E-selectin Ligand Research Articles

Preparation of E-selectin-targeting nanoparticles and preliminary in vitro evaluation

Targeted delivery aims to concentrate therapeutic agents at their site of action and thereby enhance treatment and limit side-effects. E-selectin on endothelial cells is markedly up-regulated by cytokine stimulation of inflamed and some tumoral tissues, promoting the adhesion of leukocytes and metastatic tumor cells, thus making it an interesting molecular target for drug delivery systems.We report here the preparation of targeted nanoparticles from original amphiphilic block copolymers functionalized with an analog of sialyl Lewis X (SLEx), the physiological ligand of E-selectin. Nanoparticles, prepared by nanoprecipitation, caused no significant cytotoxicity. Ligand-functionalized nanoparticles were specifically recognized and internalized better by tumor necrosis factor α (TNF-α)-activated human umbilical vein endothelial cells (HUVECs) than control nanoparticles or HUVECs with low E-selectin expression. These nanoparticles are designed to carry the ligand at the end of a PEG spacer to improve accessibility. This system has potential for the treatment of inflammation, inhibition of tumor metastasis, and for molecular imaging.

Expression of E-selectin ligands on circulating tumor cells: cross-regulation with cancer stem cell regulatory pathways?

Although significant progress has been made in the fight against cancer, successful treatment strategies have yet to be developed to combat those tumors that have metastasized to distant organs. Poor characterization of the molecular mechanisms of cancer spread is a major impediment to designing predictive diagnostics and effective clinical interventions against late stage disease. In hematogenous metastasis, it is widely suspected that circulating tumor cells (CTCs) express specific adhesion molecules that actively initiate contact with the vascular endothelium lining the vessel walls of the target organ. This “tethering” is mediated by ligands expressed by CTCs that bind to E-selectin expressed by endothelial cells. However, it is currently unknown whether expression of functional E-selectin ligands on CTCs is related to cancer stem cell regulatory or maintenance pathways, particularly epithelial-to-mesenchymal transition and the reverse, mesenchymal-to-epithelial transition. In this hypothesis and theory article, we explore the potential roles of these mechanisms on the dynamic regulation of selectin ligands mediating CTC trafficking during metastasis.

Three to Tango: MUC1 as a Ligand for Both E-Selectin and ICAM-1 in the Breast Cancer Metastatic Cascade

Cancer cell tethering and rolling on the vascular wall is facilitated by various selectin: glycoprotein interactions which lead to eventual extravasation and metastases. The aberrantly underglycosylated mucin MUC1 has been shown to both abundantly express selectin binding moieties (sialyl Lewis x and a) and to consistently expose its core epitope. Flow cytometry was used to determine MUC1 expression on ZR-75-1 and MCF7 cells, while immunofluorescence microscopy was used to confirm the aberrant form of MUC1 and MUC1:ICAM-1 interactions. Each cell line was then perfused through combined E-selectin and ICAM-1 coated microtubes, as a model of the microvascular endothelium. ZR-75-1 and MCF7 were found to express abundant and low levels of underglycosylated MUC1, respectively. The rolling/adhesion profiles showed that ZR-75-1 cells, when compared to MCF7 cells, interact with E-selectin more efficiently resulting in sufficiently slow rolling velocities to form MUC1:ICAM-1 interactions thereby facilitating firm adhesion. The purpose and novelty of this work is the demonstration of the synergistic adhesion capabilities of MUC1 in the metastatic adhesion cascade, where the observed differential adhesion is consistent with the relative metastatic potential of the ZR-75-1 (highly metastatic) and MCF7 (weakly metastatic) cell lines.

Novel Dual E-Selectin-CXCR4 Inhibitors Mobilize Human Acute Myeloid Leukemia (AML) Cells in the NODscid IL2Rγc−/− Xenograft and Confer Susceptibility to Cytarabine

