Laminar Flow Assays Research Articles

A Genetically Engineered Biofilm Material for SARS‐CoV‐2 Capturing and Isolation (Adv. Mater. Interfaces 30/2022)

Sars-CoV-2 Diagnostics A strategy for capturing, isolating, and concentrating SARS-CoV-2 virus particles from clinical samples with relatively lower viral load via a genetically engineered bacterial biofilm complex to effectively detect using Laminar Flow Assays (LFA) is proposed by Urartu Özgür Şafak Şeker and co-workers in article number 2201126. The complex structure harboring Griffithsin, showed strong capability to concentrate lower virus concentrations so that they can be detected in antigen tests (LFA) following treatment with biofilm complex.

A comparison of two methods for estimating critical swimming speed (Ucrit) in larval fathead minnows: the laminar flow assay and the spinning task assay.

Critical swimming speed (Ucrit) is considered a good predictor of swimming capabilities in fish. To estimate Ucrit, a fish is exposed to an incrementally increasing laminar flow of water until it cannot maintain its position against the current. The spinning task assay has been proposed as an alternative method to traditional laminar flow methods; however, these methods have not been directly compared. Thus, the goal of this study was to determine whether the spinning task assay is a suitable alternative to traditional laminar flow assays. To that end, the performance of fathead minnows in each assay was compared at three time points (14, 19 and 24 days post-fertilization, dpf). In 14 dpf fish, Ucrit estimates were similar regardless of the assay used. However, at 19 and 24 dpf, Ucrit estimates derived from the two assay types were significantly different. This indicates that the assays are not equivalent to one another and that the spinning task assay is not a suitable alternative to the laminar flow assay for the determination of Ucrit.

Abstract 1609: The epithelial to mesenchymal transition regulates the expression of E-selectin ligands on breast cancer cell lines

Abstract Breast cancer cells garner invasive and stem-like characteristics through the epithelial to mesenchymal transition (EMT). Consequently, the EMT has been implicated in cancer metastasis. While many of the effects of the EMT have been elucidated, it is currently unknown how the EMT modifies the expression of E-selectin ligands on breast cancer cells. These ligands are often sialofucosylated glycans that mediate cell adhesion with E-selectin molecules presented by endothelial cells lining the blood vessel walls at the metastatic site, which is commonly bone marrow. Although E-selectin is widely recognized for its role in promoting adhesion during tumor cell extravasation, it also maintains the quiescence of hematopoietic stem cells in the vascular niche of the bone marrow. Thus in this investigation, we studied the effects of the EMT on the E-selectin ligand activities of breast cancer cell lines and their behavior in an E-selectin rich environment. The EMT was induced in breast cancer cell lines by ectopic expression of either the Snail or Twist transcription factors. Through the EMT breast cancer cells upregulated the expression of vimentin and N-cadherin, yet reduced the expression of CD24, E-cadherin, and E-selectin ligands. Breast cancer cell lines were assayed for E-selectin ligand activity using laminar flow assays, flow cytometry, qPCR, and were cultured on E-selectin-coated tissue culture plates. In laminar flow assays breast cancer cells were perfused over E-selectin substrates using physiological flow conditions. Breast cancer cells expressing Snail or Twist adhered to E-selectin from the free fluid stream in significantly fewer numbers and demonstrated fewer E-selectin ligands in flow cytometry than the vector controls. Additionally, qPCR revealed that in comparison to vector controls breast cancer cells expressing Snail or Twist downregulated the expression of α-(1,3) fucosyltransferase and α-(1,4) fucosyltransferase, which catalyze terminal fucosylations necessary for E-selectin ligand function. Interestingly, on E-selectin coated plates breast cancer cells expressing Snail or Twist avoided prolonged exposure to E-selectin by forming mammospheres, yet vector controls did not. Collectively, these data demonstrate that the EMT regulates the expression of E-selectin ligands on breast cancer cells, and causes the cells to become sensitive to culture in an E-selectin rich environment. Citation Format: Grady Earl Carlson, Venktesh Shirure, Alexander O. Ostermann, Emily A. Blaha, Louis F. Delgadillo, David F.J. Tees, Fabian Benencia, Monica Burdick. The epithelial to mesenchymal transition regulates the expression of E-selectin ligands on breast cancer cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1609.

