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Kashin-Beck disease (KBD) is an endemic osteochondropathy. Due to a lack of suitable animal or cellular disease models, the research progress on KBD has been limited. Our goal was to establish the first disease-specific human induced pluripotent stem cell (hiPSC) cellular disease model of KBD, and to explore its etiology and pathogenesis exploiting transcriptome sequencing. HiPSCs were reprogrammed from dermal fibroblasts of two KBD and one healthy control donor via integration-free vectors. Subsequently, hiPSCs were differentiated into chondrocytes through three-week culture. Gene expression profiles in KBD, normal primary chondrocytes, and hiPSC-derived chondrocytes were defined by RNA sequencing. A Venn diagram was constructed to show the number of shared differentially expressed genes (DEGs) between KBD and normal. Gene oncology and Kyoto Encyclopedia of Genes and Genomes annotations were performed, and six DEGs were further validated in other individuals by RT-qPCR. KBD cellular disease models were successfully established by generation of hiPSC lines. Seventeen consistent and significant DEGs present in all compared groups (KBD and normal) were identified. RT-qPCR validation gave consistent results with the sequencing data. Glycosaminoglycan biosynthesis-heparan sulfate/heparin; PPAR signaling pathway; and cell adhesion molecules (CAMs) were identified to be significantly altered in KBD. Differentiated chondrocytes derived from KBD-origin hiPSCs provide the first cellular disease model for etiological studies of KBD. This study also provides new sights into the pathogenesis and etiology of KBD and is likely to inform the development of targeted therapeutics for its treatment.

Drug delivery to the brain is greatly hampered by the presence of the blood–brain barrier (BBB) which tightly regulates the passage of molecules from blood to brain and vice versa. Nanocarriers, in which drugs can be encapsulated, can move across the blood–brain barrier (BBB) via the process of transcytosis, thus showing promise to improve drug delivery to the brain. Here, we demonstrate the use of natural nanovesicles, that is, exosomes, derived from C17.2 neural stem cells (NSCs) to efficiently carry a protein cargo across an in vitro BBB model consisting of human brain microvascular endothelial cells. We show that the exosomes are primarily taken up in brain endothelial cells via endocytosis, while heparan sulfate proteoglycans (HSPGs) act as receptors. Taken together, our data support the view that NSC exosomes may act as biological nanocarriers for efficient passage across the BBB. Nanomedicines that target HSPGs may improve their binding to brain endothelial cells and, possibly, show subsequent transcytosis across the BBB., Neural stem cell‐derived exosomes interact with heparan sulfate proteoglycans on endothelial cells at the apical (blood) side and are taken up by dynamin dependent endocytosis. II. Exosomes are transported across the endothelial cell and reach the basal (brain) side.

Background and Purpose The TNF family ligands, B cell activating factor of the TNF family (BAFF, also known as B lymphocyte stimulator, BLyS) and a proliferation‐inducing ligand (APRIL), share the transmembrane activator and calcium‐modulator and cyclophilin ligand (CAML)‐interactor (TACI) as one of their common receptors. Atacicept, a chimeric recombinant TACI/IgG1‐Fc fusion protein, inhibits both ligands. TACI and APRIL also bind to proteoglycans and to heparin that is structurally related to proteoglycans. It is unknown whether the portion of TACI contained in atacicept can bind directly to proteoglycans, or indirectly via APRIL, and whether this could interfere with the anti‐coagulant properties of heparin. Experimental Approach Binding of atacicept and APRIL to proteoglycan‐positive cells was measured by FACS. Activities of heparin and atacicept were measured with activated factor Xa inhibition and cell‐based assays. Effects of heparin on circulating atacicept was monitored in mice. Key Results Atacicept did not bind to proteoglycan‐positive cells, but when complexed to APRIL could do so indirectly via APRIL. Multimers of atacicept obtained after exposure to cysteine or BAFF 60‐mer bound directly to proteoglycans. Atacicept alone, or in complex with APRIL, or in a multimeric form did not interfere with heparin activity in vitro. Conversely, heparin did not influence inhibition of BAFF and APRIL by atacicept and did not change circulating levels of atacicept. Conclusions and Implications Lack of detectable interference of APRIL‐bound or free atacicept on heparin activity makes it unlikely that atacicept at therapeutic doses will interfere with the function of heparin in vivo.

Anoikis is a form of programmed cell death induced by loss of contact from neighboring cells or from their extracellular matrix (ECM). Many tumorigenic cells are anoikis resistant, facilitating cancer progression and metastasis. Trastuzumab is a monoclonal antibody used for the treatment of breast and gastric cell cancer, but its mechanism of action is not well elucidated and its target molecules not well defined. Heparan sulfate proteoglycans (HSPGs) and glycosaminoglycans (GAGs) play important roles in tumor development and in response of cancer cells to drugs. This study investigates the effect of trastuzumab on the expression of HSPGs and sulfated glycosaminoglycans (SGAGs) in anoikis-resistant endothelial cells. After trastuzumab treatment, endothelial cells resistant to anoikis show an increase in adhesion to fibronectin followed by a decrease in invasion, proliferation, and angiogenic capacity. In addition, a significant increase in the number of cells in the S phase of the cell cycle was also observed. In relation to HSPGs and SGAGs expression, we observed a decrease in syndecan-4 and perlecan expression, as well as in the heparan sulfate biosynthesis in anoikis-resistant endothelial cells after exposure to trastuzumab. Our results suggest that trastuzumab interacts with GAGs and proteoglycans of the cell surface and ECM and through this interaction controls cellular events in anoikis-resistant endothelial cells.

Cleavage of heparan sulfate proteoglycans (HSPGs) by the enzyme heparanase modulates tumour-related events including angiogenesis, cell invasion, and metastasis. Metalloshielding of heparan sulfate (HS) by positively charged polynuclear platinum complexes (PPCs) effectively inhibits physiologically critical HS functions. Studies using bacterial P. heparinus heparinase II showed that a library of Pt complexes varying in charge and nuclearity and the presence or absence of a dangling amine inhibits the cleavage activity of the enzyme on the synthetic pentasaccharide, Fondaparinux (FPX). Charge-dependent affinity of PPC for FPX was seen in competition assays with methylene blue and ethidium bromide. The dissociation constant (Kd ) of TriplatinNC for FPX was directly measured by isothermal titration calorimetry (ITC). The trend in DFT calculated interaction energies with heparin fragments is consistent with the spectroscopic studies. Competitive inhibition of TAMRA-R9 internalization in human carcinoma (HCT116) cells along with studies in HCT116, wildtype CHO and mutant CHO-pgsA745 (lacking HS/CS) cells confirm that HSPG-mediated interactions play an important role in the cellular accumulation of PPCs.