Abstract 579High expression of CXCR4, a chemokine receptor for CXCL12 (SDF1), is associated with poor prognosis in AML. CXCR4 inhibitors mobilize AML and enhance chemotherapy sensitivity in laboratory studies. They are currently under study as single inhibitors in combination with standard chemotherapy regimens for induction or salvage in AML. Antibody to CD44, an E-selectin ligand when properly glycosylated [HCELL (Sackstein et al 2004)], eliminates leukemia stem cell engraftment in NODscid animals (Jin et al 2006). High level expression of CD44 contributes to leukemia relapse (Quéré et al 2011). In addition, bcr-abl positive leukemia stem cells are dependent on CD44 for homing to the marrow, (Krause et al 2006) and Nalm6 ALL cell line homes to E-selectin+SDF1+ regions of the bone marrow microvasculature, dependent on both E-selectin and CXCR4 (Sipkins et al 2005).Our preclinical studies demonstrate very high level expression of CD44 (99.8% ± 1.7%) by AML blasts from patients, and by putative CD34+CD38−CD123+ leukemia stem cells (99.8% ± 0.6%). The mean fluorescence intensity (MFI) for CD44 expression by AML blasts is one to two logs higher than the MFI for 16 other adhesion receptors. We also have found that blocking of CD44 inhibits adhesion of AML blasts to a stromal cell line in vitro. Eighty percent of the AML blast samples tested demonstrated binding of E-selectin-IgG chimeric protein by flow cytometry, and nearly all AML blast samples exhibited functional adhesion to immobilized E-selectin. We then tested the hypothesis that functional inhibition of CD44 by an E-selectin inhibitor together with mobilization and inhibition of binding to CXCR4 would enhance chemotherapy sensitivity. To target these overlapping pathways, GMI-1215 and GMI-1257 were each rationally designed to inhibit both E-selectin and CXCR4. GMI-1215 and GMI-1257 inhibit E-selectin in vitro with IC50 values of 4.1μM and 3.6μM, respectively. Both molecules also target CXCR4 as determined by inhibiting antibody (12G5) binding to CXCR4 or inhibiting SDF-1-mediated transwell migration of CCRF-CEM cells (GMI-1215 IC50 = 31nM; GMI-1257 IC50 = 1.1μM, respectively). We have tested these novel dual inhibitors of both E-selectin and CXCR4 in a murine xenograft model. The inhibitor GMI-1257 inhibits E-selectin and SDF-1 mediated chemotaxis in a transwell-based assay for CXCR4 activity. Both E-selectin-CXCR4 bifunctional inhibitors GMI-1215 and GMI-1257 successfully mobilize primary human leukemia cells in the NODscid IL2Rγc−/− mouse. The peak rise occurs at 3 hours after IV injection of 5 mg/kg. After IV injection of GMI-1215, the total white blood count rose from 4.1 × 109/L ±1.1 to 11.4 × 109/L ±0.9 (2.8 fold), and human CD34+ leukemia count from 2.5 × 109/L ±0.8 to 7.7 × 109/L ±1.0 (3.1 fold), p=0.0024 and p=0.0045, respectively. After injection of GMI-1257, the human CD45+ leukemia count rose from 0.08 to 0.27 (3.4 fold). Administration of these inhibitors daily for 3 days, followed by cytarabine 300 mg/kg/day by intraperitoneal injection, 3 hours later each day, reduced circulating leukemia in the xenograft model without affecting recovery of normal mouse neutrophil counts. For example, at 7 days post treatment with GMI-1215, the human CD34+ leukemia count fell by 86%, and at 7 days post treatment with GMI-1257, it fell by 94%. For the control mice that received only cytarabine alone, but not GMIO-1257, the leukemia number fell by only 38% by day 7. Remarkably, by 15 days post treatment with the combination of GMI-1257 and cytarabine, human CD34+ leukemia cells were largely depleted from the bone marrow (by 90%) compared to treatment with cytarabine alone. Thus, these novel dual inhibitors of E-selectin and CXCR4 effectively mobilize leukemia from the marrow and reduce the bone marrow burden of AML in combination with chemotherapy. As both E-selectin and CXCR4 play roles in the sequestration of leukemic cells in the bone marrow, an antagonist that targets both adhesion mechanisms may have added benefits in blocking these interactions and enhancing the efficacy of chemotherapy. Disclosures:Patton:GlycoMimetics, Inc.: Employment, Equity Ownership. Smith:Glycomimetics, Inc.: Employment, Equity Ownership. Sarkar:Glycomimetics, Inc.: Employment, Equity Ownership. Magnani:GlycoMimetics, Inc.: Employment, Equity Ownership. Becker:GlycoMimetics, Inc.: Research Funding.

Glycoengineering of HCELL, the Human Bone Marrow Homing Receptor: Sweetly Programming Cell Migration

