ABSTRACT
Osteoarthritis (OA) represents one major cause of disability worldwide still evading efficient pharmacological or cellular therapies. Severe degeneration of extracellular cartilage matrix precedes the loss of mobility and disabling pain perception in affected joints. Recent studies showed that a reduced heparan sulfate (HS) content protects cartilage from degradation in OA-animal models of joint destabilization but the underlying mechanisms remained unclear. We aimed to clarify whether low HS-content alters the mechano-response of chondrocytes and to uncover pathways relevant for HS-related chondro-protection in response to loading. Tissue-engineered cartilage with HS-deficiency was generated from rib chondrocytes of mice carrying a hypomorphic allele of Exostosin 1 (Ext1), one of the main HS-synthesizing enzymes, and wildtype (WT) littermate controls. Engineered cartilage matured for 2 weeks was exposed to cyclic unconfined compression in a bioreactor. The molecular loading response was determined by transcriptome profiling, bioinformatic data processing, and qPCR. HS-deficient chondrocytes expressed 3-6% of WT Ext1-mRNA levels. Both groups similarly raised Sox9, Col2a1 and Acan levels during maturation. However, HS-deficient chondrocytes synthesized and deposited 50% more GAG/DNA. TGFß and FGF2-sensitivity of Ext1gt/gt chondrocytes was similar to WT cells but their response to BMP-stimulation was enhanced. Loading induced similar activation of mechano-sensitive ERK and P38-signaling in WT and HS-reduced chondrocytes. Transcriptome analysis reflected regulation of cell migration as major load-induced biological process with similar stimulation of common (Fosl1, Itgα5, Timp1, and Ngf) as well as novel mechano-regulated genes (Inhba and Dhrs9). Remarkably, only Ext1-hypomorphic cartilage responded to loading by an expression signature of negative regulation of apoptosis with pro-apoptotic Bnip3 being selectively down-regulated. HS-deficiency enhanced BMP-sensitivity, GAG-production and fostered an anti-apoptotic expression signature after loading, all of which may protect cartilage from load-induced erosion.
Subject(s)
Chondrocytes/physiology , Heparitin Sulfate/deficiency , Animals , Bone Morphogenetic Proteins/metabolism , MAP Kinase Signaling System , Mice, Transgenic , Primary Cell Culture , Weight-BearingABSTRACT
OBJECTIVE: Exostosin-1 (EXT1) and EXT2 are the major genetic etiologies of multiple hereditary exostoses and are essential for heparan sulfate (HS) biosynthesis. Previous studies investigating HS in several mouse models of multiple hereditary exostoses have reported that aberrant bone morphogenetic protein (BMP) signaling promotes osteochondroma formation in Ext1-deficient mice. This study examined the mechanism underlying the effects of HS deficiency on BMP/Smad signaling in articular cartilage in a cartilage-specific Ext-/- mouse model. METHOD: We generated mice with a conditional Ext1 knockout in cartilage tissue (Ext1-cKO mice) using Prg4-Cre transgenic mice. Structural cartilage alterations were histologically evaluated and phospho-Smad1/5/9 (pSmad1/5/9) expression in mouse chondrocytes was analyzed. The effect of pharmacological intervention of BMP signaling using a specific inhibitor was assessed in the articular cartilage of Ext1-cKO mice. RESULTS: Hypertrophic chondrocytes were significantly more abundant (P = 0.021) and cartilage thickness was greater in Ext1-cKO mice at 3 months postnatal than in control littermates (P = 0.036 for femur; and P < 0.001 for tibia). However, osteoarthritis did not spontaneously occur before the 1-year follow-up. matrix metalloproteinase (MMP)-13 and adamalysin-like metalloproteinases with thrombospondin motifs(ADAMTS)-5 were upregulated in hypertrophic chondrocytes of transgenic mice. Immunostaining and western blotting revealed that pSmad1/5/9-positive chondrocytes were more abundant in the articular cartilage of Ext1-cKO mice than in control littermates. Furthermore, the BMP inhibitor significantly decreased the number of hypertrophic chondrocytes in Ext1-cKO mice (P = 0.007). CONCLUSIONS: HS deficiency in articular chondrocytes causes chondrocyte hypertrophy, wherein upregulated BMP/Smad signaling partially contributes to this phenotype. HS might play an important role in maintaining the cartilaginous matrix by regulating BMP signaling.
