Perlecan is a specific heparan sulfate proteoglycan that accumulates in the fibrillar beta-amyloid (A beta) deposits of Alzheimer's disease. Perlecan purified from the Engelbreth-Holm-Swarm tumor was used to define perlecan's interactions with A beta and its effects on A beta fibril formation. Using a solid-phase binding immunoassay, freshly solubilized full-length A beta peptides bound immobilized perlecan at two sites, representing both high-affinity [K(D) = approximately 5.8 x 10(-11) M for A beta (1-40); K(D) = approximately 6.5 x 10(-12) M for A beta (1-42)] and lower-affinity [K(D) = 3.5 x 10(-8) M for A beta (1-40); K(D) = 4.3 x 10(-8) M for A beta (1-42)] interactions. An increase in the binding capacity of A beta (1-40) to perlecan correlated with an increase in A beta amyloid fibril formation during a 1-week incubation period. The high-capacity binding of A beta (1-40) to perlecan was similarly observed using perlecan heparan sulfate glycosaminoglycans and was completely abolished by heparin, but not by chondroitin-4-sulfate. Using a thioflavin T fluorometry assay, perlecan accelerated the rate of A beta (1-40) amyloid fibril formation, causing a significant increase in A beta fibril assembly over a 2-week incubation period at 1 h (2.8-fold increase), 1 day (3.6-fold increase), and 3 days (2.8-fold increase) in comparison with A beta (1-40) alone. Perlecan also initially accelerated the formation of A beta (1-42) fibrils within 1 h and maintained significantly higher levels of A beta (1-42) thioflavin T fluorescence throughout a 2-week experimental period in comparison with A beta (1-42) alone, suggesting perlecan's ability to maintain amyloid fibril stability. Perlecan's effects on A beta (1-40) fibril formation and maintenance of A beta (1-42) fibril stability occurred in a dose-dependent manner and was also mediated primarily by perlecan's glycosaminoglycan chains. Perlecan was the most effective enhancer and accelerator of A beta fibril formation when compared directly with other amyloid plaque components, including apolipoprotein E, alpha1-antichymotrypsin, P component, C1q, and C3. This study, therefore, demonstrates that perlecan not only binds to the predominant isoforms of A beta, but also accelerates A beta fibril formation and stabilizes amyloid fibrils once formed, confirming pivotal roles for perlecan in the pathogenesis of A beta amyloidosis in Alzheimer's disease.

Osmotically inactive skin Na(+) storage is characterized by Na(+) accumulation without water accumulation in the skin. Negatively charged glycosaminoglycans (GAGs) may be important in skin Na(+) storage. We investigated changes in skin GAG content and key enzymes of GAG chain polymerization during osmotically inactive skin Na(+) storage. Female Sprague-Dawley rats were fed a 0.1% or 8% NaCl diet for 8 wk. Skin GAG content was measured by Western blot analysis. mRNA content of key dermatan sulfate polymerization enzymes was measured by real-time PCR. The Na(+) concentration in skin was determined by dry ashing. Skin Na(+) concentration during osmotically inactive Na(+) storage was 180-190 mmol/l. Increasing skin Na(+) coincided with increasing GAG content in cartilage and skin. Dietary NaCl loading coincided with increased chondroitin synthase mRNA content in the skin, whereas xylosyl transferase, biglycan, and decorin content were unchanged. We conclude that osmotically inactive skin Na(+) storage is an active process characterized by an increased GAG content in the reservoir tissue. Inhibition or disinhibition of GAG chain polymerization may regulate osmotically inactive Na(+) storage.

Invariant chain (Ii) is a nonpolymorphic glycoprotein that associates with major histocompatibility complex class II molecules and has been shown to mediate several functions in class II-restricted antigen presentation. A small proportion of Ii is modified by the addition of chondroitin sulfate (Ii-CS), and this form of Ii is associated with class II on the surface of antigen-presenting cells. In this report we show that expression of Ii-CS dramatically enhanced the ability of class II-positive EL4 transfectants to stimulate class II-dependent allogeneic and mitogenic T cell responses. Antibody blocking studies and the ability of CD44 to bind directly to Ii-CS suggest that Ii-CS can function as an accessory molecule during T cell responses through interactions with CD44.