Abstract 1920: Stem-like triple negative breast cancer cells exhibit a distinct response to selectin/selectin ligand interactions

Abstract Circulating tumor cells (CTCs) often over-express sialofucosylated glycans, which bind to E-selectin presented on vascular endothelial cells. This interaction facilitates the initial tethering and rolling adhesion events in CTC extravasation during cancer metastasis. Though selectins and their ligands are well-established cell adhesion molecules, relatively little is known about their roles as cell signaling molecules for regulating growth, migration, and apoptosis in breast cancer (BC). To investigate the effects of selectin/selectin ligand interactions on BC cell signaling and adhesion, Hs578T, and MDA-MB-231 stem-like triple negative cell lines and BT-20, MCF-7, and ZR-75-1 non-stem-like hormone-receptor-positive cell lines were grown on tissue culture plates pre-treated with recombinant E-selectin or perfused over E-selectin-presenting substrate at physiologically relevant laminar flow conditions. In culture, cell lines demonstrated a dose dependent response on substrates pre-treated with increasing concentrations (1-10 µg/ml) of recombinant E-selectin. More specifically, cell lines exhibited little change on substrate treated with low concentrations of recombinant E-selectin (1-5 µg/ml) relative to human Fc fragment and fibronectin controls, yet displayed varying degrees of membrane blebbing and, in some cases, aggregate formation and reduced proliferation on substrate treated with 10 µg/ml of recombinant E-selectin. Notably, the stem-like triple negative cell lines, specifically Hs578T cells, had the most pronounced response to culture on E-selectin substrate as defined by blebbing and formation of cell aggregates. Furthermore, when Hs578T cells were cultured on substrate with adjacent areas treated with either recombinant E-selectin (10 µg/ml) or fibronectin (molar equivalent), cells failed to attach and grow on E-selectin but successfully seeded and proliferated in culture on fibronectin. In the laminar flow assay, Hs578T and MDA-MB-231 stem-like triple negative cell lines had four fold fewer adhesions on E-selectin substrate relative to the adhesion of BT-20, MCF-7, and ZR-75-1 non-stem-like hormone-receptor-positive cell lines. Moreover, the average rolling velocity of the stem-like triple negative cell lines on E-selectin substrate was significantly greater than the average rolling velocities of the other cell lines, which illustrates that under dynamic flow conditions stem-like triple negative BC cell lines have relatively fewer and weaker interactions with E-selectin. These data show that BC cells, especially Hs578T stem-like triple negative BC cells, differ in response to selectin/selectin ligand interaction under static and dynamic conditions, and illustrate that threshold levels of selectin/selectin ligand interaction may regulate cell signaling in certain types of BC cell lines. Thus targeting these interactions may lead to novel BC therapeutics. Citation Format: Grady E. Carlson, Luis F. Delgadillo, Emily A. Blaha, Fabian Benencia, Monica M. Burdick. Stem-like triple negative breast cancer cells exhibit a distinct response to selectin/selectin ligand interactions. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1920. doi:10.1158/1538-7445.AM2014-1920

SAT0555 Transmigration and Mobility of Rheumatoid Arthritis Synovial Fibroblasts is Dependent on Distinct Interaction Processes with Endothelial Cells

BackgroundSynovial fibroblasts (SF) from patients with rheumatoid arthritis (RA) are involved in cartilage destruction. The behavior of RASF to migrate over long distances could be demonstrated in the SCID mouse...

E- and P-Selectins Are Essential for Repopulation of Chronic Myelogenous and Chronic Eosinophilic Leukemias in a Scid Mouse Xenograft Model

In chronic myelogenous (CML) and chronic eosinophilic leukemia (CEL), neoplastic cells spread via the circulation into various extramedullary organs. As E- and P-selectin constitute the starting point for the leucocyte adhesion/invasion cascade, and CEL and CML cells share many properties with normal granulocytes, we investigated the role of these selectins in CEL and CML cell expansion and organ invasion in a xenotransplantation model using scid mice. Using two human leukemic cell lines (EOL-1 and K562), we were able to show that E- and P-selectins mediate leukemia cell tethering and adherence in a laminar flow assay. While E-selectin binding depended on sialylated carbohydrate moieties, P-selectin binding was completely (K562) or partially (EOL-1) independent of these carbohydrates indicating the involvement of non-canonical selectin ligands. In a xenograft model in scid mice, both cell lines invaded the bone marrow and other organs, formed chloromas, and ultimately produced an overt leukemia. In contrast, in E- and P-selectin knockout scid mice, the cells failed to show engraftment in 8 out of 10 animals and even if they did engraft, they produced only little organ invasion and chloroma formation. Together, these data suggest that E- and P-selectins play an important role in leukemic dissemination in CML and CEL.