Subject(s)
Bone Morphogenetic Proteins/metabolism , Cartilage, Articular/metabolism , Chondrocytes/metabolism , Heparitin Sulfate/deficiency , Osteoarthritis, Knee/metabolism , ADAMTS5 Protein/metabolism , Animals , Bone Morphogenetic Proteins/antagonists & inhibitors , Cartilage, Articular/cytology , Chondrocytes/pathology , Disease Models, Animal , Hypertrophy , Matrix Metalloproteinase 13/metabolism , Mice , Mice, Knockout , Mice, Transgenic , N-Acetylglucosaminyltransferases/genetics , Osteoarthritis, Knee/genetics , Osteoarthritis, Knee/pathology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , Smad1 Protein/metabolism , Smad5 Protein/metabolism , Smad8 Protein/metabolismABSTRACT
Proteinuria develops when specific components in the glomerular filtration barrier have impaired function. Although the precise components involved in maintaining this barrier have not been fully identified, heparan sulfate proteoglycans are believed to play an essential role in maintaining glomerular filtration. Although in situ studies have shown that a loss of heparan sulfate glycosaminoglycans increases the permeability of the glomerular filtration barrier, recent studies using experimental models have shown that podocyte-specific deletion of heparan sulfate glycosaminoglycan assembly does not lead to proteinuria. However, tubular reabsorption of leaked proteins might have masked an increase in glomerular permeability in these models. Furthermore, not only podocytes but also glomerular endothelial cells are involved in heparan sulfate synthesis in the glomerular filtration barrier. Therefore, we investigated the effect of a global heparan sulfate glycosaminoglycan deficiency on glomerular permeability. We used a zebrafish embryo model carrying a homozygous germline mutation in the ext2 gene. Glomerular permeability was assessed with a quantitative dextran tracer injection method. In this model, we accounted for tubular reabsorption. Loss of anionic sites in the glomerular basement membrane was measured using polyethyleneimine staining. Although mutant animals had significantly fewer negatively charged areas in the glomerular basement membrane, glomerular permeability was unaffected. Moreover, heparan sulfate glycosaminoglycan-deficient embryos had morphologically intact podocyte foot processes. Glomerular filtration remains fully functional despite a global reduction of heparan sulfate.
Subject(s)
Embryo, Nonmammalian/physiology , Heparitin Sulfate/deficiency , Kidney Glomerulus/physiology , Animals , Gene Expression Regulation , Heparitin Sulfate/metabolism , Mutation , N-Acetylglucosaminyltransferases/genetics , N-Acetylglucosaminyltransferases/metabolism , Zebrafish , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
The stem cell niche normally prevents aberrant stem cell behaviors that lead to cancer formation. Recent studies suggest that some cancers are derived from endogenous populations of adult stem cells that have somehow escaped from normal control by the niche. However, the molecular mechanisms by which the niche retains stem cells locally and tightly controls their divisions are poorly understood. Here, we demonstrate that the presence of heparan sulfate (HS), a class glygosaminoglycan chains, in the Drosophila germline stem cell niche prevents tumor formation in the testis. Loss of HS in the niche, called the hub, led to gross changes in the morphology of testes as well as the formation of both somatic and germline tumors. This loss of hub HS resulted in ectopic signaling events in the Jak/Stat pathway outside the niche. This ectopic Jak/Stat signaling disrupted normal somatic cell differentiation, leading to the formation of tumors. Our finding indicates a novel non-autonomous role for niche HS in ensuring the integrity of the niche and preventing tumor formation.
