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Smooth-muscle-cell (SMC) replication and extracellular matrix protein expression are two vital and interrelated processes necessary for normal development of the vasculature. To understand better the nature of this relationship in the developing rat lung, we investigated the relationship between SMC proliferation and the expression of perlecan, a basement membrane (BM) heparan sulfate proteoglycan implicated in the control of SMC growth and differentiation, and tropoelastin (TE), a structural matrix protein not known to influence directly the replicative state of SMCs. Using bromodeoxyuridine (BrdU) incorporation to assess DNA synthesis, we first established the time course of SMC proliferation in the hilar pulmonary artery (PA) from embryonic to adult life. We found a labeling index of > 80% during the embryonic period (embryonic Day 13 [e13] to fetal Day 18 [f18]), a dramatic decline to approximately 40% during the fetal period of development, and a steady decrease in proliferation rates following birth such that, by 30 d of age, a labeling index of < 2% was noted. Using in situ hybridization, we found that although peak expression of both perlecan and TE messenger RNA (mRNA) occurred in the fetal and early postnatal periods following the major decrease in cell replication, TE mRNA expression was clearly observed in the PA as early as embryonic Day 14, whereas perlecan transcripts were virtually undetectable until fetal Day 19. Therefore, to evaluate further the relationship between cell replication and perlecan and/or TE gene expression, we used a combined in situ hybridization/BrdU immunohistochemistry technique and demonstrated that, on an individual cell basis, perlecan message was predominantly expressed by nonreplicating (BrdU-negative) PA, whereas TE mRNA was equally expressed in replicating and nonreplicating PA SMCs. Interestingly, a very similar pattern of replication and relationship to perlecan and TE mRNA expression was noted in airway SMCs and epithelial cells. Thus, in the lung as a whole, maximal expression of both the BM protein perlecan and the interstitial matrix protein TE occurs coordinately and follows the period of maximal SMC proliferation. However, in individual SMCs, perlecan mRNA expression varies inversely with DNA synthesis, whereas TE mRNA expression appears independent of the proliferative state of the cell.

Extracellular matrix (ECM) and basement membrane (BM) components were studied by immunohistological methods in native rat thyroid tissue, and in rat thyroid tissue and FRTL-5 cells cultured in a three-dimensional alginate bead system. In all three situations, the presence of collagen IV, laminin, perlecan, and fibronectin was demonstrated. There were marked differences between rat thyroid tissue and FRTL-5 cells in culture. Rat thyroid tissue maintained a follicular structure, whereas FRTL-5 cells did not form follicles. Rat thyroid cells multiplied more slowly than FRTL-5 cells and thyroglobulin (Tg) was visible in the follicular lumen, while in FRTL-5 cells Tg was only seen intracellularly. Tg iodination was much lower in FRTL-5 cells than in rat cells. In rat thyroid cells, positive staining for collagen IV, laminin, and perlecan was seen in thin membranes around individual follicles, and for fibronectin around groups of follicles. In FRTL-5 cells, these ECM/BM components could be identified, but were not organized into equally regular networks around groups of cells. These results demonstrate that of the two types of cells examined, primary cultures of rat thyroid cells in alginate beads maintain structural and functional similarities to native thyroid tissue and would therefore be suitable for future in vitro studies of thyroidal ECM/BM and their interrelationship with growth and function of this organ. FRTL-5 cells cultured in alginate beads show some functional, but not structural similarities to native thyroid tissue and so would be less valuable for use in such studies.

Syndecan-1 binds basic fibroblast growth factor (bFGF), modulates neovascularization, plays a role in epithelial differentiation and is up-regulated by WT1. Malignant mesothelioma of the pleura is one of the most aggressive tumours known and expresses high levels of angiogenic growth factors. This study has analysed syndecan-1 expression in mesothelioma tumours and cell lines by immunohistochemistry and immunoblotting, using anti-syndecan-1 antibody directed against the core protein, and has examined its relation to morphology, bFGF, WT1, and intra-tumoural microvascular density (IMD). Shedding of syndecan-1 in the conditioned medium of mesothelioma cell lines was detected in variable amounts. These studies indicate that (1) there is no correlation of syndecan-1 with either bFGF expression or IMD in mesotheliomas in vivo; (2) syndecan-1 is strongly expressed in the epithelial type of mesothelioma and in the epithelial component of biphasic mesotheliomas and the expression is reduced or lost in sarcomatoid differentiation; together with the finding that (3) syndecan-1 correlates with WT1 immuno-expression, this suggests that syndecan-1 might relate to the differentiation state of mesothelial/mesothelioma cells; and (4) syndecan-1-positive tumours are associated with a longer survival (p = 0.02) than mesotheliomas with no or little syndecan-1 expression, on univariate analysis. These findings therefore indicate that syndecan-1 can be an important prognostic indicator in mesotheliomas and its loss may be important in the epithelial-mesenchymal transformation of mesothelioma cells.

The effect of topically applied transforming growth factor beta1 (TGF-beta1) on the rat gingival wound healing process after flap surgery was evaluated by immunohistochemistry for extracellular matrix molecules (ECM), such as tenascin, heparan sulfate proteoglycan (HSPG) and type IV collagen, and for proliferating cell nuclear antigen (PCNA) in fibroblasts. TGF-beta1 solution was applied to the surgical wound experimental sites. Two microg/microl were applied at the time of the operation, and 1 microg/microl at days 1 and 2 after surgery, with contralateral control sites receiving the vehicle alone. Periodontal tissues were histologically examined at 3 and 7 days post-surgery. Tenascin was found to be more strongly stained in the granulation tissue from experimental sites at 3 days post-surgery. At 7 days postsurgery, HSPG-positive areas in granulation tissue had become smaller and there was a prominent proliferation of PCNA-positive fibroblast-like cells and type IV collagen-positive blood vessels. These results suggest that TGF-beta1 applied to surgical wounds influences early proliferation of gingival fibroblast-like cells, the formation of blood vessels, and ECM remodelling. In conclusion, TGF-beta1 application appears to promote granulation tissue formation in periodontal wound healing.

Purpose: In vivo and in vitro observations strongly suggest that marked differences exist in the phenotype, growth, and matrix-producing capabilities of distinct smooth muscle cell subpopulations. An earlier study from our laboratory showed differences in matrix metalloproteinase expression patterns in cultures of medial smooth muscle cells from tissue affected by abdominal aortic aneurysm (AAA) or atherosclerotic occlusive disease and from normal arterial tissue. In this study we were interested in ascertaining whether smooth muscle cells from the same sample groups also synthesized different proteoglycan profiles that correlated with vascular disease., Methods: Proteoglycans from smooth muscle cell monolayer cultures from tissue affected by AAA or atherosclerotic occlusive disease and from normal arterial tissue were examined by means of immunoblotting and affinity-blotting composite agarose polyacrylamide gel electrophoresis (CAPAGE) and sodium dodecyl sulphate PAGE. Enzyme-linked immunosorbent assay (ELISA) was used to quantitate perlecan levels in smooth muscle cell monolayer media samples., Results: Versican, perlecan, and biglycan levels were significantly elevated in AAA smooth muscle cell cultures. Two populations of smooth muscle cell versican were identified by means of CAPAGE-immunoblotting and by means of a novel affinity-blotting technique with biotinylated hyaluronan. A small keratan sulfate-substituted proteoglycan was present in similar levels in all smooth muscle cell cultures. This proteoglycan had a free core protein of about 55 kd after keratanase digestion and had a relatively high charge-to-mass ratio, as was evident from its electrophoretic mobility in CAPAGE; this proteoglycan was tentatively identified as keratocan. Immunoblotting with monoclonal antibodies 3-G-10 (anti-delta heparan sulfate, heparan sulfate stubs generated by heparitinase treatment) and 10-E-4 (anti-native heparan sulfate chains) helped identify several smooth muscle cell heparan sulfate-substituted proteoglycans. Elevated levels of intact and processed perlecan core protein were identified in AAA cultures by means of immunoblotting with a monoclonal antibody to perlecan core protein (A76). ELISA measurements confirmed that perlecan levels were significantly higher in AAA smooth muscle cell cultures compared with the normal arterial tissue and tissue affected by atherosclerotic occlusive disease., Conclusions: Because heparan sulfate proteoglycans can bind growth factors, their elevated synthesis by AAA smooth muscle cells in combination with an increased expression of matrix metalloproteinases may at least partly explain the differential proliferative capacity of the AAA smooth muscle cells examined and may govern the pattern of abnormal cellular proliferation and matrix protein synthesis observed in the pathogenesis of vascular disease.