Structural characterization and in vitro inhibitory activities in P-selectin-mediated leukocyte adhesion of polysaccharide fractions isolated from the roots of Physalis alkekengi

Selectin-mediated leukocyte initial attachment and rolling over vessel endothelial surface are crucial steps for inflammatory responses. As P-selectin is a promising target for anti-inflammation therapeutic strategy, recent works have focused on searching for more potent and non-toxic P-selectin antagonists among various natural carbohydrate products. Here, we isolated three water-soluble polysaccharide fractions (PPS-1, PPS-2 and PPS-3) from the roots of Physalis alkekengi by DEAE-cellulose and Sephacryl S-200 chromatography. Their physicochemical and structural characterizations were determined by chemical methods, GC (gas chromatography), HPLC (high performance liquid chromatography), FT-IR (Fourier transform infrared spectrometry), partial acid hydrolysis, methylation and GC–MS (gas chromatography–mass spectrometry) analyses. The inhibitory capacity of the polysaccharide fractions in P-selectin-mediated leukocyte adhesion was evaluated by flow cytometric, static adhesion and laminar flow assays. Results showed that different polysaccharide fractions possess distinct physicochemical and structural properties, including carbohydrate, protein and uronic acid contents, molecular weight, monosaccharide composition and glycosidic linkage type. Among the polysaccharide fractions, PPS-2 could effectively block the interaction between P-selectin and its native ligand.

Physiological Levels of Jak2 V617F Result in Enhanced Megakaryocyte Differentiation, Proplatelet Formation and Platelet Reactivity.

Abstract 226The major clinical challenge in treating patients with myeloproliferative diseases (MPD) such as essential thrombocythaemia (ET) and polycythaemia vera (PV) is to prevent thrombotic events. Despite adequate red cell and platelet count control by means of venesection or cytoreductive therapy and the use of anti-platelet agents, patients with ET and PV have a three-fold risk-increase of cardiovascular and cerebral ischaemic events. A V617F mutation in the Janus kinase 2 (Jak2) that causes constitutive activation of Jak2 has been shown to be present in >90% and ∼50% of PV and ET patients, respectively. The effect of this activation on the biology of primary megakaryocytic cells is not known and neither is it clear whether the thrombotic risk conferred by this mutation reflects the increase in red cell/platelet counts, an intrinsic increase in platelet reactivity or both. Studies in humans are hampered by the co-existence of normal and clonal haematopoiesis with significant clonal and phenotypic heterogeneity between patients. Chimaeric mouse models based on retroviral transduction of Jak2 V617F have marked overexpression of the mutant Jak2 and show a PV phenotype with normal platelet counts. Recently, Jak2 V617F transgenic mice models have been published but the transgenes have multiple (and variable) insertions and are therefore subject to position effects with varying degrees of expression of the mutant Jak2 compared to the wild-type allele. We have generated a Cre-inducible mouse model where the human Jak2 V617F gene has been knocked into one allele of the mouse endogenous Jak2 gene. Upon Cre induction, the mice exhibit an ET phenotype (platelet count increased by approximately 30% but with a normal haematocrit) that is stable for over 26 weeks. Bone marrow histology shows classical features of ET with increased numbers of megakaryocytes (MKs) and clusters with no fibrosis. Quantitative RT-PCR shows stable expression of Jak2 V617F in the MKs at a similar level to that of endogenous mouse Jak2 therefore reflecting the physiological situation found in human ET patients. In liquid cultures, bone marrow-derived MKs show increased ploidy in response to suboptimal concentrations of thrombopoietin (TPO) in keeping with the increased number of MKs found in the bone marrow histology. Crucially, in vitro proplatelet formation in mutant MKs was increased two-fold compared to MKs derived from litter-match control animals showing that platelet production may not only relate to the increased number of MKs but to intrinsic differences in MK biology and platelet production. Platelet aggregation studies and P-selectin expression in response to an array of agonists was not significantly different between mutant and controls but in vitro laminar flow assays show increased thrombus formation on collagen. Although Jak2V617F expression was down-regulated at protein level in mature MKs and platelets, analysis of downstream signalling pathways showed alteration of the phosphorylation status of Src kinases. This mouse model therefore provides a unique opportunity to understand the biological mechanism of increased platelet production and thrombotic risk in ET patients as well as unravelling the signalling pathways downstream of the Jak2 V617F in primary cells, which will be crucial in the context of specific therapeutic Jak2 inhibitors currently in clinical development.This work was supported by the British Heart Foundation Disclosures:No relevant conflicts of interest to declare.

