Heparanase-induced shedding of syndecan-1/CD138 in myeloma and endothelial cells activates VEGFR2 and an invasive phenotype: prevention by novel synstatins.

Multiple myeloma arises when malignant plasma cells invade and form multiple tumors in the bone marrow. High levels of heparanase (HPSE) correlate with poor prognosis in myeloma patients. A likely target of the enzyme is the heparan sulfate (HS) proteoglycan syndecan-1 (Sdc1, CD138), which is highly expressed on myeloma cells and contributes to poor prognosis in this disease. We find that HPSE promotes an invasive phenotype mediated by the very late antigen-4 (VLA-4, or α4ß1 integrin) in myeloma cells plated on either fibronectin (FN) or vascular endothelial cell adhesion molecule-1 (VCAM-1), ligands that are prevalent in the bone marrow. The phenotype depends on vascular endothelial cell growth factor receptor-2 (VEGFR2), which is aberrantly expressed in myeloma, and is characterized by a highly protrusive lamellipodium and cell invasion. HPSE-mediated trimming of the HS on Sdc1 and subsequent matrix metalloproteinase-9-mediated shedding of the syndecan exposes a juxtamembrane site in Sdc1 that binds VEGFR2 and VLA-4, thereby coupling VEGFR2 to the integrin. Shed Sdc1 can be mimicked by recombinant Sdc1 ectodomain or by a peptide based on its binding motif, which causes VLA-4 to re-orient from the lagging edge (uropod) to the leading edge of migrating cells, couple with and activate VEGFR2. Peptides (called 'synstatins') containing only the VLA-4 or VEGFR2 binding sites competitively inhibit invasion, as they block coupling of the receptors. This mechanism is also utilized by vascular endothelial cells, in which it is also activated by HPSE, during endothelial cell tube formation. Collectively, our findings reveal for the first time the mechanism through which HPSE modulates Sdc1 function to promote both tumor cell invasion and angiogenesis, thereby driving multiple myeloma progression. The inhibitory synstatins, or inhibitors of HPSE enzyme activity, are likely to show promise as therapeutics against myeloma extravasation and spread.

Modification, crosslinking and reactive electrospinning of a thermoplastic medical polyurethane for vascular graft applications.

Thermoplastic polyurethanes are used in a variety of medical devices and experimental tissue engineering scaffolds. Despite advances in polymer composition to improve their stability, the correct balance between chemical and mechanical properties is not always achieved. A model compound (MC) simulating the structure of a widely used medical polyurethane (Pellethane) was synthesized and reacted with aliphatic and olefinic acyl chlorides to study the reaction site and conditions. After adopting the conditions to the olefinic modification of Pellethane, processing into flat sheets, and crosslinking by thermal initiation or ultraviolet radiation, mechanical properties were determined. The modified polyurethane was additionally electrospun under ultraviolet light to produce a crosslinked tubular vascular graft prototype. Model compound studies showed reaction at the carbamide nitrogen, and the modification of Pellethane with pentenoyl chloride could be accurately controlled to up to 20% (correlation: rho=0.99). Successful crosslinking was confirmed by insolubility of the materials. Initiator concentrations were optimized and the crosslink densities shown to increase with increasing modification. Crosslinking of Pellethane containing an increasing number of pentenoyl groups resulted in decreases (up to 42%, p<0.01) in the hysteresis and 44% in creep (p<0.05), and in a significant improvement in degradation resistance in vitro. Modified Pellethane was successfully electrospun into tubular grafts and crosslinked using UV irradiation during and after spinning to render them insoluble. Prototype grafts had sufficient burst pressure (>550 mm Hg), and compliances of 12.1+/-0.8 and 6.2+/-0.3%/100 mm Hg for uncrosslinked and crosslinked samples, respectively. It is concluded that the viscoelastic properties of a standard thermoplastic polyurethane can be improved by modification and subsequent crosslinking, and that the modified material may be electrospun and initiated to yield crosslinked scaffolds. Such materials hold promise for the production of vascular and other porous scaffolds, where decreased hysteresis and creep may be required to prevent aneurismal dilation.