Glycosaminoglycan attachment to perlecan domain I (173 residues) was completely prevented by site-directed mutagenesis of Ser-65, Ser-71 and Ser-76 as shown by recombinant production in mammalian cells. This did not interfere with the proper folding of the domain's SEA module but enhanced its sensitivity to neutral proteases. Lack of substitution also abolished binding to the two major heparin binding sites of laminin-1.

Heparan sulfate proteoglycans (HSPGs) are components of the basement membrane of various tissues. They are composed of a core protein and of the negatively charged glycosaminoglycan side chain heparan sulfate, which is covalently bound to the core protein. We previously found that in both human and murine lupus nephritis, heparan sulfate staining in the basement membrane of the glomerulus is almost completely absent, and that there was an inverse correlation between heparan sulfate staining and glomerular immunoglobulin deposits. As immunoglobulin deposits are also present in the skin of systemic lupus erythematosus patients, we investigated the heparan sulfate staining pattern in the basement membrane of the dermal-epidermal junction. furthermore, we studied whether there was a correlation between heparan sulfate staining and deposition of immunoglobulin in the basement membrane of this junction, and between heparan sulfate staining and the presence of anti-DNA antibodies in the serum. Biopsies of non-lesional skin of 21 systemic lupus erythematosus patients (15 anti-DNA positive and 6 anti-DNA negative patients at the time of biopsy) were stained for the HSPG-core protein (mAb JM-72), highly sulfated stretches within heparan sulfate (JM-13), the low sulfated regions of heparan sulfate (mAb JM-403) and for immunoglobulin depositions. Abnormal and discontinuous staining of the low sulfated parts of heparan sulfate using mAb JM-403 in the basement membrane was found in skin biopsies of 4 out of 15 systemic lupus erythematosus patients with anti-DNA antibodies. In contrast, all specimens of anti-DNA negative patients showed normal continuous staining. Staining with JM-13 and JM-72 showed normal linear staining in both groups. The decreased heparan sulfate staining was correlated significantly with the presence of immunoglobulin deposits in the basement membrane. The subgroup could not be identified by its clinical picture. Our results suggest similar but not identical pathways in systemic lupus erythematosus nephritis and skin of systemic lupus erythematosus patients.

Perlecan is a major heparan-sulfate proteoglycan (HSPG) within the basement membrane surrounding skeletal muscle fibers. The C-terminus of its core protein contains three globular domain modules which are also found in laminin and agrin, two proteins that bind to dystroglycan (DG, cranin) on the muscle surface with these modules. In this study, we examined whether perlecan can also bind to DG and is involved in signaling the formation of the neuromuscular junction (NMJ). By labeling cultured muscle cells with a polyclonal anti-perlecan antibody, this protein is found both within the extracellular matrix in a fibrillar network and at the cell surface in a punctate pattern. In Xenopus muscle cells, the cell-surface perlecan is precisely colocalized with DG. Both perlecan and DG are clustered at ACh receptor clusters induced by spinal neurons or by beads coated with HB-GAM, a heparin-binding growth factor. Blot overlay assays have shown that perlecan binds alpha-DG in a calcium and heparin-sensitive manner. Furthermore, perlecan is present in muscle lysate immunoprecipitated with an anti-DG antibody. Immunolabeling also showed colocalization between HB-GAM and perlecan and between HB-GAM and DG. These data suggest that perlecan is anchored to muscle surface via DG-dystrophin complex. Since DG is also a site of agrin binding, the neural agrin secreted by motoneurons during NMJ formation may compete with the pre-existing perlecan for cell surface binding. This competition may result in the presentation of perlecan-bound growth factors such as HB-GAM to effect synaptic induction.

In the present study we compared the localization of major basement membrane (BM) components and their mRNAs between invasive carcinomas of the breast (adenocarcinomas) and larynx carcinomas (squamous cell carcinomas, SCC), in order to determine the extent of BM production and deposition in malignant tumors of biologically different behaviour. Thus, breast carcinomas usually show a rapid locoregional/systemic spread, while the laryngeal SCCs normally show a more locally restricted growth pattern. While normal mammary glands and laryngeal mucosa revealed an intact epithelial BM as evidenced by a continuous linear staining for collagen IV, laminin-1, heparan sulfate proteoglycan (perlecan) and fibronectin-as well as collagen VII in the larynx mucosa-, this continuous staining was lost in the invasive carcinomas, however, affecting the two tumor types differently. In the breast carcinomas, a complete loss was seen even in well differentiated tumors affecting the various BM components similarly, while in the SCCs well differentiated carcinomas had retained significantly more BM material than poorly differentiated ones. In the SCCs, an "early" loss of collagen VII contrasted with a "later" loss of collagen IV, laminin, perlecan and fibronectin the extent of which was, however, associated with a decreasing degree of differentiation. In contrast to the protein findings, by use of the in-situ hybridization we observed a significant expression of mRNA for collagen IV, perlecan and fibronectin. The resulting pattern was comparable between both tumor types and not significantly related to the tumor cell differentiation. Both tumor cells and stroma cells were positively labelled with a more extensive labelling of the stroma cells. Our observations indicate a similar upregulation of the mRNAs for BM-components in breast and larynx carcinomas, but significant differences in the BM-protein deposition so that either major differences in presumed BM-proteolysis or further translational defects are suggested. Furthermore, it can be speculated that the far lesser amount of BM-material in the breast carcinomas may be linked to the more aggressive metastatic spread of those tumors, particularly when compared to the SCCs.

Heparan sulphate proteoglycans (HSPGs) play important biological roles in cell-matrix adhesion processes and are essential regulators of growth factor actions (e.g., as co-receptor for hepatocyte growth factor). Since in liver carcinogenesis, interactions between cells, the matrix, and growth factors play a major role, the aim of this study was to investigate whether the distribution pattern of HSPGs is altered in human primary liver tumours. Twenty-two primary liver tumours and five normal liver biopsies were studied, using specific monoclonal antibodies against syndecans-1, -2, -3, and -4; glypican; perlecan; and heparan sulphate chains. Cholangiocarcinomas as well as hepatocellular carcinomas showed an altered immunoreactivity pattern of the different HSPGs in comparison with normal liver parenchyma, probably reflecting the growth regulatory roles of HSPGs. Intracellular positivity for integral membrane HSPGs syndecan-1 and especially syndecan-4 was a constant finding in most tumours, suggesting increased synthesis or internalization of these HSPGs. Syndecan-3 and perlecan expression in tumours was found in an expected distribution pattern. The strong reactivity for syndecan-3 and perlecan in tumoral stromal vessels might suggest a role for these HSPGs in tumoral angiogenesis. In addition, perlecan probably exerts its known growth factor reservoir function also in the stroma of primary liver tumours.

The tumor matrix of salivary pleomorphic adenoma (PA) is characteristically rich in glycosaminoglycans (GAGs), which contribute to its complex histoarchitecture. This study evaluated the microscopic localization of various GAGs in 17 PAs, using a panel of anti-GAG monoclonal antibodies and biotinylated hyaluronic acid (HA)-binding protein. Both epithelial and mesenchymal-like tissues were confirmed to contain GAGs. Luminal epithelial cells mostly lacked GAGs, whereas GAGs were seen both in the cytoplasm and cell membrane of non-luminal epithelial cells. In addition, small intercellular accumulations of GAGs were often present in solid epithelial areas, implying the epithelial origin of GAGs. GAGs did not appear to be a main component of the hyaline matrix. The myxoid region was consistently stained for both chondroitin 6-sulfate (CS-6) and HA but variably for chondroitin 4-sulfate (CS-4), dermatan sulfate (DS) and keratan sulfate (KS); heparan sulfate (HS) was not detected. The chondroid region showed increased staining for CS-6 but reduced staining for HA when compared with the myxoid region. In addition, CS-4, DS and KS were seen both in chondroid cells and the territorial matrix, whereas HS was present only in the cells. It is suggested that GAGs in PA are mainly produced by non-luminal cells and influence the proliferation, differentiation, secretory activity and shape of tumor cells, thus contributing to the morphological diversity of this tumor.

