Molecular events during leukocyte diapedesis

The recruitment of leukocytes from the circulation into tissues requires leukocyte migration through the vascular endothelium. The mechanisms by which leukocytes attach and firmly adhere to the endothelial cell surface have been studied in detail. However, much less is known about the last step in this process, the diapedesis of leukocytes through the vascular endothelium. This minireview focuses on the interactions between leukocyte and endothelial cell adhesion molecules that are important during leukocyte extravasation. In the past few years a series of endothelial cell surface and adhesion molecules have been identified that are located at endothelial cell contacts and found to participate in leukocyte diapedesis. These junctional cell adhesion molecules are believed to have an active role in controlling the opening and closure of endothelial cell contacts to allow the passage of leukocytes between adjacent endothelial cells. Alternatively, leukocytes can cross the endothelium at nonjunctional locations, with leukocytes migrating through a single endothelial cell. Further work is clearly needed to understand, in greater detail, the molecular mechanisms that allow leukocytes to cross the endothelium via either the paracellular or the transcellular pathway.     

Vimentin function in lymphocyte adhesion and transcellular migration

Although the adhesive interactions of leukocytes with endothelial cells are well understood, little is known about the detailed mechanisms underlying the actual migration of leukocytes across the endothelium (diapedesis). Leukocytes have been shown to use both paracellular and transcellular routes for transendothelial migration. Here we show that peripheral blood mononuclear cells (PBMCs; T- and B-lymphocytes) preferentially use the transcellular route. The intermediate filaments of both endothelial cells and lymphocytes formed a highly dynamic anchoring structure at the site of contact between these two cell types. The initiation of this process was markedly reduced in vimentin-deficient (vim(-/-)) PBMCs and endothelial cells. When compared with wild-type PBMCs, vim(-/-) PBMCs showed a markedly reduced capacity to home to mesenteric lymph nodes and spleen. Furthermore, endothelial integrity was compromised in vim(-/-) mice, demonstrating that intermediate filaments also regulate the barrier that governs leukocyte extravasation. Absence of vimentin resulted in highly aberrant expression and distribution of surface molecules critical for homing (ICAM-1 and VCAM-1 on endothelial cells and integrin-beta1 on PBMCs). These data show that intermediate filaments are active in lymphocyte adhesion and transmigration.     

Crossing the endothelium

Diapedesis is a vital part of tumour metastasis, whereby tumour cells attach to and cross the endothelium to enter the circulation. Specific adhesion molecules, expressed by both the tumour and endothelial cells, mediate this process. This review summarises recent findings regarding the mechanisms by which colon cancer cells migrate through the endothelium under flow conditions mediated by E-selectin. Using a laminar flow chamber and a tissue engineered human blood vessel, E-selectin was found to regulate initial attachment and rolling of colon cancer cells and also the subsequent diapedesis through the endothelium. Three different mechanisms of diapedesis were reported to be regulated by E-selectin; the formation of a mosaic chimeric layer of tissue, paracellular diapedesis between endothelial cells and transcellular diapedesis, in which tumour cells were transported via large vacuoles within the endothelial cells. Moreover activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase by E-selectin was further required for paracellular diapedesis. This study is the first to report these observations under dynamic and shear stress conditions.     

Endothelial activation drives lateral migration and diapedesis of leukocytes

To invade a tissue, leukocytes have to overcome the endothelial barrier. Prior to trans-endothelial migration, leukocytes move laterally on the endothelial surface-searching for an emigration site. It is still unclear, how the actual diapedesis step is initiated and whether the endothelium has a decisive role. Here, video-microscopy was employed to investigate, whether lateral migration of leukocytes is correlated to their diapedesis rate. To address the contribution of each cell type, selective stimulation of either leukocytes or endothelial cells with TNFα was performed. Stimulation of endothelial cells alone was sufficient for maximal effects, thereby underlining their decisive role for leukocyte diapedesis. Concomitant to the TNFα-enhanced diapedesis rate, leukocyte adhesion was intensified and, unexpectedly, the lateral leukocyte migration was accelerated.     