Subject(s)
Drosophila melanogaster/cytology , Drosophila melanogaster/metabolism , Germ Cells/pathology , Heparitin Sulfate/deficiency , Stem Cell Niche , Testis/pathology , Animals , Germ Cells/metabolism , Male , Testis/metabolismABSTRACT
Heparan sulfate (HS) is an essential component of cell surface and matrix proteoglycans (HS-PGs) that include syndecans and perlecan. Because of their unique structural features, the HS chains are able to specifically interact with signaling proteins -including bone morphogenetic proteins (BMPs)- via their HS-binding domain, regulating protein availability, distribution and action on target cells. Hereditary Multiple Exostoses (HME) is a rare pediatric disorder linked to germline heterozygous loss-of-function mutations in EXT1 or EXT2 that encode Golgi-resident glycosyltransferases responsible for HS synthesis, resulting in a systemic HS deficiency. HME is characterized by cartilaginous/bony tumors -called osteochondromas or exostoses- that form within perichondrium in long bones, ribs and other elements. This review examines most recent studies in HME, framing them in the context of classic studies. New findings show that the spectrum of EXT mutations is larger than previously realized and the clinical complications of HME extend beyond the skeleton. Osteochondroma development requires a somatic "second hit" that would complement the germline EXT mutation to further decrease HS production and/levels at perichondrial sites of osteochondroma induction. Cellular studies have shown that the steep decreases in local HS levels: derange the normal homeostatic signaling pathways keeping perichondrium mesenchymal; cause excessive BMP signaling; and provoke ectopic chondrogenesis and osteochondroma formation. Data from HME mouse models have revealed that systemic treatment with a BMP signaling antagonist markedly reduces osteochondroma formation. In sum, recent studies have provided major new insights into the molecular and cellular pathogenesis of HME and the roles played by HS deficiency. These new insights have led to the first ever proof-of-principle demonstration that osteochondroma formation is a druggable process, paving the way toward the creation of a clinically-relevant treatment.
Subject(s)
Exostoses, Multiple Hereditary/metabolism , Heparitin Sulfate/deficiency , Mutation , Animals , Bone Morphogenetic Proteins/metabolism , Disease Models, Animal , Exostoses, Multiple Hereditary/genetics , Heparitin Sulfate/chemistry , Humans , Mice , N-Acetylglucosaminyltransferases/genetics , Signal TransductionABSTRACT
Cell-surface engineering strategies that permit long-lived display of well-defined, functionally active molecules are highly attractive for eliciting desired cellular responses and for understanding biological processes. Current methodologies for the exogenous introduction of synthetic biomolecules often result in short-lived presentations, or require genetic manipulation to facilitate membrane attachment. Herein, we report a cell-surface engineering strategy that is based on the use of a CMP-Neu5Ac derivative that is modified at C-5 by a bifunctional entity composed of a complex synthetic heparan sulfate (HS) oligosaccharide and biotin. It is shown that recombinant ST6GAL1 can readily transfer the modified sialic acid to N-glycans of glycoprotein acceptors of living cells resulting in long-lived display. The HS oligosaccharide is functionally active, can restore protein binding, and allows activation of cell signaling events of HS-deficient cells. The cell-surface engineering methodology can easily be adapted to any cell type and is highly amenable to a wide range of complex biomolecules.
Subject(s)
Antigens, CD/metabolism , Cell Engineering/methods , Cytidine Monophosphate/analogs & derivatives , Sialic Acids/metabolism , Sialyltransferases/metabolism , Animals , Biotin/metabolism , Cells, Cultured , Cytidine Monophosphate/metabolism , Glycoproteins/metabolism , Glycosylation , Heparitin Sulfate/deficiency , Heparitin Sulfate/metabolism , Humans , Mice , Oligosaccharides/metabolism , Protein Binding , Signal TransductionABSTRACT
Autism Spectrum Disorders (ASD) are the second most common developmental cause of disability in the United States. ASDs are accompanied with substantial economic and emotional cost. The brains of ASD patients have marked structural abnormalities, in the form of increased dendritic spines and decreased long distance connections. These structural differences may be due to deficiencies in Heparin Sulfate (HS), a proteoglycan involved in a variety of neurodevelopmental processes. Of particular interest is its role in the Slit/Robo pathway. The Slit/Robo pathway is known to be involved in the regulation of axonal guidance and dendritic spine formation. HS mediates the Slit/Robo interaction; without its presence Slit's repulsive activity is abrogated. Slit/Robo regulates dendritic spine formation through its interaction with srGAPs (slit-robo GTPase Activating Proteins), which leads to downstream signaling, actin cytoskeleton depolymerization and dendritic spine collapse. Through interference with this pathway, HS deficiency can lead to excess spine formation.