Ribosomal Protein S19 Interacts with Macrophage Migration Inhibitory Factor and Attenuates Its Pro-inflammatory Function

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine that has been implicated in the pathogenesis of inflammatory disorders such as infection, sepsis, and autoimmune disease. MIF exists preformed in cytoplasmic pools and exhibits an intrinsic tautomerase and oxidoreductase activity. MIF levels are elevated in the serum of animals and patients with infection or different inflammatory disorders. To elucidate how MIF actions are controlled, we searched for endogenous MIF-interacting proteins with the potential to interfere with key MIF functions. Using in vivo biotin-tagging and endogenous co-immunoprecipitation, the ribosomal protein S19 (RPS19) was identified as a novel MIF binding partner. Surface plasmon resonance and pulldown experiments with wild type and mutant MIF revealed a direct physical interaction of the two proteins (K(D) = 1.3 x 10(-6) m). As RPS19 is released in inflammatory lesions by apoptotic cells, we explored whether it affects MIF function and inhibits its binding to receptors CD74 and CXCR2. Low doses of RPS19 were found to strongly inhibit MIF-CD74 interaction. Furthermore, RPS19 significantly compromised CXCR2-dependent MIF-triggered adhesion of monocytes to endothelial cells under flow conditions. We, therefore, propose that RPS19 acts as an extracellular negative regulator of MIF.

Abstract 620: Platelet CD40L: a Powerful Leukocyte and Endothelial Cell Activator in Atherosclerosis

Here we investigated whether platelet CD40L plays a role in atherosclerosis. Platelets were obtained from donor ApoE−/− or CD40L−/− ApoE−/− mice, activated with thrombin, neutralized by hirudin and injected intravenously into n=30 ApoE−/− recipients (3E10 7 platelets) every 5 days. Infusion of activated CD40L−/− platelets in ApoE−/− mice from age 5–17wks caused a decrease in atherosclerosis (predominantly early lesions) in the aortic arch and its major branch points (ApoE−/− platelets 2.01E10 5 ± 3.74E10 4 μm 2 vs CD40L−/−ApoE−/− platelets 1.01E10 5 ± 1.57E10 4 μm 2 ; p<0.05) to levels that were comparable to the sham (NaCl) group (1.17E10 5 ± 1.61E10 4 μm 2 ). To induce advanced atherosclerosis, a silastic non-constrictive collar was placed around both carotid arteries in diet fed mice, and activated platelets were injected every 5 days during 5 wks. Infusion of activated CD40L−/−ApoE−/− platelets caused a 35.4% decrease in plaque volume (ApoE−/− platelets 2.08E10 8 ± 2.55E10 7 μm 3 vs CD40L−/−ApoE−/− platelets 1.36E10 8 ± 3.45E10 7 μm 3 , Sham 1.17E10 8 ± 2.71E10 7 μm 3 p<0.05), indicating an important role for activated platelet CD40L in both early and advanced atherosclerosis. Laminar flow assays showed that activated CD40L−/−ApoE−/− platelets lose their capacity of binding CD40L+/+ApoE−/− leukocytes, especially monocytes, with 30.1% compared to activated ApoE−/− platelets. Moreover, FACS analysis (CD45, CD11b,CD41) showed that activated ApoE−/−platelets bind 24.8% less to CD40−/−ApoE−/− leukocytes, and activated CD40L−/−ApoE−/−platelets bind 40.0% less to CD40−/−ApoE−/− leukocytes, thereby pointing out leukocyte CD40 as the receptor of platelet CD40L. Intravital microscopy in carotid arteries of mice, injected with activated CD40L−/− platelets, showed a 3-fold decrease in leukocyte adhesion. Even more, deficiency of CD40L on platelets causes a 58.4% decrease in thrombus formation compared to wild type. We demonstrate that CD40L on activated platelets plays an important role in atherosclerosis by promoting platelet aggregation, the formation of platelet-leukocyte aggregates, and leukocyte-endothelial interactions. This shows that platelet CD40L has a bridging function between the leukocyte and the endothelium.

