Membrane-associated Heparan Sulfate Proteoglycan Research Articles

Effect of Syndecan-1, Syndecan-4, and Glypican-1 on Turkey Muscle Satellite Cell Proliferation, Differentiation, and Responsiveness to Fibroblast Growth Factor 2

The membrane-associated heparan sulfate proteoglycan families consisting of the syndecans and glypicans are low-affinity receptors for fibroblast growth factor 2 (FGF2). Fibroblast growth factor 2 is a potent stimulator of skeletal muscle cell proliferation and a strong inhibitor of differentiation. Because syndecan-1, syndecan-4, and glypican-1 potentially play unique, but pivotal, roles in muscle cell proliferation and differentiation, these proteoglycans were examined for their effect on muscle cell proliferation and differentiation and FGF2 responsiveness. In the present study, turkey Randombred Control 2 line myogenic satellite cells were transfected with expression vector constructions of syndecan-1, syndecan-4, or glypican-1 to assay their role during muscle development and the effect on FGF2 responsiveness. During proliferation, only syndecan-1 increased proliferation. Both syndecan-4 and glypican-1 decreased proliferation at 72 h but generally did not affect the proliferation process. There was no interaction between the transfected gene and cell proliferation response to FGF2. Glypican-1 increased differentiation early in the process (24 h), and at later times differentiation was decreased by glypican-1. Both syndecan-1 and syndecan-4 overexpression decreased differentiation. During differentiation, except for glypican-1 at 48 h of differentiation, there was no interaction between gene treatment and FGF2 responsiveness. This result indicates that FGF2 responsiveness was not affected by the overexpression of syndecan-1, syndecan-4, and glypican-1 during differentiation. These data demonstrate that syndecan-1, syndecan-4, or glypican-1 differentially affect the processes of turkey muscle cell proliferation and differentiation, and can regulate these developmental stages in an FGF2-independent manner.

A Two-hybrid Screen Identifies Cathepsins B and L as Uncoating Factors for Adeno-associated Virus 2 and 8

Vectors based on different serotypes of adeno-associated virus hold great promise for human gene therapy, based on their unique tissue tropisms and distinct immunological profiles. A particularly interesting candidate is AAV8, which can efficiently and rapidly transduce a wide range of tissues in vivo. To further unravel the mechanisms behind AAV8 transduction, we used yeast two-hybrid analyses to screen a mouse liver complementary DNA library for cellular proteins capable of interacting with the viral capsid proteins. In total, we recovered approximately 700 clones, comprising over 300 independent genes. Sequence analyses revealed multiple hits for over 100 genes, including two encoding the endosomal cysteine proteases cathepsins B and L. Notably, these two proteases also physically interacted with the corresponding portion of the AAV2 capsid in yeast, but not with AAV5. We demonstrate that cathepsins B and L are essential for efficient AAV2- and AAV8-mediated transduction of mammalian cells, and document the ability of purified cathepsin B and L proteins to bind and cleave intact AAV2 and AAV8 particles in vitro. These data suggest that cathepsin-mediated cleavage could prime AAV capsids for subsequent nuclear uncoating, and indicate that analysis of additional genes recovered in our screen may help to further elucidate the mechanisms behind transduction by AAV8 and related serotypes.

Membrane-associated heparan sulfate is not required for rAAV-2 infection of human respiratory epithelia

BackgroundAdeno-associated virus type 2 (AAV-2) attachment and internalization is thought to be mediated by host cell membrane-associated heparan sulfate proteoglycans (HSPG). Lack of HSPG on the apical membrane of respiratory epithelial cells has been identified as a reason for inefficient rAAV-2 infection in pulmonary applications in-vivo. The aim of this investigation was to determine the necessity of cell membrane HSPG for efficient infection by rAAV-2.ResultsRates of transduction with rAAV2-CMV-EGFP3 in several different immortalized airway epithelial cell lines were determined at different multiplicities of infection (MOI) before and after removal of membrane HSPG by heparinase III. Removal of HSPG decreased the efficacy of infection with rAAV2 by only 30–35% at MOI ≤ 100 for all of respiratory cell lines tested, and had even less effect at an MOI of 1000. Studies in mutant Chinese Hamster Ovary cell lines known to be completely deficient in surface HSPG also demonstrated only moderate effect of absence of HSPG on rAAV-2 infection efficacy. However, mutant CHO cells lacking all membrane proteoglycans demonstrated dramatic reduction in susceptibility to rAAV-2 infection, suggesting a role of membrane glycosaminoglycans other than HSPG in mediating rAAV-2 infection.ConclusionLack of cell membrane HSPG in pulmonary epithelia and other cell lines results in only moderate decrease in susceptibility to rAAV-2 infection, and this decrease may be less important at high MOIs. Other cell membrane glycosaminoglycans can play a role in permitting attachment and subsequent rAAV-2 internalization. Targeting alternative membrane glycosaminoglycans may aid in improving the efficacy of rAAV-2 for pulmonary applications.