Collagen-fibronectin complexes, formed by binding of fibronectin to gelatin or collagen insolubilized on Sepharose, were found to bind 20-40% of radioactivity in [35S]heparin. Fibronectin attached directly to Sepharose also bound [35S]heparin, while gelatin-Sepharose without fibronectin did not. Unlabeled heparin and highly sulfated heparan sulfate efficiently inhibited the binding of [35S]heparin, hyaluronic acid and dermatan sulfate were slightly inhibitory, while chondroitin sulfates and heparan sulfate with a low sulfate content did not inhibit. The interaction of heparin with fibronectin bound to gelatin resulted in complexes which required higher concentrations of urea to dissociate than complexes of fibronectin and gelatin alone. Heparin as well as highly sulfate heparan sulfate and hyaluronic acid brought about agglutination of plastic beads coated with gelatin when fibronectin was present. Neither fibronectin nor glycosaminoglycans alone agglutinated the beads. It is proposed that the multiple interactions of fibronectin, collagen and glycosaminoglycans revealed in these assays could play a role in the deposition of these substances as an insoluble extracellular matrix. Alterations of the quality or quantity of any one of these components could have important effects on cell surface interactions, including the lack of cell surface fibronectin in malignant cells.

Platelets participate in a variety of responses of the blood to injury. An emerging body of evidence suggests that these cells express an intrinsic capacity to interact with and trigger both classical and alternative pathways of complement. This activity requires cell activation with biochemical agonists and/or shear stress, and is associated with the expression of P-selectin, gC1qR, and chondroitin sulfate. Platelet mediated complement activation measurably increases soluble inflammatory mediators (C3a and C5a). Platelets may also serve as targets of classical complement activation in autoimmune conditions such as antiphospholipid syndromes (APS) and immune thrombocytopenia purpura (ITP). Retrospective correlation with clinical data suggests that enhanced platelet associated complement activation correlates with increased arterial thrombotic events in patients with lupus erythematosus and APS, and evidence of enhanced platelet clearance from the circulation in patients with ITP. Taken together, these data support a role for platelet mediated complement activation in vascular inflammation and thrombosis.

While recent work has implicated Tbx20 in myocardial maturation and proliferation, the role of Tbx20 in heart valve development remains relatively unknown. Tbx20 expression was manipulated in primary avian endocardial cells in order to elucidate its function in developing endocardial cushions. Tbx20 gain of function was achieved with a Tbx20-adenovirus, and endogenous Tbx20 expression was inhibited with Tbx20-specific siRNA in cultured endocardial cushion cells. With Tbx20 gain of function, the expression of chondroitin sulfate proteoglycans (CSPG), including aggrecan and versican, was decreased, while the expression of the matrix metalloproteinases (MMP) mmp9 and mmp13 was increased. Consistent results were observed with Tbx20 loss of function, where the expression of CSPG genes increased and MMP genes decreased. In addition, cushion mesenchyme proliferation increased with infection of a Tbx20-adenovirus and decreased with transfection of Tbx20-specfic siRNA. Furthermore, BMP2 treatment resulted in increased Tbx20 expression in endocardial cushion cells, and loss of Tbx20 led to increased Tbx2 and decreased N-myc gene expression. Taken together, these data support a role for Tbx20 in repressing extracellular matrix remodeling and promoting cell proliferation in mesenchymal valve precursor populations in endocardial cushions during embryonic development.

The metastatic spread of tumor cells occurs through a complex series of events, one of which involves the adhesion of tumor cells to extracellular matrix (ECM) components. Multiple interactions between cell surface receptors of an adherent tumor cell and the surrounding ECM contribute to cell motility and invasion. The current studies evaluate the role of a cell surface chondroitin sulfate proteoglycan (CSPG) in the adhesion, motility, and invasive behavior of a highly metastatic mouse melanoma cell line (K1735 M4) on type I collagen matrices. By blocking mouse melanoma cell production of CSPG with p-nitrophenyl beta-D-xylopyranoside (beta-D-xyloside), a compound that uncouples chondroitin sulfate from CSPG core protein synthesis, we observed a corresponding decrease in melanoma cell motility on type I collagen and invasive behavior into type I collagen gels. Melanoma cell motility on type I collagen could also be inhibited by removing cell surface chondroitin sulfate with chondroitinase. In contrast, type I collagen-mediated melanoma cell adhesion and spreading were not affected by either beta-D-xyloside or chondroitinase treatments. These results suggest that mouse melanoma CSPG is not a primary cell adhesion receptor, but may play a role in melanoma cell motility and invasion at the level of cellular translocation. Furthermore, purified mouse melanoma cell surface CSPG was shown, by affinity chromatography and in solid phase binding assays, to bind to type I collagen and this interaction was shown to be mediated, at least in part, by chondroitin sulfate. Additionally we have determined that mouse melanoma CSPG is composed of a 110-kD core protein that is recognized by anti-CD44 antibodies on Western blots. Collectively, our data suggests that interactions between a cell surface CD44-related CSPG and type I collagen in the ECM may play an important role in mouse melanoma cell motility and invasion, and that the chondroitin sulfate portion of the proteoglycan seems to be a critical component in mediating this effect.