Chemokine Fractalkine Mediates Leukocyte Recruitment to Inflammatory Endothelial Cells in Flowing Whole Blood

The membrane-bound chemokine fractalkine (CX3CL1) is expressed on various cell types such as activated endothelial cells and has been implicated in the inflammatory process of atherosclerosis. The aim of the present study was to dissect the role of fractalkine in leukocyte recruitment to inflamed endothelium under arterial shear forces. With the use of immunofluorescence and laminar flow assays, the present study shows that human umbilical vein endothelial cells stimulated with tumor necrosis factor-alpha and interferon-gamma abundantly express CX3CL1 and promote substantial leukocyte accumulation under arterial flow conditions. In the presence of high shear, firm adhesion of leukocytes to inflamed endothelial cells is reduced by approximately 40% by a function-blocking anti-fractalkine antibody or by an antibody directed against the fractalkine receptor (CX3CR1). With the use of intravital video-fluorescence microscopy we demonstrate that inhibition of fractalkine signaling attenuates leukocyte adhesion to the atherosclerotic carotid artery of apolipoprotein E-deficient mice, which suggests that the CX3CL1-CX3CR1 axis is critically involved in leukocyte adhesion to inflamed endothelial cells under high shear forces both in vitro and in vivo. Surprisingly, platelets were strictly required for fractalkine-induced leukocyte adhesion at high shear rates. Correspondingly, specific inhibition of platelet adhesion to inflamed endothelial cells also significantly reduced leukocyte accumulation. We show that both soluble and membrane-bound fractalkine induces platelet degranulation and subsequent surface expression of P-selectin, which thereby promotes direct platelet-leukocyte interaction. Fractalkine expressed by inflamed endothelial cells triggers P-selectin exposure on adherent platelets, which thereby initiates the local accumulation of leukocytes under arterial shear, an essential step in the development of atherosclerotic lesions.

Effects of down-regulating the Id genes in human colorectal cancer cells on early steps of haematogenous metastasis

Id genes (inhibitor of DNA binding/differentiation) play important roles in tumour growth. We have previously described crucial roles of Id gene over-expression in endothelial cells for tumour angiogenesis. Here, we have evaluated direct effects of Id gene down-regulation on tumour cells, namely on cell proliferation, motility, and adhesion to lung microvasculature during haematogenous metastasis. For this purpose, Id genes were stably down-regulated by RNA interference in human colorectal cancer cells. These cells showed delayed proliferation, inhibited motility and decreased expression of integrin alpha6 and consequently reduced adhesion to lung microvasculature in mice. Static adhesion assays and laminar flow assays revealed decreased laminin binding capacity of these cells, and blocking experiments confirmed that it could be attributed to decreased expression of integrin alpha6. The present results indicate important roles of Id genes in tumour cells during early steps of haematogenous metastasis and suggest dual effects from their therapeutic inhibition.

Dynamics of fluorescence dequenching of ostrich-quenched fluorescein biotin: A multifunctional quantitative assay for biotin

We describe a simple and rapid quantitative assay for biotin and biotin conjugates. The assay is based on the kinetic analysis of the enhancement of fluorescence of streptavidin/fluorescein biotin complexes in the presence of biotin. The kinetic response of fluorescence enhancement is proportional to the concentration of biotin. Standard calibration curves based on the kinetic response are obtained and detection limits of ≈10 −9 M are established. Because the assay is amenable for use in small volumes of 5–50 μL or bead-based assays, the detection limits can be extended to the femtomole range. Since the assay depends on kinetic analysis, routine quantitation can be achieved without reference to standard curves. The dynamic aspects allow the assay to be extended to a broader range of applications including its use as an indicator of reagent mixing in laminar-flow assays carried out in microfluidic devices.