Crossing the endothelium: E-selectin regulates tumor cell migration under flow conditions

Diapedesis is a vital part of tumor metastasis, whereby tumor cells attach to and cross the endothelium to enter the circulation. Specific adhesion molecules, expressed by both the tumor and endothelial cells, mediate this process. This review summarizes recent findings regarding the mechanisms by which colon cancer cells migrate through the endothelium under flow conditions mediated by E-selectin. Using a laminar flow chamber and a tissue engineered human blood vessel, E-selectin was found to regulate initial attachment and rolling of colon cancer cells and also the subsequent diapedesis through the endothelium. Three different mechanisms of diapedesis were reported to be regulated by E-selectin; the formation of a mosaic chimeric layer of tissue, paracellular diapedesis between endothelial cells and transcellular diapedesis, in which tumor cells were transported via large vacuoles within the endothelial cells. Moreover activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase by E-selectin was further required for paracellular diapedesis. This study is the first to report these observations under dynamic and shear stress conditions.Key words: E-selectin, endothelium, tumor cells, diapedesis, site-specific metastasisDuring metastasis, tumor cells must complete a number of stages to successfully migrate from one site in the body to another. These include detachment of cells from the primary tumor, degradation and migration through the surrounding extra cellular matrix, extravasation and survival in the circulation and arrest in distant capillaries.¹ Once at the secondary site, they must undergo the reverse process before initiating growth and angiogenesis to support their development. This is a highly inefficient process, with few cells completing all stages to establish metastases.²The process of extravasation involves the diapedesis of tumor cells across the endothelium (‘dia’ meaning through and ‘pèdan’ meaning to leap). Similar to neutrophil extravasation at sites of inflammation, tumor cells initially adhere to the endothelium forming loose interactions via adhesion molecules before rolling and finally forming firm attachments, prior to diapedesis. Site-specific metastasis has been described for a number of tumors, with specific interactions between the tumor and endothelial cells potentially playing a pivotal role. Expression of E-selectin by endothelial cells following activation by inflammatory cytokines for example, is reported to be involved with the homing of colon cancer cells to the liver.^3–5 This review summarizes recent findings by Tremblay et al. (2008), published in Cancer Research, in which three distinct mechanisms of transendothelial migration by colon cancer cells are described, ultilizing E-selectin adhesion molecules on the endothelium (Fig. 1): (1) paracellular diapedesis at endothelial junctions, (2) formation of mosaic chimeric tissue of tumor and endothelial cells and (3) transcellular diapedesis.⁶Open in a separate windowFigure 1Schematic representation of the three mechanisms that colon cancer cells use during diapedesis of the endothelium. Under flow conditions, tumor cells initially form loose attachments with the endothelium, prior to rolling and forming firm attachments, mediated by specific adhesion molecules such as E-selectin. Following firm adhesion, tumor cells initiate diapedesis by (1) migrating between the junctions of endothelial cells (paracellular diapedesis), (2) forming a mosaic chimeric layer of both cell types (although this may not lead to complete diapedesis) and (3) passaging through the endothelial cells in large vacuoles (transcellular diapedesis).To demonstrate the importance of E-selectin in the adhesion of colon cancer cells to the endothelium, a laminar flow chamber was initially used. Human umbilical vein endothelial cells (HUVECs), expressing WT or a truncated form of E-selectin (with a cytoplasmic domain deleted) (ES-ΔICD) grown as a monolayer on glass slides, were treated for four hours with IL-1β to induce E-selectin expression. Cultures were then placed in a laminar flow chamber with culture medium and shear stress applied. HT29 colon cancer cells were then injected into the flow chamber, video sequences were recorded after 10 and 30 minutes and the subsequent HT29 cells that had adhered to the HUVECs was counted. The results demonstrated that colon cancer cells adhered to the endothelium under flow conditions using E-selectin, but only following treatment with IL-β. Blocking E-selectin and mutating the intracellular domain of E-selectin both prevented tumor cell adhesion. Similar results were also generated using a tissue-engineered human blood vessel, to more closely mimic the in vivo situation.⁷ Using these systems, the authors verified that E-selectin activation by circulating colon cancer cells is involved in p38 and ERK activation, via the intracellular domain.Using HT29 cells labeled with Vybrant Di1, the authors next used time-lapse microscopy over a 24-hour period to investigate the mechanisms by which colon cancer cells migrate through the endothelium under flow conditions. With endothelial cells expressing WT and truncated E-selectin, E-selectin was found to mediate initial attachment and rolling of the HT29 cells on the endothelial layer, in support of their previous results. Interestingly, most adhered tumor cells penetrated the endothelium, with 75% inserting themselves within the endothelial cell layer, creating a chimeric mosaic (Fig. 1). Many of these cells however did not complete migration through the endothelium and this was especially the case when using endothelial cells expressing E-selectin with the truncated intracellular domain, suggesting that this region was required for tumor cells to complete diapedesis.Of the cells that penetrated the endothelium, only 25% of the colon cancer cells completed diapedesis. This process was observed by two different mechanisms (Fig. 1). Firstly, at the junction of three endothelial cells, HT29 cells migrated between the cells (i.e., paracellular diapedesis); this required E-selectin signaling to ERK. Secondly, transcellular migration was also observed, in which tumor cells attached to the endothelial cells (not at the cell junctions) and induced endothelial cell retraction and blebbing. As a result tumor cells were engulfed within large vacuoles in the endothelial cells and transported across the cell. This was accompanied by loss of endothelial contact with the extracellular matrix. Interestingly this process was not fatal for all endothelial cells and some remained in the culture medium.In conclusion, this is the first paper to demonstrate three different mechanisms by which colon cancer cells migrate through the endothelium under conditions of shear stress, in an E-selectin-dependent fashion. Although a large proportion of tumor cells remained within the endothelium as a mosaic layer and did not complete diapedesis, the authors concluded that such cells form a subpopulation of tumor cells that are capable of initiating, but not completing extravasation, potentially due to nature of the tight junctions between the endothelial cells.^8,9 If proliferation is then stimulated, such cells may further be responsible for the development of local metastases within the vessel. A small percentage of tumor cells however completed diapedesis. This occurred either at the junction of endothelial cells (paracellular) or of particular interest whereby tumor cells crossed through the endothelial cells (transcellular). Similar data have been described previously for neutrophils,¹⁰ but this is the first study to demonstrate this phenomenon with tumor cells via E-selectin. This work provides new insights in understanding the mechanisms by which tumor cells cross blood vessel walls and the metastatic process.     