Differential expression of membrane-associated heparan sulfate proteoglycans in the skeletal muscle of turkeys with different growth rates

Heparan sulfate proteoglycans (HSPG) are key components of the cell membrane and extracellular matrix of skeletal muscle cells. Two major groups of membrane-associated HSPG found in skeletal muscle are syndecans (SYN) and glypicans (GPC), both of which can regulate growth factor activities and, thus, modulate cell proliferation and differentiation. In the current study, the mRNA expression of a group of membrane-associated HSPG (SYN 2 through 4 and GPC 1) was investigated in embryonic pectoralis major muscle [embryonic days (ED) 14, 16, 18, 20, 22, 24] and myogenic satellite cells isolated from males of a turkey genetic line selected for increased 16-wk BW (F line) and an unselected randombred control (RBC2 line) from which the F line was developed. The mRNA expression was measured by a real-time quantitative PCR approach. The SYN 2 and SYN 4 expression exhibited a similar pattern during embryonic p. major muscle development, which remained constant from ED 14 to ED 22 and declined sharply from ED 22 to ED 24 to a very low level. In contrast, the SYN 3 and GPC 1 expression showed a continuous decline from ED 14 to ED 24. The F line had higher SYN 2 (ED 14, 18, 20, 22), SYN 3 (ED 22), and SYN 4 (ED 22) expression than the RBC2 line. In myogenic satellite cells, initiating differentiation resulted in a decrease in SYN 2 expression and an increase in GPC 1 expression. Both SYN 3 and SYN 4 expression stayed almost constant through both the proliferation and differentiation stages. The proliferating satellite cells from the F line displayed higher SYN 4 expression than those from the RBC2 line. Collectively, the results from the current study suggest that membrane-associated HSPG are differentially expressed in both embryonic p. major muscle tissue and satellite cells isolated from F-line and RBC2-line male turkeys, implying their distinct roles in myogenesis and differing influence on muscle growth properties.

246. Efficient Gene Transfer to Peripheral Nervous System Via Systemic Delivery of AAV Vectors

Top of pageAbstract Gene transfer to the peripheral nervous system has great therapeutic potential for the treatment of many neurological diseases, such as spinal muscular atrophy, amyotrophic lateral sclerosis, hereditary axonal motor neuropathies, and so on. Here we describe evidence that AAV vectors can be efficiently delivered to peripheral nervous system by a single intraperitoneal injection into neonatal mice. We delivered AAV 8-LacZ vector encoding beta-gal, and AAV8- mini-agrin vector encoding secreatable agrin protein, which is a basement membrane-associated heparan sulphate proteoglycan, to the neonates respectively. In AAV8-LacZ injected mice, we observed robust beta-gal positive cells in sensory neuron cells and peripheral nerve roots. In AAV8-mini-agrin injected mice, we noticed extensive presence of mini-agrin protein surrounding individual axon-Schwann cell units. Additionally, mini-agrin protein co-localized with laminin |[gamma]|1, a component of extracellular matrix, in the basal lamina of peripheral nerve roots. Our results demonstrate that AAV vector can be efficiently delivered to the peripheral nervous system systemically. This study should facilitate the development of gene therapy based strategy for treatment of peripheral neuronal diseases.

