The lack of effective conventional therapies for the treatment of advanced stage melanoma has stimulated interest in the development of novel strategies for the management of patients with malignant melanoma. Among them, immunotherapy has attracted much attention because of the potential role played by immunological events in the clinical course of melanoma. For many years, T cell-based immunotherapy has been emphasized in part because of the disappointing results of the monoclonal antibody (mAb)-based clinical trials conducted in the early 1980s and in part because of the postulated major role played by T cells in tumor growth control. More recently, mAb-based therapies have gained in popularity given their clinical and commercial success for a variety of malignant diseases. As a result, there has been increased interest in identifying and characterizing antibody-defined melanoma antigens. Among them, the chondroitin sulfate proteoglycan 4 (CSPG4), also known as high molecular weight-melanoma associated antigen (HMW-MAA) or melanoma chondroitin sulfate proteoglycan (MCSP), has attracted much attention in recent years because of the growing experimental evidence that it fulfills two requirements for immunotherapy to be therapeutically effective: (1) targeting of cancer stem cells (CSC) and (2) development of combinatorial therapies to counteract the escape mechanisms driven by the genetic instability of tumor cells. With this in mind, in this chapter, we have reviewed recent information related to the distribution of CSPG4 on various types of tumors, including CSC, its expression on pericytes in the tumor microenvironment, its recognition by T cells, its role in cell biology as well as the potential mechanisms underlying the ability of CSPG4-specific immunity to control malignant cell growth.

Platelet factor 4 (PF4) is an abundant platelet alpha-granule C-X-C chemokine that has weak chemotactic potency but strongly inhibits hematopoiesis through an unknown mechanism. We find that PF4 binds to human CD34+ hematopoietic progenitor cells (HPCs) with a median effective concentration of 1 microg/mL but not after exposure to chondroitinase ABC. PF4 enhances adhesion of HPCs to intact stroma. Committed progenitors also adhere avidly to immobilized PF4. This adhesion is time-dependent, requires metabolic activity, causes cytoskeletal rearrangement, and induces cell-cycle inhibition. Using extracellular acidification rate to indicate transmembrane signaling, we find that interleukin-8 (IL-8), but not PF4, activates CD34+ progenitors, and PF4 blocks IL-8-mediated activation. Surface plasmon resonance analysis shows that PF4 binds IL-8 with high (dissociation constant [Kd] = 42 nM) affinity. Nuclear magnetic resonance analysis of IL-8 and PF4 in solution confirms this interaction. We conclude that PF4 has the capacity to influence hematopoiesis through mechanisms not mediated by a classical high-affinity, 7-transmembrane domain chemokine receptor. Instead, PF4 may modulate the hematopoietic milieu both directly, by promoting progenitor adhesion and quiescence through interaction with an HPC chondroitin sulfate-containing moiety, and indirectly, by binding to or interfering with signaling caused by other, hematopoietically active chemokines, such as IL-8.

OBJECTIVE

To compare the efficacy and safety of chondroitin sulfate plus glucosamine hydrochloride (CS+GH) versus celecoxib in patients with knee osteoarthritis and severe pain.

METHODS

Double-blind Multicentre Osteoarthritis interVEntion trial with SYSADOA (MOVES) conducted in France, Germany, Poland and Spain evaluating treatment with CS+GH versus celecoxib in 606 patients with Kellgren and Lawrence grades 2-3 knee osteoarthritis and moderate-to-severe pain (Western Ontario and McMaster osteoarthritis index (WOMAC) score ≥301; 0-500 scale). Patients were randomised to receive 400 mg CS plus 500 mg GH three times a day or 200 mg celecoxib every day for 6 months. The primary outcome was the mean decrease in WOMAC pain from baseline to 6 months. Secondary outcomes included WOMAC function and stiffness, visual analogue scale for pain, presence of joint swelling/effusion, rescue medication consumption, Outcome Measures in Rheumatology Clinical Trials and Osteoarthritis Research Society International (OMERACT-OARSI) criteria and EuroQoL-5D.

RESULTS

The adjusted mean change (95% CI) in WOMAC pain was -185.7 (-200.3 to -171.1) (50.1% decrease) with CS+GH and -186.8 (-201.7 to -171.9) (50.2% decrease) with celecoxib, meeting the non-inferiority margin of -40: -1.11 (-22.0 to 19.8; p=0.92). All sensitivity analyses were consistent with that result. At 6 months, 79.7% of patients in the combination group and 79.2% in the celecoxib group fulfilled OMERACT-OARSI criteria. Both groups elicited a reduction >50% in the presence of joint swelling; a similar reduction was seen for effusion. No differences were observed for the other secondary outcomes. Adverse events were low and similarly distributed between groups.