Lymphatic endothelium expresses PECAM-1

The expression of adhesion molecules on the lymphatic endothelium of human small intestine and submandibular lymph node was studied immunohistochemically with the antibodies for selectin family and Ig superfamily members. In both small intestine and submandibular lymph node, lymphatic endothelium did not express intercellular adhesion molecule-1 and endothelial cell-selectin but expressed platelet-endothelial cell adhesion molecule-1 (PECAM-1). Though lymphatic vessels may not have a positive function in leukocyte rolling and adhesion, lymphatic endothelium may interact with leukocytes, with PECAM-1 playing a role.     

Filamin B mediates ICAM-1-driven leukocyte transendothelial migration

During inflammation, the endothelium mediates rolling and firm adhesion of activated leukocytes. Integrin-mediated adhesion to endothelial ligands of the Ig-superfamily induces intracellular signaling in endothelial cells, which promotes leukocyte transendothelial migration. We identified the actin cross-linking molecule filamin B as a novel binding partner for intracellular adhesion molecule-1 (ICAM-1). Immune precipitation as well as laser scanning confocal microscopy confirmed the specific interaction and co-localization of endogenous filamin B with ICAM-1. Importantly, clustering of ICAM-1 promotes the ICAM-1-filamin B interaction. To investigate the functional consequences of filamin B binding to ICAM-1, we used small interfering RNA to reduce filamin B expression in ICAM-1-GFP expressing HeLa cells. We found that filamin B is required for the lateral mobility of ICAM-1 and for ICAM-1-induced transmigration of leukocytes. Reducing filamin B expression in primary human endothelial cells resulted in reduced recruitment of ICAM-1 to endothelial docking structures, reduced firm adhesion of the leukocytes to the endothelium, and inhibition of transendothelial migration. In conclusion, this study identifies filamin B as a molecular linker that mediates ICAM-1-driven transendothelial migration.     