The destruction of the epithelial basement membrane is widely regarded as a clear criterion for invasive malignant tumor growth. Since, however, defects in the basement membrane may also occur in non-invasive conditions, such as inflammatory and proliferative lesions, and since it has been shown that particularly in highly differentiated squamous cell carcinomas a continuous basement membrane is mimicked by the presence of isolated components, this criterion seems to be of minor value for the diagnosis of malignancy. Despite these drawbacks, the immunolocalization of basement membrane material may still be of differential diagnostic significance in certain situations. This holds particularly true for invasive (ductal) breast carcinomas, which usually completely lack a basement membrane. Accordingly, sclerosing adenosis can be distinguished from invasive carcinoma, as a distinction can be made between neoplastic (malignant) tubular formations and reactive lesions.

Several animal models have been developed for the mucopolysaccharidoses (MPSs), a group of lysosomal storage disorders caused by lysosomal hydrolase deficiencies that disrupt the catabolism of glycosaminoglycans (GAG). Among the MPS, the MPS-III (Sanfilippo) syndromes lacked an animal counterpart until recently. In this investigation of caprine MPS-IIID, the clinical, biochemical, morphological, and immunohistochemical studies revealed severe and mild phenotypes like those observed in human MPS III syndromes. Both forms of caprine MPS IIID result from a nonsense mutation and consequent deficiency of lysosomal N-acetylglucosamine 6-sulfatase (G6S) activity and are associated with tissue storage and urinary excretion of heparan sulfate (HS). Using special stains, immunohistochemistry, and electron microscopy, secondary lysosomes filled with GAG were identified in most tissues from affected goats. Primary neuronal accumulation of HS and the secondary storage of gangliosides were observed in the central nervous system (CNS) of these animals. In addition, morphological changes in the CNS such as neuritic expansions and other neuronal alterations that may have functional significance were also seen. The spectrum of lesions was greater in the severe form of caprine MPS IIID and included mild cartilaginous, bony, and corneal lesions. The more pronounced neurological deficits in the severe form were partly related to a greater extent of CNS dysmyelination. These findings demonstrate that caprine MPS IIID is a suitable animal model for the investigation of therapeutic strategies for MPS III syndromes.

The invasion of extravillous trophoblast cells into the maternal endometrium is one of the key events in human placentation. The ability of these cells to infiltrate the uterine wall and to anchor the placenta to it as well as their ability to infiltrate and to adjust utero-placental vessels to pregnancy depends, among other things, on their ability to secrete enzymes that degrade the extracellular matrix. Most of the latter enzymes belong to the family of matrix metalloproteinases. Their activity is regulated by the tissue inhibitors of matrix metalloproteinases. We have studied the distribution patterns of matrix metalloproteinases-1, -2, -3, and -9 and their inhibitors TIMP-1 and TIMP-2 as compared to the distribution of their substrates along the invasive pathway of extravillous trophoblast of 1st, 2nd, and 3rd trimester placentas by means of light microscopy on paraffin and cryostat sections as well as at the ultrastructural level (only 3rd trimester placenta). The comparison of different methods proved to be necessary, since the immunohistochemical distribution patterns of these soluble enzymes are considerably influenced by the pretreatment of tissues. All three methods revealed immunoreactivities of both, proteinases and their inhibitors, not only intracellularly in the extravillous trophoblast but also extracellularly in its surrounding matrix, the distribution patterns depending on the stage of pregnancy and on the degree of differentiation of trophoblast cells along their invasive pathway. Within the extracellular matrix, immunolocalization of matrix metalloproteinases as well as their inhibitors showed a specific relation to certain extracellular matrix molecules.

The distribution of major components of the basement membrane, such as type IV collagen, laminin, and heparan sulfate proteoglycan (HSPG), was investigated in the rat cochlear duct. Immunofluorescence demonstrated that type IV collagen, laminin and HSPG were distributed along capillaries in the cochlear duct, including the stria vascularis, spiral ligament, spiral prominence and spiral limbus. Additionally, type IV collagen, laminin and HSPG were found to be distributed from the basement membrane of Reissner's membrane to that of the spiral prominence in a linear pattern. The scala media was surrounded by these basement membrane components, demarcating endolymph from perilymph, along epithelial cells except at the stria vascularis. These findings suggest that type IV collagen, laminin and HSPG create the anatomical separation between endolymph and perilymph, thus indicating that they may be involved in the regulation of fluid transport between the endolymph and perilymph.

It is known from previous work that tau is the main component of paired helical filaments (PHFs) and that it can assemble in vitro into polymers resembling PHFs when high concentrations of protein are used. In the search for molecules that can facilitate tau polymerization, a component of neurofibrillary tangles, heparan sulfate (or its more sulfated form, heparin), and other glycosaminoglycans have been tested. Glycosaminoglycans, in the sulfated but not in the unsulfated form, facilitate not only tau assembly but also the formation of polymers resembling PHFs. Conversely, PHFs were found to contain heparan sulfate and chondroitin sulfate. Heparinase or chondroitinase treatment of PHFs results in the formation of straight structures. All of these results suggest a role for sulfated glycosaminoglycans in determining the helicity of PHFs.

Heparan sulfate proteoglycans (HSPG) have been shown to be involved in the activation of tyrosine kinase receptors by basic fibroblasts growth factor (bFGF), a strong inhibitor of skeletal muscle differentiation. Skeletal muscle fibers contact extracellular matrix (ECM) that surrounds individual fibers (endomysium) and bundles of several fibers (perimysium). Perlecan is a HSPG present in the majority of basement membranes. In this study we evaluated the expression and localization of perlecan during differentiation of C2C12 skeletal muscle cells. C2C12 myoblasts incubated with [35S]Na2SO4 synthesize a HSPG that can be specifically immunoprecipitated with antibodies against murine perlecan. The immunoprecipitated HSPG eluted from a Sepharose CL-4B with a Kav of 0.44. Analysis of the core protein of the HSPG immunoprecipitated from [35S]methionine-labeled C2C12 after treatment with heparitinase revealed two polypeptides of 170 and over 300 kDa. The amount of polypeptides immunoprecipitated decreased with muscle differentiation. Immunocytolocalization studies indicate that perlecan is localized on the myoblast surface and by immunogold staining we have demonstrated that it is associated with patches of incipient extracellular matrix. The expression of perlecan mRNA decreased substantially during skeletal muscle differentiation, in contrast to the increase in transcripts for specific skeletal muscle proteins such as myogenin and creatine kinase. By immunofluorescence microscopy almost no perlecan staining associated with the surface of myotubes was observed. All these results suggests that perlecan, a HSPG that binds myogenic inhibitory bFGF, normally associated with basement membranes in adult tissues is present on the surface of myoblasts and its expression is down regulated during skeletal muscle differentiation.

Proteoglycans interact with soluble proteins such as growth factors and thereby regulate extracellular signals. During B lymphocyte maturation, secretion of proteoglycans may be functionally related to the different requirements of the respective maturation stage. In order to address this question we compared structures of proteoglycans released by three B lymphocyte lines which correspond to different maturation stages. Plasma-cell type U266 cells secreted the largest proteoglycans (150 kDa), followed by mature B cells JOK-1 (130 kDa) and pre-B cells Nalm 6 (90 kDa). On average, secreted proteoglycans carried four glycosaminoglycan chains with molecular masses ranging each from 32 kDa (U266) to 23 kDa (Nalm 6). All three cell lines secreted more than 90% of their proteoglycans possessing chondroitin sulfate chains having chondroitin-4-sulfate (delta Di-4S) as the prevalent disaccharide unit. In these proteochondroitin sulfates, unsulfated chondroitin (delta Di-0S) was present in smaller quantities and chondroitin-6-sulfate (delta Di-6S)-containing proteoglycan was released only by Nalm 6 and U266 cells. Cell line Nalm 6 exclusively produced proteochondroitin sulfate, whereas in culture medium of JOK-1 and U266 a small amount of proteoheparan sulfate was found also. In all three cell lines, treatment with chondroitinase ABC released a protein of 30 kDa and chemical deglycosylation resulted in a core protein of 21 kDa. In addition to pure proteochondroitin sulfate, a small portion of proteoheparan sulfate with a protein moiety of 30 kDa was detected after heparitinase treatment in supernatants of JOK-1 and U266. Thus, our results indicate that released proteoglycans may undergo modulations in their glycosaminoglycan moieties during B-cell differentiation. This may have functional consequences at the level of growth factor regulation.