Background Dyssegmental dysplasia Silverman‐Handmaker (DDSH; MIM 224410) type is an extremely rare skeletal dysplasia caused by functional null mutations in the perlecan gene. Less than forty cases are reported in the literature, of which only four were prenatally detected. Methods We report on a dizygotic twin pregnancy from consanguineous parents for which one of the twins presented prenatally with severe micromelia, limb bowing and scoliosis, and postnatally with clinical and radiological features compatible with a diagnosis of dyssegmental dysplasia. Molecular studies were undertaken to confirm the clinical diagnosis of DDSH. Results Molecular analysis results revealed a novel homozygous variant in the HSPG2 gene (MIM 142461), NM_005529.6(HSPG2):c.4029 + 1G>A, consistent with a diagnosis of DDSH. Conclusion To the best of our knowledge, the current report is only the seventh molecularly confirmed case of DDSH.

Background The glycosylation of proteins on the surface of corneocytes is believed to play an important role in cellular adhesion in the stratum corneum (SC) of human skin. Mapping with accuracy the localization of glycans on the surface of corneocytes through traditional methods of immunohistochemistry and electron microscopy remains a challenging task as both approaches lack enough resolution or need to be performed in high vacuum conditions. Materials and methods We used an advanced mode of atomic force microscope (AFM), with simultaneous topography and recognition imaging to investigate the distribution of glycans on native (no chemical preparation) stripped samples of human SC. The AFM cantilever tips were functionalized with anti‐heparan sulfate antibody and the lectin wheat germ agglutinin (WGA) which binds specifically to N‐acetyl glucosamine and sialic acid. Results From the recognition imaging, we observed the presence of the sulfated glycosaminoglycan, heparan sulfate, and the glycans recognized by WGA on the surface of SC corneocytes in their native state. These glycans were found associated with bead‐like domains which represent corneodesmosomes in the SC layers. Glycan density was calculated to be ~1200 molecules/μm2 in lower layers of SC compared to an important decrease, (~106 molecules/μm2) closer to the surface due probably to corneodesmosome degradation. Conclusion Glycan spatial distribution and degradation is first observed on the surface of SC in native conditions and at high resolution. The method used can be extended to precisely localize the presence of other macromolecules on the surface of skin or other tissues where the maintenance of its native state is required.

We investigated the role of glycosaminoglycans (GAGs) in the regulation of endothelial nitric oxide synthase (eNOS) activity in wild-type CHO-K1 cells and in xylosyltransferase-deficient CHO-745 cells. GAGs inhibit the integrin/FAK/PI3K/AKT signaling pathway in CHO-K1 cells, decreasing the phosphorylation of eNOS at Ser1177. Furthermore, in CHO-K1 cells, eNOS and PKCα are localized at sphingolipid- and cholesterol-rich domains in the plasma membrane called caveolae. At caveolae, PKCα activation stimulates the phosphorylation of eNOS on Thr495, resulting in further inhibition of NO production in these cells. In our data, CHO-745 cells generate approximately 12-fold more NO than CHO-K1 cells. Increased NO production in CHO-745 cells promotes higher rates of protein S-nitrosylation and protein tyrosine nitration. Regarding reactive oxygen species (ROS) production, CHO-745 cells show lower basal levels of superoxide (O2- ) than CHO-K1 cells. In addition, CHO-745 cells express higher levels of GPx, Trx1, and catalase than CHO-K1 cells, suggesting that CHO-745 cells are in a constitutive nitrosative/oxidative stress condition. Accordingly, we showed that CHO-745 cells are more sensitive to oxidant-induced cell death than CHO-K1 cells. The high concentration of NO and reactive oxygen species generated by CHO-745 cells can induce simultaneous mitochondrial biogenesis and antioxidant gene expression. These observations led us to propose that GAGs are part of a regulatory mechanism that participates in eNOS activation and consequently regulates nitrosative/oxidative stress in CHO cells.

High-risk neuroblastoma is characterized by undifferentiated neuroblasts and low schwannian stroma content. The tumor stroma contributes to the suppression of tumor growth by releasing soluble factors that promote neuroblast differentiation. Here we identify heparin-binding epidermal growth factor–like growth factor (HBEGF) as a potent prodifferentiating factor in neuroblastoma. HBEGF mRNA expression is decreased in human neuroblastoma tumors compared with benign tumors, with loss correlating with decreased survival. HBEGF protein is expressed only in stromal compartments of human neuroblastoma specimens, with tissue from high-stage disease containing very little stroma or HBEGF expression. In 3 human neuroblastoma cell lines (SK-N-AS, SK-N-BE2, and SH-SY5Y), soluble HBEGF is sufficient to promote neuroblast differentiation and decrease proliferation. Heparan sulfate proteoglycans and heparin derivatives further enhance HBEGF-induced differentiation by forming a complex with the epidermal growth factor receptor, leading to activation of the ERK1/2 and STAT3 pathways and up-regulation of the inhibitor of DNA binding transcription factor. These data support a role for loss of HBEGF in the neuroblastoma tumor microenvironment in neuroblastoma pathogenesis.—Gaviglio, A. L., Knelson, E. H., Blobe, G. C. Heparin-binding epidermal growth factor-like growth factor promotes neuroblastoma differentiation.

Hexavalent sulfoglycodendrimers (SGDs) are synthesized as mimics of host cell heparan sulfate proteoglycans (HSPGs) to inhibit the early stages in viral binding/entry of HIV‐1 and SARS‐CoV‐2. Using an HIV neutralization assay, the most promising of the seven candidates are found to have sub‐micromolar anti‐HIV activities. Molecular dynamics simulations are separately implemented to investigate how/where the SGDs interacted with both pathogens. The simulations revealed that the SGDs: 1) develop multivalent binding with polybasic regions within and outside of the V3 loop on glycoprotein 120 (gp120) for HIV‐1, and consecutively bind with multiple gp120 subunits, and 2) interact with basic amino acids in both the angiotensin‐converting enzyme 2 (ACE2) and HSPG binding regions of the Receptor Binding Domain (RBD) from SARS‐CoV‐2. These results illustrate the considerable potential of SGDs as inhibitors in viral binding/entry of both HIV‐1 and SARS‐CoV‐2 pathogens, leading the way for further development of this class of molecules as broad‐spectrum antiviral agents., Hexavalent sulfoglycodendrimer (SGD) inhibitors are capable of multivalent binding with both trimeric HIV‐1 glycoprotein 120 (gp120) and to two separate sites (ACE2 and heparin binding region) of the Receptor Binding Domain of SARS‐CoV‐2. The SGDs have potential as broadly active antiviral agents.