Lymphocyte transcellular migration occurs through recruitment of endothelial ICAM-1 to caveola- and F-actin-rich domains

During inflammation, leukocytes bind to the adhesion receptors ICAM-1 and VCAM-1 on the endothelial surface before undergoing transendothelial migration, also called diapedesis. ICAM-1 is also involved in transendothelial migration, independently of its role in adhesion, but the molecular basis of this function is poorly understood. Here we demonstrate that, following clustering, apical ICAM-1 translocated to caveolin-rich membrane domains close to the ends of actin stress fibres. In these F-actin-rich areas, ICAM-1 was internalized and transcytosed to the basal plasma membrane through caveolae. Human T-lymphocytes extended pseudopodia into endothelial cells in caveolin- and F-actin-enriched areas, induced local translocation of ICAM-1 and caveolin-1 to the endothelial basal membrane and transmigrated through transcellular passages formed by a ring of F-actin and caveolae. Reduction of caveolin-1 levels using RNA interference (RNAi) specifically decreased lymphocyte transcellular transmigration. We propose that the translocation of ICAM-1 to caveola- and F-actin-rich domains links the sequential steps of lymphocyte adhesion and transendothelial migration and facilitates lymphocyte migration through endothelial cells from capillaries into surrounding tissue.     

Endothelial cells proactively form microvilli-like membrane projections upon intercellular adhesion molecule 1 engagement of leukocyte LFA-1

Specific leukocyte/endothelial interactions are critical for immunity and inflammation, yet the molecular details of this interaction interface remain poorly understood. Thus, we investigated, with confocal microscopy, the distribution dynamics of the central adhesion molecules ICAM-1 and LFA-1 in this context. Monolayers of activated HUVECs stained with fluorescent anti-ICAM-1 Fabs or Chinese hamster ovary-K1 cells expressing ICAM-1-green fluorescent protein were allowed to bind LFA-1-bearing monocytes, neutrophils, or K562 LFA-1 transfectants. ICAM-1 was rapidly relocalized to newly formed microvilli-like membrane projections in response to binding LFA-1 on leukocytes. These ICAM-1-enriched projections encircled the leukocytes extending up their sides and clustered LFA-1 underneath into linear tracks. Projections formed independently of VCAM-1/very late Ag 4 interactions, shear, and proactive contributions from the LFA-1-bearing cells. In the ICAM-1-bearing endothelial cells, projections were enriched in actin but not microtubules, required intracellular calcium, and intact microfilament and microtubule cytoskeletons and were independent of Rho/Rho kinase signaling. Disruption of these projections with cytochalasin D, colchicine, or BAPTA-AM had no affect on firm adhesion. These data show that in response to LFA-1 engagement the endothelium proactively forms an ICAM-1-enriched cup-like structure that surrounds adherent leukocytes but is not important for firm adhesion. This finding leaves open a possible role in leukocyte transendothelial migration, which would be consistent with the geometry and kinetics of formation of the cup-like structure.     

Leukocyte adhesion molecule-1 (LAM-1, L-selectin) interacts with an inducible endothelial cell ligand to support leukocyte adhesion.

The human lymphocyte homing receptor, LAM-1, mediates the adhesion of lymphocytes to specialized high endothelial venules (HEV) of peripheral lymph nodes. We now report that LAM-1 is also a major mediator of leukocyte attachment to activated human endothelium. In a novel adhesion assay, LAM-1 was shown to mediate approximately 50% of the adhesion of both lymphocytes and neutrophils to TNF-activated human umbilical vein endothelial cells at 4 degrees C. The contribution of LAM-1 to leukocyte adhesion was only detectable when the assays were carried out under rotating (nonstatic) conditions, suggesting that LAM-1 is involved in the initial attachment of leukocytes to endothelium. In this assay at 37 degrees C, essentially all lymphocyte attachment to endothelium was mediated by LAM-1, VLA-4/VCAM-1, and the CD11/CD18 complex, whereas neutrophil attachment was mediated by LAM-1, endothelial-leukocyte adhesion molecule-1, and CD11/CD18. Thus, multiple receptors are necessary to promote optimal leukocyte adhesion to endothelium. LAM-1 also appeared to be involved in optimal neutrophil transendothelial migration using a videomicroscopic in vitro transmigration model system. LAM-1-dependent leukocyte adhesion required the induction and surface expression of a neuraminidase-sensitive molecule that was expressed for at least 24 h on activated endothelium. Expression of the LAM-1 ligand by endothelium was optimally induced by LPS and the proinflammatory cytokines TNF-alpha and IL-1 beta, whereas IFN-gamma and IL-4 induced lower levels of expression. The LAM-1 ligand on HEV and cytokine treated endothelium may be similar carbohydrate-containing molecules, because phosphomannan monoester core complex from yeast Hansenula hostii cell wall blocked binding of lymphocytes to both cell types, and identical epitopes on LAM-1-mediated lymphocyte attachment to HEV and activated endothelium. Thus, LAM-1 and its inducible endothelial ligand constitute a new pair of adhesion molecules that may regulate initial leukocyte/endothelial interactions at sites of inflammation.     