Heparan sulfate proteoglycans (HSPGs) are essential components of the glomerular basement membrane (GBM) carrying a strong anionic charge. A well-characterized extracellular HSPG is perlecan, ubiquitously expressed in basement membranes. A cDNA construct encoding domains I and II of human perlecan was expressed as a fusion protein with glutathione S-transferase. This fusion protein was used to generate monoclonal antibody 95J10. We compared the staining pattern of 95J10 with that of M215, a previously prepared mAb that recognizes HSPG isolated from human GBM. In kidney cortex, the anti-perlecan mAb 95J10 showed a strong staining of the mesangium, Bowman's capsule, the tubular basement membrane, and stained the GBM only slightly. In contrast, M215 predominantly stained the GBM in a linear fashion. Immunoelectron microscopy supported these results, showing concentrations of perlecan in some regions of the GBM, whereas the unidentified M215 antigen was homogenously distributed throughout the GBM. In other human tissues, both antibodies also produced a different staining pattern. Furthermore, a polyclonal antiserum recognizing HSPG isolated from the GBM did not recognize perlecan from EHS tumors. These results provide evidence for the presence of another HSPG in the GBM that is immunologically distinct from perlecan. The absence of perlecan splice variants in the kidney suggests that this component is encoded by a different gene than perlecan. Given its marked expression in the GBM, this component could be a determining factor in the maintenance of selective glomerular permeability.

The biosynthesis and hydration of glycosaminoglycans (GAG) has been implicated in the generation of palatal shelf-elevating force(s) in mammals, although the nature of the palatal shelf extracellular matrices during cleft palate formation remains poorly understood. This study quantifies the GAG composition in the palatal shelves of Wistar rat fetuses at various periods of palatogenesis where clefts were induced experimentally using 5-fluoro-2-deoxyuridine (FUDR). For both normal and cleft palatal shelves, hyaluronan, heparan sulphate and chondroitin-4-sulphate were detected but not dermatan sulphate or chondroitin-6-sulphate. Throughout the period of cleft development studied, the total amount of GAG was significantly decreased (by approx. 30%) compared with normal development, this decrease being particularly marked at a time equivalent to post-elevation during normal development (approx. 75%). Furthermore, and unlike normal palatogenesis, no significant differences were recorded between the anterior and posterior parts of the palatal shelves during cleft formation. As for normal palatogenesis, however, the percentages of each GAG were not altered at any stage. The findings are consistent with the view that suppression of GAG biosynthesis is related to the development of cleft palate in FUDR-treated rat fetuses and can therefore be interpreted as providing evidence of a role for the mesenchymal glycoconjugates in shelf elevation during normal palatogenesis.

Previous studies have shown that expression of the heparan sulfate proteoglycan, perlecan, on the external trophectodermal cell surfaces of mouse blastocysts increases during acquisition of attachment competence. However, it is not clear if this change in perlecan protein expression also is reflected at the level of perlecan mRNA expression. In the present investigation, the spatial and temporal patterns of perlecan mRNA expression in the mouse embryo during the periimplantation period were examined by in situ hybridization and reverse transcriptase-polymerase chain reaction. In addition, a delayed implantation model was used to determine the expression of perlecan mRNA and protein in dormant and estrogen-activated hatched blastocysts. The results demonstrate that perlecan mRNA expression is low in morulae, but increases in Day 4 blastocysts, attaining maximal expression in Day 4.5 attachment-competent blastocysts. In contrast, perlecan mRNA is detected in both the dormant and estrogen-activated delayed blastocysts; however, within 12 hr of blastocyst activation by estrogen, both perlecan protein and heparan sulfate chain expression markedly increase. Taken together, these results suggest that during normal development perlecan mRNA expression increases with the acquisition of attachment competence. Moreover, perlecan protein expression also is attenuated during delayed implantation and appears to increase in response to nidatory estrogen, perhaps via the increased translation of preexisting perlecan mRNA.

Aim: To investigate the chemical pathology in the blood and lens, in cases of congenital or infantile cataract in children excreting predominantly non-reducing carbohydrates in urine., Methods: Urine samples from children with congenital or infantile cataract, and age and sex-matched controls, were analysed for (i) inherited errors of metabolism, (ii) paper chromatography of sugars, (iii) spectrophotometric assay of glycosaminoglycans (GAG), (iv) cetyl trimethyl ammonium bromide test, (v) electrophoresis using Alcian blue, (vi) ion exchange chromatography with IR 120 resin, and (vii) HPLC for xylose. Blood and lens material were also tested for GAG fragments and xylose. beta Glucuronidase was assayed in lymphocytes and urine., Results: Of 220 children of both sexes below 12 years of age, with congenital or infantile cataract treated in Sankara Nethralaya, Madras, India, during a period of 2 years, 145 excreted fragments of GAG (heparan and chondroitin sulphates) in their urine. There was no such excretion among the control group of 50 children. The same was found accumulated in the blood and lenses of affected children. In addition, xylose was present in small amounts in the urine and blood and xylitol was present in the lens. There was a significant elevation in the activity of beta glucuronidase in lymphocytes and urine, when compared with normals. All the above findings suggest deranged proteoglycan metabolism. As the urine contained mostly GAG fragments and very little xylose, Benedict's reagent was not reduced. This ruled out galactosaemia., Conclusion: An increase of beta glucuronidase activity might have caused extensive fragmentation of GAG with resultant accumulation in the blood and lens and excretion in urine. Small amounts of xylose may have come from xylose links between GAG and core protein of proteoglycans. Owing to their polyanionic nature, GAG fragments in the lens might abstract sodium, and with it water, thereby increasing the hydration of the lens. Excessive hydration and the osmotic effect of xylitol from xylose might cause cataract. While corneal clouding has been reported in inborn acid mucopolysaccharidosis, congenital or infantile cataract with deranged metabolism of proteoglycans (acid mucopolysaccharide-xylose-protein complex) is reported in children for the first time.

General agreement exists on the correlation of renal insufficiency with the severity of tubulointerstitial abnormalities in the biopsy. This could not be shown for the severity of glomerular pathology by semiquantitative methods. The relation between renal function and glomerular pathology was therefore evaluated in patients with various kidney diseases using quantitative measurements. Fifty-five patients and ten controls were studied. Histomorphometric measurements of renal biopsies were obtained for both frozen and paraffin sections and were correlated with serum creatinine values. The frozen sections were stained with an indirect immunoperoxidase technique using antibodies against collagen types I, IV, V, VI, laminin, fibronectin, decorin and heparansulphate proteoglycan core protein. The contribution of each component to the composition of the mesangial extracellular matrix was scored with a semiquantitative technique. All glomerular histomorphometric indices correlated with the severity of renal insufficiency expressed as serum creatinine at the time of biopsy. However, quantitative estimates of the glomerular deposition of periodic acid-Schiff positive extracellular matrix seemed to be the most important structural correlate of renal function (r = 0.524, p = 0.0001). Semiquantitative estimates of interstitial extracellular matrix accumulation were less correlated with renal function (r = 0.370, p = 0.01). The composition of the mesangial extracellular matrix did not differ in patients and controls, with the exception of the extent of laminin staining, which was significantly higher in patients. The extent of fibronectin staining in patients correlated with the severity of glomerular structural abnormalities. This study demonstrates that the severity of renal insufficiency in a variety of renal diseases correlates with the severity of glomerular pathology, when quantitative scoring is applied.

The protein encoded by ras and src protooncogenes are frequently activated in a constitutive state in human colorectal cancer. In this study, we investigated the effect of oncogenic p21ras and Py-MT/pp60c-src on the synthesis of syndecan-1, a membrane anchored proteoglycan playing a role in cell-matrix interaction and neoplastic growth control. To this end, we used Caco-2 cells transfected with an activated (Val-12) human Ha-ras gene or the polyoma middle T (Py-MT) oncogene, a constitutive activator of pp60c-src tyrosine kinase activity. As compared to control vector-transfected Caco-2 cells, both oncogene-transfected cells exhibited: (1) a decrease in syndecan-1 specific activity; (2) a decrease in size and sulfation of syndecan-1 ectodomain glycosaminoglycan side chains; and (3) an active heparanase specifically degrading the heparan sulfate chains. In conclusion, the tumorigenic progression induced by oncogenic p21ras or Py-MT/pp60c-src is associated with marked alterations of syndecan-1 at the post-translational level.