CONCLUSIONS

CS+GH has comparable efficacy to celecoxib in reducing pain, stiffness, functional limitation and joint swelling/effusion after 6 months in patients with painful knee osteoarthritis, with a good safety profile.

BACKGROUND

NCT01425853.

Pregnancy-associated malaria (PAM) is characterized by the placental sequestration of Plasmodium falciparum-infected erythrocytes (IEs) with the ability to bind to chondroitin sulfate A (CSA). VAR2CSA is a leading candidate for a pregnancy malaria vaccine, but its large size ( approximately 350 kDa) and extensive polymorphism may pose a challenge to vaccine development. In this study, rabbits were immunized with individual VAR2CSA Duffy binding-like (DBL) domains expressed in Pichia pastoris or var2csa plasmid DNA and sera were screened on different CSA-binding parasite lines. Rabbit antibodies to three recombinant proteins (DBL1, DBL3, and DBL6) and four plasmid DNAs (DBL1, DBL3, DBL5, and DBL6) reacted with homologous FCR3-CSA IEs. By comparison, antibodies to the DBL4 domain were unable to react with native VAR2CSA protein unless it was first partially proteolyzed with trypsin or chymotrypsin. To investigate the antigenic relationship of geographically diverse CSA-binding isolates, rabbit immune sera were screened on four heterologous CSA-binding lines from different continental origins. Antibodies did not target conserved epitopes exposed in all VAR2CSA alleles; however, antisera to several DBL domains cross-reacted on parasite isolates that had polymorphic loops in common with the homologous immunogen. This study demonstrates that VAR2CSA contains common polymorphic epitopes that are shared between geographically diverse CSA-binding lines.

A common pathological characteristic of Plasmodium falciparum infection is the cytoadhesion of mature-stage-infected erythrocytes (IE) to host endothelium and syncytiotrophoblasts. Massive accumulation of IE in the brain microvasculature or placenta is strongly correlated with severe forms of malaria. Extensive binding of IE to placental chondroitin sulfate A (CSA) is associated with physiopathology during pregnancy. The adhesive phenotype of IE correlates with the appearance of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) at the erythrocyte surface (approximately 16 h after merozoite invasion), so that only early blood-stage (ring-stage) IE appear in the peripheral blood. Here, we describe results that challenge the existing view of blood-stage IE biology by demonstrating the specific adhesion of IE, during the early ring-stage, to endothelial cell lines from the brain and lung and to placental syncytiotrophoblasts. Later, during blood-stage development of these IE, trophozoites switch to an exclusively CSA cytoadhesion phenotype. Therefore, adhesion to an individual endothelial cell or syncytiotrophoblast may occur throughout the blood-stage cycle, indicating the presence in malaria patients of noncirculating (cryptic) parasite subpopulations. We detected two previously unknown parasite proteins on the surface of ring-stage IE. These proteins disappear shortly after the start of PfEMP1-mediated adhesion.

Recent findings indicate that glycosaminoglycans can play important roles in animal development. The genes sqv-3, -7, and -8, which are necessary for vulval morphogenesis in Caenorhabditis elegans, affect the biosynthesis of chondroitin and heparan sulfate glycosaminoglycans. We cloned sqv-1 and showed that the SQV-1 protein is a type II transmembrane protein that functions as a UDP-glucuronic acid decarboxylase. SQV-1 localizes to punctate cytoplasmic compartments and colocalizes with the SQV-7 nucleotide-sugar transporter, which probably acts in the Golgi apparatus. SQV-1 and SQV-7 are both expressed in the vulva and in oocytes, where they likely act in vulval morphogenesis and embryonic development, respectively. Progeny of sqv-7 and sqv-1 null mutants fail to initiate cytokinesis, possibly because they are unable to separate the plasma membrane from the eggshell, a defect analogous to that of incomplete vulval invagination.