Synthesis and characterization of new cationic quaternary ammonium polymerizable surfactants.

Two new polymerizable surfactants (surfmers), (11-acryloyloxyundecyl)dimethylethylammonium bromide (ethyl surfmer) and (11-acryloyloxyundecyl)dimethyl(2-hydroxyethyl)ammonium bromide (hydroxyethyl surfmer), were synthesized and characterized. The binary phase diagrams of both surfmer/water systems are described. Both surfmers can form isotropic solutions and lamellar lyotropic liquid crystalline phases. The hydroxyethyl surfmer/water system forms a lamellar phase for weight concentrations of surfmer between 70 and 90% relative to between 75 and 85% for the ethyl surfmer/water system. The differences in the self assembly of these surfmers were attributed to the ability of hydroxyethyl surfmer to form hydrogen bonds (between two head groups and with water), whereas no such interactions can occur with the ethyl surfmer system.

Development of an ultrasonic technique for in situ investigating the properties of deposited protein during crossflow ultrafiltration.

Although an amount of research has reported that a flux minimum occurs at the isoionic/isoelectric points (pH 4.6-5.0) in the absence of salts in the ultrafiltration of bovine serum albumin (BSA), the real mechanism remains incompletely understood due to the lack of additional techniques in real time to detect the properties of deposited BSA (gel) layers formed during ultrafiltration (UF). An ultrasonic technique was developed as an analytical noninvasive tool to in situ investigate the properties of deposited BSA layers at pH 4.9 (isoionic or isoelectric point, IEP) and 6.9 during crossflow ultrafiltration. The membrane was a polysulfone (PSf) UF membrane with molecular weight cut-off (MWCO) 35 kDa. The feed used was 0.5 g/l BSA solution. Results show good correspondence between the ultrasonic signal responses and the development of BSA gel layers on the membranes. The deposit is thicker at pH 6.9 than at pH 4.9. However, the deposited gel layers are more compressible at pH 4.9 than at pH 6.9. The flux decline is mainly controlled by the density (packing) of the deposit layer. At pH 6.9, protein mainly deposits on the membrane surface. Around the isoelectric point, protein absorbs within and on the membranes. A functional relationship between acoustic signals and fouling resistance exists. The fouling resistance is mainly attributed to pore blocking or pore constriction.

Initialisation in RAFT-mediated polymerisation of methyl acrylate.

Inhibition in Reversible Addition-Fragmentation chain Transfer (RAFT)-mediated polymerisations is currently a controversial issue; here we provide evidence that the slow "propagation" of the initiating and leaving group radicals during the early part (the period of consumption of the initial RAFT agent) of methyl acrylate RAFT-mediated polymerisation has characteristics similar to inhibition.

Syndecan-1 in B lymphoid malignancies.

Syndecans are heparan sulfate-bearing proteoglycans that are found on the surface of most cells. Syndecan-1 is expressed predominantly on epithelia, but is also present on pre-B cells and plasma cells. The syndecans act to bind various effector molecules via their heparan sulfate chains, including both soluble and insoluble molecules within the extracellular milieu. These interactions promote cell adhesion to extracellular matrix and to adjacent cells. In addition, the syndecans can bind to and affect the biological activity of a number of heparin-binding growth factors. Thus, syndecan-1 can play a dramatic role in regulating cell behavior. In this review we discuss the expression of syndecan-1 on malignant B lymphoid cells as well as specific structure-function relationships of the molecule. Emphasis is placed on the important role that syndecan-1 has in regulating the growth of B lymphoid malignancies, particularly multiple myeloma.

Syndecan-1 (CD138) immunoreactivity in bone marrow biopsies of multiple myeloma: shed syndecan-1 accumulates in fibrotic regions.

