecipitation experiments. Moreover, ANX1 competed with the endothelial integrin counterreceptor, VCAM-1, for binding to alpha 4 integrin. These results indicate that ANX1 plays an important physiological role in modulating monocyte firm adhesion to the endothelium.

The effect of carotenoids on the expression of cell surface adhesion molecules and binding of monocytes to human aortic endothelial cells

Several large epidemiological studies have shown a correlation between elevated plasma carotenoid levels and decreased risk of cardiovascular disease (CVD). One proposed mechanism for the beneficial effect of carotenoids is through functional modulation of potentially atherogenic processes associated with the vascular endothelium. To test this, we incubated confluent human aortic endothelial cell (HAEC) cultures (passages 4–8) for 24 h with each of the five most prevalent carotenoids in human plasma, which are α-carotene, β-carotene, β-cryptoxanthin, lutein, and lycopene, at an approximate concentration of 1 μmol/l. Carotenoids were solubilized in 0.7% (v/v) tetrahydrofuran and incorporated into FBS before adding to cell culture medium. Due to disparate solubilities in aqueous medium, final concentrations of α-carotene, β-carotene, β-cryptoxanthin, lutein, and lycopene were 1.7, 1.1, 0.7, 0.9, and 0.3 μmol/l and monolayers accumulated 647, 158, 7, 113, and 9 pmol/mg protein, respectively. Monolayers were then stimulated with IL-1β (5 ng/ml) for 6 h with subsequent determination of cell surface expression of adhesion molecules as measured by an enzyme-linked immunosorbent assay (ELISA). To assess endothelial cell adhesion to monocytes, IL-1β-stimulated monolayers were incubated for 10 min with 51Cr-labeled U937 monocytic cells and adhesion determined by isotope counting. Pre-incubation of HAEC with β-carotene, lutein and lycopene significantly reduced VCAM-1 expression by 29, 28, and 13%, respectively. Pre-incubation with β-carotene and lutein significantly reduced E-selectin expression by 38 and 34%, respectively. Pre-treatment with β-carotene, lutein and lycopene significantly reduced the expression of ICAM-1 by 11, 14, and 18%, respectively. While other carotenoids were ineffective, lycopene attenuated both IL-1β-stimulated and spontaneous HAEC adhesion to U937 monocytic cells by 20 and 25%, respectively. Thus, among the carotenoids, lycopene appears to be most effective in reducing both HAEC adhesion to monocytes and expression of adhesion molecules on the cell surface.

Effect of selective proteasome inhibitors on TNF-induced activation of primary and transformed endothelial cells.

The objective of this study was to assess the effects of two structurally distinct yet selective proteasome inhibitors (PS-341 and lactacystin) on leukocyte adhesion, endothelial cell adhesion molecule (ECAM) expression, and nuclear factor-kappaB (NF-kappaB) activation in tumor necrosis factor (TNF)-alpha-stimulated human umbilical vein endothelial cells (HUVEC) and the transformed, HUVEC-derived, ECV cell line. We found that TNF (10 ng/ml) significantly enhanced U-937 and polymorphonuclear neutrophil (PMN) adhesion to HUVEC but not to ECV; TNF also significantly enhanced surface expression of vascular cell adhesion molecule 1 and E-selectin (in HUVEC only), as well as intercellular adhesion molecule 1 (ICAM-1; in HUVEC and ECV). Pretreatment of HUVEC with lactacystin completely blocked TNF-stimulated PMN adhesion, partially blocked U-937 adhesion, and completely blocked TNF-stimulated ECAM expression. Lactacystin attenuated TNF-stimulated ICAM-1 expression in ECV. Pretreatment of HUVEC with PS-341 partially blocked TNF-stimulated leukocyte adhesion and ECAM expression. These effects of lactacystin and PS-341 were associated with inhibitory effects on TNF-stimulated NF-kappaB activation in both HUVEC and ECV. Our results demonstrate the importance of the 26S proteasome in TNF-induced activation of NF-kappaB, ECAM expression, and leukocyte-endothelial adhesive interactions in vitro.

Alpha-Tocopherol enrichment of monocytes decreases agonist-induced adhesion to human endothelial cells.