Human UDP-d-xylose:proteoglycan core protein beta-d-xylosyltransferase (EC 2.4.2.26, XT-I) initiates the biosynthesis of glycosaminoglycan chains in proteoglycans by transferring xylose from UDP-xylose to specific serine residues of the core protein. Based on the partial amino acid sequence of the purified enzyme from human JAR choriocarcinoma cell culture supernatant we isolated a cDNA encoding XT-I using the degenerate reverse transcriptase-polymerase chain reaction method. This enzyme, which is involved in chondroitin sulfate, heparan sulfate, heparin and dermatan sulfate biosynthesis, belongs to a novel family of glycosyltransferases with no homology to proteins known so far. 5' and 3'-RACE were performed to isolate a novel cDNA fragment of 3726 bp with a single open reading frame encoding at least 827 amino acid residues with a molecular mass of 91 kDa. The human XT-I gene was located on chromosome 16p13.1 using radiation hybrid mapping, and extracts from CHO-K1 cells transfected with the XT-I cDNA in an expression vector exhibited marked XT activity. A new 3608 bp cDNA fragment encoding a protein of 865 amino acid residues was also isolated by PCR using degenerate primers based on the amino acid sequence of human XT-I. The amino acid sequence of this XT-II isoform displayed 55% identity to the human XT-I. The XT-II gene was located on chromosome 17q21.3-17q22, and the exon/intron structure of the 15 kb gene was determined. RT-PCR analyses of XT-I and XT-II mRNA from various tissues confirmed that both XT-I and XT-II transcripts are ubiquitously expressed in the human tissues, although with different levels of transcription. Furthermore, the cDNAs encoding XT-I and XT-II from rat were cloned. The deduced amino acid sequences of rat xylosyltransferases displayed 94% identity to the corresponding human enzyme.

OBJECTIVE

To determine the influence of low oxygen tension on the redifferentiation and matrix production of dedifferentiated articular chondrocytes in monolayer and alginate bead culture.

METHODS

Bovine articular chondrocytes were isolated enzymatically. After multiplication and dedifferentiation in a 2-week monolayer culture under 21% oxygen, the cells were subcultured in monolayer or alginate bead culture and subjected to 21% or 5% O(2)for 2 or 3 weeks in order to redifferentiate. Controls consisted of primary cultures in alginate. Matrix production was monitored immunocytochemically [collagen types I, II, IX, and GAGs (keratan sulfate, chondroitin-4- and -6-sulfate)] and collagen type II additionally assayed by Western blotting. Biosynthetic activity was measured by [(3)H]-proline incorporation and cell-viability by the trypan blue exclusion method.

RESULTS

The cell number increased more than four-fold during dedifferentiation. Collagen type II was not produced by dedifferentiated chondrocytes under 5% or 21% oxygen in the monolayers or under 21% in alginate. However, dedifferentiated cells in alginate subjected to 5% oxygen exhibited a strong collagen type II expression indicating a redifferentiation. Additionally, collagen type IX and GAGs were also higher and [(3)H]-proline incorporation increased significantly. Primary cultures in alginate displayed a stronger collagen type II expression under 5% but no significant differences for other extracellular matrix components, or [(3)H]-proline incorporation. Viability was approximately 90% for all alginate cultures.

CONCLUSIONS

A combination of alginate and high oxygen tension might not be suitable for redifferentiation or culturing of dedifferentiated chondrocytes. However, low oxygen tension promotes or induces a redifferentiation of dedifferentiated cells in alginate, stimulates their biosynthetic activity, and increases collagen type II production in primary alginate cultures.