Syndecan-1 (CD138) mediates myeloma cell adhesion, and loss of syndecan-1 from the cell surface may contribute to myeloma proliferation and dissemination. Flow cytometry analysis of myeloma cells in bone marrow specimens shows heterogeneity in cell surface syndecan-1 expression. It is not known whether weaker expression correlates with more aggressive disease. However, recent reports suggest that variations in syndecan-1 staining intensity on myeloma cells may be an artifact of specimen handling. In this study, we evaluate syndecan-1 expression in bone marrow biopsy sections from 28 multiple myeloma patients, to elucidate the heterogeneity of syndecan-1 expression in situ. Immunoreactivity for syndecan-1, using the antibody B-B4 (CD138), was found in more than 95% of multiple myeloma cells in 27 of 28 biopsies. However, one biopsy had more than 50% CD138-negative cells and cells with weak CD138 expression were identified in the majority of cases. Loss of syndecan-1 did not appear to relate to myeloma cell differentiation. In addition, syndecan-1 was detected on intravascular and intrasinusoidal myeloma cells suggesting that loss of syndecan-1 may not be required for extramedullary dissemination. Bone marrow biopsies from nine additional patients, with variable CD138 staining intensity on myeloma cells as determined by flow cytometry, were studied by immunohistochemistry. The heterogeneous CD138 expression was confirmed in situ, with weakly positive cells concentrated in areas of reticulin fibrosis. These cells had a disrupted pattern of membrane staining in contrast to the strong linear membrane staining seen in the other multiple myeloma cells. In addition, the fibrotic stroma stained intensely for syndecan-1. Accumulation of syndecan-1 within the extracellular matrix of the marrow likely is derived by shedding of the molecule from the surface of myeloma cells. Because syndecan-1 can act to regulate the activity of heparan-binding growth factors, these reservoirs of syndecan-1 may play a critical role in promoting myeloma pathogenesis, or in regeneration of the tumor after chemotherapy.

Sperm protein 17 is expressed on normal and malignant lymphocytes and promotes heparan sulfate-mediated cell-cell adhesion.

Sperm protein 17 (Sp17) is a highly conserved mammalian protein present on acrosome-reacted sperm that is thought to promote fertilization by binding sulfated carbohydrates of the oocyte zona pellucida. Although Sp17 was originally described as a testis-specific antigen, emerging evidence indicates that it may be more ubiquitously expressed than was previously thought. With the use of a specific antiserum, Sp17 was found to be present on the surface of malignant lymphoid cells, including B- and T-lymphoid cell lines, and on the surface of primary cells isolated from 2 patients having B-lymphoid tumors. Surprisingly, circulating B lymphocytes isolated from healthy volunteers also expressed Sp17, while circulating T lymphocytes exhibited only very weak expression. The role of Sp17 in promoting lymphoid cell adhesion was addressed with the use of recombinant Sp17 (rSp17). The rSp17 binds to the surface of myeloma cells but not to cells pretreated with heparitinase, an enzyme that removes heparan sulfate from the cell surface. Moreover, rSp17 promotes extensive aggregation of cells that express the syndecan-1 heparan sulfate proteoglycan, but in contrast, cells lacking syndecan-1 expression fail to aggregate in the presence of rSp17. These findings suggest that Sp17 promotes heparan sulfate-mediated cell aggregation and thereby plays a role in regulating adhesion and migration of normal and malignant lymphocytes.

Heparan sulfate proteoglycans in invasion and metastasis.

Because heparan sulfate proteoglycans mediate cell adhesion and control the activities of numerous growth and motility factors, they play a critical role in regulating the metastatic behavior of tumor cells. Due to their utilitarian nature, heparan sulfate proteoglycans may at times act as inhibitors of cell invasion and at other times as promoters of cell invasion, with their function being determined by their location (cell surface or extracellular matrix), the heparin-binding molecules they associate with, the presence of modifying enzymes (proteases, heparanases) and the precise structural characteristics of the proteoglycan. Also, the tissue type and pathophysiological state of the tumor influence proteogylcan function. This review summarizes our current knowledge of the role heparan sulfate proteoglycans play in regulating tumor cell metastasis, proposes mechanisms of how these molecules function and examines the potential for discovery of new therapeutic approaches designed to block metastatic cancer.

High levels of soluble syndecan-1 in myeloma-derived bone marrow: modulation of hepatocyte growth factor activity.