Neointimal smooth muscle cells display a proinflammatory phenotype resulting in increased leukocyte recruitment mediated by P-selectin and chemokines.

Leukocyte recruitment is crucial for the response to vascular injury in spontaneous and accelerated atherosclerosis. Whereas the mechanisms of leukocyte adhesion to endothelium or matrix-bound platelets have been characterized, less is known about the proadhesive role of smooth muscle cells (SMCs) exposed after endothelial denudation. In laminar flow assays, neointimal rat SMCs (niSMCs) supported a 2.5-fold higher arrest of monocytes and "memory" T lymphocytes than medial SMCs, which was dependent on both P-selectin and VLA-4, as demonstrated by blocking antibodies. The increase in monocyte arrest on niSMCs was triggered by the CXC chemokine GRO-alpha and fractalkine, whereas "memory" T cell arrest was mediated by stromal cell-derived factor (SDF)-1alpha. This functional phenotype was paralleled by a constitutively increased mRNA and surface expression of P-selectin and of relevant chemokines in niSMCs, as assessed by real-time PCR and flow cytometry. The increased expression of P-selectin in niSMCs versus medial SMCs was associated with enhanced NF-kappaB activity, as revealed by immunofluorescence staining for nuclear p65 in vitro. Inhibition of NF-kappaB by adenoviral IkappaBalpha in niSMCs resulted in a marked reduction of increased leukocyte arrest in flow. Furthermore, P-selectin expression by niSMCs in vivo was confirmed in a hypercholesterolemic mouse model of vascular injury by double immunofluorescence and by RT-PCR after laser microdissection. In conclusion, we have identified a NF-kappaB-mediated proinflammatory phenotype of niSMCs that is characterized by increased P-selectin and chemokine expression and thereby effectively supports leukocyte recruitment.

Cell Membrane Alignment along Adhesive Surfaces: Contribution of Active and Passive Cell Processes

Cell adhesion requires nanometer scale membrane alignment to allow contact between adhesion receptors. Little quantitative information is presently available on this important biological process. Here we present an interference reflection microscopic study of the initial interaction between monocytic THP-1 cells and adhesive surfaces, with concomitant determination of cell deformability, using micropipette aspiration, and adhesiveness, using a laminar flow assay. We report that 1), during the first few minutes after contact, cells form irregular-shaped interaction zones reaching ∼100 μm 2 with a margin extension velocity of 0.01–0.02 μm/s. This happens before the overall cell deformations usually defined as spreading. 2), These interference reflection microscopic-detected zones represent bona fide adhesion inasmuch as cells are not released by hydrodynamic forces. 3), Alignment is markedly decreased but not abolished by microfilament blockade with cytochalasin or even cell fixation with paraformaldehyde. 4), In contrast, exposing cells to hypotonic medium increased the rate of contact extension. 5), Contacts formed in presence of cytochalasin, after paraformaldehyde fixation or in hypotonic medium, were much more regular-shaped than controls and their extension matched cell deformability. 6), None of the aforementioned treatments altered adhesiveness to the surface. It is concluded that adhesive forces and passive membrane deformations are sufficient to generate initial cell alignment to adhesive surfaces, and this process is accelerated by spontaneous cytoskeletally-driven membrane motion.

Deposition of platelet RANTES triggering monocyte recruitment requires P-selectin and is involved in neointima formation after arterial injury.