Evidence That the Cellular Ligand for the Human NK Cell Activation Receptor NKp30 Is Not a Heparan Sulfate Glycosaminoglycan

NKp30 (NCR3, CD337) is a natural cytotoxicity receptor, expressed on subsets of human peripheral blood NK cells, involved in NK cell killing of tumor cells and immature dendritic cells. The cellular ligand for NKp30 has remained elusive, although evidence that membrane-associated heparan sulfate (HS) proteoglycans are involved in the recognition of cellular targets by NKp30 was recently reported. The data presented in this report show conclusively that HS glycosaminoglycans (GAG) are not ligands for NKp30. We show that removing HS completely from the cell surface of human 293-EBNA cells with mammalian heparanase does not affect binding of rNKp30/human IgG1 Fc chimera complexes or binding of multimeric liposome-rNKp30 complexes. Removing HS from 293-EBNA cells, culture-generated DC, MM-170 malignant melanoma cells, or HeLa cells does not affect the NKp30-dependent killing of these cells by NK cells. We show further that the GAG-deficient hamster pgsA-745 cells that lack HS and the GAG-expressing parent CHO-K1 cells are both killed by NK cells, with killing of both cell lines inhibited to the same extent by anti-NKp30 mAb. From these results we conclude that HS GAG are not ligands for NKp30, leaving open the question as to the nature of the cellular ligand for this important NK cell activation receptor.

Membrane-associated heparan sulfate proteoglycans are involved in the recognition of cellular targets by NKp30 and NKp46.

Lysis of virus-infected and tumor cells by NK cells is mediated via natural cytotoxicity receptors (NCRs). We have recently shown that the NKp44 and NKp46 NCRs, but not the NKp30, recognize viral hemagglutinins. In this study we explored the nature of the cellular ligands recognized by the NKp30 and NKp46 NCRs. We demonstrate that target cell surface heparan sulfate proteoglycans (HSPGs) are recognized by NKp30 and NKp46 and that 6-O-sulfation and N-acetylation state of the glucose building unit affect this recognition and lysis by NK cells. Tumor cells expressing cell surface heparanase, CHO cells lacking membranal heparan sulfate and glypican-1-suppressed pancreatic cancer cells manifest reduced recognition by NKp30 and NKp46 and are lysed to a lesser extent by NK cells. Our results are the first clue for the identity of the ligands for NKp30 and NKp46. Whether the ligands are particular HSPGs, unusual heparan sulfate epitopes, or a complex of HSPGs and either other protein or lipid moieties remains to be further explored.

Role of glypican 4 in the regulation of convergent extension movements during gastrulation in Xenopus laevis.

Coordinated morphogenetic cell movements during gastrulation are crucial for establishing embryonic axes in animals. Most recently, the non-canonical Wnt signaling cascade (PCP pathway) has been shown to regulate convergent extension movements in Xenopus and zebrafish. Heparan sulfate proteoglycans (HSPGs) are known as modulators of intercellular signaling, and are required for gastrulation movements in vertebrates. However, the function of HSPGs is poorly understood. We analyze the function of Xenopus glypican 4 (Xgly4), which is a member of membrane-associated HSPG family. In situ hybridization revealed that Xgly4 is expressed in the dorsal mesoderm and ectoderm during gastrulation. Reducing the levels of Xgly4 inhibits cell-membrane accumulation of Dishevelled (Dsh), which is a transducer of the Wnt signaling cascade, and thereby disturbs cell movements during gastrulation. Rescue analysis with different Dsh mutants and Wnt11 demonstrated that Xgly4 functions in the non-canonical Wnt/PCP pathway, but not in the canonical Wnt/beta-catenin pathway, to regulate gastrulation movements. We also provide evidence that the Xgly4 protein physically binds Wnt ligands. Therefore, our results suggest that Xgly4 functions as positive regulator in non-canonical Wnt/PCP signaling during gastrulation.

Early neutrophilic expression of vascular endothelial growth factor after traumatic brain injury