Cellular recognition and adhesion to the extracellular matrix (ECM) has a complex molecular basis, involving both integrins and cell surface proteoglycans (PG). The current studies have used specific inhibitors of chondroitin sulfate proteoglycan (CSPG) synthesis along with anti-alpha 4 integrin subunit monoclonal antibodies to demonstrate that human melanoma cell adhesion to an A-chain derived, 33-kD carboxyl-terminal heparin binding fragment of human plasma fibronectin (FN) involves both cell surface CSPG and alpha 4 beta 1 integrin. A direct role for cell surface CSPG in mediating melanoma cell adhesion to this FN fragment was demonstrated by the identification of a cationic synthetic peptide, termed FN-C/H-III, within the fragment. FN-C/H-III is located close to the amino terminal end of the fragment, representing residues #1721-1736 of intact FN. FN-C/H-III binds CSPG directly, can inhibit CSPG binding to the fragment, and promotes melanoma cell adhesion by a CSPG-dependent, alpha 4 beta 1 integrin-independent mechanism. A scrambled version of FN-C/H-III does not inhibit CSPG binding or cell adhesion to the fragment or to FN-C/H-III, indicating that the primary sequence of FN-C/H-III is important for its biological properties. Previous studies have identified three other synthetic peptides from within this 33-kD FN fragment that promote cell adhesion by an arginyl-glycyl-aspartic acid (RGD) independent mechanism. Two of these synthetic peptides (FN-C/H-I and FN-C/H-II) bind heparin and promote cell adhesion, implicating cell surface PG in mediating cellular recognition of these two peptides. Additionally, a third synthetic peptide, CS1, is located in close proximity to FN-C/H-I and FN-C/H-II and it promotes cell adhesion by an alpha 4 beta 1 integrin-dependent mechanism. In contrast to FN-C/H-III, cellular recognition of these three peptides involved contributions from both CSPG and alpha 4 integrin subunits. Of particular importance are observations demonstrating that CS1-mediated melanoma cell adhesion could be inhibited by interfering with CSPG synthesis or expression. Since CS1 does not bind CSPG, the results suggest that CSPG may modify the function and/or activity of alpha 4 beta 1 integrin on the surface of human melanoma cells. Together, these results support a model in which the PG and integrin binding sites within the 33-kD fragment may act in concert to focus these two cell adhesion receptors into close proximity on the cell surface, thereby influencing initial cellular recognition events that contribute to melanoma cell adhesion on this fragment.

Proteoglycans are constituents of the cell surface that may play important roles in the regulation of cell behavior. Here we report that the 250-kDa receptor subunit that binds the multifunctional protein, transforming growth factor-beta 1 (TGF-beta 1), contains chains of heparan sulfate and chondroitin sulfate and thus is a proteoglycan. Digestion of TGF-beta 1-receptor complexes with glycosaminoglycan (GAG)-specific degradative enzymes yield core proteins of 115-140 kDa. Cell monolayers that had been predigested with GAG-specific degradative enzymes were capable of binding high levels of TGF-beta 1, but the size of the binding components was shifted from the high molecular weight species to the lower molecular weight core proteins, indicating that GAG chains are not necessary for TGF-beta 1 binding to the cell. The presence of GAG chains on the receptor subunit indicates that it has the potential for interaction with the extracellular matrix.

The 165-amino acid form of vascular endothelial growth factor (VEGF165) is a mitogen for vascular endothelial cells and a potent angiogenic factor. Expression of a chimeric receptor containing the extracellular domain of the flk-1 receptor fused to the transmembrane and intracellular domains of the human c-fms receptor in NIH-3T3 cells, resulted in the appearance of high affinity binding sites for 125I-VEGF165 on transfected cells. The binding of 125I-VEGF165 to the flk-1/fms chimeric receptor of the transfected cells as well as the VEGF165-induced autophosphorylation of the chimeric receptors were inhibited in the presence of low concentrations of heparin (1-10 micrograms/ml). In contrast, similar concentrations of heparin potentiated the binding of 125I-VEGF165 to the endogenous VEGF receptors of the transfected cells, indicating that to some extent, the effect of heparin on 125I-VEGF165 binding is receptor type-dependent. A soluble fusion protein containing the extracellular domain of flk-1 fused to alkaline phosphatase (flk-1/SEAP) was used to study the effects of heparin on the binding of 125I-VEGF165 to flk-1 in a cell-free environment. The fusion protein specifically inhibited VEGF165-induced proliferation of vascular endothelial cells, but bound 125I-VEGF165 inefficiently in the absence of heparin. Addition of low concentrations of heparin or heparan sulfate (0.1-1 microgram/ml) resulted in a strong potentiation of 125I-VEGF165 binding, whereas higher heparin or heparan sulfate concentrations inhibited the binding. The effect of heparin on the binding of 125I-VEGF165 to flk-1/SEAP could not be mimicked by desulfated heparin or by chondroitin sulfate. Both bFGF and aFGF inhibited the binding when low concentrations of heparin were added to the binding reaction. However, higher concentrations of heparin abolished the inhibition, indicating that the inhibition is probably caused by competition for available heparin. Taken as a whole, these results indicate that heparin-like molecules regulate the binding of VEGF165 to its receptors in complex ways which depend on the heparin binding properties of VEGF165, on the specific VEGF receptor type involved, and on the amount and composition of heparin-like molecules that are present on the cell surface of VEGF receptor containing cells.