Subject(s)
Autism Spectrum Disorder/metabolism , Autism Spectrum Disorder/pathology , Brain/abnormalities , Dendritic Spines/pathology , GTPase-Activating Proteins/metabolism , Heparitin Sulfate/deficiency , Nerve Tissue Proteins/metabolism , Receptors, Immunologic/metabolism , Animals , Brain/metabolism , Dendritic Spines/metabolism , Humans , Phenotype , Signal Transduction , Roundabout ProteinsABSTRACT
This was an observational study where we examined the role of perlecan HS on the deposition of TGF-ß1 in C57BL/6 and Hspg2(∆3-/∆3-) perlecan exon 3 null mouse skin. Despite its obvious importance in skin repair and tissue homeostasis no definitive studies have immunolocalised TGF-ß1 in skin in WT or Hspg2(∆3-/∆3-) perlecan exon 3 null mice. Vertical parasagittal murine dorsal skin from 3, 6 and 12 week old C57BL/6 and Hspg2(∆3-/∆3-) mice were fixed in neutral buffered formalin, paraffin embedded and 4 µm sections stained with Mayers haematoxylin and eosin (H & E). TGF-ß1 was immunolocalised using a rabbit polyclonal antibody, heat retrieval and the Envision NovaRED detection system. Immunolocalisation of TGF-ß1 differed markedly in C57BL/6 and Hspg2(∆3-/∆3-) mouse skin, ablation of exon 3 of Hspg2 resulted in a very severe reduction in the deposition of TGF-ß1 in skin 3-12 weeks postnatally. The reduced deposition of TGF-ß1 observed in the present study would be expected to impact detrimentally on the remodelling and healing capacity of skin in mutant mice compounding on the poor wound-healing properties already reported for perlecan exon 3 null mice due to an inability to signal with FGF-2 and promote angiogenic repair processes. TGF-ß1 also has cell mediated effects in tissue homeostasis and matrix stabilisation a reduction in TGF-ß1 deposition would therefore be expected to detrimentally impact on skin homeostasis in the perlecan mutant mice.
Subject(s)
Exons , Heparan Sulfate Proteoglycans/deficiency , Heparan Sulfate Proteoglycans/genetics , Heparitin Sulfate/deficiency , Skin/metabolism , Transforming Growth Factor beta1/metabolism , Animals , Genotype , Heparan Sulfate Proteoglycans/metabolism , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Knockout , Skin/pathologyABSTRACT
Heparan sulfate (HS) proteoglycans on stromal and hematopoietic stem/progenitor cells (HSPC) help form the stem cell niche, co-localize molecules that direct stem cell fate, and modulate HSPC homing and retention. Inhibition of HS function mobilizes marrow HSPC. In vitro, HSPC maintenance is influenced by stromal HS structure and concentration. Because inhibition of HS activity or synthesis may be developed for HSPC transplantation, it is important to examine if systemic HS deficiency influences hematopoiesis in vivo. In a transgenic mouse model of HS haploinsufficiency, we examined endogenous hematopoiesis and engraftment of allogeneic bone marrow. Endogenous hematopoiesis was normal except gender-specific alterations in peripheral blood monocyte and platelet counts. Donor engraftment was achieved in all mice following myeloablative irradiation, but HS deficiency in the stromal microenvironment, on HSPC, or both (the 3 test conditions), was associated with a trend towards lower donor engraftment percentage in the bone marrow. Following non-myeloablative irradiation, competitive engraftment was achieved in 22% of mice in the test conditions, vs 50% of control animals (P = 0.03). HS deficiency did not re-direct donor engraftment from bone marrow to spleen or liver. Normal HS levels in the stromal microenvironment and HSPC are required for HSPC engraftment following non-myeloablative conditioning.