Syndecan-1 is a heparan sulfate proteoglycan expressed on the surface of, and actively shed by, myeloma cells. Hepatocyte growth factor (HGF) is a cytokine produced by myeloma cells. Previous studies have demonstrated elevated levels of syndecan-1 and HGF in the serum of patients with myeloma, both of negative prognostic value for the disease. Here we show that the median concentrations of syndecan-1 (900 ng/mL) and HGF (6 ng/mL) in the marrow compartment of patients with myeloma are highly elevated compared with healthy controls and controls with other diseases. We show that syndecan-1 isolated from the marrow of patients with myeloma seems to exist in an intact form, with glucosaminoglycan chains. Because HGF is a heparan-sulfate binding cytokine, we examined whether it interacted with soluble syndecan-1. In supernatants from myeloma cells in culture as well as in pleural effusions from patients with myeloma, HGF existed in a complex with soluble syndecan-1. Washing myeloma cells with purified soluble syndecan-1 could effectively displace HGF from the cell surface, suggesting that soluble syndecan-1 can act as a carrier for HGF in vivo. Finally, using a sensitive HGF bioassay (interleukin-11 production from the osteosarcoma cell line Saos-2) and intact syndecan-1 isolated from the U-266 myeloma cell line, we found that the presence of high concentrations of syndecan-1 (more than 3 microg/mL) inhibited the HGF effect, whereas lower concentrations potentiated it. HGF is only one of several heparin-binding cytokines associated with myeloma. These data indicate that soluble syndecan-1 may participate in the pathology of myeloma by modulating cytokine activity within the bone marrow.

Syndecan-1 is targeted to the uropods of polarized myeloma cells where it promotes adhesion and sequesters heparin-binding proteins.

Syndecan-1 (CD138) is a heparan sulfate-bearing proteoglycan present on the surface of myeloma cells where it mediates myeloma cell-cell and cell-extracellular matrix adhesion. In this study, we examined myeloma cell lines for cell membrane localization of syndecan-1. On some cells we note a striking localization of syndecan-1 to a single small membrane protrusion, with the remainder of the cell surface being mostly negative for syndecan-1. Examination of cell morphology reveals that a proportion of cells from myeloma cell lines, as well as primary myeloma cells, are polarized, with a uropod on one end and lamellipodia on the other end. On these polarized cells, syndecan-1 is specifically targeted to the uropod, but in contrast, on nonpolarized cells syndecan-1 is evenly distributed over the entire cell surface. In addition to syndecan-1, several other cell surface molecules localize specifically to the uropod, including CD44 and CD54. Functional assays reveal that myeloma cell lines with a high proportion of polarized cells have a much higher migratory potential than cell lines with few polarized cells. Moreover, the uropod is the cell pole preferentially involved in aggregation of myeloma cells and in adhesion of myeloma cells to osteoblast-like cells. When polarized myeloma cells are incubated with heparin-binding proteins, like hepatocyte growth factor or osteoprotegerin, they concentrate in the uropod. These data indicate that syndecan-1 is targeted to the uropod of polarized myeloma cells and that this targeting plays a role in promoting cell-cell adhesion and may also regulate the biological activity of heparin-binding cytokines.

The cysteine-rich domain of human ADAM 12 supports cell adhesion through syndecans and triggers signaling events that lead to beta1 integrin-dependent cell spreading.

The ADAMs (a disintegrin and metalloprotease) family of proteins is involved in a variety of cellular interactions, including cell adhesion and ecto- domain shedding. Here we show that ADAM 12 binds to cell surface syndecans. Three forms of recombinant ADAM 12 were used in these experiments: the cys-teine-rich domain made in Escherichia coli (rADAM 12-cys), the disintegrin-like and cysteine-rich domain made in insect cells (rADAM 12-DC), and full-length human ADAM 12-S tagged with green fluorescent protein made in mammalian cells (rADAM 12-GFP). Mesenchymal cells specifically and in a dose-dependent manner attach to ADAM 12 via members of the syndecan family. After binding to syndecans, mesenchymal cells spread and form focal adhesions and actin stress fibers. Integrin beta1 was responsible for cell spreading because function-blocking monoclonal antibodies completely inhibited cell spreading, and chondroblasts lacking beta1 integrin attached but did not spread. These data suggest that mesenchymal cells use syndecans as the initial receptor for the ADAM 12 cysteine-rich domain-mediated cell adhesion, and then the beta1 integrin to induce cell spreading. Interestingly, carcinoma cells attached but did not spread on ADAM 12. However, spreading could be efficiently induced by the addition of either 1 mM Mn(2+) or the beta1 integrin-activating monoclonal antibody 12G10, suggesting that in these carcinoma cells, the ADAM 12-syndecan complex fails to modulate the function of beta1 integrin.