Tissue-engineered skin is a significant advance in the field of wound healing and was developed due to limitations associated with the use of autografts. These limitations include the creation of a donor site which is at risk of developing pain, scarring, infection and/or slow healing. A number of products are commercially available and many others are in development. Cultured epidermal autografts can provide permanent coverage of large area from a skin biopsy. However, 3 weeks are needed for graft cultivation. Cultured epidermal allografts are available immediately and no biopsy is necessary. They can be cryopreserved and banked, but are not currently commercially available. A nonliving allogeneic acellular dermal matrix with intact basement membrane complex (Alloderm) is immunologically inert. It prepares the wound bed for grafting allowing improved cultured allograft 'take' and provides an intact basement membrane. A nonliving extracellular matrix of collagen and chondroitin-6-sulfate with silicone backing (Integra) serves to generate neodermis. A collagen and glycosaminoglycan dermal matrix inoculated with autologous fibroblasts and keratinocytes has been investigated but is not commercially available. It requires 3 to 4 weeks for cultivation. Dermagraft consists of living allogeneic dermal fibroblasts grown on degradable scaffold. It has good resistance to tearing. An extracellular matrix generated by allogeneic human dermal fibroblasts (TransCyte) serves as a matrix for neodermis generation. Apligraf is a living allogeneic bilayered construct containing keratinocytes, fibroblasts and bovine type I collagen. It can be used on an outpatient basis and avoids the need for a donor site wound. Another living skin equivalent, composite cultured skin (OrCel), consists of allogeneic fibroblasts and keratinocytes seeded on opposite sides of bilayered matrix of bovine collagen. There are limited clinical data available for this product, but large clinical trials are ongoing. Limited data are also available for 2 types of dressing material derived from pigs: porcine small intestinal submucosa acellular collagen matrix (Oasis) and an acellular xenogeneic collagen matrix (E-Z-Derm). Both products have a long shelf life. Other novel skin substitutes are being investigated. The potential risks and benefits of using tissue-engineered skin need to be further evaluated in clinical trials but it is obvious that they offer a new option for the treatment of wounds.

The structure and function of the tumor microvasculature is of great interest for cancer biology, diagnosis, and therapy. The distribution of endothelial cells, pericytes, and basal lamina in tumors is not well documented. In this study, the authors investigated the distribution of markers for these different components in a series of malignant human tumors and in human granulation tissue, both situations with extensive angiogenesis. Their results show a striking heterogeneity in the expression of markers for pericytes and endothelial cells between different tumors, but also within a single tumor lesion. To be able to distinguish between these two adjacent cell types decisively, all marker studies were carried out both on the light and the electron microscopical level and compared with staining results in granulation tissue of cutaneous wounds in healthy volunteers and of decubitus lesions. In granulation tissue of decubitus lesions, well-defined zones with increasing levels of maturation can be delineated. It was found that antibodies recognizing von Willebrand factor often failed to stain the tumor capillaries. Of the pericyte markers, alpha-smooth muscle actin was only locally expressed by pericytes in the tumor vasculature, whereas the high-molecular-weight melanoma-associated antigen, a chondroitin sulfate proteoglycan, stained the microvasculature broadly. Staining of the basal lamina components collagen type IV and laminin was, within the tumor, not restricted to the microvasculature. From their findings the authors conclude that 1) for the visualization of the tumor vasculature, antibodies recognizing endothelial markers, especially monoclonal antibodies PAL-E and BMA 120, are preferable to those recognizing pericytes or basal lamina; 2) within the microvasculature of tumors and granulation tissue, a heterogeneity of expression of endothelial and pericyte markers is observed; 3) during the formation of granulation tissue, all three microvascular components can be demonstrated already in the histologically earliest stage, suggesting not only an involvement of endothelial cells but also of pericytes and basal lamina in the initial steps of angiogenesis in wound healing.

Patients with clinically localized prostate cancer who might be cured by aggressive management are not easily identified using current clinical information. Additional, more accurate, biomarkers of tumor behavior need to be identified to improve clinical outcome. Our previous studies indicated that the concentration of the glycosaminoglycan chondroitin sulfate in prostatic stroma might be a useful biomarker of disease progression in early-stage prostate cancer. In this study, two chondroitin sulfate proteoglycans, versican and decorin, were investigated. Versican and decorin were immunolocalized to the periacinar and peritumoral fibromuscular stroma in sections of nonmalignant and malignant human prostate tissues. Video image measurements indicated that the concentrations of both proteoglycans were increased in the prostatic tissue of men with early-stage prostate cancer compared with tissue from men without cancer (P = 0.0006). Cox's univariate analysis indicated that increases in versican concentration but not in that of decorin were associated with increased risk of prostate-specific antigen (PSA) progression. Versican concentration was compared with other clinical or biological features of prognosis in two-variable regression analyses. Versican and serum PSA concentrations were independent predictors of PSA progression. Versican was a stronger prognostic factor than tumor grade, and it could predict outcome for patients with moderately differentiated tumors. Patients with low versican concentration had significantly better progression-free survival than patients with high levels of versican (Kaplan-Meier plot, 89% versus 27% PSA progression-free at 5 years, respectively; P = 0.0001). We conclude that the measurement of prostatic concentrations of versican, a molecule with reported anticellular adhesive properties, may be a useful marker of disease progression in patients with early-stage prostate cancer and that further study of versican in other patient cohorts is warranted.