The successful clinical implementation of adoptive cell therapeutics, including bone marrow transplantation and other stem cell-based treatments, depends critically on the ability to deliver cells to sites where they are needed. E-selectin, an endothelial C-type lectin, binds sialofucosylated carbohydrate determinants on its pertinent ligands. This molecule is expressed in a constitutive manner on bone marrow and dermal microvascular endothelium, and inducibly on post-capillary venules at all sites of tissue injury. Engagement of E-selectin with relevant ligand(s) expressed on circulating cells mediates initial "tethering/rolling" endothelial adhesive interactions prerequisite for extravasation of blood-borne cells at any target tissue. Most mammalian cells express high levels of a transmembrane glycoprotein known as CD44. A specialized glycoform of CD44 called "Hematopoietic Cell E-/L-selectin Ligand" (HCELL) is a potent E-selectin ligand expressed on human cells. Under native conditions, HCELL expression is restricted to human hematopoietic stem/progenitor cells. We have developed a technology called "Glycosyltransferase-Programmed Stereosubstitution" (GPS) for custom-modifying CD44 glycans to create HCELL on the surface of living cells. GPS-based glycoengineering of HCELL endows cell migration to endothelial beds expressing E-selectin. Enforced HCELL expression targets human mesenchymal stem cell homing to marrow, licensing transendothelial migration without chemokine signaling via a VLA-4/VCAM-1-dependent "Step 2-bypass pathway." This review presents an historical framework of the homing receptor concept, and will describe the discovery of HCELL, its function as the bone marrow homing receptor, and how enforced expression of this molecule via chemical engineering of CD44 glycans could enable stem cell-based regenerative medicine and other adoptive cell therapeutics.

Coexpression of chemokine receptors CCR5, CXCR3, and CCR4 and ligands for P‐ and E‐selectin on T lymphocytes of patients with juvenile idiopathic arthritis

To investigate P- and E-selectin ligand coexpression with chemokine receptors (CKRs) on T cells in the synovial fluid (SF) and blood of children with juvenile idiopathic arthritis (JIA). Sixteen patients with polyarticular or persistent oligoarticular JIA (ages 5.3-15.1 years) were studied. SF and venous blood were collected, and immunostaining for the expression of CCR4, CCR5, CXCR3, and P- or E-selectin ligands was performed. Compared to blood, SF was greatly enriched for CD4+ T cells bearing CCR5, CCR4, CXCR3, and both P- and E-selectin ligand. Twenty-five percent of the CD4+ T cells in SF expressed both CCR5 and CCR4, some also coexpressing CXCR3. Such cells were rare in blood. Half of the few CCR5+ T cells in blood coexpressed P- or E-selectin ligand, a phenotype that was enriched up to 50-fold in SF. A minority of CCR4+ and CXCR3+ cells in blood (∼25%) coexpressed selectin ligand; these were enriched 4-8-fold in SF. Most CCR4-expressing CD4+ T cells expressed both E-selectin ligand and cutaneous lymphocyte antigen. CCR4-, CCR5-, CXCR3-, and selectin ligand-expressing CD4+ T cells preferentially accumulate in the joints of children with JIA. The marked enrichment of CCR5+ T cells coexpressing P-selectin and/or E-selectin ligand in CD4+ SF T cells suggests that the few such cells in blood selectively migrate to inflamed joints via endothelial P- and E-selectin- and CCR5-activating chemokines. The predominance of CCR4-expressing CD4+ T cells coexpressing E-selectin ligand suggests that such cells migrate not only to areas of cutaneous inflammation, as previously reported, but also to the joints in JIA. Combined targeting of CCR5- and E-selectin-dependent mechanisms may be a relevant treatment strategy.

Analysis of glycoprotein E-selectin ligands on human and mouse marrow cells enriched for hematopoietic stem/progenitor cells

AbstractAlthough well recognized that expression of E-selectin on marrow microvessels mediates osteotropism of hematopoietic stem/progenitor cells (HSPCs), our knowledge regarding the cognate E-selectin ligand(s) on HSPCs is incomplete. Flow cytometry using E-selectin-Ig chimera (E-Ig) shows that human marrow cells enriched for HSPCs (CD34+ cells) display greater E-selectin binding than those obtained from mouse (lin−/Sca-1+/c-kit+ [LSK] cells). To define the relevant glycoprotein E-selectin ligands, lysates from human CD34+ and KG1a cells and from mouse LSK cells were immunoprecipitated using E-Ig and resolved by Western blot using E-Ig. In both human and mouse cells, E-selectin ligand reactivity was observed at ∼ 120- to 130-kDa region, which contained two E-selectin ligands, the P-selectin glycoprotein ligand-1 glycoform “CLA,” and CD43. Human, but not mouse, cells displayed a prominent ∼ 100-kDa band, exclusively comprising the CD44 glycoform “HCELL.” E-Ig reactivity was most prominent on CLA in mouse cells and on HCELL in human cells. To further assess HCELL's contribution to E-selectin adherence, complementary studies were performed to silence (via CD44 siRNA) or enforce its expression (via exoglycosylation). Under physiologic shear conditions, CD44/HCELL-silenced human cells showed striking decreases (> 50%) in E-selectin binding. Conversely, enforced HCELL expression of LSK cells profoundly increased E-selectin adherence, yielding > 3-fold more marrow homing in vivo. These data define the key glycoprotein E-selectin ligands of human and mouse HSPCs, unveiling critical species-intrinsic differences in both the identity and activity of these structures.