Syndecan-1 expression is decreased with increasing aggressiveness of basal cell carcinoma.

Syndecans, a family of cell-surface proteoglycans of which syndecan-1 is the prototypical member, play an important role in limiting tumor growth and invasive capacity through their actions as receptors for growth factors and extracellular matrix. Cutaneous biopsy specimens of basal cell carcinoma, including superficial, nodular, infiltrative, and morpheic subtypes, were assessed regarding the pattern of syndecan-1 expression. We found that with increasing aggressiveness of basal cell carcinomas, syndecan-1 expression is lost from the surface of the neoplastic cells. However, within the dermis, which is normally devoid of syndecan-1 expression, immunopositivity for syndecan-1 is present in areas adjacent to aggressive tumors. This pattern of staining indicates that syndecan-1 expression is produced by stromal cells rather than being shed by the carcinoma cells into the stroma.

Matrix metalloproteinases in multiple myeloma.

Multiple myeloma is a deadly malignancy characterized by plasma cell infiltration of bones. The resulting effect is painful "punched-out" lesions where bone is eroded and filled with myeloma cells that suppress and replace the normal marrow components. Recently it has been shown that myeloma cells produce matrix-metalloproteinase-9 (MMP-9) and MMP-2 and that accumulation of MMP-9 protein is suppressed upon expression of the heparan sulfate proteoglycan, syndecan-1. In this review, we briefly consider the potential roles for MMPs in the pathogenesis of multiple myeloma. MMPs likely have major roles in: 1) the infiltration of bone and other tissues by the myeloma cells; 2) the osteolytic bone destruction caused by overly active osteoclasts, 3) extracellular matrix remodeling by bone marrow stromal cells; 4) promoting the invasion of the endothelial cells that form neoangiogenic blood vessels necessary to sustain tumor foci; and 5) promoting the growth of myeloma cells. Effective and safe synthetic inhibitors of MMPs are available and these may prove useful in limiting the growth and spread of myeloma cells. In addition, recent insights into the suppression of MMP-9 by syndecan-1 may suggest new strategies for treatment of myeloma.

Syndecan-1 expression is diminished in acantholytic cutaneous squamous cell carcinoma.

Syndecan-1 is a cell surface proteoglycan predominantly expressed on the surface of adult epithelial cells, and is normally present in all epidermal layers except for the most superficial terminally differentiated cells. Syndecan-1 mediates cell-cell and cell-extracellular matrix adhesion, thereby influencing cell morphology and growth characteristics. In addition, in vitro studies have shown that expression of syndecan-1 on tumor cells inhibits their invasion into the extracellular matrix. A total of 23 cutaneous biopsies of squamous cell carcinoma, including acantholytic squamous cell carcinoma, invasive squamous cell carcinoma which was not acantholytic, and squamous cell carcinoma in situ were examined for syndecan-1 immunoreactivity. The level of syndecan-1 expression was related to the degree of squamous cell dyshesion, with expression being greatest in the in situ lesions and least in the acantholytic lesions. The loss of syndecan-1 expression with increasing dyshesion of squamous cell carcinoma may be a mechanism for loosening of intercellular and cell-extracellular matrix attachments, thereby promoting the invasion of neoplastic cells into the dermis.

Syndecan-1 expression is induced in the stroma of infiltrating breast carcinoma.