Genetic transfer of fusion proteins effectively inhibits VCAM-1-mediated cell adhesion and transmigration via inhibition of cytoskeletal anchorage

The adhesion of leukocytes to endothelium plays a central role in the development of atherosclerosis and thus represents an attractive therapeutic target for anti-atherosclerotic therapies. Vascular cell adhesion molecule-1 (VCAM-1) mediates both the initial tethering and the firm adhesion of leukocytes to endothelial cells. Our work evaluates the feasibility of using the cytoskeletal anchorage of VCAM-1 as a target for gene therapy. As a proof of concept, integrin α_IIbβ₃-mediated cell adhesion with clearly defined cytoskeletal anchorage was tested. We constructed fusion proteins containing the intracellular domain of β₃ placed at various distances to the cell membrane. Using cell adhesion assays and immunofluorescence, we established fusion constructs with competitive and dominant negative inhibition of cell adhesion. With the goal being the transfer of the dominant negative mechanism towards VCAM-1 inhibition, we constructed a fusion molecule containing the cytoplasmic domain of VCAM-1. Indeed, VCAM-1 mediated leukocyte adhesion can be inhibited via transfection of DNA encoding the designed VCAM-1 fusion protein. This is demonstrated in adhesion assays under static and flow conditions using CHO cells expressing recombinant VCAM-1 as well as activated endothelial cells. Thus, we are able to describe a novel approach for dominant negative inhibition of leukocyte adhesion to endothelial cells. This approach warrants further development as a novel gene therapeutic strategy that aims for a locally restricted effect at atherosclerotic areas of the vasculature.     

Lipoxin A4 inhibits immune cell binding to salivary epithelium and vascular endothelium

Lipoxins are formed by leukocytes during cell-cell interactions with epithelial or endothelial cells. Native lipoxin A(4) (LXA(4)) binds to the G protein-coupled lipoxin receptors formyl peptide receptor 2 (FPR2)/ALX and CysLT1. Furthermore, LXA(4) inhibits recruitment of neutrophils, by attenuating chemotaxis, adhesion, and transmigration across vascular endothelial cells. LXA(4) thus appears to serve as an endogenous "stop signal" for immune cell-mediated tissue injury (Serhan CN; Annu Rev Immunol 25: 101-137, 2007). The role of LXA(4) has not been addressed in salivary epithelium, and little is known about its effects on vascular endothelium. Here, we determined that interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) receptor activation in vascular endothelium and salivary epithelium upregulated the expression of adhesion molecules that facilitates the binding of immune cells. We hypothesize that the activation of the ALX/FPR2 and/or CysLT1 receptors by LXA(4) decreases this cytokine-mediated upregulation of cell adhesion molecules that enhance lymphocyte binding to both the vascular endothelium and salivary epithelium. In agreement with this hypothesis, we observed that nanomolar concentrations of LXA(4) blocked IL-1β- and TNF-α-mediated upregulation of E-selectin and intercellular cell adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVECs). Binding of Jurkat cells to stimulated HUVECs was abrogated by LXA(4). Furthermore, LXA(4) preincubation with human submandibular gland cell line (HSG) also blocked TNF-α-mediated upregulation of vascular cell adhesion molecule-1 (VCAM-1) in these cells, and it reduced lymphocyte adhesion. These findings suggest that ALX/FPR2 and/or CysLT1 receptor activation in endothelial and epithelial cells blocks cytokine-induced adhesion molecule expression and consequent binding of lymphocytes, a critical event in the pathogenesis of Sj?gren's syndrome (SS).     