We have identified cDNA clones encoding a chondroitin sulfate proteoglycan of rat brain (previously designated 3F8 and now named phosphacan) that binds to neurons and neural cell-adhesion molecules. A sequence of 1616 amino acids deduced from a 4.8-kb open reading frame contains the N-terminal amino acid sequence of the 3F8 core glycoprotein as well as four internal CNBr, tryptic, and endoproteinase Lys-C peptide sequences from the proteoglycan. The deduced amino acid sequence, beginning with a 24-amino acid signal peptide, reveals an N-terminal domain of 255 amino acids homologous to carbonic anhydrases. The entire amino acid sequence deduced from our cDNA clones corresponds to the extracellular portion of a human receptor-type protein tyrosine phosphatase (RPTP zeta/beta) with which it has 76% identity, and the proteoglycan may represent an mRNA splicing variant of the larger transmembrane protein. RNA analysis demonstrated that a probe to the N-terminal carbonic anhydrase domain of the proteoglycan hybridizes with rat brain mRNA of 9.5, 8.4, and 6.4 kb, whereas probes to the phosphatase domains hybridize with only the 9.5-kb message and with the 6.4-kb message (which corresponds to a previously identified variant of the transmembrane protein in which half of the extracellular domain is deleted). The 30 N-terminal amino acids of the 3H1 chondroitin/keratan sulfate proteoglycan of brain are identical to those of the 3F8 proteoglycan, and six internal tryptic peptide sequences also matched those found in sequenced peptides of the 3F8 proteoglycan and/or amino acid sequences deduced from the cDNA clones. We therefore conclude that the 3H1 chondroitin/keratan sulfate proteoglycan and the 3F8 chondroitin sulfate proteoglycan represent glycosylation and possible extracellular splicing variants of a receptor-type protein tyrosine phosphatase. These proteoglycans may modulate cell interactions and other developmental processes in nervous tissue through heterophilic binding to cell-surface and extracellular matrix molecules, and by competition with ligands of the transmembrane phosphatase.

Many parts of the life cycle of hepatitis B virus (HBV) infection of hepatocytes have been unravelled, but the attachment and entry process leading to infection is largely unknown. Using primary Tupaia hepatocyte cultures as an in vitro infection system, we determined that HBV uses cell-surface heparan sulfate proteoglycans as low-affinity receptor, because HBV infection was inhibited by heparin (IC50: 5 microg ml(-1)) or other higher-sulfated polymers, but not by lower-sulfated glycosaminoglycans, such as chondroitin sulfate. Pretreatment of primary hepatocytes with heparinase decreased viral binding and inhibited HBV infection completely. Interestingly, after preS1-dependent viral binding at 16 degrees C to the cell surface, subsequent infection could still be inhibited by HBV preS1-lipopeptides, but not by heparin any more, suggesting a shift of the virus to a high-affinity receptor. In summary, we suggest following multistep attachment process: in vivo, HBV is initially trapped within the liver in the space of Dissé by heparan sulfate proteoglycans. Thereafter, HBV binds via its preS1 attachment site and the N-terminal myristic acid to a yet unknown, high-affinity receptor that confers uptake in a yet unknown compartment.

BACKGROUND

The cell surface proteoglycan, chondroitin sulfate proteoglycan 4 (CSPG4), is a potential target for monoclonal antibody (mAb)-based immunotherapy for many types of cancer. The lack of effective therapy for triple-negative breast cancer (TNBC) prompted us to examine whether CSPG4 is expressed in TNBC and can be targeted with CSPG4-specific mAb.

METHODS

CSPG4 protein expression was assessed in 44 primary TNBC lesions, in TNBC cell lines HS578T, MDA-MB-231, MDA-MB-435, and SUM149, and in tumor cells in pleural effusions from 12 metastatic breast cancer patients. The effect of CSPG4-specific mAb 225.28 on growth, adhesion, and migration of TNBC cells was tested in vitro. The ability of mAb 225.28 to induce regression of tumor metastases (n = 7 mice) and to inhibit spontaneous metastasis and tumor recurrence (n = 12 mice per group) was tested in breast cancer models in mice. The mechanisms responsible for the antitumor effect of mAb 225.28 were also investigated in the cell lines and in the mouse models. All statistical tests were two-sided.