Loss of expression of the heparan sulfate proteoglycan syndecan-1 leads to reduced cell adhesion, increased invasive potential, and dysregulated growth of mammary epithelial cells in vitro. We compared syndecan-1 expression in malignant and nonmalignant breast tissues using immunohisto-chemistry with monoclonal antibody B-B4. Staining for syndecan-1 is greatly diminished on malignant cells within infiltrating ductal carcinomas (n = 20) as compared with ductal epithelium of both normal breast (n = 14) and stromal-epithelial neoplasms (n = 10), which exhibit extensive basolateral epithelial staining. Surprisingly, comparison of malignant and nonmalignant breast tissue also reveals a striking difference in expression of syndecan-1 within the stromal compartment. In infiltrating ductal carcinomas, strong staining for syndecan-1 is present both within the connective tissue and on stromal cell surfaces, whereas syndecan-1 expression is absent in the stroma of both normal breast and stromal-epithelial neoplasms. Because syndecan-1 interacts with heparin-binding growth factors such as FGF-2, accumulation of syndecan-1 within the tumor stroma may contribute to the extensive angiogenesis and stromal proliferation characteristic of infiltrating breast carcinoma. Moreover, the induction of syndecan-1 within the stroma, coupled with the loss of syndecan-1 on malignant cells, suggests that changes in syndecan-1 expression are critical in promoting the metastatic phenotype of infiltrating ductal carcinoma of the breast.

Syndecan-1 (CD 138) in myeloma and lymphoid malignancies: a multifunctional regulator of cell behavior within the tumor microenvironment.

Syndecan-1 is a transmembrane proteoglycan expressed on the surface of tumor cells of various origins including myeloma, Hodgkin's disease, and certain human immunodeficiency virus (HIV) associated lymphomas. Functional studies in myeloma reveal that syndecan-1 may act as a multifunctional regulator of cell behavior in the tumor microenvironment; it mediates cell-cell adhesion, binding of myeloma cells to type I collagen, and inhibits tumor cell invasion into collagen gels. In addition, syndecan-1 is released from the surface of myeloma cells and this shed form of the molecule inhibits growth and induces apoptosis of myeloma cells and may modulate myeloma bone disease by inhibiting osteoclast formation and promoting osteoblast formation. In view of its effects on tumor cell growth, survival, adhesion and invasion and on bone cell differentiation, syndecan-1 may be an important potentially beneficial regulator of myeloma pathobiology. Further studies are needed to define the clinical significance of syndecan-1 in myeloma and to examine its functional significance in other lymphoid malignancies.

Syndecan-1 expression suppresses the level of myeloma matrix metalloproteinase-9.

ARH-77 human myeloma cells invade into type I collagen gels but become non-invasive when engineered to express syndecan-1, a heparan sulphate proteoglycan that promotes cell adhesion to collagen. To determine if syndecan-1 expression influences the activity of proteases that may facilitate invasion, we analysed media harvested from syndecan-1 expressing and non-expressing cells. High levels of a 92 kD gelatinase accumulated in serum-free growth medium of both parental and control-transfected ARH-77, but much less 92 kD gelatinase accumulated in the medium of ARH-77 transfectants expressing syndecan-1. The gelatinase was identified as matrix metalloproteinase (MMP)-9 because its activity was immunoprecipitated with a MMP-9-specific monoclonal antibody. Gelatinase activity and Western blot analyses revealed 2-3-fold less MMP-9 in medium from syndecan-1 transfected cells than in medium from parental cells. Decreased MMP-9 was not due to increased association of MMP-9 with cells expressing syndecan-1. An inverse correlation between the syndecan 1 level and the level of MMP-9 accumulation in the media was observed using a panel of ARH-77 transfectants expressing syndecan-1. Investigation of six unrelated human myeloma cell lines confirmed that high gelatinase levels were recovered from conditioned media of those that did not express syndecan-1 (ARH-77, Mer and Col) and one line that expressed a low level of syndecan-1 (RPMI-8226), but low gelatinase levels were recovered from media of lines that expressed high levels of syndecan-1 (ARK and clone 2+). Therefore syndecan-1 may play a dual role in inhibiting the metastasis of tumour cells by promoting cell adhesion to the extracellular matrix and suppressing the proteolytic activity needed for invasion.