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.

In the present paper immunocytochemical analysis at the fluorescence microscopical level has been performed of neural cell adhesion. molecule (NCAM) immunoreactivity in the adult rat tel- and diencephalon in order to further substantiate the highly selective neuronal localization of NCAM immunoreactivity, using an affinity purified rabbit antiserum recognizing homologous NCAM proteins from rat brain. Also, double immunolabelling experiments were performed with monoclonal antibodies specific for heparan sulfate related epitopes or gamma-aminobutyric acid (GABA) to establish in which cell populations a colocalization existed with immunoreactive heparan sulfate proteoglycans of GABA. Within the neocortex NCAM immunoreactivity was exclusively localized to the area of the cell membrane of soma and proximal dendrites of subsets of large pyramidal nerve cells of the layer 5 of the frontoparietal cortex. Within the dorsal hippocampus, the NCAM immunoreactivity was exclusively located to the cell surface area of the pyramidal cell bodies of area CA2. Two colour immunofluorescence procedures demonstrated a colocalization of NCAM and 3G10 but not 10E4 immunoreactivities in the cell surface area of many of the NCAM-positive nerve cell bodies of these two regions. Within the thalamus, strong NCAM immunoreactivity was exclusively demonstrated at all rostrocaudal levels of the reticular thalamic nucleus. The horizontal band of NCAM immunoreactivity was not continuous, but split up into patches of NCAM immunoreactivity within groups of nerve cell bodies. When analysing the number of cells per unitary square in the rostrocaudal direction, a significant increase of positive cells was found in the rostral and middle thirds versus the caudal third of the reticular thalamic nucleus. Many of the cell bodies with NCAM immunoreactivity in their cell surface are showed cytoplasmic GABA immunoreactivity. In the three regions shown to contain NCAM immunoreactivity, proteins of the NCAM type may play a special role for the maintenance of the synaptic structure. The findings also suggest that the sulfated proteoglycans and NCAM can interact in the regulation of cell-cell interaction via adhesion. In the reticular thalamic nucleus NCAM molecules may be part of a set of cell-adhesion molecules involved in a structural organization of the nucleus, which allows it to play a key role in relating cortical maps to thalamic maps.

Cell-surface proteoglycans (PGs) were extracted with Triton X-100 from rabbit articular chondrocytes cultured in monolayers and in alginate beads. They were first purified on DEAE-Trisacryl columns and the proportion of hydrophobic PGs was determined by both Octyl-Sepharose chromatography and partitioning in Triton X-114. These two methods revealed that the proportion of hydrophobic PGs was higher in monolayer culture system as compared to alginate beads (24 and 15%, respectively). Characterization of the PGs by Sepharose CL 6B gel filtration followed by electrophoresis indicated that the PGs isolated from monolayers were composed of three chondroitin sulfate (CS) PGs (core proteins of 180, 100 and 50 kDa) and a heparan sulfate (HS) PG (core protein of 60 kDa). In the alginate system. CSPGs with core proteins of 180, 45 and 32 kDa were observed, but no HSPG was present. In parallel, the effect of TGF-beta on the distribution of membrane-associated PGs was studied. The results showed that the synthesis of cell-surface PGs was stimulated by TGF-beta in monolayers whereas it was inhibited in alginate beads, but the amount of hydrophobic PGs was not altered by the growth factor. These data clearly indicate that TGF-beta induces a differential expression of the PG families present at the cell surface. Taken together, the results reveal the complex regulation of cell-surface PG distribution, which obviously depends on the culture method used and suggest that rabbit articular chondrocytes may differentially respond to extracellular ligands according to their morphological state and environment.

A decrease in anionic change and the loss of heparan sulfate proteoglycan have previously been observed in the glomerular basement membrane (GBM) during diabetic glomerulosclerosis. We studied the chronological changes in the anionic character and the glycosaminoglycan content in the GBM of WBN/ Kob rats with spontaneous diabetes. Two types of cationic probes were used: polyethyleneimine (PEI) and cationic colloidal gold (CCG). Immunogold labeling was performed with anti-monoclonal-heparan-sulfate-glycosaminoglycan (HS-GAG) and anti-chondroitin-sulfate-glycosaminoglycan (CS-GAG) antibodies. The GBM width, the anionic sites and the GAG sites were investigated in diabetic WBN/Kob rats at 2, 10 and 19 months, compared with control rats. Diabetes was confirmed in WBN/Kob rats after 8 months in this study. The GBM width gradually thickened with age. The PEI anionic sites significantly decreased in the lamina rara externa (LRE) at 19 months (vs. 2 and 10 months). The HS-GAG sites also significantly decreased in the LRE at 10 and 19 months (vs. 2 months). However, the CCG anionic sites and the CS-GAG sites significantly increased in the LRE and the lamina densa at 10 months (vs. 2 months) and, after 19 months, returned to the level seen at 2 months. Results indicate that there is an early transient increase in CS-GAG in the GBM while HS-GAG decreases. We noticed a transient increase in the CCG anionic sites at this early stage of diabetic glomerulosclerosis as well. The increase in CS-GAG may provide a marker for early diabetic changes in the GBM.

The possible involvement of proteoglycans in the pathogenesis of emphysema was studied in rats by a single intratracheal instillation of p-nitrophenyl-beta-D-xylopyranoside (beta-D-xyloside), an inhibitor of proteoglycan synthesis. The first 3 days after instillation are characterized by mild hemorrhages, some infiltration of inflammatory cells, and edema. After 1 wk, lung morphology is normal again. Forty days after instillation, considerable parenchymal destruction has occurred as determined by the mean linear intercept (81 +/- 12 microns versus 57 +/- 5 microns for control [P < 0.001]). Pulmonary fibrosis is not observed. Instillation with p-nitrophenyl-alpha-D-xylopyranoside and p-nitrophenol do not induce parenchymal destruction, indicating the specificity of beta-D-xyloside action. Urinary glycosaminoglycan (GAG) content of the beta-D-xyloside-treated rats is increased 15-fold during the first day after instillation, mainly due to elevated levels of chondroitin sulfate and dermatan sulfate. The increase is correlated to the extent of parenchymal destruction after 40 days (r = 0.68; P < 0.002). At day 2 and thereafter, levels are normal again. A short-term increase in dermatan and chondroitin sulfate content is also observed in serum, bronchoalveolar lavage (BAL) fluid, and lung tissue. Heparan sulfate content is decreased in BAL fluid and lung tissue. Instillation with p-nitrophenyl-alpha-D-xylopyranoside and p-nitrophenol do not induce elevated GAG concentration in urine. We suggest that a disturbance in proteoglycan synthesis accompanied by an increase of (beta-D-xyloside-primed) free GAGs results in loss of stability and integrity of the alveolar wall, leading to parenchymal destruction and emphysematous lesions. beta-D-xyloside treatment may be an alternative experimental method for inducing emphysema.

In the mammalian eye, FGF plays a key role in the induction of lens fibre differentiation and, in other systems, heparan sulphate proteoglycans (HSPGs) have been shown to modulate FGF activity. HSPGs were isolated from the anterior and posterior rat and bovine lens capsule and assessed in terms of their ability to bind FGF-1 and FGF-2. In the rat, at least four HSPGs were identified with molecular weights of 142, 166, 200 and approximately 250 kD, the latter species predominating. The capsule HSPGs bound both FGF-1 and FGF-2. There appeared to be little, if any, competition for binding between FGF-1 and FGF-2. The capsule contained substantial amounts of core protein, which did not bind FGF, with a higher core protein/HSPG ratio in the anterior than in the posterior capsule. This was the only major HSPG-related difference noted between anterior and posterior capsule.

An enzyme- linked immunosorbent assay (ELISA) for heparan sulfate proteoglycan (HSPG) was developed based on the high affinity binding profile of HSPG to lipoprotein lipase (LPL). LPL was shown to bind to precoated HSPG in dose dependent manner and was determined spectrophotometrically using specific anti- LPL antibody. This ELISA allowed to evaluate HSPG produced by PC12 cell with clear linearity at range of 10 - 500 ng/ml. Soluble chondroitin sulfate proteoglycan (CSPG) from rat brain, which was not detectable by this method, did not exhibit any inhibitory effects on affinity binding of HSPG to LPL, even if 8 times higher concentrations of CSPG to HSPG was added. The sensitivity of this ELISA was about 100 times higher than that of conventional carbazole reaction method. These findings indicated its potential usefulness of this method for measuring small amounts of HSPG capable of binding to LPL and for studying biological implications of HSPG - LPL interaction.