The pharmacokinetics of chondroitin sulfate (CS, Condrosulf, IBSA, Lugano, Switzerland) were investigated in rats and in healthy volunteers using CS tritiated at the reducing end and CS labeled with 131I or 99mTc respectively. A rapid absorption of orally administered CS is observed in rats and in humans when the drug is dissolved in water. Lower and delayed absorption is observed when CS is administered in gastroresistant capsules. The absolute bio-availability is 15 and 12% for rats and humans respectively. The CS shows a tropism for cartilagineous tissues in rats and for knee tissues in humans as demonstrated by scintigraphic analysis with 99mTc-CS. Monomers, oligo and polysaccharides produced by enzymatic hydrolysis of CS appear in the blood and tissues together with native CS. The effects of partially depolymerized (m.m. 3 to 15 kD) and desulfated fractions on human leukocytes were investigated. CS and its fractions inhibit the directional chemotaxis induced by zymosan-activated serum, are able to decrease the phagocytosis and the release of lysozyme induced by zymosan and to protect the plasma membrane from oxygen reactive species. In rats the oral administration of CS significantly decreases granuloma formation due to sponge implants and cell migration and lysosomal enzyme release in carrageenan pleurisy. Compared with nonsteroidal anti-inflammatory drugs (indomethacin, ibuprofen), CS appears to be more effective on cellular events of inflammation than on edema formation. It is noteworthy that CS is devoid of dangerous effects on the stomach, platelets and kidneys. In synovial fluid of patients requiring joint aspiration, treated orally for 10 days with CS (800 mg/day) the hyaluronate concentration and the intrinsic viscosity significantly increased, while collagenolytic activity, phospholipase A2 and N-acetylglucosaminidase (NAG) decreased. These results give an insight into the mechanism of the anti-inflammatory and chondroprotective actions demonstrated by this drug in a number of clinical trials in patients with osteoarthritis.

Cytotactin is an extracellular matrix glycoprotein with a restricted distribution during development. In electron microscopic images, it appears as a hexabrachion with six arms extending from a central core. Cytotactin binds to other extracellular matrix proteins including a chondroitin sulfate proteoglycan (CTB proteoglycan) and fibronectin. Although cytotactin binds to a variety of cells including fibroblasts and neurons, in some cases it causes cells in culture to round up and it inhibits their migration. To relate these various effects of cytotactin on cell behavior to its binding regions, we have examined its ability to support cell-substrate adhesion and have mapped its cell-binding function onto its structure. In a cell-substrate adhesion assay, fibroblasts bound to cytotactin but remained round. In contrast, they both attached and spread on fibronectin. Neither neurons nor glia bound to cytotactin in this assay. In an assay in which cell-substrate contact was initiated by centrifugation, however, neurons and glia bound well to cytotactin; this binding was blocked by specific anti-cytotactin antibodies. The results suggest that neurons and glia can bind to cytotactin-coated substrates and that these cells, like fibroblasts, possess cell surface ligands for cytotactin. After applying methods of limited proteolysis and fractionation, these assays were used to map the binding functions of cytotactin onto its structure. Fragments produced by limited proteolysis were fractionated into two major pools: one (fraction I) contained disulfide-linked oligomers of a 100-kD fragment and two minor related fragments, and the second (fraction II) contained monomeric 90- and 65-kD fragments. The 90- and 65-kD fragments in fraction II were closely related to each other and were structurally and immunologically distinct from the fragments in fraction I. Only components in fraction I were recognized by mAb M1, which binds to an epitope located in the proximal portion of the arms of the hexabrachion and by a polyclonal antibody prepared against a 75-kD CNBr fragment of intact cytotactin. A mAb (1D8) and a polyclonal antibody prepared against a 35-kD CNBr fragment of cytotactin only recognized components present in fraction II. In cell-binding experiments, fibroblasts, neurons, and glia each adhered to substrates coated with fraction II, but did not adhere to substrates coated with fraction I. Fab fragments of the antibody to the 35-kD CNBr fragment strongly inhibited the binding of cells to cytotactin, supporting the conclusion that fraction II contains a cell-binding region. In addition, Fab fragments of this antibody inhibited the binding of cytotactin to CTB pr

Fibrillin-1 assembly into microfibrils and elastic fiber formation involves interactions with glycosaminoglycans. We have used BIAcore technology to investigate fibrillin-1 interactions with heparin and with heparin saccharides that are analogous to S-domains of heparan sulfate. We have identified four high affinity heparin-binding sites on fibrillin-1, localized three of these sites, and defined their binding kinetics. Heparin binding to the fibrillin-1 N terminus has particularly rapid kinetics. Hyaluronan and chondroitin sulfate did not interact significantly with fibrillin-1. Heparin saccharides with more than 12 monosaccharide units bound strongly to all four fibrillin-1 sites. Heparin did not inhibit fibrillin-1 N- and C-terminal interactions or RGD-dependent cell attachment, but heparin and MAGP-1 competed for binding to the fibrillin-1 N terminus, and heparin and tropoelastin competed for binding to a central fibrillin-1 sequence. By regulating these key interactions, heparin can profoundly influence microfibril and elastic fiber assembly.