Leukocyte – endothelial interactions in inflammation

At sites of inflammation, infection or vascular injury local proinflammatory or pathogen-derived stimuli render the luminal vascular endothelial surface attractive for leukocytes. This innate immunity response consists of a well-defined and regulated multi-step cascade involving consecutive steps of adhesive interactions between the leukocytes and the endothelium. During the initial contact with the activated endothelium leukocytes roll along the endothelium via a loose bond which is mediated by selectins. Subsequently, leukocytes are activated by chemokines presented on the luminal endothelial surface, which results in the activation of leukocyte integrins and the firm leukocyte arrest on the endothelium. After their firm adhesion, leukocytes make use of two transmigration processes to pass the endothelial barrier, the transcellular route through the endothelial cell body or the paracellular route through the endothelial junctions. In addition, further circulating cells, such as platelets arrive early at sites of inflammation contributing to both coagulation and to the immune response in parts by facilitating leukocyte–endothelial interactions. Platelets have thereby been implicated in several inflammatory pathologies. This review summarizes the major mechanisms and molecules involved in leukocyte–endothelial and leukocyte-platelet interactions in inflammation.     

Transendothelial migration of monocytes in rat aorta: distribution of F-actin, alpha-catnin, LFA-1, and PECAM-1.

To determine changes in the distribution of cell adhesion molecules during diapedesis of monocytes in situ, we labeled aortic whole mounts from hypercholesterolemic rats with Texas red-phalloidin and antibodies to LFA-1, PECAM-1, or alpha-catenin, and analyzed them by laser scanning confocal microscopy. Monocytes transmigrated through circular openings (transmigration passages) formed by pseudopodia that penetrated between adjacent endothelial cells. Transmigrating monocytes remained spherical above the endothelium, while spreading beneath it. The transmigration passage was lined by F-actin and partially by alpha-catenin, suggesting cadherin-mediated heterotypic interactions. LFA-1 was present in clusters at the monocyte cell surface throughout diapedesis, but was concentrated at the margin of the transmigration passage. PECAM-1 was enriched in the endothelial contact regions where the monocytes transmigrated. PECAM-1 was barely detectable in monocytes before and after diapedesis, but appeared during diapedesis at the cell surface in the parts of the monocyte located above the endothelium. PECAM-1 was enriched near the endothelial cell-cell junctions, but was not detected in parts that spread beneath the endothelium. Our results suggest a major role for LFA-1 during diapedesis and reveal dynamic changes in the distribution of PECAM-1, the actin cytoskeleton, and alpha-catenin during monocyte diapedesis in situ.     

Transendothelial Migration of Monocytes in Rat Aorta: Distribution of F-actin, α-Catenin,LFA-1, and PECAM-1

To determine changes in the distribution of cell adhesion molecules during diapedesis of monocytes in situ, we labeled aortic whole mounts from hypercholesterolemic rats with Texas red-phalloidin and antibodies to LFA-1, PECAM-1, or α-catenin, and analyzed them by laser scanning confocal microscopy. Monocytes transmigrated through circular openings (transmigration passages) formed by pseudopodia that penetrated between adjacent en-dothelial cells. Transmigrating monocytes remained spherical above the endothelium, while spreading beneath it. The transmigration passage was lined by F-actin and partially by α-catenin, suggesting cadherin-mediated heterotypic interactions. LFA-1 was present in clusters at the monocyte cell surface throughout diapedesis, but was concentrated at the margin of the transmigration passage. PECAM-1 was enriched in the endothelial contact regions where the monocytes transmigrated. PECAM-1 was barely detectable in monocytes before and after diapedesis, but appeared during diapedesis at the cell surface in the parts of the monocyte located above the endothelium. PECAM-1 was enriched near the endothelial cell-cell junctions, but was not detected in parts that spread beneath the endothelium. Our results suggest a major role for LFA-1 during diapedesis and reveal dynamic changes in the distribution of PECAM-1, the actin cytoskeleton, and α-catenin during monocyte diapedesis in situ.     