Chemokines expressed on atherosclerotic endothelium or deposited by activated platelets have been implicated in monocyte recruitment during atherogenesis and restenosis. Although the involvement of P-selectin in these processes is evident from studies in knockout mice, it has not been elucidated whether delivery of platelet chemokines requires P-selectin, thus serving as a P-selectin-dependent effector function. Using immunofluorescence and laminar flow assays, we found that the deposition of the platelet-derived chemokine RANTES and monocyte arrest subsequently triggered by RANTES immobilized on inflamed endothelium are more efficient after preperfusion than after static preincubation of platelets and appear to depend on interactions of platelet but not endothelial P-selectin. This was revealed by the effects of P-selectin antibodies and comparison of P-selectin-deficient and wild-type platelets. Immunohistochemistry detected a substantial luminal expression of RANTES on neointimal lesions in wire-injured carotid arteries of apolipoprotein E (apoE)-deficient mice but not of mice with a combined deficiency in apoE and P-selectin (or platelet P-selectin). As assessed by histomorphometry, treatment of apoE-deficient mice with the RANTES receptor antagonist Met-RANTES markedly reduced neointimal plaque area and macrophage infiltration. Our data suggest that RANTES deposition and subsequent monocyte arrest are promoted by platelet P-selectin and involved in wire-induced intimal hyperplasia, and that blocking RANTES receptors attenuates neointima formation and macrophage infiltration. This mechanism represents an important component explaining the protection against neointimal growth in P-selectin-deficient mice and may represent a novel approach to the treatment of restenosis or atherosclerosis by the administration of chemokine receptor antagonists.

Hyaluronan Anchoring and Regulation on the Surface of Vascular Endothelial Cells Is Mediated through the Functionally Active Form of CD44

CD44 on lymphocytes binding to its carbohydrate ligand hyaluronan can mediate primary adhesion (rolling interactions) of lymphocytes on vascular endothelial cells. This adhesion pathway is utilized in the extravasation of activated T cells from the blood into sites of inflammation and therefore influences patterns of lymphocyte homing and inflammation. Hyaluronan is a glycosaminoglycan found in the extracellular matrix and is involved in a number of biological processes. We have shown that the expression of hyaluronan on the surface of endothelial cells is inducible by proinflammatory cytokines. However, the manner through which hyaluronan is anchored to the endothelial cell surface so that it can resist shear forces and the mechanism of the regulation of the level of hyaluronan on the cell surface has not been investigated. In order to characterize potential hyaluronan receptors on endothelial cells, we performed analyses of cell surface staining by flow cytometry on intact endothelial cells and ligand blotting assays using membrane fractions. Hyaluronan binding activity was detected as a major species corresponding to the size of CD44, and this was confirmed to be the same by Western blotting and immunoprecipitation. Moreover, alterations in the surface level of hyaluronan after tumor necrosis factor-alpha stimulation is regulated primarily by changes in the cell surface levels of the hyaluronan-binding form of CD44. In laminar flow assays, lymphoid cells specifically roll on hyaluronan anchored by purified CD44 coated on glass tubes, indicating that the avidity of the endothelial CD44/hyaluronan interaction is sufficient to support rolling adhesions under conditions mimicking physiologic shear forces. Together these studies show that CD44 serves to anchor hyaluronan on endothelial cell surfaces, that activation of CD44 is a major regulator of endothelial surface hyaluronan expression, and that the non-covalent interaction between CD44 and hyaluronan is sufficient to provide resistance to shear under physiologic conditions and thereby support the initial steps of lymphocyte extravasation.

Vinculin and cell-cell adhesion.

Vinculin, a 117-kDa protein, is a constituent of adhesion plaques and adherence junctions in non-muscle cells. We investigated the role of vinculin on the physical strength of cell-cell adhesion by conducting disaggregation assays on aggregates of parental wild-type F9 mouse embryonal carcinoma cells (clone BIM), two vinculin-depleted F9 cell lines, gamma 227 and gamma 229, and a reconstituted gamma 229 cell line (R3) that re-express vinculin. Immunoblotting demonstrated that the four cell lines used in the study had similar expressions of the cell-cell adhesion molecule E-cadherin and associated membrane proteins alpha- and beta-catenin. Double immunofluorescence analysis showed that, in contrast to the vinculin-null cell lines. BIM and R3 cells expressed abundant vinculin at the cell margins in adhesion plaques and in cell-cell margins that also contained actin. Laminar flow assays showed that both the vinculin-positive and vinculin-negative cell aggregates that were formed in culture in the course of 24 to 48 hours largely remained intact despite the imposition of shear flow at high shear rates. Since laminar flow imposed on cell aggregates act to separate cells from each other, our data indicate that F9 cells that were adherent to a substrate formed strong cell-cell adhesion bonds independent of vinculin expression. On the other hand, aggregates of vinculin-depleted gamma 229 and gamma 227 cells that were formed in suspension during a two-hour static incubation at 37 degrees C were desegregated more easily with the imposition of shear flow than the BIM and R3 cell aggregates formed under identical conditions. Loss of vinculin was associated with a reduction in cell-cell adhesion strength only among those cells lacking contact to a substrate. Overall, the results indicate that vinculin is not needed for forming strong cell-cell adhesion bonds between neighboring carcinoma cells which are adherent to the basal lamina.