We have demonstrated previously that chick embryo fibroblasts synthesize and secrete a large chondroitin sulfate proteoglycan (designated PG-M) that binds to fibronectin. We now report the possibility that PG-M interactions with cell surfaces can modulate cell-substrate adhesion. When PG-M was added to the medium, various types of trypsinized cells failed to adhere not only to fibronectin-coated substrates but also to collagen- or vitronectin-coated substrates. Adhesion of the cells to laminin or glycyl-arginyl-glycyl-aspartyl-serine derivatized serum albumin (arginyl-glycyl-aspartic acid-containing molecules with no capacity to bind PG-M) was also inhibited by PG-M. Treatment of the proteoglycan with either proteolytic enzymes or chondroitinase abolished its inhibitory effects on the cell adhesion. These results suggest that direct binding between PG-M and fibronectin, if any, is not a cause of the inhibition by PG-M and that only the proteoglycan form is responsible for the activity. When the immobilization of added PG-M to available plastic surfaces of coated dishes was blocked by pretreating the dishes with serum albumin, the inhibitory effect of PG-M was abolished, suggesting that the immobilized fraction of PG-M can act as a cell adhesion inhibitor. In immobilized form, both cartilage chondroitin sulfate proteoglycan (designated PG-H) and chondroitin sulfate-derivatized serum albumin also inhibited cell adhesion. In contrast, heparan sulfate proteoglycan form LD and heparan sulfate-derivatized serum albumin had far lower inhibitory activities, indicating that the active site for the interaction between cells and PG-M is on the chondroitin sulfate chains.

Hydrogels are highly swollen, insoluble networks which can entrap chondrocytes and provide a 3-D environment necessary for the re-growth of cartilaginous tissue. In this study, hydrogels were formulated with a synthetic poly(ethylene glycol) (PEG) component to provide control over the macroscopic gel properties and from a cartilage specific compound, chondroitin sulfate (ChSA), to capture features of the chondrocytes' native environment. PEG was chosen as the base hydrogel chemistry, because it forms a 3-D environment that maintains chondrocyte function. ChSA, a highly negatively charged main component of proteoglycans, was then selectively incorporated into the PEG gel. Macroscopic gel properties were manipulated to obtain high compressive moduli coupled with a high degree of swelling by formulating copolymer gels with these chemistries. The gel compressive modulus of cell-free PEG gels increased from 34 to 140 kPa with the incorporation of ChSA for similar degrees of swelling. When chondrocytes were encapsulated in pure ChSA gels, synthesis of collagen and glycosaminoglycans was inhibited. However, when PEG was introduced into the copolymer gels, both extracellular matrix components were stimulated. Total collagen content increased from non-detectable in the pure ChSA gels to 0.48+/-0.05 mg/g wet weight in the copolymer gels (40/60 ChSA/PEG). Gene expression for collagen type II was also enhanced by the incorporation of PEG into the gel, illustrating an important influence of gel chemistry on chondrocyte function; however, aggrecan gene expression was unaffected. This study demonstrates that the macroscopic properties of chondrocyte gel carriers can be controlled through the incorporation of charge into networks by ChSA, but the neutral, non-interactive base PEG chemistry facilitates extracellular matrix deposition.

CCL5 (RANTES (regulated on activation normal T cell expressed and secreted)) and its cognate receptor, CCR5, have been implicated in T cell activation. CCL5 binding to glycosaminoglycans (GAGs) on the cell surface or in extracellular matrix sequesters CCL5, thereby immobilizing CCL5 to provide the directional signal. In two CCR5-expressing human T cell lines, PM1.CCR5 and MOLT4.CCR5, and in human peripheral blood-derived T cells, micromolar concentrations of CCL5 induce apoptosis. CCL5-induced cell death involves the cytosolic release of cytochrome c, the activation of caspase-9 and caspase-3, and poly(ADP-ribose) polymerase cleavage. CCL5-induced apoptosis is CCR5-dependent, since native PM1 and MOLT4 cells lacking CCR5 expression are resistant to CCL5-induced cell death. Furthermore, we implicate tyrosine 339 as a critical residue involved in CCL5-induced apoptosis, since PM1 cells expressing a tyrosine mutant receptor, CCR5Y339F, do not undergo apoptosis. We show that CCL5-CCR5-mediated apoptosis is dependent on cell surface GAG binding. The addition of exogenous heparin and chondroitin sulfate and GAG digestion from the cell surface protect cells from apoptosis. Moreover, the non-GAG binding variant, (44AANA47)-CCL5, fails to induce apoptosis. To address the role of aggregation in CCL5-mediated apoptosis, nonaggregating CCL5 mutant E66S, which forms dimers, and E26A, which form tetramers at micromolar concentrations, were utilized. Unlike native CCL5, the E66S mutant fails to induce apoptosis, suggesting that tetramers are the minimal higher ordered CCL5 aggregates required for CCL5-induced apoptosis. Viewed altogether, these data suggest that CCL5-GAG binding and CCL5 aggregation are important for CCL5 activity in T cells, specifically in the context of CCR5-mediated apoptosis.