Monocyte-endothelium adhesion is a crucial early event in atherogenesis. Several reports indicate that alpha-tocopherol (AT) is a potent antioxidant in plasma and LDL and also has intracellular effects that are antiatherogenic. Recently, it has been shown that AT supplementation results in decreased monocyte-endothelial cell adhesion. However, there is a paucity of data on the mechanisms by which AT inhibits adhesion of monocytes. We studied the effect of AT enrichment of a human monocytic cell line, U937, on adhesion to human umbilical vein endothelial cells (HUVECs). Both lipopolysaccharide (LPS)- and N-formyl-methionyl-leucyl-phenylalanine (FMLP)-stimulated U937 adhesion to HUVECs were studied. AT (50 and 100 micromol/L) significantly decreased adhesion of both LPS- and FMLP-stimulated U937 cells to HUVECs (LPS-treated cells, P<0.0125; FMLP-treated cells, P<0.05). Expression of the adhesion molecules CD11a, CD11b, CD11c, very late antigen-4 (VLA-4), and L-selectin, as assessed by flow cytometry, was increased in the stimulated U937 cells, and AT resulted in significant reduction in the expression of CD11b and VLA-4. In addition, activation of the transcription factor nuclear factor-kappaB (NF-kappaB), as assessed by gel shift assays, was inhibited by pretreatment with AT in LPS-treated U937 cells. AT significantly decreases adhesion of activated monocytes to endothelial cells by decreasing expression of CD11b and VLA-4 on monocytes, possibly by inhibiting the activation of NF-kappaB.

Inhibitory effect of fluvastatin, an HMG-CoA reductase inhibitor, on the expression of adhesion molecules on human monocyte cell line

The effect of fluvastatin, an HMG-CoA reductase inhibitor, was investigated on the adhesive interaction between U937 cells, the human monocyte cell line, and human umbilical vein endothelial cells (HUVEC), focusing on the expression of adhesion molecules. U937 treated with fluvastatin lowered the capacity for binding to HUVEC. Fluvastatin at 0.1 μM or more inhibited the expression of lymphocyte function associated antigen-I (LFA-1) on U937 and intercellular adhesion molecule-1 (ICAM-I) on U937. The expression of ICAM-1 on HUVEC was not inhibited by fluvastatin. The inhibitory effects of fluvastatin on the expression of adhesion molecules on U937 were completely reversed by the addition of mevalonate. Because fluvastatin did not affect the expression of other cell surface markers, CD4 and CD71, the inhibitory effects of fluvastatin on adhesion molecule expression could not be attributed to the non-specific suppression of the cell. It is conceivable that cellular interaction between monocytes and endothelial cells is inhibited by fluvastatin, mediated via reducing the expression of adhesion molecules, particularly in the side of monocyte.

Effects of protein tyrosine kinase inhibitors on cytokine‐induced adhesion molecule expression by human umbilical vein endothelial cells

1. Endothelial cells can be stimulated by the pro-inflammatory cytokines interleukin (IL)-1 alpha and tumour necrosis factor (TNF) alpha to express the leukocyte adhesion molecules E-selectin, vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 but the intracellular signalling mechanisms leading to this expression are incompletely understood. We have investigated the role of protein tyrosine kinases (PTK) in adhesion molecule expression by cytokine-activated human umbilical vein endothelial cells (HUVEC) using the PTK inhibitors genistein and herbimycin A, and the protein tyrosine phosphatase (PTP) inhibitor sodium orthovanadate. 2. Maximal E-selectin expression induced by incubation of HUVEC for 4 h with IL-1 alpha (100 u ml-1) and TNF alpha (100 u ml-1) was dose-dependently inhibited by genistein and herbimycin A. Although similar effects were seen on phorbol 12-myristate, 13-acetate (PMA)-induced expression, this was not due to inhibition of protein kinase C (PKC) activity as the selective inhibitors of PKC, bisindolylmaleimide (BIM), Ro31-7549 or Ro31-8220 did not affect IL-1 alpha- or TNF alpha-induced E-selectin expression at concentrations which maximally inhibited PMA-induced expression. 3. Genistein inhibited VCAM-1 expression induced by incubation of HUVEC for 24 h with TNF alpha or IL-1 alpha whereas it did not affect ICAM-1 expression induced by 24 h incubation with either of these cytokines. Herbimycin A inhibited both VCAM-1 and ICAM-1 expression induced by TNF alpha. 4. Basal expression of E-selectin, VCAM-1 and ICAM-1 was dose-dependently enhanced by sodium orthovanadate. In contrast, vanadate differentially affected TNF alpha-induced expression of these molecules with maximal E-selectin and ICAM-1 expression being slightly enhanced and VCAM-1 expression dose-dependently reduced. 5. We also studied the effects of PTK and PTP inhibitors on adhesion of the human pre-myeloid cell line U937 to TNF alpha-stimulated HUVEC. Adhesion of U937 cells to HUVEC pretreated for 4 or 24 h with TNF alpha was dose-dependently inhibited by genistein and herbimycin A but unaffected by daidzein. Adhesion of U937 cells after 4 h was partially inhibited by blocking antibodies against both E-selectin and VCAM-1 but after 24 h was only inhibited by anti-VCAM-1. 6. Sodium orthovanadate had no effect on TNF alpha-induced U937 adhesion but dose-dependently enhanced adhesion to unstimulated HUVEC. Vanadate-induced adhesion was inhibited by an antibody against VCAM-1. 7. These results demonstrate that PTK-mediated phosphorylation events are important for the regulation of adhesion molecule expression by human endothelial cells, and additionally show that PTK inhibitors differentially affect upregulation of different adhesion molecules, implicating divergent regulatory pathways for cytokine-induced adhesion molecule expression.