To gain insight into the contribution of immunologic and hemodynamic factors in the progressive demise of structure and function in chronic renal allograft dysfunction, we studied the histological changes, the immunostainable glomerular anionic sites, and glomerular capillary hydrostatic pressures of rat renal allografts with chronic rejection. Recipient animals were left untreated, received 8 weeks of treatment with the immunosuppressive drug cyclosporine, or received antihypertensive drugs consisting of the combination of reserpine, hydralazine and hydrochlorothiazide, the angiotensin-converting enzyme inhibitor cilazapril, or the angiotensin II receptor blocker L-158,809. Grafts in untreated recipients developed chronic interstitial inflammation, as well as vascular and glomerular lesions consistent with chronic rejection. These lesions were associated with immunohistochemical loss of the negatively charged heparan sulfate proteoglycan side chain. All treatment regimens decreased the systemic and glomerular capillary pressures and were associated with no loss of function, decreased proteinuria, and a tendency to improved graft function. Cyclosporine prevented all histological manifestations of rejection, and antihypertensive drugs decreased the extent of glomerular mesangiolysis and glomerulosclerosis; L-158,809 and cilazapril also inhibited graft atherosclerosis and tubular atrophy. We conclude that chronic rejection is primarily an immune-mediated process, but hemodynamic and angiotensin II-mediated effects may play a pivotal role in the expression of immune-mediated lesions.

Although drug-induced gingival hyperplasia has been extensively studied, the pathogenesis of this disorder has not been clarified to date. Transforming growth factor beta (TGF beta) and basic fibroblast growth factor (bFGF) have been shown to be implicated in diverse fibrotic and hyperplastic diseases. Heparan sulphate proteoglycan (HSPG), which is composed of heparan sulphate glycosaminoglycan (HSGAG), has also been shown to play an important role in the pathogenesis of tissue overgrowth by enhancing the functions of bFGF. However, the possible implication of these growth factors in gingival hyperplasia has not been studied. Immunohistochemical localization of TGF beta, bFGF, their receptors and HSGAG was studied in 4 nifedipine-induced and 5 phenytoin-induced hyperplastic gingival tissues, and 5 non-hyperplastic control gingival tissues to elucidate the pathogenesis of this disease. Significant immunostaining against TGF beta, bFGF, the receptors of these two growth factors and HSGAG was observed in the lamina propria of hyperplastic gingival tissues while less immunostaining was observed in the controls. The mean numbers of immunostained cells against TGF beta, bFGF, their receptors in a square unit (0.1 x 0.1 mm) of the lamina propria, which were counted to 10 units of each hyperplastic gingival tissue, were significantly higher than those of the controls. The results suggest that the increased synthesis of TGF beta, bFGF, their receptors and HSGAG may be related to the pathogenesis of drug-induced gingival hyperplasia.

The qualitative and quantitative pattern of GAGs was examined by electrophoresis in aging chicken brain in four different stages starting from day 1 to 30 months. GAG content referred to defatted dry tissue exhibits constant decrease. Four main GAGs have been identified with a mobility corresponding to hyaluronate, condroitin sulfate, heparan sulfate and dermatan sulfate controls. Hyaluronate appears the main GAG represented while dermatan sulfate the minor one. Our data show that in chicken brain GAG percentage undergoes age-related changes.

Leukocyte migration from circulation into tissue depends on leukocyte integrin-mediated adhesion to endothelium, but integrins cannot function until activated. However, it remains to be understood how tumor cells adhere to endothelium and infiltrate into underlying tissue. We studied mechanisms of extravasation of leukemic cells using adult T cell leukemia (ATL) cells and report the following novel features of cell surface heparan sulfate proteoglycan on ATL cells in ATL cell adhesion to endothelium: ATL cells adhere to endothelial cells through already activated integrins without exogenous stimulation; different from any other hematopoietic cells, ATL cells express a characteristic heparan sulfate capable of immobilizing heparin-binding chemokine macrophage inflammatory protein (MIP)-1 beta, a potent T cell integrin trigger, produced by the cells themselves; competitive interruption of endogenous heparan sulfate proteoglycan synthesis reduces cell surface MIP-1 beta and prevents ATL cells from integrin-mediated adhesion to endothelial cells or intercellular adhesion molecule-1 triggered through G-protein. We propose that leukemic cells adhere to endothelial cells through the adhesion cascade, similar to normal leukocyte, and that the cell surface heparan sulfate, particularly on ATL cells, is pivotally involved in chemokine-dependent autocrine stimulation of integrin triggering by immobilizing the chemokine on them.

The submandibular gland proteoglycans were investigated biochemically and immunohistochemically in male Sprague-Dawley rats. Proteoglycans were extracted with 4 M guanidine-HCl, followed by ultracentrifugation in a CsCl density gradient, and fractionated by ion-exchange chromatography and gel filtration. The molecular weight of PGs was estimated by SDS-PAGE and immunoblot analysis with monoclonal antibodies (HepSS-1 or 6-B-6). The glycosaminoglycan side-chains in the proteoglycan fractions were identified by electrophoresis on cellulose acetate membrane. Three proteoglycan fractions were obtained. One was a heparan sulphate proteoglycan that migrated as a diffuse band of about 210 kDa. The other two fractions contained at least two dermatan sulphate proteoglycans of 70-85 kDa and 40-50 kDa. Digestion of these two proteoglycans with chondroitinase ABC, but not heparitinase, produced two bands of 50 and 21 kDa, which were core proteins. The smaller dermatan sulphate proteoglycan may be a portion of the other, as the core protein of both bound to 6-B-6 antibody, and sugar chains of both were the same (20-30 kDa). Heparan sulphates recognized by antibody HepSS-1 were observed widely in the basement membrane, fibrous connective tissue, and striated and excretory ductal cells, while dermatan sulphate proteoglycans recognized by antibody 6-B-6 were located in the connective tissue surrounding striated and excretory ducts.

With advancing age clusters of abnormal granules positive for periodic acid-Schiff appear in the hippocampus of C57BL/6 (B6) mice and the senescence-accelerated mouse (SAM) P8. The granules can also be visualized with a polyclonal antibody to a 110,000 mol. wt laminin-binding protein and stain specifically with a monoclonal antibody to heparan sulfate proteoglycan. The present study used light and electron-microscopic analysis to compare the staining and morphological properties of these granules in SAM P8 hippocampus with those in B6 hippocampus at different ages. The results of the light-microscopic analysis revealed that granules in SAM P8 and B6 had similar morphology, staining characteristics and distribution patterns, and appeared to have a close association with astrocytic process. The onset of granules in SAM P8 mice (at two to three months of age) was earlier than that observed in B6 mice (at four to six months of age), but the maximum incidence was similar in both strains. Electron-microscopic analysis revealed that the granules in SAM P8 and B6 mice also had a very similar ultrastructure. Granules in both strains were surrounded by a discontinuous membrane and contained mostly crystalline-like, degenerated material. The successive ultrastructural changes from the exterior to interior of the granules suggest that the degenerative process was initiated outside the granules and that degenerative structures migrate inward. Astrocytes and heparan sulfate proteoglycan are closely associated with beta-amyloid deposits in Alzheimer's disease. The presence of astrocyte-associated heparan sulfate proteoglycan-positive material in aged SAM P8 and B6 mice might model age-related alterations in glia function possibly involved in human cerebral amyloidogenesis.