The formation of edema after traumatic brain injury (TBI) is in part associated with the disruption of the blood–brain barrier. However, the molecular and cellular mechanisms underlying these phenomena have not been fully understood. One possible factor involved in edema formation is vascular endothelial growth factor (VEGF). This growth factor has previously been demonstrated to increase the blood–brain barrier permeability to the low molecular weight markers and macromolecules. In this study, we analyzed the temporal changes in VEGF expression after TBI in rats. In the intact brain, VEGF was expressed at relatively low levels and was found in the cells located close to the cerebrospinal fluid space. These were the astrocytes located under the ependyma and the pia-glial lining, as well as the epithelial cells of the choroid plexus. In addition, several groups of neurons, including those located in the frontoparietal cortex and in all hippocampal regions, were VEGF-positive. The pattern of VEGF-immunopositive staining of neurons and choroidal epithelium suggested that in these cells, VEGF binds to the cell membrane-associated heparan sulfate proteoglycans. Following TBI, there was an early (within 4 h post-injury) increase in VEGF expression in the traumatized parenchyma associated with neutrophilic invasion. The ipsilateral choroid plexus appeared to play a role in facilitating the migration of neutrophils from blood into the cerebrospinal fluid space, from where many of these cells infiltrated the brain parenchyma. VEGF-immunopositive staining of neutrophils resembled haloes and was found ipsilaterally within the frontoparietal cortex and around the velum interpositum, a part of the subarachnoid space. These haloes likely represent the deposition of neutrophil-derived VEGF within the extracellular matrix, from where this growth factor may be gradually released during an early post-traumatic period. The maximum number of VEGF-secreting neutrophils was observed between 8 h and 1 day after TBI. In addition, from 4 h post-TBI, there was a progressive increase in the number of VEGF-immunoreactive astrocytes in the ipsilateral frontoparietal cortex. The maximum number of astrocytes expressing VEGF was observed 4 days after TBI, and then the levels of astroglial VEGF expression declined gradually. Early invasion of brain parenchyma by VEGF-secreting neutrophils together with a delayed increase in astrocytic synthesis of this growth factor correlate with the biphasic opening of the blood–brain barrier and formation of edema previously observed after TBI. Therefore, these findings suggest that VEGF plays an important role in promoting the formation of post-traumatic brain edema.

NCAM regulates cell motility.

Cell migration is required during development of the nervous system. The regulatory mechanisms for this process, however, are poorly elucidated. We show here that expression of or exposure to the neural cell adhesion molecule (NCAM) strongly affected the motile behaviour of glioma cells independently of homophilic NCAM interactions. Expression of the transmembrane 140 kDa isoform of NCAM (NCAM-140) caused a significant reduction in cellular motility, probably through interference with factors regulating cellular attachment, as NCAM-140-expressing cells exhibited a decreased attachment to a fibronectin substratum compared with NCAM-negative cells. Ectopic expression of the cytoplasmic part of NCAM-140 also inhibited cell motility, presumably via the non-receptor tyrosine kinase p59(fyn) with which NCAM-140 interacts. Furthermore, we showed that the extracellular part of NCAM acted as a paracrine inhibitor of NCAM-negative cell locomotion through a heterophilic interaction with a cell-surface receptor. As we showed that the two N-terminal immunoglobulin modules of NCAM, which are known to bind to heparin, were responsible for this inhibition, we presume that this receptor is a heparan sulfate proteoglycan. A model for the inhibitory effect of NCAM is proposed, which involves competition between NCAM and extracellular components for the binding to membrane-associated heparan sulfate proteoglycan.

Mechanical stress regulates syndecan-4 expression and redistribution in vascular smooth muscle cells.

Syndecan-4 is a unique membrane-associated heparan sulfate proteoglycan that colocalizes with integrin heterodimers in focal adhesion complexes. Because focal adhesions serve as a putative mechanotransduction system, we postulated that physical forces that are sensed by focal adhesions may regulate the expression and intracellular distribution of syndecan-4 and thereby modulate cell movement and orientation. In this report, syndecan-4 was identified as a transcriptionally regulated, immediate-early gene in response to the application of oscillatory stress. This fluctuation was associated with coordinate changes in the concentration and compartmentalization of syndecan-4 proteins. Specifically, syndecan-4 was lost from the dorsal aspect of the cell membrane and translocated from its intracellular pool to the ventral cell surface. Dissociation of syndecan-4 and vinculin from focal adhesions may contribute to promoting cell motility, because overexpression of syndecan-4, in part, blocked this dissociation and also retarded mechanical stretch-induced cell migration. These studies suggest that mechanical stress induces cell locomotion, in part, by the dynamic regulation of syndecan-4 expression and relocation.