Modulators of Oxidized LDL-Induced Hyperadhesiveness in Human Endothelial Cells

The adherence of monocytes to the endothelium is an early event in atherogenesis. Our previous studies have demonstrated that oxidized LDL induced U937 cells-endothelial interactions and that HDL prevented oxidized LDL effects. Here, we provide evidence that treatment of endothelial cells with the anti-inflammatory agent indomethacin abolished oxidized LDL as well as interleukin 1- and lipopolysaccharide-stimulated U937 adhesion. It is noteworthy that HDL, which is known to be protective against atherosclerosis, was effective only in negating U937 adhesion induced by oxidized LDL, while it did not affect interleukin 1- and lipopolysaccaride-induced hyperadhesiveness in endothelial cells. Since indomethacin inhibits cyclooxygenase which is the key enzyme in the synthesis of prostanoids, we have studied the effect of oxidized LDL on the expression of cyclooxygenase type 2 and demonstrated that oxidized LDL induces a sustained increase in the expression of cylooxygenase mRNA.

Antioxidants inhibit monocyte adhesion by suppressing nuclear factor-kappa B mobilization and induction of vascular cell adhesion molecule-1 in endothelial cells stimulated to generate radicals.

Cell adhesion to endothelial cells stimulated by tumor necrosis factor-alpha (TNF) is due to induction of surface receptors, such as vascular cell adhesion molecule-1 (VCAM-1). The antioxidant pyrrolidine dithiocarbamate (PDTC) specifically inhibits activation of nuclear factor-kappa B (NF-kappa B). Since kappa B motifs are present in VCAM-1 and intercellular adhesion molecule-1 (ICAM-1) promoters, we used PDTC to study the regulatory mechanisms of VCAM-1 and ICAM-1 induction and subsequent monocyte adhesion in TNF-treated human umbilical vein endothelial cells (HUVECs). PDTC or N-acetylcysteine dose dependently reduced TNF-induced VCAM-1 but not ICAM-1 surface protein (also in human umbilical arterial endothelial cells) and mRNA expression (by 70% at 100 mumol/L PDTC) in HUVECs as assessed by flow cytometry and polymerase chain reaction. Gel-shift analysis in HUVECs demonstrated that PDTC prevented NF-kappa B mobilization by TNF, suggesting that only VCAM-1 induction was controlled by NF-kappa B. Since HUVECs released superoxide anions in response to TNF, and H2O2 induces VCAM-1, PDTC may act as a radical scavenger. Although ICAM-1 induction was unaffected, inhibitors of NADPH oxidase (apocynin) or cytochrome P-450 (SKF525a) suppressed VCAM-1 induction by TNF, revealing that several radical-generating systems are involved in its regulation. PDTC, apocynin, or SKF525a decreased adhesion of monocytic U937 cells to TNF-treated HUVECs (by 75% at 100 mumol/L PDTC). Inhibition by anti-VCAM-1 monoclonal antibody 1G11 indicated that U937 adhesion was VCAM-1 dependent and suppression by antioxidants was due to reduced VCAM-1 induction.(ABSTRACT TRUNCATED AT 250 WORDS)