The goal of the study was to investigate bone morphogenetic protein 2 (BMP-2) and transforming growth factor beta (TGF-beta) control of the expression of beta1,3-glucuronosyl transferase 1 (GlcAT-1), an important regulator of chondroitin sulfate synthesis in cells of the nucleus pulposus. Treatment with both growth factors resulted in induction of GlcAT-1 expression and promoter activity. Deletion analysis indicated that promoter constructs lacking AP1 and TonE sites were unresponsive to growth factor treatment. Experiments using dominant-negative proteins showed that these transcription factors along with Sp1 were required for induction of GlcAT-1 promoter activity. Moreover, when either AP1 or TonE binding sites were mutated, induction was suppressed. Both BMP-2 and TGF-beta increased c-Jun and TonEBP expression and phosphorylation of transactivation domains. We investigated the role of the mitogen-activated protein kinase (MAPK) signaling pathway following growth factor treatment; a robust and transient activation of ERK1/2, p38, and JNK was noted. Treatment with MAPK inhibitors blocked BMP-2- and TGF-beta-induced AP1 reporter function, GlcAT-1 expression, and GAG accumulation. We found that DN-ERK1 but not DN-ERK2 resulted in suppression of growth factor-mediated induction of GlcAT-1 promoter activity; we also showed that p38 delta was important in GlcAT-1 activation. Results of these studies demonstrate that BMP-2 and TGF-beta regulate GlcAT-1 expression in nucleus pulposus cells through a signaling network comprising MAPK, AP1, Sp1, and TonEBP. It is concluded that by controlling both GAG and aggrecan synthesis, these growth factors positively influence disk cell function.

NG2 (nerve/glia antigen-2) is a type I transmembrane glycoprotein and also known as chondroitin sulfate proteoglycan 4. In the parenchyma of the central nervous system, NG2-expressing (NG2(+) ) cells have been identified as a novel type of glia with a strong potential to generate oligodendrocytes (OLs) in the developing white matter. However, the differentiation potential of NG2 glia remained controversial, largely attributable to shortcomings of transgenic mouse models used for fate mapping. To minimize these restrictions and to more faithfully mimic the endogenous NG2 expression in vivo, we generated a mouse line in which the open reading frame of the tamoxifen-inducible form of the Cre DNA recombinase (CreERT2) was inserted into the NG2 locus by homologous recombination. Results from this novel mouse line demonstrate that at different developmental stages of the brain, NG2(+) cells either stayed as NG2 glia or differentiated into OLs during the whole life span. Interestingly, when Cre activity was induced at embryonic stages, a significant number of reporter(+) astrocytes could be detected in the gray matter after birth. However, in other brain regions, such as olfactory bulb, brain stem, and cerebellum, all of the NG2 glia was restricted to the OL lineage. In addition, tamoxifen-sensitive and NG2 gene locus-dependent gene recombination could be detected in a small, but persistent population of cortical NeuN(+) neurons starting from the second postnatal week.

Although monocyte- and macrophage-derived molecules are known to promote extracellular matrix (ECM) disruption and destabilization, it is less appreciated that they also synthesize molecules contributing to ECM formation, stabilization, and function. We have identified and characterized the synthesis of proteoglycans and related proteins, some not previously known to be associated with macrophages. Proteoglycan extracts of [(35)S]sulfate- and (35)S-trans amino acid-radiolabeled culture media from THP-1 monocytes induced to differentiate by treatment with phorbol myristate acetate revealed three major proteins of ~25, 90, and 100 kDa following chondroitin ABC lyase digestion. The 25-kDa protein was predominant for monocytes, whereas the 90- and 100-kDa proteins were predominant for macrophages. Tandem mass spectrometry identified (i) the 25-kDa core protein as serglycin, (ii) the 90-kDa core protein as inter-α-inhibitor heavy chain 2 (IαIHC2), and (iii) the 100-kDa core as amyloid precursor-like protein 2 (APLP2). Differentiation was also associated with (i) a >500-fold increase in mRNA for TNF-stimulated gene-6, an essential cofactor for heavy chain-mediated matrix stabilization; (ii) a >800-fold increase in mRNA for HAS2, which is responsible for hyaluronan synthesis; and (iii) a 3-fold increase in mRNA for versican, which interacts with hyaluronan. Biochemical evidence is also presented for an IαIHC2-APLP2 complex, and immunohistochemical staining of human atherosclerotic lesions demonstrates similar staining patterns for APLP2 and IαIHC2 with macrophages, whereas serglycin localizes to the underlying glycosaminoglycan-rich region. These findings indicate that macrophages synthesize many of the molecules participating in ECM formation and function, suggesting a novel role for these molecules in the differentiation of macrophages in the development of atherosclerosis.