The biology of CD44 and HCELL in hematopoiesis: the ‘step 2-bypass pathway’ and other emerging perspectives

The homing and egress of hematopoietic stem and progenitor cells (HSPCs) to and from marrow, respectively, and the proliferation and differentiation of HSPCs within marrow are complex processes critically regulated by the ordered expression and function of adhesion molecules that direct key cell-cell and cell-matrix interactions. The integral membrane molecule CD44, known primarily for its role in binding hyaluronic acid, is characteristically expressed on HSPCs. Conspicuously, human HSPCs uniquely display a specialized glycoform of CD44 known as hematopoietic cell E-/L-selectin ligand (HCELL), which is the most potent ligand for both E-selectin and L-selectin expressed on human cells. This review focuses on recent advances in our understanding of the biology of CD44 and HCELL in hematopoiesis. New data indicate that CD44-mediated events in hematopoiesis are more complex than previously imagined. Ex-vivo glycan engineering has established that HCELL serves as a 'bone marrow homing receptor'. Moreover, biochemical studies now show that CD44 forms bimolecular complexes with a variety of membrane proteins, one of which is VLA-4. Engagement of CD44 or of HCELL directly induces VLA-4 activation via G-protein-dependent signaling, triggering a 'step 2-bypass pathway' of cell migration, and extravascular lodgment, in absence of chemokine receptor engagement. Recent studies have further clarified the roles of CD44 and its glycoform HCELL in hematopoietic processes, providing key insights on how targeting these molecules may be beneficial in promoting hematopoiesis and in treating hematologic malignancies.

Effect of the E-selectin Gene Polymorphism (S149R) on Platelet Activation and Adverse Events After Coronary Artery Surgery

A common single nucleotide polymorphism in the E-selectin (SELE) gene S149R results in the loss of E-selectin ligand binding specificity. The 149R allele has been associated with severe cardiovascular diseases. We hypothesized that S149R may regulate platelet activation after stimuli associated with perioperative procedures in patients undergoing isolated coronary artery bypass grafting (CABG). Associations between the S149R polymorphism and an increased risk of perioperative acute thrombotic events with regards to a platelet count and activity were analyzed. In elective CABG patients (n= 152) we analyzed associations between S149R polymorphism and an increased risk of perioperative acute thrombotic events with regard to platelet count and activity. The S149R SELE gene polymorphism was determined by real-time polymerase chain reaction. Platelet count and activation marker (β-thromboglobulin-βTG) were evaluated. Prevalence of the S149R genotypes was as follows: 81.8% (n= 121) of homozygotes (149SS), 15.5% (n= 23) of heterozygotes and 2.7% (n= 4) of homozygotes (149RR). The 149R allele carriers had significantly higher postoperative βTG levels thanthe homozygotes (97 [79-120] vs. 76 [66-91] IU/mL, p= 0.03). Sixteen patients had adverse events: myocardial infarction (n= 14), stroke (n= 1) and fatal pulmonary embolism (n= 1). Twelve patients were carriers of the 149R allele. Relative risk (RR) ofpostoperative adverse events in the 149R allele carriers was 2.03 with 95% CI (1.05-3.03). Postoperative platelet activation is related to the S149R polymorphism, which enhances the risk of adverse events after CABG.

Peracetylated 4-Fluoro-glucosamine Reduces the Content and Repertoire of N- and O-Glycans without Direct Incorporation

Prior studies have shown that treatment with the peracetylated 4-fluorinated analog of glucosamine (4-F-GlcNAc) elicits anti-skin inflammatory activity by ablating N-acetyllactosamine (LacNAc), sialyl Lewis X (sLeX), and related lectin ligands on effector leukocytes. Based on anti-sLeX antibody and lectin probing experiments on 4-F-GlcNAc-treated leukocytes, it was hypothesized that 4-F-GlcNAc inhibited sLeX formation by incorporating into LacNAc and blocking the addition of galactose or fucose at the carbon 4-position of 4-F-GlcNAc. To test this hypothesis, we determined whether 4-F-GlcNAc is directly incorporated into N- and O-glycans released from 4-F-GlcNAc-treated human sLeX (+) T cells and leukemic KG1a cells. At concentrations that abrogated galectin-1 (Gal-1) ligand and E-selectin ligand expression and related LacNAc and sLeX structures, MALDI-TOF and MALDI-TOF/TOF mass spectrometry analyses showed that 4-F-GlcNAc 1) reduced content and structural diversity of tri- and tetra-antennary N-glycans and of O-glycans, 2) increased biantennary N-glycans, and 3) reduced LacNAc and sLeX on N-glycans and on core 2 O-glycans. Moreover, MALDI-TOF MS did not reveal any m/z ratios relating to the presence of fluorine atoms, indicating that 4-F-GlcNAc did not incorporate into glycans. Further analysis showed that 4-F-GlcNAc treatment had minimal effect on expression of 1200 glycome-related genes and did not alter the activity of LacNAc-synthesizing enzymes. However, 4-F-GlcNAc dramatically reduced intracellular levels of uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc), a key precursor of LacNAc synthesis. These data show that Gal-1 and E-selectin ligand reduction by 4-F-GlcNAc is not caused by direct 4-F-GlcNAc glycan incorporation and consequent chain termination but rather by interference with UDP-GlcNAc synthesis.