Cell-surface heparan sulfate proteoglycan–mediated regulation of human neutrophil migration by the serpin antithrombin III

AbstractThe serpin antithrombin III (AT III) is reported to have hemostasis-regulating and anti-inflammatory properties. To determine its ability to influence thrombin-independent leukocyte responses, the direct effects of the AT III concentrate Kybernin P and a monoclonal antibody-purified AT III on neutrophil migration were studied. Chemotactic activity of human neutrophils isolated from the blood of healthy donors was determined in modified Boyden microchemotaxis chambers, and binding studies were performed according to standard experimental protocols. Preincubation in vitro of neutrophils with Kybernin P or immune-adsorbed AT III significantly deactivated migration toward fMet-Leu-Phe, or interleukin-8 (IL-8), in a concentration-dependent manner. In the absence of additional attractants, neutrophils exhibited a migratory response toward gradients of AT III preparations. True chemotaxis was confirmed in checkerboard assays. Analyses revealed that the AT III heparin-binding site interacts with neutrophil membrane–associated heparan sulfate proteoglycan receptors. Mechanisms of intracellular signaling differed; the deactivation of IL-8–induced chemotaxis resulted from tyrphostin-sensitive interactions of AT III-signaling with the IL-8 signal transduction pathway, whereas AT III–induced chemotaxis involved protein kinase C and phosphodiesterases. Signaling similarities between AT III and the proteoglycan syndecan-4 may suggest the binding of AT III to this novel type of membrane receptor. Under physiological conditions, AT III may prevent neutrophils from premature activation. Moreover, the systemic administration of AT III concentrate could have beneficial effects in combating systemic inflammation.

Location, substructure, and composition of basal laminar drusen compared with drusen associated with aging and age-related macular degeneration

PURPOSE: To determine whether basal laminar drusen differ in their location, ultrastructure, or composition from drusen associated with aging and age-related macular degeneration. METHODS: A paraffin-embedded block from an eye of a patient with basal laminar drusen was obtained. Sections were examined immunohistochemically using a battery of antibodies and lectins directed against drusen-associated proteins and glycoconjugates, respectively. Thin sections were examined by electron microscopy and compared with eyes with age-related macular degeneration. RESULTS: Drusen in the eye with basal laminar drusen are located between the basal lamina of the retinal pigment epithelium and the inner collagenous layer of Bruch membrane, just as they are in age-related macular degeneration. Two distinct ultrastructural phenotypes are observed in the eye with basal laminar drusen; their substructure is indistinguishable from drusen phenotypes in age-related macular degeneration. Both basal laminar drusen and drusen associated with age-related macular degeneration are bound by the lectins Ricinis communis agglutinin and Arachis hypogea agglutinin (after neuraminidase digestion) and by antivitronectin, anti–HLA-DR, anti–serum amyloid P, and anti-C5 antibodies, but not by antibodies directed against basement membrane–associated heparan sulfate proteoglycan, laminin, fibrinogen, or collagen type IV. CONCLUSIONS: These data support the notion that cuticular or basal laminar drusen are similar to, and perhaps indistinguishable from, drusen associated with age-related macular degeneration and are not nodular or diffuse thickenings of Bruch membrane, as previously suggested. Thus, we suggest basal laminar drusen is a misnomer. This clinical phenotype should be identified as “early adult onset, grouped drusen” or by the eponym “Gass syndrome.” Features of basal laminar drusen, such as uniform drusen size, clustered distribution, and angiographic features, do not appear to be related to differences in drusen location, composition, or substructure.

Agrin Is a Major Heparan Sulfate Proteoglycan Accumulating in Alzheimer's Disease Brain

Heparan sulfate proteoglycans (HSPGs) have been suggested to play an important role in the formation and persistence of senile plaques and neurofibrillary tangles in dementia of the Alzheimer's type (DAT). We performed a comparative immunohistochemical analysis of the expression of the HSPGs agrin, perlecan, glypican-1, and syndecans 1-3 in the lesions of DAT brain neocortex and hippocampus. Using a panel of specific antibodies directed against the protein backbone of the various HSPG species and against the glycosaminoglycan (GAG) side-chains, we demonstrated the following. The basement membrane-associated HSPG, agrin, is widely expressed in senile plaques, neurofibrillary tangles and cerebral blood vessels, whereas the expression of the other basement membrane-associated HSPG, perlecan, is lacking in senile plaques and neurofibrillary tangles and is restricted to the cerebral vasculature. Glypican and three different syndecans, all cell membrane-associated HSPG species, are also expressed in senile plaques and neurofibrillary tangles, albeit at a lower frequency than agrin. Heparan sulfate GAG side chains are also associated with both senile plaques and neurofibrillary tangles. Our results suggest that glycosaminoglycan side chains of the HSPGs agrin, syndecan, and glypican, but not perlecan, may play an important role in the formation of both senile plaques and neurofibrillary tangles. In addition, we speculate that agrin, because it contains nine protease-inhibiting domains, may protect the protein aggregates in senile plaques and neurofibrillary tangles against extracellular proteolytic degradation, leading to the persistence of these deposits.

Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions.

The human parvovirus adeno-associated virus (AAV) infects a broad range of cell types, including human, nonhuman primate, canine, murine, and avian. Although little is known about the initial events of virus infection, AAV is currently being developed as a vector for human gene therapy. Using defined mutant CHO cell lines and standard biochemical assays, we demonstrate that heparan sulfate proteoglycans mediate both AAV attachment to and infection of target cells. Competition experiments using heparin, a soluble receptor analog, demonstrated dose-dependent inhibition of AAV attachment and infection. Enzymatic removal of heparan but not chondroitin sulfate moieties from the cell surface greatly reduced AAV attachment and infectivity. Finally, mutant cell lines that do not produce heparan sulfate proteoglycans were significantly impaired for both AAV binding and infection. This is the first report that proteoglycan has a role in cellular attachment of a parvovirus. Together, these results demonstrate that membrane-associated heparan sulfate proteoglycan serves as the viral receptor for AAV type 2, and provide an explanation for the broad host range of AAV. Identification of heparan sulfate proteoglycan as a viral receptor should facilitate development of new reagents for virus purification and provide critical information on the use of AAV as a gene therapy vector.

Development of an enzyme immunoassay specific for a core protein epitope of a novel small basement membrane associated heparan sulphate proteoglycan from human kidney.

Heparan sulphate proteoglycans are major components of the glomerular basement membrane and play a key role in their molecular organization and function. Moreover, their presence is essential for the maintenance of the selective permeability of the glomerular basement membrane. Recently, we have isolated and characterized a novel, small basement membrane associated heparan sulphate proteoglycan from human aorta and kidney. Using specific monoclonal antibodies we have shown that the novel heparan sulphate proteoglycan is predominantly located in the glomerular basement membrane, to a lesser extent in the basement membrane of tubuli, and also in the mesangium. Turnover or, in the course of kidney diseases, degradation of heparan sulphate proteoglycan from glomerular basement membranes may lead to urinary excretion of heparan sulphate proteoglycan. Therefore, changes in the structure and function of glomerular basement membranes may be directly detected by measuring the excretion of a component of this basement menbrane, e. g. heparan sulphate proteoglycan into urine. Here we describe the establishment of an enzyme immunoassay for the sensitive detection of the novel, small heparan sulphate proteoglycan in urine. In this assay the specific monoclonal antibody 1F10/B8, which recognizes a core protein epitope, was used to detect the polyanionic heparan sulphate proteoglycan bound to the surface of a cationic charge modified microtitre plate. This assay allows the sensitive and specific detection of the small heparan sulphate proteoglycan, which is released from the glomerular basement membrane into urine during normal turnover and also in the course of kidney diseases.

Developmental and FGF-2-Mediated Regulation of Syndecans (1–4) and Glypican in Oligodendrocytes

Differentiating cells undergo developmentally regulated changes in cell–cell and cell–matrix adhesion that control migration through microenvironments, proliferation, and differentiation. The diversity of the patterns of expression of heparan sulfate proteoglycans (HSPGs), coupled with their interactions with extracellular matrix, cell adhesion molecules, and growth factors, has emphasized their critical importance in the regulation of these events. Syndecans (1–4), glypican, and cerebroglycan are membrane-associated HSPGs that have been implicated in these events in various tissues and several tumor cell lines. We have examined the developmental expression and FGF-2-mediated regulation of these HSPGs during differentiation within a specific lineage of primary cells, oligodendrocytes (OL). Northern analyses of highly purified, developmentally synchronized populations of OL-lineage cells at three stages of differentiation (early and late progenitors and mature OLs) showed that the expression of individual forms of these syndecans and glypican are developmentally regulated. Specifically, the level of expression of syndecan-2 and -4 and glypican mRNAs increased as the cells differentiated from proliferative late progenitors to postmitotic mature cells. The expression of syndecan-1 and -3 had the inverse developmental pattern. Therefore, these two sets of molecules may have different roles in regulating the onset of terminal differentiation in OLs. The levels of mRNA expression were regulated by FGF-2: in late progenitors, FGF-2 induced a doubling of the mRNA levels of syndecan-2, -3, and -4, while those for syndecan-1 and glypican remained unaffected; in mature OLs, the levels of syndecan-1 mRNA were up-regulated, the levels of syndecan-2 and -4 and glypican were down-regulated. These results suggest that the individual syndecan molecules have distinct functions during the differentiation process and that multiple levels of regulation must exist, leading to a changing repertoire of these molecules during OL lineage progression and myelinogenesis.