Purpose The aim of this pilot study was to conduct a non-targeted exploratory proteomics profiling analysis on sera obtained from patients diagnosed with diffuse large B-cell lymphoma (DLBCL) with the goal of identifying disease-specific biomarkers. Experimental design Sera from 87 participants (57 chemotherapy-naive diffuse DLBCL patients, 30 controls frequency-matched by age group and World Health Organization (WHO) BMI categories) that were part of a large San Francisco Bay Area case-control study of non-Hodgkin lymphoma were analyzed by liquid chromatography combined with tandem mass spectrometry. Results Thirty-five proteins (p-adjusted

Neural epidermal growth factor-like (NEL)-like 1 (NELL1) is a secretory osteogenic protein comprising an N-terminal thrombospondin-1-like (TSPN) domain, four von Willebrand factor type C domains, and six epidermal growth factor-like repeats. NELL1 shows heparin-binding activity; however, the biological significance remains to be explored. In this report, we demonstrate that NELL1 binds to cell surface proteoglycans through its TSPN domain. Major heparin-binding sites were identified on the three-dimensional structural model of the TSPN domain of NELL1. Mutant analysis of the heparin-binding sites indicated that the heparin-binding activity of the TSPN domain is involved in interaction of NELL1 with cell surface proteoglycans.

Elastin-like-peptides (ELPs) are stimulus-responsive protein-based polymers and are attractive biomaterials due to their biocompatibility and unique properties. This study shows that in addition to their physical properties, ELPs have biological activities that are conducive to tissue regeneration. Specifically, we found that ELPs induce fibroblast proliferation via cell surface heparan sulfate proteoglycans (HSPG). Furthermore, our data suggests that ELP based materials with differential proliferative potential can be designed by controlling the interaction of ELPs with HSPGs by incorporating either hydrophobic or positively charged residues within the ELP sequence. Fibroblast proliferation is important for granulation tissue formation which is important in chronic wounds as well as in healing of other tissues. The customizable biological activity of ELPs coupled with their unique physical properties will enable us to design novel, sustainable and cost effective therapies for different tissue regeneration applications. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 697-706, 2016.

The pathogenesis of cancer is often driven by local invasion and metastasis. Recently, mechanical properties of the tumor microenvironment have been identified as potent regulators of invasion and metastasis, while matrix metalloproteinases (MMPs) are classically known as significant enhancers of cancer cell migration and invasion. Here we have been able to sensitively measure MMP activity changes in response to specific extracellular matrix (ECM) environments and cell contractility states. Cells of a pancreatic cancer cell line, Panc-1, up-regulate MMP activities between 3- and 10-fold with increased cell contractility. Conversely, they down-regulate MMP activities when contractility is blocked to levels seen with pan-MMP activity inhibitors. Similar, albeit attenuated, responses are seen in other pancreatic cancer cell lines, BxPC-3 and AsPC-1. In addition, MMP activity was modulated by substrate stiffness, collagen gel concentration, and the degree of collagen cross-linking, when cells were plated on collagen gels ranging from 0.5 to 5 mg/ml that span the physiological range of substrate stiffness (50-2000 Pa). Panc-1 cells showed enhanced MMP activity on stiffer substrates, whereas BxPC-3 and AsPC-1 cells showed diminished MMP activity. In addition, eliminating heparan sulfate proteoglycans using heparinase completely abrogated the mechanical induction of MMP activity. These results demonstrate the first functional link between MMP activity, contractility, and ECM stiffness and provide an explanation as to why stiffer environments result in enhanced cell migration and invasion.

The patterning of many developing tissues is orchestrated by gradients of morphogens through a variety of elaborate regulatory interactions. Such interactions are thought to make gradients robust, that is, resistant to changes induced by genetic or environmental perturbations; but just how this might be done is a major unanswered question. Recently extensive numerical simulations suggest that robustness of signaling gradients cannot be attained by negative feedback (of the Hill's function type) on signaling receptors but can be achieved through binding with nonsignaling receptors (or nonreceptors for short) such as heparan sulfate proteoglycans with the resulting complexes degrading after endocytosis. These were followed by a number of analytical and numerical studies in support of the aforementioned observations. However, evidence of feedback regulating signaling gradients has been reported in literature. The present paper undertakes a different approach to the role of feedback in robust signaling gradients. The overall goal of the project is to investigate the effectiveness of feedback mechanisms on ligand synthesis, receptor synthesis, nonreceptor synthesis, and other regulatory processes in the morphogen gradient system. As a first step, we embark herein a proof-of-concept examination of a new spatially uniform feedback process that is distinctly different from the conventional spatially nonuniform Hill function approach.

The patterning of many developing tissues is orchestrated by gradients of signaling morphogens. Included among the molecular events that drive the formation of morphogen gradients are a variety of elaborate regulatory interactions. Such interactions are thought to make gradients robust, i.e. insensitive to change in the face of genetic or environmental perturbations. But just how this is accomplished is a major unanswered question. Recently extensive numerical simulations suggest that robustness of signaling gradients can be achieved through morphogen degradation mediated by cell surface bound non-signaling receptor molecules (or nonreceptors for short) such as heparan sulfate proteoglycans (HSPG). The present paper provides a mathematical validation of the results from the aforementioned numerical experiments. Extension of a basic extracellular model to include reversible binding with nonreceptors synthesized at a prescribed rate and mediated morphogen degradation shows that the signaling gradient diminishes with increasing concentration of cell-surface nonreceptors. Perturbation and asymptotic solutions obtained for i) low (receptor and nonreceptor) occupancy, and ii) high nonreceptor concntration permit more explicit delineation of the effects of nonreceptors on signaling gradients and facilitate the identification of scenarios in which the presence of nonreceptors may or may not be effective in promoting robustness.