We have investigated the role of glycosaminoglycans in fibronectin matrix assembly and the incorporation of tenascin-C into matrix fibrils. Chinese hamster ovary cell mutants with a total block in heparan and chondroitin sulfate production failed to assemble a fibronectin matrix, and incorporated no tenascin-C. Another mutant with reduced heparan sulfate produced a normal fibronectin matrix but failed to incorporate tenascin-C. Excess soluble glycosaminoglycans inhibited the binding of tenascin-C to purified fibronectin in ELISA, and completely blocked incorporation into matrix fibrils. Treating cultured cells with xyloside, which interferes with glycosaminoglycan attachment to proteoglycans, also completely blocked their ability to incorporate tenascin-C into matrix fibrils. We conclude that proteoglycans bound to fibronectin fibrils play a major role in binding tenascin-C to these fibrils. We examined more closely the large heparan sulfate proteoglycan, perlecan, and found that it co-localizes with tenascin-C and fibronectin in the matrix. The perlecan binding site in tenascin-C was mapped to the fibronectin type III domains 3-5, but this binding was strongly enhanced for the small splice variant, which is the major form incorporated into the matrix. Apparently when the alternative splice segment is inserted after domain 5 it inhibits perlecan binding. Thus heparan sulfate glycosaminoglycans, and perlecan in particular, may play a role in incorporation of the small splice variant of tenascin-C into fibronectin matrix fibrils.

Recovery of function after damage to the CNS is limited due to the absence of axon regeneration and relatively low levels of plasticity. Plasticity in the CNS can be reactivated in the adult CNS by treatment with chondroitinase ABC, which removes glycosaminoglycan (GAG) chains from chondroitin sulfate proteoglycans (CSPGs). Plasticity in the adult CNS is restricted by perineuronal nets (PNNs) around many neuronal cell bodies and dendrites, which appear at the closure of critical periods and contain several inhibitory CSPGs. Formation of these structures and the turning off of plasticity is triggered by impulse activity in neurons. Expression of a link protein by neurons is the event that triggers the formation of PNNs. Treatment with chondroitinase removes PNNs and other inhibitory influences in the damaged spinal cord and promotes sprouting of new connections. However, promoting plasticity by itself does not necessarily bring back useful behavior; this only happens when useful connections are stabilized and inappropriate connections removed, driven by behavior. Thus after rodent spinal cord injury, combining a daily rehabilitation treatment for skilled paw function with chondroitinase produces much greater recovery than either treatment alone. The rehabilitation must be specific for the behavior that is to be enhanced because non-specific rehabilitation improves locomotor behavior but not skilled paw function. Plasticity-enhancing treatments may therefore open up a window of opportunity for successful rehabilitation.

In the fetal ductus arteriosus (DA) disruption in the assembly of elastin fibers is associated with intimal thickening and we previously reported that fetal lamb DA smooth muscle cells incubated with endothelial conditioned medium produce two-fold more chondroitin sulfate (CS) compared with aorta (Ao) cells (Boudreau, N., and M. Rabinovitch. 1991. Lab. Invest. 64:187-199). We hypothesized that CS or dermatan sulfate (DS), both N-acetylgalactosamine glycosaminoglycans (GAGs), may be similar to free galactosugars in causing release of the 67-kD elastin binding protein (EBP) from the smooth muscle cell surfaces and impaired elastin fiber assembly. Using immunohistochemistry, immunoelectron microscopy, and western immunoblot we demonstrated a reduction in the 67-kD EBP in fetal lamb DA smooth muscle in tissue and in cultured cells. Also, reduced EBP was observed in fetal lamb and neonatal rat Ao smooth muscle cells incubated with N-acetylgalactosamine GAGs, CS, and DS, but not with N-acetylglucosamine containing GAGs, heparan sulfate (HS), or hyaluronan. Reduction in EBP was related to shedding from cell surfaces into the conditioned medium. This was associated with impaired elastin fiber assembly in cultured cells, assessed both morphologically and by a relative increase in tropoelastin and decrease in desmosines. The EBP extracted from smooth muscle cell membranes binds to an elastin affinity gel and can be eluted from it with CS but not with HS. Moreover, the amount of EBP extractable from smooth muscle cell membranes correlated with the morphologic assessment. We propose that increased CS or DS, may impair assembly of newly synthesized elastin in the media of the ductus arteriosus associated with the development of intimal thickening.