Senescence Stimulates U937-Endothelial Cell Interactions

Progressive pathophysiologic modifications of endothelial cells are associated with aging. In vitro, endothelial cell senescence is accompanied by the failure to proliferate as well as by perturbations in gene expression. Here we show that (i) senescence enhances monoblastoid U937 cell adhesion to the endothelial monolayer; (ii) the enhanced interaction between senescent endothelial cells and U937 cells is mediated, at least in part, by the overexpression of ICAM-1; and (iii) LPS and interleukin 1 α, but not tumor necrosis factor α, are unable to stimulate the adhesion of U937 to senescent endothelial cells. Since monocyte adhesion to the endothelium is an early event in atherosclerosis, the altered adhesive properties observed in senescent cells could give insights into the formation of atherosclerotic lesions.

Synergism of interleukin 6 and 1 alpha,25-dihydroxyvitamin D3 in induction of myeloid differentiation of human leukemic cell lines.

Interleukin 6 (IL-6) is a multifunctional cytokine with an important role in immunity. We analyzed the effect of recombinant human IL-6 in combination with 1 alpha,25-dihydroxyvitamin D3 (Vit. D3) on differentiation of the human myeloid leukemic cell lines U937 and HL-60 with respect to alterations in antigen expression and functional activity. Of a panel of antigens analyzed, only CD11b (the alpha chain of CR3), and CD14 (a cell surface protein recognizing the lipopolysaccharide-binding protein-lipopolysaccharide complex) had significantly increased expression. Expression of ICAM-1 (CD54), a ligand for LFA-1, was also found to be enhanced with a concomitant increase of ICAM-1 mRNA levels. Enhanced nonspecific esterase levels and induction of respiratory burst activity confirmed that cell differentiation was induced. Furthermore, IL-6 and Vit. D3 had a profound effect on functional activities, as shown by enhancement of rosetting between sheep erythrocytes, sensitized with C3bi (EAC), and either U937 or HL-60 cells. Also, phorbol myristate acetate-induced homotypic adhesion of U937, which is ICAM-1 dependent, was markedly induced by these agents. These results indicate an important role of IL-6 and Vit. D3 in myeloid cell function and development.

A role for urokinase in mediating phorbol ester induced macrophagelike maturation and adhesion of u937 and other myeloid cells

The phorbol ester, phorbol myristate acetate (PMA), induces myeloid cells to differentiate towards the macrophage lineage. Polyclonal antibodies to the urokinase plasminogen activator (u-PA) interfere with this process as defined by the inability of U937 cells to adhere on tissue culture surfaces. This block in differentiation is overcome by a 21 amino acid synthetic peptide from the u-PA growth factor domain which has residues of the receptor binding sequence. Moreover, a 40 amino acid peptide from the u-PA growth factor domain is more potent and enhances by 4-fold the PMA stimulated adhesion of a similar cell line, HL60. These observations indicate that the growth factor domain of u-PA plays an important role in PMA mediated myeloid cell differentiation and adhesion.

Evidence for the involvement of carbohydrate moieties in the adhesion of U937 cells to tumor necrosis factor-α (TNFα)-stimulated vascular endotheliumin vitro

Treatment of U937 cells with fructose 1-phosphate (P) and fucoidan dose-dependently inhibited the adhesion of these monocytic cells to TNF alpha-stimulated human umbilical vein endothelial cells (HUVEC) (IC50 = 1 mM and 10 micrograms/ml respectively). These carbohydrates (CHO) failed to inhibit U937 adhesion to unstimulated (basal) HUVEC or phorbol 12, 13 dibutyrate (PdBu)-stimulated HUVEC. At 10 mM concentration, both fucose 1-P and lactose 1-P inhibited TNF alpha-stimulated adhesion while the latter also inhibited basal adhesion. Fructose 6-P, fucose, galactose 1-P, glucose 1-P, glucose 6-P, glucuronic acid, beta-glycerol 1-P, mannose 1-P, mannose 6-P, ribose 1-P and ribose 5-P tested at 10 mM did not inhibit U937 cells adhesion to basal or TNF alpha-stimulated HUVEC. These data suggest that CHO may play an important role in modulating monocytes adhesion to cytokine-induced adhesion molecule(s) on the surface of HUVEC.