RESULTS

CSPG4 protein was preferentially expressed in 32 of the 44 (72.7%) primary TNBC lesions tested, in TNBC cell lines, and in tumor cells in pleural effusions from 12 metastatic breast cancer patients. CSPG4-specific mAb 225.28 statistically significantly inhibited growth, adhesion, and migration of TNBC cells in vitro. mAb 225.28 induced 73.1% regression of tumor metastasis in a TNBC cell-derived experimental lung metastasis model (mAb 225.28 vs control, mean area of metastatic nodules = 44590.8 vs 165950.8 μm(2); difference of mean = 121360.0 μm(2), 95% confidence interval = 91010.7 to 151709.4 μm(2); P < .001). Additionally, mAb 225.28 statistically significantly reduced spontaneous lung metastases and tumor recurrences in an orthotopic xenograft mouse model. The mechanisms responsible for antitumor effect included increased apoptosis and reduced mitotic activity in tumor cells, decreased blood vessel density in the tumor microenvironment, and reduced activation of signaling pathways involved in cell survival, proliferation and metastasis.

CONCLUSIONS

This study identified CSPG4 as a new target for TNBC. The antitumor activity of CSPG4-specific mAb was mediated by multiple mechanisms, including the inhibition of signaling pathways crucial for TNBC cell survival, proliferation, and metastasis.

We previously reported that the transplantation of neural stem/progenitor cells (NSPCs) can contribute to the repair of injured spinal cord in adult rats and monkeys. In some cases, however, most of the transplanted cells adhered to the cavity wall and failed to migrate and integrate into the host spinal cord. In this study we focused on chondroitin sulfate proteoglycan (CSPG), a known constituent of glial scars that is strongly expressed after spinal cord injury (SCI), as a putative inhibitor of NSPC migration in vivo. We hypothesized that the digestion of CSPG by chondroitinase ABC (C-ABC) might promote the migration of transplanted cells and neurite outgrowth after SCI. An in vitro study revealed that the migration of NSPC-derived cells was inhibited by CSPG and that this inhibitory effect was attenuated by C-ABC pre-treatment. Consistently, an in vivo study of C-ABC treatment combined with NSPC transplantation into injured spinal cord revealed that C-ABC pre-treatment promoted the migration of the transplanted cells, whereas CSPG-immunopositive scar tissue around the lesion cavity prevented their migration into the host spinal cord in the absence of C-ABC pre-treatment. Furthermore, this combined treatment significantly induced the outgrowth of a greater number of growth-associated protein-43-positive fibers at the lesion epicentre, compared with NSPC transplantation alone. These findings suggested that the application of C-ABC enhanced the benefits of NSPC transplantation for SCI by reducing the inhibitory effects of the glial scar, indicating that this combined treatment may be a promising strategy for the regeneration of injured spinal cord.

NO has been shown to mediate angiogenesis; however, its role in vessel morphogenesis and maturation is not known. Using intravital microscopy, histological analysis, alpha-smooth muscle actin and chondroitin sulfate proteoglycan 4 staining, microsensor NO measurements, and an NO synthase (NOS) inhibitor, we found that NO mediates mural cell coverage as well as vessel branching and longitudinal extension but not the circumferential growth of blood vessels in B16 murine melanomas. NO-sensitive fluorescent probe 4,5-diaminofluorescein imaging, NOS immunostaining, and the use of NOS-deficient mice revealed that eNOS in vascular endothelial cells is the predominant source of NO and induces these effects. To further dissect the role of NO in mural cell recruitment and vascular morphogenesis, we performed a series of independent analyses. Transwell and under-agarose migration assays demonstrated that endothelial cell-derived NO induces directional migration of mural cell precursors toward endothelial cells. An in vivo tissue-engineered blood vessel model revealed that NO mediates endothelial-mural cell interaction prior to vessel perfusion and also induces recruitment of mural cells to angiogenic vessels, vessel branching, and longitudinal extension and subsequent stabilization of the vessels. These data indicate that endothelial cell-derived NO induces mural cell recruitment as well as subsequent morphogenesis and stabilization of angiogenic vessels.

Increased extracellular matrix (ECM) deposition in the airway wall contributes to the airway wall remodeling observed in asthmatics. Although alterations in collagen have been well described, less is known about changes in other components of the ECM, particularly proteoglycans (PGs). Endobronchial biopsies were obtained from seven patients with mild atopic asthma and six normal control subjects. Tissues were blocked in OCT and frozen in isopentane. Sections were immunostained with antibodies for the small leucine-rich PGs, lumican, biglycan, decorin, and fibromodulin and for versican, a large chondroitin sulfate PG. We calculated the area of positive staining in the subepithelial layer, correcting for basement membrane length. Lumican, biglycan, and versican were localized predominantly in the subepithelial layer of the airway wall in all groups. PG deposition was significantly increased in asthmatics as compared with that in control subjects. Furthermore, the degree of PG immunoreactivity was significantly correlated with airway responsiveness in the asthmatics (lumican; r = -0.77, p < 0.05; biglycan: r = -0.76, p < 0.05; versican: r = -0.74, p = 0.06). Our results suggest that PGs may play a role in airway wall remodeling and thereby, airway mechanics in asthma.