High-temporal-resolution analysis demonstrates that ICAM-1 stabilizes WEHI 274.1 monocytic cell rolling on endothelium

Leukocyte rolling, adhesion, and migration on vascular endothelium involve several sets of adhesion molecules that interact simultaneously. Each of these receptor-ligand pairs may play multiple roles. We examined the role of ICAM-1 in adhesive interactions with mouse aortic endothelial cells (MAECs) in an in vitro flow system. Average rolling velocity of the monocytic cell line WEHI 274.1 was increased on ICAM-1-deficient MAECs compared with wild-type MAECs, both with and without TNF- stimulation. High-temporal-resolution analysis provided insights into the underlying basis for these differences. Without TNF- stimulation, average rolling velocity was slower on wild-type than on ICAM-1-deficient endothelium because of brief (<1 s) pauses. On TNF--stimulated ICAM-1-deficient endothelium, cells rolled faster because of transient accelerations, producing "jerky" rolling. Firm adhesion to ICAM-1-deficient MAECs was significantly reduced compared with wild-type MAECs, although the number of rolling cells was similar. These results demonstrate directly that ICAM-1 affects rolling velocity by stabilizing leukocyte rolling. intercellular adhesion molecule-1; cell adhesion; leukocytes; vascular endothelium; videomicroscopy     

Characterization of porcine arterial endothelial cells cultured on amniotic membrane, a potential matrix for vascular tissue engineering

The existing of basement membrane improves the development of endothelium while constructing blood vessel equivalent. The amniotic membrane (AM) provides a natural basement membrane and has been used in ocular surface reconstruction. This study evaluated the molecular and cellular characteristics of porcine vascular endothelial cells (ECs) cultured on AM. ECs cultured on AM expressed the endothelial marker vWF and exhibited normal endothelial morphology. Here, we demonstrated that AM enhanced the expression of intercellular molecules, platelet-endothelial cell adhesion molecule-1 (PECAM-1), and adhesion molecule VE-cadherin at the intercellular junctions. The expression level of integrin was markedly higher in ECs cultured on AM than on plastic dish. Furthermore, the AM downregulated the expression of E-selectin and P-selectin in both LPS-activated and non-activated ECs. Consistently, adhesion of leukocytes to both activated and non-activated cells was decreased in ECs cultured on AM. Our results suggest that AM is an ideal matrix to develop a functional endothelium in blood vessel equivalent construction.     

The effect of short-term, high glucose concentration on endothelial cells and leukocytes in a 3D in vitro human vascular tissue model

Efforts to determine a link between diabetes and atherosclerosis have involved examining the effect of high glucose levels on the adhesion and migration of circulating leukocytes, mostly monocytes and T lymphocytes. Leukocyte differentiation and proliferation within the subendothelial space can also be investigated by the use of a 3D in vitro human vascular tissue model. This model was used to study the effect of short-term, high glucose concentration on certain cell behavior associated with the early stages of atherosclerosis. Samples were exposed to either a 30- or 5.6-mM glucose concentration for 9 h to represent either hyperglycemic or normoglycemic conditions, respectively. There was a significant increase in vascular cell adhesion molecule-1 expression on the endothelial cells exposed to a 30-mM compared to a 5.6-mM glucose concentration. There was no significant difference in either intercellular adhesion molecule-1 or E-selectin expression on the endothelial cells exposed to a 30-mM compared to a 5.6-mM glucose concentration. After the endothelium was exposed to 30 mM glucose concentration, there was a 70% increase in the number of monocytes (CD14⁺) migrating across the endothelium and a 28% increase in the number of these monocytes differentiating into macrophages, compared to cell migration and differentiation across the endothelium exposed to 5.6 mM glucose concentration. Also, for the endothelium exposed to 30 mM glucose concentration, there were nearly 2.5 times more T lymphocytes that migrated across the endothelium, along with significant cell proliferation, compared to cell migration across the endothelium exposed to 5.6 mM glucose concentration.