Subject(s)
Bone Marrow Transplantation , Graft Survival/genetics , Haploinsufficiency/immunology , Hematopoiesis/genetics , Heparitin Sulfate/genetics , N-Acetylglucosaminyltransferases/genetics , Animals , Blood Platelets/immunology , Blood Platelets/pathology , Bone Marrow Cells/cytology , Bone Marrow Cells/immunology , Cell Count , Female , Gene Expression , Hematopoiesis/radiation effects , Hematopoietic Stem Cells/cytology , Hematopoietic Stem Cells/immunology , Heparitin Sulfate/deficiency , Male , Mice , Mice, Inbred C57BL , Monocytes/immunology , Monocytes/pathology , N-Acetylglucosaminyltransferases/deficiency , Sex Factors , Stem Cell Niche , Transplantation Chimera , Transplantation Conditioning , Transplantation, Homologous , Whole-Body IrradiationABSTRACT
UNLABELLED: Filoviruses, including both Ebola virus (EBOV) and Marburg virus (MARV), can infect humans and other animals, causing hemorrhagic fever with a high mortality rate. Entry of these viruses into the host is mediated by a single filoviral glycoprotein (GP). GP is composed of two subunits: GP1, which is responsible for attachment and binding to receptor(s) on susceptible cells, and GP2, which mediates viral and cell membrane fusion. Although numerous host factors have been implicated in the entry process, the initial attachment receptor(s) has not been well defined. In this report, we demonstrate that exostosin 1 (EXT1), which is involved in biosynthesis of heparan sulfate (HS), plays a role in filovirus entry. Expression knockdown of EXT1 by small interfering RNAs (siRNAs) impairs GP-mediated pseudoviral entry and that of infectious EBOV and MARV in tissue cultured cells. Furthermore, HS, heparin, and other related glycosaminoglycans (GAGs), to different extents, can bind to and block GP-mediated viral entry and that of infectious filoviruses. These results strongly suggest that HS and other related GAGs are attachment receptors that are utilized by filoviruses for entry and infection. These GAGs may have therapeutic potential in treating EBOV- and MARV-infected patients. IMPORTANCE: Infection by Ebola virus and Marburg virus can cause severe illness in humans, with a high mortality rate, and currently there is no FDA-approved vaccine or therapeutic treatment available. The ongoing 2014 outbreak in West Africa underscores a lack of our understanding in the infection and pathogenesis of these viruses and the urgency of drug discovery and development. In this study, we provide several pieces of evidence that demonstrate that heparan sulfate and other closely related glycosaminoglycans are the molecules that are used by filoviruses for initial attachment. Furthermore, we demonstrate that these glycosaminoglycans can block entry of and infection by filoviruses. Thus, this work provides mechanistic insights on the early step of filoviral infection and suggests a possible therapeutic option for diseases caused by filovirus infection.
Subject(s)
Filoviridae/physiology , Glycosaminoglycans/physiology , N-Acetylglucosaminyltransferases/physiology , Virus Internalization , Animals , Cell Line , Ebolavirus/pathogenicity , Ebolavirus/physiology , Filoviridae/pathogenicity , Filoviridae Infections/etiology , Filoviridae Infections/virology , Gene Knockdown Techniques , HEK293 Cells , Heparin/physiology , Heparitin Sulfate/biosynthesis , Heparitin Sulfate/deficiency , Host-Pathogen Interactions , Humans , Marburgvirus/pathogenicity , Marburgvirus/physiology , Mice , N-Acetylglucosaminyltransferases/antagonists & inhibitors , N-Acetylglucosaminyltransferases/genetics , Receptors, Virus/physiology , Viral Proteins/physiology , VirulenceABSTRACT
Congenital diaphragmatic hernia (CDH) is a common birth malformation with a heterogeneous etiology. In this study, we report that ablation of the heparan sulfate biosynthetic enzyme NDST1 in murine endothelium (Ndst1ECKO mice) disrupted vascular development in the diaphragm, which led to hypoxia as well as subsequent diaphragm hypoplasia and CDH. Intriguingly, the phenotypes displayed in Ndst1ECKO mice resembled the developmental defects observed in slit homolog 3 (Slit3) knockout mice. Furthermore, introduction of a heterozygous mutation in roundabout homolog 4 (Robo4), the gene encoding the cognate receptor of SLIT3, aggravated the defect in vascular development in the diaphragm and CDH. NDST1 deficiency diminished SLIT3, but not ROBO4, binding to endothelial heparan sulfate and attenuated EC migration and in vivo neovascularization normally elicited by SLIT3-ROBO4 signaling. Together, these data suggest that heparan sulfate presentation of SLIT3 to ROBO4 facilitates initiation of this signaling cascade. Thus, our results demonstrate that loss of NDST1 causes defective diaphragm vascular development and CDH and that heparan sulfate facilitates angiogenic SLIT3-ROBO4 signaling during vascular development.