High levels of hepatocyte growth factor (HGF), a healing factor with regenerative and cytoprotective effects, are associated with inflammatory diseases, including periodontitis. HGF biologic activity requires binding to its receptors, the proto-oncogene c-Met and heparan sulfate proteoglycan (HSPG). This study investigates HGF expression and its relationship to subgingival microbiota in medically healthy individuals with and without periodontitis.Saliva, gingival crevicular fluid (GCF), and blood samples from 30 patients with severe periodontitis and 30 healthy controls were analyzed for HGF concentration using enzyme-linked immunosorbent assay and binding affinity for HSPG and c-Met using surface plasmon resonance. The regenerative effects of saliva from three patients and controls were analyzed in an in vitro model of cell injury. Subgingival plaques were analyzed for the presence of 18 bacterial species.Patients with periodontitis showed higher HGF concentrations in saliva, GCF, and serum (P0.001); however, the binding affinities for HSPG and c-Met were reduced in GCF and saliva (P0.002). In contrast to the controls, saliva from patients showed no significant regenerative effect over time on gingival epithelial cells. Compared with controls, patients had a higher prevalence of periodontally related bacteria.Higher circulatory HGF levels indicate a systemic effect of periodontitis. However, the HGF biologic activity at local inflammation sites was reduced, and this effect was associated with the amount of periodontal bacteria. Loss of function of healing factors may be an important mechanism in degenerative processes in periodontally susceptible individuals.

Objectives: Fractones are extracellular matrix structures that form a niche for neural stem cells and their immediate progeny in the subventricular zone of the lateral ventricle (SVZa), the primary neurogenic zone in the adult brain. We have previously shown that heparan sulphates (HS) associated with fractones bind fibroblast growth factor‐2 (FGF‐2), a powerful mitotic growth factor in the SVZa. Here, our objective was to determine whether the binding of FGF‐2 to fractone‐HS is implicated in the mechanism leading to cell proliferation in the SVZa. Materials and methods: Heparitinase‐1 was intracerebroventricularly injected with FGF‐2 to N‐desulfate HS proteoglycans and determine whether the loss of HS and of FGF‐2 binding to fractones modifies FGF‐2 effect on cell proliferation. We also examined in vivo the binding of Alexa‐Fluor‐FGF‐2 in relationship with the location of HS immunoreactivity in the SVZa. Results: Heparatinase‐1 drastically reduced the stimulatory effect of FGF‐2 on cell proliferation in the SVZa. Alexa‐Fluor‐FGF‐2 binding was strictly co‐localized with HS immunoreactivity in fractones and adjacent vascular basement membranes in the SVZa. Conclusions: Our results demonstrate that FGF‐2 requires HS to stimulate cell proliferation in the SVZa and suggest that HS associated with fractones and vascular basement membranes are responsible for activating FGF‐2. Therefore, fractones and vascular basement membranes may function as a HS niche to drive cell proliferation in the adult neurogenic zone.

Heparan sulfate proteoglycans (HSPGs) have essential functions during embryonic development and throughout postnatal life. To exert these functions, they undergo a series of processing reactions by heparan-sulfate-modifying enzymes (HSMEs), which endows them with highly modified heparan sulfate (HS) domains that provide specific docking sites for a large number of bioactive molecules. The development and antigen-dependent differentiation of normal B lymphocytes, as well as the growth and progression of B-lineage malignancies, are orchestrated by an array of growth factors, cytokines and chemokines many of which display HS binding. As discussed in this review, tightly regulated HSPG expression is a requirement for normal B cell maturation, differentiation and function. In addition, the HSPG syndecan-1 functions as a versatile co-receptor for signals from the bone marrow microenvironment, essential for the survival of long-lived plasma cells and multiple myeloma (MM) plasma cells. Targeting of HSMEs or HS chains on MM cells increases their sensitivity to drugs currently used in MM treatment, including bortezomib, lenalidomide or dexamethasone. Taken together, these findings render the HS biosynthetic machinery a promising target for MM treatment.

Aging poses one of the largest risk factors for the development of cardiovascular disease. The increased propensity toward vascular pathology with advancing age maybe explained, in part, by a reduction in the ability of circulating endothelial progenitor cells to contribute to vascular repair and regeneration. Although there is evidence to suggest that colony forming unit-Hill cells and circulating angiogenic cells are subject to age-associated changes that impair their function, the impact of aging on human outgrowth endothelial cell (OEC) function has been less studied. We demonstrate that OECs isolated from cord blood or peripheral blood samples from young and old individuals exhibit different characteristics in terms of their migratory capacity. In addition, age-related structural changes were discovered in OEC heparan sulfate (HS), a glycocalyx component that is essential in many signalling pathways. An age-associated decline in the migratory response of OECs toward a gradient of VEGF significantly correlated with a reduction in the relative percentage of the trisulfated disaccharide, 2-O-sulfated-uronic acid, N, 6-O-sulfated-glucosamine (UA[2S]-GlcNS[6S]), within OEC cell surface HS polysaccharide chains. Furthermore, disruption of cell surface HS reduced the migratory response of peripheral blood-derived OECs isolated from young subjects to levels similar to that observed for OECs from older individuals. Together these findings suggest that aging is associated with alterations in the fine structure of HS on the cell surface of OECs. Such changes may modulate the migration, homing, and engraftment capacity of these repair cells, thereby contributing to the progression of endothelial dysfunction and age-related vascular pathologies.

Objective CXCL12γ is an alternative splicing isoform of CXCL12 with enhanced affinity for heparan sulfate (HS) proteoglycans. This study was undertaken to investigate the distribution and potential function of CXCL12γ in rheumatoid arthritis (RA) synovium and normal lymphoid tissue, where its immobilization to HS may be relevant in pathologic or homeostatic immune cell migration and activation. Methods Expression of CXCL12 or CXCL12γ was immunodetected in RA and normal synovium, lymphoid tissue, and cultured cells with anti–pan-CXCL12 or anti-CXCL12γ–specific monoclonal antibodies. CXCL12α and CXCL12γ messenger RNA expression was analyzed by quantitative reverse transcription–polymerase chain reaction. Binding of wild-type CXCL12 isoforms or their HS binding–defective mutants to monocyte-derived dendritic cells (DCs) was analyzed by flow cytometry. The effect of DC-bound CXCL12α and CXCL12γ on T cell activation was analyzed in DC/T cell allogeneic cultures. Results CXCL12γ expression was increased in RA compared to normal synovium and preferentially located in endothelia and DC-SIGN–positive cells. This distribution was also observed in lymphoid organs. Surface-bound CXCL12γ was detected in a fraction of freshly isolated DCs. Monocyte-derived DCs, but not monocytes, showed a high capacity to bind CXCL12γ in an HS-dependent manner. Surface-bound CXCL12α and CXCL12γ on monocyte-derived DCs were potent inhibitors of allogeneic T cell activation, in contrast to the T cell–stimulatory effects of soluble CXCL12 proteins. Conclusion CXCL12γ shows a specific and similar distribution in RA synovium and lymphoid tissue, consistent with its higher HS binding affinity. Presentation of CXCL12 to T cells on membrane HS in DCs can play a distinct regulatory role in T cell activation.