Intracellular mechanisms involved in leukotriene C 4-stimulated adhesion of U-937 cells

Leukotrienes C 4 and D 4 (LTC 4 and LTD 4) stimulated, 5- to 6-fold, the adhesion of the monoblastoid cell line U-937 to plastic. Half-maximal effects were observed around 1 nM. Leukotrienes E 4 and B 4 (LTE 4 and LTB 4) were less effective. The adhesive response to LTC 4 was inhibited by pertussis toxin and was completely dependent on the presence of extracellular Ca 2+. The LTC 4-stimulated increases in inositol-phosphates and in intracellular Ca 2+-concentration were insensitive to pertussis toxin. Activation of leukocyte adhesion is a novel action of cysteinyl-leukotrienes and the present study suggests that control of U-937-cell adhesion by LTC 4 involves two pathways; one pertussis toxin insensitive pathway regulating intracellular Ca 2+ in a manner partly dependent on extracellular Ca 2+ and one pertussis toxin sensitive pathway not concerned with Ca i 2+-regulation.

The role of CD44, CD45, CD45RO, CD46 and CD55 as potential anti‐adhesion molecules involved in the binding of human tonsillar T cells to phorbol 12‐myristate 13‐acetate‐differentiated U‐937 cells

In this study, we have shown that human tonsillar T cells adhere to phorbol 12-myristate 13-acetate(PMA)-differentiated U-937 cells. To examine the molecular mechanisms involved, the effect of a panel of monoclonal antibodies upon this adhesion was assessed in a quantitative binding assay. Antibodies against LFA-1 and ICAM-1 inhibited binding, directly implicated these molecules in T cell-PMA-induced U-937 adhesion. Furthermore, the adhesion was magnesium but not calcium dependent. Of the remaining antibodies that were tested, none of those against CD2, LFA-3, Mac-1, p150,95, CD43, CD45RA or CD56 affected binding. However, antibodies against CD44, CD45, CD45RO, CD46 and CD55 enhanced binding suggesting an anti-adhesive role for these molecules during U-937-T cell interaction.

Characterization of the adhesion of the human monocytic cell line U937 to cultured endothelial cells.

Adhesion of blood-borne monocytes to the vascular endothelium is the first step in the infiltration of this leukocyte into the vessel wall or the interstitial space during inflammation. A significant role for the monocyte in both wound healing and atherogenesis is now well accepted. The molecular interactions involved in monocyte attachment to the endothelium are unknown. To study this phenomenon we have developed an in vitro system that uses the human monocytic tumor cell line U937 as a model for the blood-borne monocyte. 51Cr-labeled U937 cells were found to adhere with high affinity to cultured endothelial cells (ECs) from several sources. Much less binding was observed to either smooth muscle cells or fibroblasts from several species. Conditioned medium and cocultivation experiments ruled out the possibility that target cells could affect U937 cell binding by secretion of factors. Binding of U937 cells to porcine aortic ECs reached equilibrium after 30 min at 37 degrees C and 90 min at 4 degrees C with similar extent of binding at the two temperatures. Binding of U937 to the endothelium reached saturation at 9-12 U937 per porcine aortic EC (semi-confluent) with half-maximal binding at 1.5 X 10(6) U937 cells/ml. Bound cells dissociated with a half-life of 20 h at 37 degrees C. Adhesion of U937 cells was blocked by prior incubation of ECs with normal monocytes but not with platelets, lymphocytes, or neutrophils. Trypsin treatment or detergent solubilization of ECs inhibited U937 cell binding. A striking effect of EC density on monocytic cell adhesion was observed with bovine, rat, and porcine ECs. Confluent cultures of these cells exhibited negligible binding of U937, but when plated sparsely, the same cells were excellent targets for U937 cell adhesion. In addition, when confluent cultures of bovine aortic ECs were "wounded" with a cotton swab and then allowed to recover for 24 h at 37 degrees C, U937 cells were found to adhere most readily to the ECs migrating into the wound and neighboring the wound but not to ECs in the confluent monolayer away from the wound edge. These latter results may have implications for the focal adhesion of monocytes to the vessel wall in vivo.