Elevated serum levels of Sialyl Lewis X (sLeX) and inflammatory cytokines in patients with nonmetastatic breast cancer.

e11061 Background: Sialyl Lewis X (sLeX), a carbohydrate antigen on tumor cells and ligand for E-selectin, mediates tethering and rolling along vascular endothelium. sLeX on tumor cells is purported to correlate with metastasis and poor prognosis. Inflammatory cytokines promote tumorigenesis and modulate metastasis. We assessed serum levels of sLeX and cytokines in patients (pts) with ductal carcinoma in situ (DCIS/stage 0), early invasive breast cancer (EBC/stages I/IIA) and locally advanced breast cancer (LABC/stages IIB/IIIA-C). Methods: Sera from 183 pts [26 DCIS, 83 EBC, 74 LABC including 23 pts with inflammatory breast cancer (IBC)] and 43 healthy donors (HD) were assessed for sLeX by CSLEX immunoassay (Nittobo Medical, Japan) and cytokines by Milliplex kits (Millipore Corp, Billerica, MA). The Kruskal-Wallis and Mann-Whitney tests determined differences in cytokine levels between patient groups and healthy donors (HD). Spearman’s Rank Correlation test determined relationship between sLeX and cytokines. Results: Compared with the sLeX level of HD (1.57 U/mL), those of DCIS (2.305 U/mL), EBC (2.830 U/mL), or LABC (3.300 U/mL) are significantly higher (all P < 0.05). sLeX levels increased with stage of disease (DCIS < EBC < LABC), and LABC is more likely than EBC to have a sLeX level above the mean ± 2SD of HD (χ2=4.8, P=0.03). IBC and non-IBC had similar sLeX levels. DCIS, EBC, or LABC pts have significantly higher median levels of cytokines [interleukin (IL)-1β, IL-1RA, IL-2, IL-6, IL-8, and IL-10; all P values < 0.05] than HD; whereas EBC and LABC pts had similar median levels. Moreover, there were positive correlations between serum levels of sLeX and inflammatory cytokines IL-1RA, IL-2, IL-6, and IL-8. Conclusions: Serum sLeX levels increase from premalignant conditions to invasive disease and appears to increase with stage of disease suggesting a potential role as a tumor marker with application for early detection. Moreover, our preliminary data support a role for inflammatory cytokines in upregulating serum sLeX to promote tumor progression. Additional analyses will be performed to determine the prognostic value of serum sLeX and the role of inflammatory cytokines in primary breast cancer.

Abstract 3338: Capacity of breast cancer cell homing to distant organs is altered by acquisition of stem cell properties

Abstract It is hypothesized that migration of tumor cells to distant organs is mediated by E-selectin expressed on vascular endothelium of a secondary tissue and its counter receptors expressed on circulating breast tumor cells. Previously, our group has characterized glycosphingolipids and CD44 as E-selectin binding molecules expressed on BT-20 and MDA-MB-468 breast cancer cell lines. We now report that the ZR-75-1 breast cancer cell line possesses E-selectin ligand activity, yet does not express any of the known E-selectin ligands found on colon cancer cells, leukocyte, or BT-20 and MDA-MB-468 breast cancer cells. A systematic investigation to identify novel E-selectin ligands expressed by ZR-75-1 cells was performed. Molecules isolated from whole cell lysate by immunoprecipitation with mouse E-selectin/ human Fc chimera (E-Ig chimera) were analyzed by mass spectrometry, which revealed that a glycoprotein known as Mac-2 binding protein (Mac-2bp) is an E-selectin ligand. Immunoprecipitation of Mac-2bp from whole cell lysate by using anti-Mac-2bp antibody followed by western blotting with E-Ig chimera and HECA-452 antibody demonstrated that Mac-2bp is a sialofucosylated E-selectin ligand. Dual staining of ZR-75-1 cells with anti-Mac-2bp antibody and E-Ig chimera imaged by fluorescence microscopy showed that Mac-2bp is a cell surface glycoprotein that is co-localized with E-selectin binding epitopes. Collectively, these results demonstrated that Mac-2bp is a novel E-selectin ligand expressed by ZR-75-1 breast cancer cells. In addition to the three breast cancer cell lines above, we tested four other breast cancer cell lines for their E-selectin ligand activity. Among the panel of cell lines examined, MDA-MB-231 and Hs-578t cells showed very weak activity for E-selectin binding as compared to the other five cell lines. Surprisingly, these were the only cells among all tested that were characterized by the CD44+/CD24-/low phenotype (&amp;gt;80% of cells). Staining with E-Ig chimera with subsequent detection by flow cytometry revealed differential E-selectin ligand activities of cancer stem versus non-stem cell phenotypes. Further, BT-20 cells (predominantly CD44+/CD24+) were treated with the metastatic niche factor TGF-β to force EMT and transformation into cancer stem cells, and change(s) in E-selectin ligand expression and function were assessed. Based on these results, we propose a new paradigm for the metastasis cascade, whereby breast cancer cells home to distant organs via an E-selectin-mediated pathway followed by transformation into breast cancer stem cells using metastatic niche factors. This model is consistent with the notion that cancer cell plasticity might have a significant role in metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3338. doi:10.1158/1538-7445.AM2011-3338