Summary Heparan sulfate proteoglycans (HSPGs) have been shown to regulate signaling in many systems and are of increasing interest in cancer. While these are not the only sugars to drive melanoma metastasis, HSPGs play important roles in driving metastatic signaling cascades in melanoma. The ability of these proteins to modulate ligand–receptor interactions in melanoma has been quite understudied. Recent data from several groups indicate the importance of these ligands in modulating key signaling pathways including Wnt and fibroblast growth factor (FGF) signaling. In this review, we summarize the current knowledge regarding the structure and function of these proteoglycans and their role in melanoma. Understanding how HSPGs modulate signaling in melanoma could lead to new therapeutic approaches via the dampening or heightening of key signaling pathways.

It has been shown that shear stress plays a critical role in promoting endothelial cell (EC) differentiation from embryonic stem cell (ESC)-derived EC. However, the underlying mechanisms mediating shear stress effect in this process have yet to be investigated. It has been reported that the glycocalyx component heparan sulfate proteoglycan (HSPG) mediates shear stress mechanotransduction in mature EC. In this study, we investigated whether cell surface HSPG plays a role in shear stress modulation of EC phenotype. ESC-derived endothelial cells were subjected to shear stress (5 dyn/cm2) for 8 hours with or without heparinase III (Hep III) that digests heparan sulfate. Immunostaining showed that ESC-derived EC surfaces contain abundant HSPG, which could be cleaved by Hep III. We observed that shear stress significantly increased the expression of vascular endothelial cell-specific marker genes (vWF, VE-cadherin, PECAM-1). The effect of shear stress on expression of tight junction protein genes (ZO-1, OCLD, CLD5) was also evaluated. Shear stress increased the expression of ZO-1 and CLD5, while it didn’t alter the expression of OCLD. Shear stress increased expression of vasodilatory genes (eNOS, COX-2), while it decreased the expression of the vasoconstrictive gene ET1. After reduction of HSPG with Hep III, the shear stress-induced expression of vWF, VE-cadherin, ZO-1, eNOS, and COX-2, were abolished, suggesting that shear stress-induced expression of these genes depends on HSPG. These findings indicate for the first time that HSPG is a mechanosensor mediating shear stress-induced EC differentiation from ESC-derived EC cells.

The HSulf-1 gene encodes an extracellular 6-O-endosulfatase and regulates the sulfation status of heparan sulfate proteoglycans (HSPG). We have demonstrated that promoter hypermethylation is correlated with the HSulf-1 silencing in gastric cancer. To investigate the functional importance of HSulf-1 silencing in gastric cancer, we restored HSulf-1 expression in the gastric cancer cell line MKN28, which lacks endogenous HSulf-1. Following restoration of expression, HSulf-1 inhibited cell proliferation, motility, and invasion in vitro, as well as significantly suppressing the MKN28 xenograft model (P

Most coinhibitory receptors regulate T-cell responses through an ITIM that recruits protein tyrosine phosphatases (PTPs) to mediate inhibitory function. Because syndecan-4 (SD-4), the coinhibitor for DC-associated heparan sulfate proteoglycan integrin ligand (DC-HIL), lacks such an ITIM, we posited that SD-4 links with a PTP in an ITIM-independent manner. We show that SD-4 associates constitutively with the intracellular protein syntenin but not with the receptor-like PTP CD148 on human CD4(+) T cells. Binding to DC-HIL allowed SD-4 to assemble with CD148 through the help of syntenin as a bridge, and this process upregulated the PTP activity of CD148, which is required for SD-4 to mediate DC-HIL's inhibitory function. Using a mouse model, we found SD-4 to be located away from the immunological synapse formed between T cells and APCs during activation of T cells. These findings indicate that SD-4 is unique among known T-cell coinhibitors, in employing CD148 to inhibit T-cell activation at a site distal from the synapse.

We measured the gene expression, glycocalyx content, and surface properties of human coronary artery endothelial cells (HCAECs) cultured on poly(sodium p-styrene sulfonate) (PNaSS) hydrogels with various levels of elasticity ranged in 3-300 kPa. We found that all HCAECs reached confluence on these hydrogels while retaining the similar expression of EC-specific markers to that on polystyrene (PS), a widely used scaffold in cell culture in vitro. Real-time polymerase chain reaction (PCR) and glycosaminoglycan (GAG) assay showed that the amount of EC-specific glycocalyx secreted by HCAECs cultured on PNaSS gels was higher than that cultured on PS, and it increased with an increase of gel elasticity. Furthermore, the HCAECs cultured on PNaSS gels showed excellent property against platelet adhesion and lower surface friction than that on PS. The platelet adhesion and surface friction of HCAECs cultured on PNaSS gels also depend on the elasticity of gels. The largest amount of EC-specific glycocalyx, excellent blood compatibility, and the lowest friction were observed when the elastic modulus of the gel was larger than 60 kPa. Overall, HCAECs cultured on these hydrogels have better properties than those cultured on PS scaffold, demonstrating the PNaSS gels can be used as potential tissue engineering material for blood vessels.

Basement membranes (BMs) are specializations of the extracellular matrix that act as key mediators of development and disease. Their sheet like protein matrices typically serve to separate epithelial or endothelial cell layers from underlying mesenchymal tissues, providing both a biophysical support to overlying tissue as well as a hub to promote and regulate cell-cell and cell-protein interactions. In the latter context, the BM is increasingly being recognized as a mediator of growth factor interactions during development. In this review, we discuss recent findings regarding the structure of the BM and its roles in mediating the normal development of the embryo, and we examine congenital diseases affecting the BM which impact embryonic development and health in later life.