Lateral dimerization is required for the homophilic binding activity of C-cadherin.

Regulation of cadherin-mediated adhesion can occur rapidly at the cell surface. To understand the mechanism underlying cadherin regulation, it is essential to elucidate the homophilic binding mechanism that underlies all cadherin-mediated functions. Therefore, we have investigated the structural and functional properties of the extracellular segment of Xenopus C-cadherin using a purified, recombinant protein (CEC 1-5). CEC 1-5 supported adhesion of CHO cells expressing C-cadherin. The extracellular segment was also capable of mediating aggregation of microspheres. Chemical cross-linking and gel filtration revealed that CEC 1-5 formed dimers in the presence as well as absence of calcium. Analysis of the functional activity of purified dimers and monomers demonstrated that dimers retained substantially greater homophilic binding activity than monomers. These results demonstrate that lateral dimerization is necessary for homophilic binding between cadherin extracellular segments and suggest multiple potential mechanisms for the regulation of cadherin activity. Since the extracellular segment alone possessed significant homophilic binding activity, the adhesive activity of the extracellular segment in a cellular context was analyzed. The adhesion of CHO cells expressing a truncated version of C-cadherin lacking the cytoplasmic tail was compared to cells expressing the wild-type C-cadherin using a laminar flow assay on substrates coated with CEC 1-5. CHO cells expressing the truncated C-cadherin were able to attach to CEC 1-5 and to resist detachment by low shear forces, demonstrating that tailless C-cadherin can mediate basic, weak adhesion of CHO cells. However, cells expressing the truncated C-cadherin did not exhibit the complete adhesive activity of cells expressing wild-type C-cadherin. Cells expressing wild-type C-cadherin remained attached to CEC 1-5 at high shear forces, while cells expressing the tailless C-cadherin did not adhere well at high shear forces. These results suggest that there may be two states of cadherin-mediated adhesion. The first, relatively weak state can be mediated through interactions between the extracellular segments alone. The second strong adhesive state is critically dependent on the cytoplasmic tail.

Sialomucin CD34 is the major L-selectin ligand in human tonsil high endothelial venules.

Peripheral node addressin (PNAd) is a complex mixture of glycoproteins with L-selectin ligand activity that functions in lymphocyte homing. We have investigated the contribution of the sialomucin CD34 relative to other components of PNAd in lymphocyte tethering and rolling in in vitro laminar flow assays. PNAd was isolated with MECA-79 mAb-Sepharose from tonsillar stroma, and the CD34 component (PNAd,CD34+) and CD34-negative component (PNAd,CD34-) separated on CD34 mAb-Sepharose. Lymphocytes on the PNAd,CD34- fraction tether less efficiently, roll faster and are less resistant to shear detachment than on PNAd. The PNAd,CD34+ fraction constitutes about half the total functional activity. These studies show that CD34 is a major functional component of PNAd. Ligand activity in both the PNAd,CD34+ and PNAd,CD34- fractions is expressed on mucin-like domains, as shown with O-sialoglycoprotease. The CD34 component of PNAd has about four times higher tethering efficiency than total tonsillar CD34. CD34 from spleen shows no lymphocyte tethering. Although less efficient than the PNAd,CD34+ fraction from tonsil, CD34 from the KG1a hematopoietic cell line is functionally active as an L-selectin ligand despite lack of reactivity with MECA-79 mAb, which binds to a sulfation-dependent epitope. All four forms of CD34 are active in binding to E-selectin. KG1a CD34 but not spleen CD34 are active as L-selectin ligands, yet both lack MECA-79 reactivity and possess E-selectin ligand activity. This suggests that L-selectin ligands and E-selectin ligands differ in more respects than presence of the MECA-79 epitope.