Thromboembolism is a prominent but poorly understood feature of eosinophilic, or Loeffler's endocarditis. Eosinophil (EO) specific granule proteins, in particular major basic protein (MBP), accumulate on endocardial surfaces in the course of this disease. We hypothesized that these unusually cationic proteins promote thrombosis by binding to the anionic endothelial protein thrombomodulin (TM) and impairing its anticoagulant activities. We find that MBP potently (IC50 of 1-2 microM) inhibits the capacity of endothelial cell surface TM to generate the natural anticoagulant activated protein C (APC). MBP also inhibits APC generation by purified soluble rabbit TM with an IC50 of 100 nM without altering its apparent Kd for thrombin or Km for protein C. This inhibition is reversed by polyanions such as chondroitin sulfate E and heparin. A TM polypeptide fragment comprising the extracellular domain that includes its naturally occurring anionic glycosaminoglycan (GAG) moiety (TMD-105) is strongly inhibited by MBP, whereas its counterpart lacking the GAG moiety (TMD-75) is not. MBP also curtails the capacity of TMD-105 but not TMD-75 to prolong the thrombin clotting time. Thus, EO cationic proteins potently inhibit anticoagulant activities of the glycosylated form of TM, thereby suggesting a potential mechanism for thromboembolism in hypereosinophilic heart disease.

Slow vascularization rate is considered one of the main drawbacks of scaffolds used in wound healing. Several efforts, including cellular and acellular technologies, have been made to induce vascular growth in scaffolds. However, thus far, there is no established technology for inducing vascular growth. The aim of this study was to promote the vascularization capacities of scaffolds by seeding adipose-derived stem cells (ADSCs) on them and to compare the vascularization capacities of different scaffolds seeded with ADSCs. Two kinds of extracellular matrix scaffolds (small intestinal submucosa [SIS] and acellular dermal matrix [ADM]) and a kind of composite scaffold (collagen-chondroitin sulfate-hyaluronic acid [Co-CS-HA]) were selected. Subcutaneous implantation analysis showed that the vascularization capacity of SIS and ADM was greater than that of Co-CS-HA. ADSCs seeded in SIS and ADM secreted greater amounts of vascular endothelial growth factor than those seeded in Co-CS-HA. In a murine skin injury model, ADSC-seeded scaffolds enhanced the angiogenesis and wound healing rate compared with the nonseeded scaffolds. Moreover, ADSC-SIS and ADSC-ADM had greater vascularization capacity than that of ADSC-Co-CS-HA. Taken together, these results suggest that ADSCs could be used as a cell source to promote the vascularization capacities of scaffolds. The vascularization capacities of ADSC-seeded scaffolds were influenced by both the vascularization capacities of the scaffolds themselves and their effects on the angiogenic potential of ADSCs; the combination of extracellular matrix scaffolds and ADSCs exhibited synergistic angiogenesis promoting effects.

OBJECTIVE

The inhibitory role of secreted chondroitin sulfate proteoglycans on oncolytic viral (OV) therapy was examined. Chondroitinase ABC (Chase-ABC) is a bacterial enzyme that can remove chondroitin sulfate glycosaminoglycans from proteoglycans without any deleterious effects in vivo. We examined the effect of Chase-ABC on OV spread and efficacy.

METHODS

Three-dimensional glioma spheroids placed on cultured brain slices were utilized to evaluate OV spread. Replication-conditional OV-expressing Chase-ABC (OV-Chase) was engineered using HSQuik technology and tested for spread and efficacy in glioma spheroids. Subcutaneous and intracranial glioma xenografts were utilized to compare antitumor efficacy of OV-Chase, rHsvQ (control), and PBS. Titration of viral particles was performed from OV-treated subcutaneous tumors. Glioma invasion was assessed in collagen-embedded glioma spheroids in vitro and in intracranial tumors. All statistical tests were two sided.

RESULTS

Treatment with Chase-ABC in cultured glioma cells significantly enhanced OV spread in glioma spheroids grown on brain slices (P < 0.0001). Inoculation of subcutaneous glioma xenografts with Chase-expressing OV significantly increased viral titer (>10 times, P = 0.0008), inhibited tumor growth, and significantly increased overall animal survival (P < 0.006) compared with treatment with parental rHsvQ virus. Single OV-Chase administration in intracranial xenografts also resulted in longer median survival of animals than rHsvQ treatment (32 vs. 21 days, P < 0.018). Glioma cell migration and invasion were not increased by OV-Chase treatment.

CONCLUSIONS

We conclude that degradation of glioma extracellular matrix with OV-expressing bacterial Chase-ABC enhanced OV spread and antitumor efficacy.