The Escherichia coli K5 capsular polysaccharide has the same (GlcUA-->GlcNAc)n structure as the nonsulfated heparan sulfate/heparin precursor polysaccharide. A monoclonal antibody (mAb 865) against the K5 polysaccharide has been described (Peters, H., Jürs, M., Jann, B., Jann, K., Timmis, K. N., and Bitter-Sauermann, D. (1985) Infect. Immun. 50, 459-466). In this report, we demonstrate the binding of anti-K5 mAb 865 to N-acetylated sequences in heparan sulfates and heparan sulfate proteoglycans but not to heparin. This is shown by direct binding and fluid phase inhibition of mAb 865 in an enzyme-linked immunosorbent assay. In this system we found that the binding of the mAb decreased with increasing sulfate content of the polysaccharide. By testing chemically modified K5 and heparin polysaccharides, we found that each of the modifications that occur during heparan sulfate (HS) synthesis (N-sulfation, C-5 epimerization, and O-sulfation) prevents recognition by mAb 865. Samples of heparan sulfate from human aorta (HS-II) were selectively degraded so as to allow the separate isolation of N-sulfated and N-acetylated block structures. N-Sulfated oligosaccharides (obtained after N-deacetylation by hydrazinolysis followed by nitrous acid deamination at pH 3.9) were not recognized by mAb 865, in contrast to N-acetylated oligosaccharides (obtained after nitrous acid deamination at pH 1.5), although the reactivity was lower than for intact HS-II. Analysis of the latter's pH 1.5 deamination products by gel filtration indicated that a minimal size of 18 saccharide units was necessary for antibody binding. These results lead us to propose bivalent antibody-heparan sulfate interaction, in which both F(ab) domains of the mAb interact with their epitopes, both of which are present in a single large (>/=18 saccharide units) N-acetylated domain and additionally with single epitopes present in two N-acetylated sequences (each <18 saccharide units) bridged by a short N-sulfated domain. Immunohistochemistry with mAb 865 on cryostat sections of normal human kidney tissue, revealed its binding to most but not all renal basement membranes. However, all renal basement membranes contain heparan sulfate, as shown by a mAb against heparitinase-digested heparan sulfate stubs (mAb 3G10). This finding indicates that not all heparan sulfate chains present in basement membranes express the mAb 865 epitopes. Besides the normal distribution, mAb 865 staining was found in fibrotic and sclerotic lesions in vessels, interstitium, and mesangium in transplant kidneys with chronic vascular rejection. Occasionally, a decrease of staining was observed within tubulo-interstitium and glomeruli. These findings show that N-acetylated sequences in heparan sulfates can be demonstrated by anti-K5 mAb 865 in normal and diseased kidneys.

It has been proposed that basic fibroblast growth factor (basic FGF) mediates the neovascular response in a variety of conditions, including diabetic retinopathy and branch retinal vein occlusion. To test the hypothesis that basic FGF was released from retinal stores as a result of retinal ischaemia, transient retinal ischaemia was induced, followed by 48 h of reperfusion, in the rat by combined central retinal vasculature and optic nerve ligation. The immunolocalization of basic FGF was studied in the retina. We found that basic FGF in the normal retina is present around the deeper retinal vessels and in the neuronal tissue of the outer plexiform layer. In the eyes that had ischaemia followed by reperfusion, there was moderate cellular oedema with retinal swelling, and mitoses in the inner nuclear and plexiform layers. There were no changes evident at the immunohistochemical level either in the intensity or distribution of stores of basic FGF. We conclude from these data that stores of basic FGF are not altered dramatically under the conditions of transient experimental ischaemia and reperfusion in the rat, despite the presence of cellular proliferation.

In order to reconstruct the characteristic three-dimensional architecture of adenoid cystic carcinoma, we cultured ACC2 cells, a cell system established from a human adenoid cystic carcinoma of the palate, in collagen gel matrix and transplanted them in SCID mice. In the collagen gel culture, the cells formed spherical colonies measuring 75.6 +/- 14.6 microns in diameter by 6 days after seeding. The tumor cell nests contained vacuolar structures that were immunopositive for heparan sulfate proteoglycan, type III collagen, type IV collagen, and fibronectin. The rim of the nests was argyrophilic and immunopositive for type I collagen, type IV collagen, laminin, and fibronectin. Transplants of ACC2 cells in SCID mice grew to form tumor masses in which pseudocysts were formed. The results indicate that our collagen gel culture system provides physiological conditions for ACC2 cells to secrete particular extracellular matrix molecules and form pseudocystic spaces.

Primary olfactory neurons arise from placodal neuroepithelium that is separate from the neuroepithelial plate that forms the neural tube and crest. The axons of these neurons course along a stereotypical pathway and invade the rostral telencephalic vesicle where they induce the formation of the olfactory bulb. In the present study we examined the expression of several extracellular matrix constituents during formation of the olfactory nerve pathway in order to identify putative developmentally significant molecules. Double-label immunofluorescence was used to simultaneously map the trajectory of growing primary olfactory axons by expression of growth associated protein 43 (GAP-43) and the distribution of either laminin, heparan sulfate proteoglycans (HSPG), or chondroitin sulfate proteoglycans (CSPG). At embryonic day 12.5 (E12.5) primary olfactory axons have exited the olfactory neuroepithelium of the nasal pit and formed a rudimentary olfactory nerve. These axons together with migrating neural cells form a large mass outside the rostral surface of the telencephalon. This nerve pathway is clearly defined by a punctate distribution of laminin and HSPG. CSPG is selectively present in the mesenchyme between the olfactory nerve pathway and the nasal pit and in the marginal zone of the telencephalon. At E14.5 primary olfactory axons pierce the telencephalon through gaps that have emerged in the basement membrane. At this age both laminin and HSPG are colocalized with the primary olfactory axons that have entered the marginal zone of the telencephalon. CSPG expression becomes downregulated in this same region while it remains highly expressed in the marginal zone adjacent to the presumptive olfactory bulb. By E16.5 most of the basement membrane separating the olfactory nerve from the telencephalon has degraded, and there is direct continuity between the olfactory nerve pathway and the central nervous system. This strict spatiotemporal regulation of extracellular matrix constituents in the olfactory nerve pathway supports an important role of these molecules in axon guidance. We propose that laminin and HSPG are expressed by migrating olfactory Schwann cells in the developing olfactory nerve pathway and that these molecules provide a conducive substrate for axon growth between the olfactory neuroepithelium and the brain. CSPG in the surrounding mesenchyme may act to restrict axon growth to within this pathway. The regional degradation of the basement membrane of the telencephalon and the downregulation of CSPG within the marginal zone probably facilitates the passage of primary olfactory axons into the brain to form the presumptive nerve fiber layer of the olfactory bulb.

Invasive extravillous trophoblast cells of the human placenta are embedded in a self-secreted extracellular matrix, the matrix-type fibrinoid. The ultrastructure and molecular composition of the matrix-type fibrinoid of the term human placenta were studied by transmission electron microscopy and immunogold labelling. We used antibodies directed against different matrix proteins such as collagen type IV, laminin, vitronectin, heparan sulfate, various fibronectin isoforms, and against the oncofetal blood group antigen, "i". Immunogold labelling patterns of matrix proteins are the basis for the subdivision of the trophoblast-derived matrix-type fibrinoid into mosaic-like patches of structurally and immunocytochemically different compartments. Firstly, fine granular patches with structural similarities to basal lamina material are composed solely of collagen type IV and laminin. Secondly, an ultrastructurally amorphous glossy substance shows reactivity with antibodies against heparan sulfate and vitronectin. A third type of patches, fine fibrillar networks embedded in the above-mentioned glossy matrix, are reactive with antibodies against normal fibronectin isoforms (IST-4, IST-6, IST-9) and oncofetal isoforms (BC-1, FDC-6). The blood group precursor antigen "i" was not only expressed on the surfaces of the extravillous trophoblast cells but was associated with the fibronectin-positive fibrils. In conclusion, within this extracellular matrix, clear compartments of different composition can be distinguished from each other. Glycosylation with "i" in this matrix may be involved in immunological masking, thus preventing rejection of placenta and fetus.

The mammary gland underwent morphological changes starting from a simple tubular structure, developing into a rich alveolar formation during lactation and ultimately a terminally differentiated tubular complex in retired breeders. Early stages of development, male and virgin mouse glands, were rich in hyaluronic acid but also contained smaller amounts of a low sulfate heparan. During lactation, there was a dramatic increase in sulfated glycosaminoglycan, in particular dermatan sulfate. Retired breeders were characterized by a highly sulfated heparan. These changes in glycosaminoglycan composition were analogous to changes seen in many embryologic tissues during morphogenesis and reflect the alterations in tissue modeling. The changes in glycosaminoglycans were reflective of the change observed in solubilized membrane associated proteoglycan. During lactation there was a dramatic increase in the content of dermatan sulfate proteoglycan. Both the dermatan sulfate and the proteoglycan which predominate during lactation was isolated as a complex with a small RNA, called pgRNA. The pgRNA was found only in-the lactating stage and not in virgin mice or retired breeders.