Adhesive PfEMP1 proteins are displayed on the surface of malaria-infected red blood cells. They play a critical role in the disease, tethering infected cells away from destruction by the spleen and causing many severe symptoms. A molecular understanding of how these domains maintain their binding properties while evading immune detection will be important in developing therapeutics. In malaria of pregnancy, domains from the var2csa-encoded PfEMP1 protein interact with chondroitin sulfate on the placenta surface. This causes accumulation of infected red blood cells, leading to placental inflammation and block of blood flow to the developing fetus. This is associated with maternal anemia, low birth weight, and premature delivery and can lead to the death of mother and child. Here I present the structure of the chondroitin sulfate-binding DBL3X domain from a var2csa-encoded PfEMP1 protein. The domain adopts a fold similar to malarial invasion proteins, with extensive loop insertions. One loop is flexible in the unliganded structure but observed in the presence of sulfate or disaccharide, where it completes a sulfate-binding site. This loop, and others surrounding this putative carbohydrate-binding site, are flexible and polymorphic, perhaps protecting the binding site from immune detection. This suggests a model for how the domain maintains ligand binding while evading the immune response and will guide future drug and vaccine development.

Approximately 50% of the weight of a mature mast cell (MC) consists of varied neutral proteases stored in the cell's secretory granules ionically bound to serglycin proteoglycans that contain heparin and/or chondroitin sulfate E/diB chains. Mouse MCs express the exopeptidase carboxypeptidase A3 and at least 15 serine proteases [designated as mouse MC protease (mMCP) 1-11, transmembrane tryptase/tryptase gamma/protease serine member S (Prss) 31, cathepsin G, granzyme B, and neuropsin/Prss19]. mMCP-6, mMCP-7, mMCP-11/Prss34, and Prss31 are the four members of the chromosome 17A3.3 family of tryptases that are preferentially expressed in MCs. One of the challenges ahead is to understand why MCs express so many different protease-proteoglycan macromolecular complexes. MC-like cells that contain tryptase-heparin complexes in their secretory granules have been identified in the Ciona intestinalis and Styela plicata urochordates that appeared approximately 500 million years ago. Because sea squirts lack B cells and T cells, it is likely that MCs and their tryptase-proteoglycan granule mediators initially appeared in lower organisms as part of their innate immune system. The conservation of MCs throughout evolution suggests that some of these protease-proteoglycan complexes are essential to our survival. In support of this conclusion, no human has been identified that lacks MCs. Moreover, transgenic mice lacking the beta-tryptase mMCP-6 are unable to combat a Klebsiella pneumoniae infection effectively. Here we summarize the nature and function of some of the tryptase-serglycin proteoglycan complexes found in mouse and human MCs.

Chondroitin sulfate proteoglycans are synthesized and deposited in the spinal cord following injury. These proteoglycans may restrict regeneration and plasticity and contribute to the limited recovery seen after an injury. Chondroitinase, a bacterial enzyme that catalyzes the hydrolysis of the chondroitin chains on proteoglycans, has been shown to improve motor and sensory function following partial transection lesions of the spinal cord. To assess the effects of chondroitinase in a clinically relevant model of spinal cord injury, 128 female Long-Evans rats received either a severe, moderate, or mild contusion injury at the vertebral level T9/T10 with a forceps model and were treated for 2 weeks with chondroitinase ABCI at 0.06 Units per dose, penicillinase, or vehicle control via an intrathecal catheter placed near the injury. Motor behavior was measured by open-field testing of locomotion and bladder function monitored by measuring daily residual urine volumes. Animals treated with chondroitinase showed significant improvements in open-field locomotor activity as measured by the Basso, Beattie and Bresnahan scoring system after both severe and moderate SCI (p<0.05 and 0.01, respectively). No significant locomotor differences were observed in the mild injury group. In the moderate injury group, residual urine volumes were reduced with chondroitinase treatment by 2 weeks after injury (p<0.05) and in the severe injury group, by 6 weeks after injury (NS). These results demonstrate that chondroitinase is effective at promoting both somatic and autonomic motor recovery following a clinically relevant contusion spinal cord injury and is a candidate as a therapeutic for human spinal cord injury.