Physiological Contribution of CD44 as a Ligand for E-Selectin during Inflammatory T-Cell Recruitment

Endothelial selectins guide the migration of inflammatory T cells to extralymphoid tissues. Whereas P-selectin glycoprotein ligand-1 (PSGL-1) functions as the exclusive ligand for P-selectin, it acts in coordination with additional glycoproteins to mediate E-selectin binding. CD44 can act as one such ligand in neutrophils, but its contribution in inflammatory T lymphocytes remains unexplored. We have used real-time in vivo imaging of the cremasteric and dermal microcirculations to explore the dynamics of leukocyte recruitment, as well as the physiological contribution of CD44 in a model of Th1-driven inflammation. CD4(+) T-cell rolling frequency and kinetics, as well as arrest, were dependent on endothelial selectins and were markedly altered under inflammatory conditions. CD44 extracted from Th1 cells bound to soluble E-selectin in vitro and cooperated with PSGL-1 by controlling rolling velocities and promoting firm arrest. Using several competitive recruitment assays in a delayed-type hypersensitivity model, we show that the combined absence of CD44 and PSGL-1 impairs inflammatory T-cell recruitment beyond that of PSGL-1 alone. Differential expression of leukocyte fucosyltransferases in these cells may account for the differential use of E-selectin ligands relative to neutrophils. Our results identify additional mechanisms by which CD44 modulates the inflammatory response.

Gangliosides expressed on breast cancer cells are E-selectin ligands

Cancer cell adhesion to vascular endothelium is a critical process in hematogenous metastasis. We hypothesized that breast cancer cells express ligands that bind under blood flow conditions to E-selectin expressed by endothelial cells. At a hemodynamic wall shear rate, BT-20 and MDA-MB-468 breast cancer cells adhered to cytokine-activated human umbilical cord vein endothelial cells (HUVECs) but not to anti-E-selectin monoclonal antibody treated HUVECs, demonstrating that adhesion was specifically mediated by E-selectin. Characterization of glycans expressed on breast cancer cells by a panel of antibodies revealed that BT-20 cells expressed sialyl Lewis X (sLe x) and sialyl Lewis A (sLe a) but MDA-MB-468 cells did not, suggesting that the former possess classical glycans involved in E-selectin mediated adhesion while the latter have novel binding epitopes. Protease treatment of the breast cancer cells failed to significantly alter the carbohydrate expression profiles, binding to soluble E-selectin–Ig chimera, or the ability of the cells to tether and roll on E-selectin expressed by HUVECs, indicating that glycosphingolipids are functional E-selectin ligands on these cells. Furthermore, extracted breast cancer cell gangliosides supported binding of E-selectin–Ig chimera and adhesion of E-selectin transfected cells under physiological flow conditions. In summary, our results demonstrate that breast cancer cells express sialylated glycosphingolipids (gangliosides) as E-selectin ligands that may be targeted for prevention of metastasis.

Peripheral augmentation index and vascular inflammation in autosomal dominant polycystic kidney disease