Objective Development of an artificial salivary gland will benefit patients with xerostomia after radiation therapy for upper respiratory cancer. The goal is to devise a three-dimensional (3D) culture system in which salivary cells differentiate into polarized acini that express essential biomarkers and directionally secrete α-amylase. Differentiated acini-like structures in a 3D biomaterial-based scaffold will mimic salivary gland functions. Study Design Cells were seeded onto HA-based hydrogels containing PlnDIV peptide and allowed to differentiate into acini-like structures. Cell viability and phenotype were examined. Setting Laboratory-based tissue procurement study. Subjects and Methods Salivary gland tissue was obtained from patients undergoing surgery. Marker expression established the phenotype of salivary gland cells. Perlecan/HSPG2, an important component of the basement membrane, was highly expressed in salivary gland tissue. A culture system consisting of hyaluronic acid (HA) hydrogel and a coupled bioactive peptide derived from domain IV of perlecan (PlnDIV) was used. Prior studies demonstrated differentiation of acinar cells into lobular structures that mimicked intact glands when cultured on PlnDIV peptide–coated surfaces. Results Lobular acini-like structures formed on hydrogels and expressed tight junction components such as zona occludens 1. Acini-like structures were stained for the presence of α-amylase. Live/dead staining revealed the presence of apoptotic cells in the center of the acini-like structures, indicative of lumen formation. Conclusion A novel system supporting acini-like assembly in a 3D culture system was established. Presence of biomarkers and secretion of salivary enzymes confirms functionality in vitro. Future experiments will test the 3D system in an animal model.

Normal endothelial cells respond to shear stress by elongating and aligning in the direction of fluid flow. Elevated glucose concentrations have been shown to impair this response, though the precise mechanism of damage is not clear. Using an in vitro model of hyperglycemia, we tested the hypothesis that high glucose (HG) impairs the endothelial shear stress response by damaging the glycocalyx. 50 mU/mL heparinase III enzyme removes similar proportions of cell surface heparan sulfate proteoglycan (HSPG) as HG conditions and results in similar impairment of the elongation and alignment response to flow. Doubling the shear stress overcomes the inhibited flow response in HG cells, but not in enzyme treated cells. These findings may be explained by HG leading to decreased expression of full-length HSPG; whereas, heparinase results in a normal density of HSPG of shorter length. © 2010 Wiley-Liss, Inc.

Proteoglycans synthesized by osteoblasts are incorporated into bone matrix and thought to play a role in bone metabolism. Transforming growth factor (TGF) beta affects the synthesis of matrix proteins, including proteoglycans, in various stromal cells, and proteoglycans, especially decorin, are associated with matrix collagen. In the present study, the effects of TGF-beta 1 and L-ascorbate, a factor essential for collagen synthesis, on the synthesis and distribution of proteoglycans were examined using murine osteoblast-like MC3T3-E1 cells. TGF-beta 1 stimulated the synthesis of proteoglycans in MC3T3-E1 cells. Among various proteoglycans, the synthesis of decorin was preferentially enhanced by TGF-beta 1, and the effect was more pronounced on secreted decorin compared to that associated with the cell/matrix layer. TGF-beta 1 also stimulated the initiation and elongation of the dermatan sulfate glycosaminoglycan chain, resulting in a larger molecular size of decorin. TGF-beta 1 influenced the synthesis of a heparan sulfate proteoglycan only slightly. L-ascorbate had no effect on the synthesis of proteoglycans, but increased those associated with the cell/matrix layer. Furthermore, when L-ascorbate was added to the culture along with TGF-beta 1, the percentage of proteoglycans associated with the cell/matrix layer increased from 25.8 +/- 1.0 to 41.0 +/- 0.5%. These data demonstrate that TGF-beta 1 markedly stimulates the synthesis of proteoglycans, especially decorin, mainly as a secreting form, that the accumulation of decorin into matrix is enhanced by L-ascorbate, and that the effects of TGF-beta 1 and L-ascorbate are additive.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural and immunohistochemical studies were performed to clarify the localization of heparan sulfate proteoglycans (HSPG) and their role in cell-cell and/or cell-matrix interaction of bone cells. Ultrastructural observations using the freeze-substitution method revealed electron-dense undercoat structures on the cytoplasmic side of plasma membranes and extracellular matrices in the intercellular spaces where stromal cells came in contact with hematopoietic cells and/or osteoclasts. Immunohistochemical localization of HSPG in rat tibiae was examined using monoclonal antibody directed to glycosaminoglycan of HSPG by confocal laser scanning microscopy and transmission electron microscopy. Intense immunoreactivity was detected on the basement membranes of blood vessels, as well as the plasma membranes of fibroblast-like cells surrounding them. Immunoreactivity was also seen on extracellular matrices between fibroblast-like cells and osteoclast lineage cells. In addition, osteoblasts showed moderate immunoreactivity on those plasma membranes attached to bone matrix. A postembedding method revealed gold particles in Golgi vacuoles of osteoblasts, indicating the localization of HSPG. In osteoclasts, HSPG was localized in Golgi apparatus and lysosomal structures. These findings suggest that (1) osteoblasts and osteoclast lineage cells synthesize HSPG; (2) both membrane and matrix HSPG are localized in bone tissue; and (3) HSPG may play an important role in cell-cell interaction between fibroblast-like cells and osteoclast lineage cells by reserving heparin binding growth factors and/or heparin binding adhesion molecules, such as fibronectin.

In previous work, we compared the steady-state levels of specific matrix components in human bone cells derived from patients with osteogenesis imperfecta (OI) to those of age-matched controls. A remarkable finding was the observation that there was a reduction not only in the total levels of collagen, but also in osteonectin and three proteoglycans (a large chondroitin sulfate proteoglycan, biglycan, and decorin). This pattern was observed in patients with and without detectable collagen defects. More recent analysis of extracellular matrix composition have yielded that, compared with age-matched controls, bone cells from OI patients produced higher steady-state levels of fibronectin and thrombospondin. The percentage of these two proteins incorporated into the cell layer pool was also higher in OI than in age-matched controls. In addition, the steady-state levels of hyaluronan and a heparan sulfate proteoglycan were analyzed in both OI and age-matched controls. Although the total (medium + cell layer) steady-state levels of hyaluronan were reduced by 1/3, the percentage of the hyaluronan in the cell layer pool of patients with OI increased between 100-250% of age-matched control. Thus the matrix elaborated by human OI bone cells is not only quantitatively different but also qualitatively distinct from that of age-matched controls. Not only have specific bone cell matrix components (collagen, osteonectin, the large chondroitin sulfate proteoglycan, biglycan, and decorin) been found to be present in reduced levels in OI bone cells, but some matrix components (thrombospondin, fibronectin, and hyaluronan) have also been found to be present in elevated levels in the matrix of OI cells.(ABSTRACT TRUNCATED AT 250 WORDS)