The temporomandibular joint (TMJ) articular discs were removed from female Sprague-Dawley rats 3, 5, 10, 32, 90 and 130 weeks of age. Glycosaminoglycans (GAGs) were extracted from the discs by heat treatment, alkali treatment and digestion with Pronase E, and purified by precipitation with cetylpyridinium chloride and ethanol. The concentration of total GAG was highest in the 3 week extracts and tended to decrease with age. Dermatan sulphate was the predominant GAG detected in all age groups along with chondroitin sulphate, hyaluronic acid and heparan sulphate. The disaccharides obtained from chondroitin sulphate were delta Di-4S, delta Di-6S and delta Di-0S, with delta Di-4S being the predominant isomer followed by delta Di-6S for all ages of all the GAG examined. The concentration of chondroitin sulphate showed a decrease with age. Quantitative changes of GAG with age may be related to functional changes in TMJ discs.

The products of ras and src proto-oncogenes are frequently activated in a constitutive state in human colorectal cancer. In this study we attempted to establish whether the tumorigenic progression induced by oncogenic activation of p21ras and pp60c-src in human colonic Caco-2 cells is associated with specific alterations of syndecan-1, a membrane-anchored proteoglycan playing a role in cell-matrix interaction and neoplastic growth control. To this end, we used Caco-2 cells made highly tumorigenic by transfection with an activated (Val 12) human Ha-ras gene or with the polyoma middle T (Py-MT) oncogene, a constitutive activator of pp60c-src tyrosine kinase activity. Compared with control vector-transfected Caco-2 cells, both oncogene-transfected cell lines (1) contained smaller amounts of membrane-anchored PGs; (2) exhibited decreased syndecan-1 expression at the protein but not the mRNA level; (3) synthesized 35S-labelled syndecan-1 with decreased specific activity; (4) produced a syndecan-1 ectodomain with a lower molecular mass and reduced GAG chain size and sulphation; and (5) expressed heparanase degradative activity. These results show that the dramatic activation of the tumorigenic potential induced by oncogenic p21ras or Py-MT/pp60c-src in Caco-2 cells is associated with marked alterations of syndecan-1 expression at the translational and post-translational levels.

Objective: To study whether the transition to menopause is accompanied by changes in the paraurethral connective tissue and if these changes are modified by estrogen replacement therapy., Study Design: Biopsies were obtained from the paraurethral tissue from 34 women; 12 menstruating, 14 postmenopausal without estrogen treatment, and 8 with estrogen treatment. Collagen concentration and collagen extractability by pepsin digestion were measured. Proteoglycan composition and concentration were analysed using Alcian blue. The mRNA levels for collagen I and III, the small proteoglycans (PGS) decorin and biglycan, and the large proteoglycan versican, were estimated., Results: The paraurethral biopsies consisted of fibrous connective tissue, with collagen fibers as dominating structure. Several proteoglycans were identified; versican, heparansulphate proteoglycans, biglycan and decorin. The small proteoglycan decorin represented 85% of all proteoglycans. The collagen concentration was almost doubled in postmenopausal biopsies compared to premenopausal. The collagen fibril organization was also changed with higher cross-linking after menopause whereas the amount and the composition of the proteoglycans were unchanged. The proteoglycan/collagen ratio was significantly decreased. Estrogen replacement therapy resulted in decreased collagen concentration, decreased cross-linking of the collagen and reversal of the PGS/collagen ratio to almost premenopausal level. The therapy resulted in increased levels of mRNA for collagen I and III which suggests that the changes are due to an increased turnover., Conclusion: The decrease in estrogen levels at menopause results in a connective tissue with different qualities after menopause. Estrogen replacement therapy tends to restore the metabolism of the genitourinary connective tissue to premenopausal conditions.

Proteoglycans (PGs) were isolated from the dentin-predentin of fluorotic and control rat incisor teeth using demineralization in EDTA, followed by extraction with 4 M guanidinium chloride in the presence of protease inhibitors. Differences in the behaviour of fluorotic and control PG were evident during purification by anion exchange chromatography on Q-Sepharose and MONO-Q interfaced with fast protein liquid chromatography. The PG from fluorotic teeth exhibited a more anionic profile, due to changes in glycosaminoglycan characteristics, since no apparent differences were evident between the respective core proteins, both of which were 45 kDa. The constituent glycosaminoglycan chains of fluorotic dentin were of lower molecular size and showed the additional presence of dermatan sulfate and heparan sulfate by comparison to nonfluorotic controls, where only chondroitin-4-sulfate was detected.

Experiments on the S27 cell line, a variant of the C2 mouse muscle cell line that shows reduced incorporation of 35SO4 into proteoglycans, suggest that proteoglycans play a role in the clustering of acetylcholine receptors, an early step in synaptogenesis. Thus, unlike the C2 line, S27 myotubes do not form acetylcholine receptor clusters on their surface in aneural cultures and form few clusters in response to agrin. We have examined the proteoglycans synthesized by S27 myotubes to define further the biochemical defect in these cells. Gel filtration analysis of radiolabeled proteoglycans synthesized by C2 and S27 myotubes shows that both cell types express a similarly polydisperse complement of proteoglycans. Both radiolabeled heparan sulfate proteoglycans and chondroitin/dermatan sulfate proteoglycans are reduced in S27 myotubes, with the chondroitin/dermatan sulfate proteoglycans showing a distinct reduction in size. The core protein of perlecan, a major proteoglycan species in muscle, was present in S27 cells and unaltered in electrophoretic mobility. Thus a principal deficiency in S27 cells appears to be a defect in glycosaminoglycan chain elongation.

An impairment of the mucosal glycoconjugates could be an important factor in the development of bladder disorders such as interstitial cystitis. However, very little definitive biochemical information is available on the glycoco-jugate components of the mammalian bladder mucosa. In this-study, the mucosa from metabolically radiolabeled rabbit bladder was separated, delipidated, and digested with protease, and the released glycosaminoglycans and glycopeptides were fractionated. About 80 and 36% of the nondialyzable tritium and 35S activities, respectively, was associated with the sialoglycopeptide fractions. The balance of the total tritium activity in the protease digest was in glycosaminoglycans identified as hyaluronan, chondroitin sulfates, and heparan sulfate. Immunohistochemical examination using anti-heparan sulfate antibodies, including one against mouse syndecan-1, indicated the presence of heparan sulfate proteoglycan in the epithelium. In contrast, there was no significant staining of the bladder epithelium with anti-chondroitin-4- and 6-sulfate antibodies or hyaluronan-binding protein. The lamina propria and muscle layers showed strong staining with anti-chondroitin-4-sulfate antibody and hyaluronan-binding protein and weak staining with anti-chondroitin-6-sulfate antibody. The insignificant levels of glycosaminoglycans in the glycocalyx of bladder mucosa epithelium suggest that glycosaminoglycans may be less important than other glycoconjugates in maintaining normal epithelial function and in bladder disorders such as interstitial cystitis.

Proteoglycans are important components of the extracellular matrix. They are involved in liver regeneration as well as in liver fibrosis. The distribution and cellular source of proteoglycans under normal as well as pathological conditions is still under debate. Localization of decorin and perlecan was studied in normal, acutely damaged, and cirrhotic liver by histochemistry. Furthermore, their synthesis was analyzed in different liver cell populations isolated from normal rat liver. In normal liver, decorin positivity was observed in the perisinusoidal space and in the portal area. Perlecan was clearly detectable in the portal area (blood vessels and bile ducts); a moderate reaction was also seen along the sinusoids. Strong positivity for both proteoglycans was detectable in the necrotic areas of acutely damaged liver. Chronic liver damage was characterized by the deposition of decorin and perlecan in the fibrotic septa. Immunocytochemical reactions were positive for perlecan and decorin in cultured Ito and endothelial cells but not in hepatocytes and Kupffer cells. Northern hybridization confirmed the capacity of Ito cells and endothelial cells to express the two genes. Interestingly, although rat skin fibroblasts expressed strong messages for both proteoglycans, rat aortic smooth muscle cells did not synthesize decorin.