Cardiovascular disease is the leading cause of premature mortality in autosomal dominant polycystic kidney disease (ADPKD). We examined peripheral augmentation index (AIx) as a measure of systemic vascular function and circulating markers of vascular inflammation in patients with ADPKD. Fifty-two ADPKD patients with hypertension and estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m(2), 50 ADPKD patients with hypertension and eGFR ≥ 60 mL/min/1.73 m(2), 42 normotensive ADPKD patients with eGFR ≥ 60 mL/min/1.73 m(2) and 51 normotensive healthy controls were enrolled in this study. AIx was measured from peripheral artery tone recordings using finger plethysmography. Serum levels of soluble intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule-1, P-selectin, E-selectin, soluble Fas (sFas) and Fas ligand (FasL) were measured as markers of vascular inflammation. AIx was higher in all three patient groups with ADPKD compared to healthy controls (P < 0.05). AIx was similar between the normotensive ADPKD patients with eGFR ≥ 60 mL/min/1.73 m(2) and hypertensive ADPKD patients with eGFR < 60 mL/min/1.73 m(2) (P > 0.05). ICAM, P-selectin, E-selectin and sFas were higher and FasL lower in all ADPKD groups compared to controls (P < 0.05). ICAM, P-selectin and E-selectin were similar between the normotensive ADPKD patients with eGFR ≥ 60 mL/min/1.73 m(2) and hypertensive ADPKD patients with eGFR < 60 mL/min/1.73 m(2) (P > 0.05). According to multiple regression analysis, predictors of AIx in ADPKD included age, height, heart rate and mean arterial pressure (P < 0.05). Vascular inflammatory markers were not predictors of AIx in ADPKD. Systemic vascular dysfunction, manifesting as an increase in AIx and vascular inflammation is evident in young normotensive ADPKD patients with preserved renal function. Vascular inflammation is not associated with elevated AIx in ADPKD.

Enforced hematopoietic cell E- and L-selectin ligand (HCELL) expression primes transendothelial migration of human mesenchymal stem cells

According to the multistep model of cell migration, chemokine receptor engagement (step 2) triggers conversion of rolling interactions (step 1) into firm adhesion (step 3), yielding transendothelial migration. We recently reported that glycosyltransferase-programmed stereosubstitution (GPS) of CD44 on human mesenchymal stem cells (hMSCs) creates the E-selectin ligand HCELL (hematopoietic cell E-selectin/L-selectin ligand) and, despite absence of CXCR4, systemically administered HCELL(+)hMSCs display robust osteotropism visualized by intravital microscopy. Here we performed studies to define the molecular effectors of this process. We observed that engagement of hMSC HCELL with E-selectin triggers VLA-4 adhesiveness, resulting in shear-resistant adhesion to ligand VCAM-1. This VLA-4 activation is mediated via a Rac1/Rap1 GTPase signaling pathway, resulting in transendothelial migration on stimulated human umbilical vein endothelial cells without chemokine input. These findings indicate that hMSCs coordinately integrate CD44 ligation and integrin activation, circumventing chemokine-mediated signaling, yielding a step 2-bypass pathway of the canonical multistep paradigm of cell migration.

C2-O-sLeX Glycoproteins Are E-Selectin Ligands that Regulate Invasion of Human Colon and Hepatic Carcinoma Cells

Similar to mechanisms of recruitment of activated leukocytes to inflamed tissues, selectins mediate adhesion and extravasation of circulating cancer cells. Our objective was to determine whether sialyl Lewis X modified core 2 O-glycans (C2-O-sLeX) present on colon and hepatic carcinoma cells promote their adhesion and invasion. We examined membrane expression of C2-O-sLeX, selectin binding, invasion of human colon and hepatic carcinoma cell lines, and mRNA levels of alpha-2,3 fucosyltransferase (FucT-III) and core 2 beta-1,6 N-acetylglucosaminyltransferase (C2GnT1) genes, necessary for C2-O-sLeX synthesis, by quantitative reverse-transcriptase (RT) PCR. Synthesis of core 2 branched O-glycans decorated by sLeX is dependent on C2GnT1 function and thus we determined enzyme activity of C2GnT1. The cell lines that expressed C2GnT1 and FucT-III mRNA by quantitative RT-PCR were highly positive for C2-O-sLeX by flow cytometry, and colon carcinoma cells possessed highly active C2GnT1 enzyme. Cells bound avidly to E-selection but not to P- and L-selectin. Gene knock-down of C2GnT1 in colon and hepatic carcinoma cells using short hairpin RNAs (shRNA) resulted in a 40–90% decrease in C2-O-sLeX and a 30–50% decrease in E-selectin binding compared to control cells. Invasion of hepatic and colon carcinoma cells containing C2GnT1 shRNA was significantly reduced compared to control cells in Matrigel assays and C2GnT1 activity was down-regulated in the latter cells. The sLeX epitope was predominantly distributed on core 2 O-glycans on colon and hepatic carcinoma cells. Our findings indicate that C2GnT1 gene expression and the resulting C2-O-sLeX carbohydrates produced mediate the adhesive and invasive behaviors of human carcinomas which may influence their metastatic potential.