In the skin, heparin, heparan sulphate and heparan sulphate proteoglycans control the storage and release of growth factors and protect them from early degradation. We developed a cosmetic active ingredient containing Hibiscus Abelmoschus seed extract (trade name Linefactor) that can maintain the FGF-2 content in the skin by mimicking the protective effect of heparan sulphate proteoglycans. By preventing the natural degradation of FGF-2, Hibiscus Abelmoschus seed extract maintains the bioavailability of this growth factor for its target cells, i.e. skin fibroblasts. Our in vitro evaluations showed that this ingredient exhibited heparan sulphate-like properties and dose-dependently protected FGF-2 from thermal degradation. We could also show that, in turn, the protected FGF-2 could stimulate the synthesis of sulphated GAGs, the natural protective molecules for FGF-2, thus providing a double protection. Finally, the in vitro results were confirmed in vivo thanks to a clinical study in which skin biomechanical properties and reduction in wrinkles were assessed.

The extracellular sulfatases Sulf1 and Sulf2 remove specific 6-O-sulfate groups from heparan sulfate, thereby modulating numerous signalling pathways underlying development and homeostasis. In vitro data have suggested that the two enzymes show functional redundancy. To elucidate their in vivo functions and to further address the question of a putative redundancy, we have generated Sulf1- and Sulf2-deficient mice. Phenotypic analysis of these animals revealed higher embryonic lethality of Sulf2 knockout mice, which can be associated with neuroanatomical malformations during embryogenesis. Sulf1 seems not to be essential for developmental or postnatal viability, as mice deficient in this sulfatase show no overt phenotype. However, neurite outgrowth deficits were observed in hippocampal and cerebellar neurons of both mutant mouse lines, suggesting that not only Sulf2 but also Sulf1 function plays a role in the developing nervous system. Behavioural analysis revealed differential deficits with regard to cage activity and spatial learning for Sulf1- and Sulf2-deficient mouse lines. In addition, Sulf1-specific deficits were shown for synaptic plasticity in the CA1 region of the hippocampus, associated with a reduced spine density. These results reveal that Sulf1 and Sulf2 fulfil non-redundant functions in vivo in the development and maintenance of the murine nervous system.

Papillomaviruses are epitheliotropic non-enveloped double-stranded DNA viruses, whose replication is strictly dependent on the terminally differentiating tissue of the epidermis. They induce self-limiting benign tumors of skin and mucosa, which may progress to malignancy (e.g. cervical carcinoma). Prior to entry into basal cells, virions attach to heparan sulfate moieties of the basement membrane. This triggers conformational changes, which affect both capsid proteins, L1 and L2, and such changes are a prerequisite for interaction with the elusive uptake receptor. These processes are very slow, resulting in an uptake half-time of up to 14 h. This minireview summarizes recent advances in our understanding of cell surface events, internalization and the subsequent intracellular trafficking of papillomaviruses.

Angiogenesis, the formation of new blood vessels from preexisting vasculature, is required for normal physiological as well as pathological events. The angiogenic process requires endothelial cells to proliferate, migrate, and undergo tubulogenesis. These multistep processes necessitate secretion of pro-angiogenic growth factors, activation of specific intracellular signaling, and interaction of endothelial cells with basement membrane (BM) extracellular matrix components. The generation and release of angiogenic molecules are highly regulated and are influenced by numerous factors, including BM-derived fragments, proteolytic enzymes, as well as metabolites of arachidonic acid (AA). The interactions between these key modulators of angiogenesis is extremely complex, as AA metabolites can regulate the synthesis of soluble angiogenic factors, BM components, as well as enzymes capable of cleaving BM components, which result in the generation of pro- and/or anti-angiogenic products. Furthermore, some BM-derived fragments can alter the expression of AA-converting enzymes and consequently the synthesis of angiogenic factors. In this review we describe the relationship between BM components and AA metabolites with respect to the regulation of endothelial cell functions in health and disease. Copyright © 2009 John Wiley & Sons, Inc. For further resources related to this article, please visit the WIREs website.

Cells exert tension on the extracellular matrix through specific receptors that link to the actin cytoskeleton. The best characterized are the integrins, which, when activated and clustered, can link to the extracellular matrix at specialized adhesion zones, known as focal contacts or focal adhesions. However, other transmembrane receptors can also localize there, including one transmembrane proteoglycan, syndecan-4. This heparan sulfate proteoglycan can also link directly to the cytoskeleton through alpha-actinin, and can signal through protein kinase C. In turn, the pathway leads to RhoA and Rho kinases that control actomyosin contractility. Syndecan-4 may, therefore, be a sensor of tension exerted on the matrix. These processes are described here, their significance being potential roles in wound contraction, tumor-stroma interactions, fibrosis and the regulation of motility.

Osteogenic differentiation is coordinated by the exposure of cells to temporal changes in a combination of growth factors and elements within the extracellular matrix (ECM). Many of the key proteins that drive these changes share the property of being dependent on ECM glycosaminoglycans (GAGs) for their activity. Here, we examined whether GAGs isolated from proliferating, differentiating and mineralizing MG-63 osteosarcoma cells differed in their physical properties, and thus in their capacities to coordinate the osteogenic cascade both in human MG-63 osteosarcoma cells and primary human mesenchymal stem cells (hMSCs). Our results show that the size distribution of GAGs, the expression of GAG-carrying proteoglycan cores and the expression of enzymes involved in their modification systematically change as MG-63 cells mature in culture. When dosed back onto cells exogenously in soluble form, GAGs regulated MG-63 survival and growth in a dose-dependent manner, but not differentiation in either cell type. In contrast, hMSCs aggregated into distinct colonies when grown on GAG-coated substrates, while MG-63 cells did not. Heparin-coated substrates improved hMSC viability without inducing aggregation. These results suggest a complex role for GAGs in coordinating the emergence of the osteoblast phenotype, and provide further evidence for the use of heparans in bone tissue repair applications.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) regulate a multitude of biological functions in embryonic development and in adult. A major question is how does one family of growth factors and their receptors control such a variety of functions? Classically, specificity was thought to be imparted by alternative splicing of the FGFRs, resulting in isoforms that bind specifically to a subset of the FGFs, and by different saccharide sequences in the heparan sulfate proteoglycan (HSPG) co-receptor. A growing number of noncanonical co-receptors such as integrins and neural cell adhesion molecule (NCAM) are now recognized as imparting additional complexity to classic FGFR signaling. This review will discuss the noncanonical FGFR ligands and speculate on the possibility that they provide additional and alternative means to determining the functional specificity of FGFR signaling. We will also discuss how invertebrate models such as C. elegans may advance our understanding of noncanonical FGFR signaling. Developmental Dynamics 238:277–293, 2009. © 2008 Wiley-Liss, Inc.