Expression of proteoglycans and hyaluronan during wound healing.

We investigated the expression of proteoglycans (PGs) and hyaluronan (HA) during healing of human mucosal wounds. Biopsy specimens of experimental wounds were taken 1, 3, and 7 days after wounding. Frozen sections were used for immunolocalization of CD44, syndecan-1, basement membrane-associated heparan sulfate proteoglycan (BM-HSPG), decorin, and biglycan. HA was localized in paraffin sections with a specific HA-binding probe. Epithelium showed first signs of migration on Day 1, more progressive migration on Day 3, and epithelial sheets confronted on Day 7. CD44 surrounded migrating keratinocytes at all stages of wound healing. In epithelium, CD44 and HA remarkably localized to the same region. Expression of syndecan-1 was switched from the suprabasal cell layer of unwounded epithelium to the basal cell layer of the migrating wound epithelium. BM-HSPG was absent under migrating keratinocytes. It started to reappear at the basement membrane zone on Day 7. The area under the wound epithelium containing newly synthesized collagen fibers first became positive for decorin on Day 7, whereas staining of biglycan was negative. Granulation tissue was also strongly positive for CD44 and hyaluronan. Our results indicate that migrating keratinocytes express both CD44 and syndecan-1 but not BM-HSPG. During differentiation of keratinocytes, expression of CD44 preceded that of syndecan-1. The results suggest that different HSPGs have multiple functions in keratinocyte migration and differentiation during reepithelialization.

Heparin induces endothelial extracellular matrix alterations and barrier dysfunction

We investigated the influence of heparin on the composition of the subendothelial matrix and on endothelial permeability to elucidate the structure-function relationship of matrix composition and permeability. Albumin flux across the confluent endothelial monolayers was used to assess the macromolecular permeability. Lowest values were obtained when 100% serum was used as medium for permeability studies. The endothelial matrix components, fibronectin and basement membrane-associated heparan sulfate proteoglycan (HSPG), were measured by enzyme immunoassay. Treatment of proliferating endothelial cells with heparin (0-900 micrograms/ml) induced a dose-dependent decrease in endothelial HSPG content, whereas the fibronectin content was unaltered. This structural change was accompanied by an increase in albumin permeability. Both heparin effects exhibited similar dose-response curves with half-maximal effects at approximately 5 micrograms/ml heparin. Acute addition of 300 micrograms/ml heparin had no effect on permeability or HSPG content. When endothelial cells were preincubated with an HSPG antiserum, the endothelial permeability increased nearly threefold. Our results indicate that heparin-induced loss of HSPG may cause the increase in endothelial permeability. The data underline the importance of HSPG for the integrity of the endothelial barrier.

Calcium regulation of heparan sulfate proteoglycans in breast cancer cells.

Breast tumor cells have been shown to be responsive to calcium in that external calcium modifies cell calcium, shape and growth. In order to highlight some of the numerous mechanisms by which calcium is operating, we investigated its influence on the cell microenvironment and particularly its effect on membrane-associated heparan sulfate proteoglycans. The breast cancer cells MCF-7 were grown either at low (0.04 mM) or high (2.5 mM) calcium concentration. After 3 days of culture, cells were labeled with Na2(35)SO4 for 24 h and cell-associated proteoglycans extracted and purified. We showed that calcium enhances approximately twofold the synthesis of sulfated proteoglycans and, among these sulfated proteoglycans, chemical treatments indicated a specific two- to threefold increase of heparan sulfate proteoglycans. In view of the increasing implication of heparan sulfate proteoglycans in numerous mechanisms such as cell-cell contact, cell-matrix interactions and cell growth control, it appears that calcium may be a target for modulating metastatic and growth processes in breast tumor cells.