Direct Measurements on CD24-Mediated Rolling of Human Breast Cancer MCF-7 Cells on E-Selectin

Tumor cell rolling on the endothelium plays a key role in the initial steps of cancer metastasis, i.e., extravasation of circulating tumor cells (CTCs). Identification of the ligands that induce the rolling of cells is thus critical to understanding how cancers metastasize. We have previously demonstrated that MCF-7 cells, human breast cancer cells, exhibit the rolling response selectively on E-selectin-immobilized surfaces. However, the ligand that induces rolling of MCF-7 cells on E-selectin has not yet been identified, as these cells lack commonly known E-selectin ligands. Here we report, for the first time to our knowledge, a set of quantitative and direct evidence demonstrating that CD24 expressed on MCF-7 cell membranes is responsible for rolling of the cells on E-selectin. The binding kinetics between CD24 and E-selectin was directly measured using surface plasmon resonance (SPR), which revealed that CD24 has a binding affinity against E-selectin (K(D) = 3.4 ± 0.7 nM). The involvement of CD24 in MCF-7 cell rolling was confirmed by the rolling behavior that was completely blocked when cells were treated with anti-CD24. A simulated study by flowing microspheres coated with CD24 onto E-selectin-immobilized surfaces further revealed that the binding is Ca(2+)-dependent. Additionally, we have found that actin filaments are involved in the CD24-mediated cell rolling, as observed by the decreased rolling velocities of the MCF-7 cells upon treatment with cytochalasin D (an inhibitor of actin-filament dynamics) and the stationary binding of CD24-coated microspheres (the lack of actins) on the E-selectin-immobilized slides. Given that CD24 is known to be directly related to enhanced invasiveness of cancer cells, our results imply that CD24-based cell rolling on E-selectin mediates, at least partially, cancer cell extravasation, resulting in metastasis.

Identification of Thioaptamer Ligand against E-Selectin: Potential Application for Inflamed Vasculature Targeting

Active targeting of a drug carrier to a specific target site is crucial to provide a safe and efficient delivery of therapeutics and imaging contrast agents. E-selectin expression is induced on the endothelial cell surface of vessels in response to inflammatory stimuli but is absent in the normal vessels. Thus, E-selectin is an attractive molecular target, and high affinity ligands for E-selectin could be powerful tools for the delivery of therapeutics and/or imaging agents to inflamed vessels. In this study, we identified a thiophosphate modified aptamer (thioaptamer, TA) against E-selectin (ESTA-1) by employing a two-step selection strategy: a recombinant protein-based TA binding selection from a combinatorial library followed by a cell-based TA binding selection using E-selectin expressing human microvascular endothelial cells. ESTA-1 selectively bound to E-selectin with nanomolar binding affinity (KD = 47 nM) while exhibiting minimal cross reactivity to P- and L-selectin. Furthermore, ESTA-1 binding to E-selectin on the endothelial cells markedly antagonized the adhesion (over 75% inhibition) of sLex positive HL-60 cells at nanomolar concentration. ESTA-1 also bound specifically to the inflamed tumor-associated vasculature of human carcinomas derived from breast, ovarian, and skin but not to normal organs, and this binding was highly associated with the E-selectin expression level. Similarly, intravenously injected ESTA-1 demonstrated distinct binding to the tumor vasculature in a breast cancer xenograft model. Together, our data substantiates the discovery of a thioaptamer (ESTA-1) that binds to E-selectin with high affinity and specificity, thereby highlighting the potential application of ESTA-1 for E-selectin targeted delivery.

E-selectin engages PSGL-1 and CD44 through a common signaling pathway to induce integrin αLβ2-mediated slow leukocyte rolling

AbstractIn inflamed venules, neutrophils rolling on E-selectin induce integrin αLβ2-dependent slow rolling on intercellular adhesion molecule-1 by activating Src family kinases (SFKs), DAP12 and Fc receptor-γ (FcRγ), spleen tyrosine kinase (Syk), and p38. E-selectin signaling cooperates with chemokine signaling to recruit neutrophils into tissues. Previous studies identified P-selectin glycoprotein ligand-1 (PSGL-1) as the essential E-selectin ligand and Fgr as the only SFK that initiate signaling to slow rolling. In contrast, we found that E-selectin engagement of PSGL-1 or CD44 triggered slow rolling through a common, lipid raft–dependent pathway that used the SFKs Hck and Lyn as well as Fgr. We identified the Tec kinase Bruton tyrosine kinase as a key signaling intermediate between Syk and p38. E-selectin engagement of PSGL-1 was dependent on its cytoplasmic domain to activate SFKs and slow rolling. Although recruiting phosphoinositide-3-kinase to the PSGL-1 cytoplasmic domain was reported to activate integrins, E-selectin–mediated slow rolling did not require phosphoinositide-3-kinase. Studies in mice confirmed the physiologic significance of these events for neutrophil slow rolling and recruitment during inflammation. Thus, E-selectin triggers common signals through distinct neutrophil glycoproteins to induce αLβ2-dependent slow rolling.