Olfactory sensory neuron (OSN) axons follow stereotypic spatio-temporal paths in the establishment of the olfactory pathway. Extracellular matrix (ECM) molecules are expressed early in the developing pathway and are proposed to have a role in its initial establishment. During later embryonic development, OSNs sort out and target specific glomeruli to form precise, complex topographic projections. We hypothesized that ECM cues may help to establish this complex topography. The aim of this study was to characterize expression of ECM molecules during the period of glomerulogenesis, when synaptic contacts are forming. We examined expression of laminin-1, perlecan, tenascin-C, and CSPGs and found a coordinated pattern of expression of these cues in the pathway. These appear to restrict axons to the pathway while promoting axon outgrowth within. Thus, ECM molecules are present in dynamic spatio-temporal positions to affect OSN axons as they navigate to the olfactory bulb and establish synapses.

Perlecan, a highly conserved and ubiquitous basement membrane heparan sulfate proteoglycan, is essential for life, inasmuch as its absence results in embryonic lethality in mice and C. elegans, and neonatal lethality in humans. Perlecan plays an essential role in vasculogenesis and chondrogenesis, as well as in pathological states where these processes are maladapted. Although a large body of evidence supports a pro-angiogenic role for perlecan, recent findings suggests that portions of the perlecan protein core can be antiangiogenic, requiring a further evaluation of the functioning of this complex molecule. This review is focused on the genetics of mammalian and nonmammalian perlecan, the elucidation of its novel interacting partners and its role in angiogenesis. By more fully understanding perlecan’s functioning in angiogenesis, we may gain invaluable insight that could lead to therapeutic interventions in cancer and other pathologic states.

Perlecan is a ubiquitous proteoglycan of basement membrane and vascularized tissues but is also present in articular cartilage, meniscus and intervertebral disc, which are devoid of basement membrane and predominantly avascular. It is a prominent pericellular proteoglycan in the transitory matrix of the cartilaginous rudiments that develop into components of diarthrodial joints and the axial skeleton, and it forms intricate perichondrial vessel networks that define the presumptive articulating surfaces of developing joints and line the cartilage canals in cartilaginous rudiments. Such vessels have roles in the nutrition of the expanding cell numbers in the developing joint. Perlecan sequesters a number of growth factors pericellularly (FGFs, PDGF, VEGF and CTGF) and through these promotes cell signalling, cell proliferation and differentiation. Perlecan also interacts with a diverse range of extracellular matrix proteins, stabilising and organising the ECM, and promoting collagen fibrillogenesis. Perlecan is a prominent pericellular component of mesenchymal cells from their earliest developmental stages through to maturation, forming cell-cell and cell-ECM interconnections that are suggestive of a role in mechanosensory processes important to tissue homeostasis.

Basement membranes (BMs) consisting of laminins, collagens, and heparan sulfate proteoglycans (HSPGs) are vital for proper endothelial cell function, but many aspects of their role in vascular development remain unknown. Here, we demonstrate that vascular structures within differentiating embryoid bodies are wrapped in a BM composed of alpha4- and alpha5-chain laminins, fibronectin, collagen IV, and HSPGs. In sprouting angiogenesis, laminins were produced by stalk cells, as well as the leading tip cell, and deposited along the sprout length, including tip cell filopodia. In embryonic stem cells deficient in laminins, due to lamc1 (laminin gamma1) deletion, vascular development and organization were largely unaffected. However, the frequency of vessels with wide lumens was increased 4-fold. Laminin-deficient vessels were moreover characterized by increased fibronectin levels and enhanced endothelial cell proliferation. We conclude that laminins are dispensable for vascular development but that they regulate lumen formation in the absence of flow and vascular tone.

Although interleukin-2 (IL-2) is typically considered a soluble cytokine, our laboratory has shown that the availability of IL-2 in lymphoid tissues is regulated, in part, by an association with heparan sulfate glycosaminoglycan. Heparan sulfate is usually found in proteoglycan form, in which the heparan sulfate chains are covalently linked to a specific core protein. We now show that perlecan is one of the major IL-2-binding heparan sulfate proteoglycans in murine spleen. IL-2 binds perlecan via heparan sulfate chains, as enzymatic removal of heparan sulfate from splenic perlecan abolishes its ability to bind IL-2. Furthermore, we demonstrate that perlecan-bound IL-2 supports the proliferation of an IL-2-dependent cell line. Identification of perlecan as a major heparan sulfate proteoglycan that binds IL-2 has implications for both the localization and regulation of IL-2 in vivo.

Two human colon carcinoma cell lines, HT29 and Caco-2 were co-cultured with fetal rat or human skin fibroblasts. Their morphological features, ultra-structural characteristics at the heterologous cell interface, and the deposition of basement-membrane molecules [laminin, type-IV collagen, heparan sulfate proteoglycan (HSPG)] at the epithelial-stromal junction were analyzed. The 2 cell lines behaved differently. HT29 cells did not spread on the fibroblasts and grew as clusters, while Caco-2 cells formed a monolayer over the fibroblastic feeder layer. Only the latter carcinoma cells exhibited cytoplasmic processes towards the fibroblasts and, after 5 days in co-cultures, a structured basement membrane (BM). The immunocytochemical analysis of the BM constituents revealed the absence of the molecules studied at the sites of heterologous contacts in the case of HT29 cells. In contrast, in the co-cultures comprising Caco-2 cells, laminin and type-IV collagen were progressively deposited in a polar fashion at the epithelial-fibroblastic interface which, however, remained devoid of HSPG molecules. Together with earlier data indicating a dual origin of the BM molecules located at the epithelial-fibroblastic interface in normal intestine, the present study shows that the cancer cells as well as the fibroblastic ones under the influence of carcinoma cells display an altered capacity to synthesize and/or secrete BM molecules. The extent of such abnormalities correlates with the differentiation of the cells. Finally, these modifications occur concomitantly with alterations in cell interactions which vary among cell lines.