Kidneys were perfused with [35S]sulfate at 4 h in vitro to radiolabel sulfated proteoglycans. Glomeruli were isolated from the labeled kidneys, and purified fractions of glomerular basement membrane (GBM) were prepared therefrom. Proteoglycans were extracted from GBM fractions by use of 4 M guanidine-HCl at 4 degrees C in the presence of protease inhibitors. The efficiency of extraction was approximately 55% based on 35S radioactivity. The extracted proteoglycans were characterized by gel-filtration chromatography (before and after degradative treatments) and by their behavior in dissociative CsCl gradients. A single peak of proteoglycans with an Mr of 130,000 (based on cartilage proteoglycan standards) was obtained on Sepharose CL-4B or CL-6B. Approximately 85% of the total proteoglycans were susceptible to nitrous acid oxidation (which degrades heparan sulfates), and approximately 15% were susceptible to digestion with chondroitinase ABC (degrades chondroitin-4 and -6 sulfates and dermatan sulfate). The released glycosaminoglycan (GAG) chains had an Mr of approximately 26,000. Density gradient centrifugation resulted in the partial separation of the extracted proteoglycans into two types with different densities: a heparan sulfate proteoglycan that was enriched in the heavier fraction (p greater than 1.43 g/ml), and a chondroitin sulfate proteoglycan that was concentrated in the lighter fractions (p less than 1.41). The results indicate that two types of proteoglycans are synthesized and incorporated into the GBM that are similar in size and consist of four to five GAG chains (based on cartilage proteoglycan standards). The chromatographic behavior of the extracted proteoglycans and the derived GAG, together with the fact that the two types of proteoglycans can be partially separated into the density gradient, suggest that the heparan sulfate and chondroitin sulfate(s) are located on different core proteins.

The subventricular zone (SVZ) of the lateral ventricle remains mitotically active in the adult mammalian central nervous system (CNS). Recent studies have suggested that this region may contain neuronal precursors (neural stem cells) in adult rodents. A variety of neuronal and glial markers as well as three extracellular matrix (ECM) markers were examined with the hope of understanding factors that may affect the growth and migration of neurons from this region throughout development and in the adult. This study has characterized the subventricular zone of late embryonic, postnatal, and adult mice using several neuronal markers [TuJ1, nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-d), neuron-specific enolase (NSE)], glial markers [RC-2, vimentin, glial fibrillary acidic protein (GFAP), galactocerebroside (Gal-C)], ECM markers [tenascin-C (TN-C), chondroitin sulfate, a chondroitin sulfate proteoglycan termed dermatan sulfate-dependent proteoglycan-1 (DSD-1-PG)], stem-cell marker (nestin), and proliferation-specific marker [bromodeoxyuridine (BrdU)]. TuJ1+ and nestin+ cells (neurons and stem cells, respectively) persist in the region into adulthood, although the numbers of these cells become more sparse as the animal develops, and they appear to be immature compared to the cells in surrounding forebrain structures (e.g., not expressing NSE and having few, if any, processes). Likewise, NADPH-d+ cells are found in and around the SVZ during early postnatal development but become more sparse in the proliferative zone through maturity, and, by adulthood, only a few labeled cells can be found at the border between the SVZ and surrounding forebrain structures (e.g., the striatum), and even smaller numbers of positive cells can be found within the adult SVZ proper. BrdU labeling also seems to decrease significantly after the first postnatal week, but it still persists in the SVZ of adult animals. The disappearance of RC-2+ (radial) glia during postnatal development and the persistence of glial-derived ECM molecules such as tenascin and chondroitin sulfate proteoglycans (as well as other "boundary" molecules) in the adult SVZ may be associated with a persistence of immaturity, cell death, and a lack of cell emigration from the SVZ in the adult.

We have identified a receptor in hepatic endothelial and Kupffer cells that binds oligosaccharides terminating with the sequence SO4-4GalNAc beta 1,4GlcNAc beta 1,2-Man alpha (S4GGnM). This receptor can account for the rapid removal of the glycoprotein hormone lutropin, which bears unique Asn-linked oligosaccharides terminating in S4GGnM, from the circulation. Hepatic endothelial cells express 579,000 S4GGnM receptors at their surface and bind lutropin with an apparent Kd of 1.63 x 10(-7) M. Bound ligand is rapidly internalized. Binding does not require divalent cations, is reversed by incubation at pH 5.0 or below, and is inhibited by fucoidin but not by hyaluronate, heparin, chondroitin sulfate, or dextran sulfate. We propose that the S4GGnM-specific receptor represents a major mechanism for clearance of certain sulfated glycoproteins from the blood, including members of the glycoprotein hormone family.