Subject(s)
Heparitin Sulfate/deficiency , Hernias, Diaphragmatic, Congenital , Neovascularization, Physiologic , Sulfotransferases/genetics , Animals , Apoptosis , Cell Hypoxia , Cell Movement , Cell Proliferation , Cell Survival , Diaphragm/abnormalities , Diaphragm/blood supply , Diaphragm/enzymology , Endothelial Cells/enzymology , Female , Genetic Association Studies , Hernia, Diaphragmatic/enzymology , Hernia, Diaphragmatic/genetics , Male , Membrane Proteins/metabolism , Mice , Mice, Knockout , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Penetrance , Receptors, Cell Surface , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Signal Transduction , Sulfotransferases/deficiency , Tendons/abnormalities , Tendons/pathology , Vascular Endothelial Growth Factor A/metabolismABSTRACT
Heparan sulfate (HS), a constituent of HS proteoglycans (HSPGs), is a linear polysaccharide present on the cell surface. HSPGs modulate functions of several growth factors and signaling molecules. We examined whether small intestinal epithelial HS plays some roles in crypt homeostasis using intestinal epithelium cell (IEC)-specific HS-deficient C57Bl/6 mice. Survival rate after total body irradiation was significantly reduced in HS-deficient mice due to profound intestinal injury. HS-deficient IECs exhibited Wnt/ß-catenin pathway disruption, decreased levels of ß-catenin nuclear localization, and reduced expression of Wnt target genes, including Lgr5 during crypt regeneration. Moreover, epithelial HS increased Wnt binding affinity of IECs, promoted phosphorylation of Wnt coreceptor LRP6, and enhanced Wnt/ß-catenin signaling following ex vivo stimulation with Wnt3a, whereas activation of canonical Wnt signaling following direct inhibition of glycogen synthase kinase-3ß by lithium chloride was similar between HS-deficient and wild-type mice. Thus HS influences the binding affinity of IECs to Wnt, thereby promoting activation of canonical Wnt signaling and facilitating regeneration of small intestinal crypts after epithelial injury.
Subject(s)
Heparitin Sulfate/deficiency , Heparitin Sulfate/physiology , Intestinal Mucosa/pathology , Intestinal Mucosa/physiopathology , Wnt Signaling Pathway/physiology , Animals , Heparan Sulfate Proteoglycans , Homeostasis , Intestinal Mucosa/radiation effects , Intestine, Small/physiopathology , Mice , Mice, Inbred C57BL , Regeneration/physiology , Whole-Body Irradiation , Wnt3A Protein/metabolismABSTRACT
Abnormal cellular growth and organization have been characterized in postmortem tissue from brains of autistic individuals, suggestive of pathology in a critical neurogenic niche, the subventricular zone (SVZ) of the brain lateral ventricles (LV). We examined cellular organization, cell proliferation, and constituents of the extracellular matrix such as N-sulfated heparan sulfate (HS) and laminin (LAM) in postmortem brain tissue from the LV-SVZ of young to elderly individuals with autism (n=4) and age-matched typically developing (TD) individuals (n=4) using immunofluorescence techniques. Strong and systematic reductions in HS immunofluorescence were observed in the LV-SVZ of the TD individuals with increasing age. For young through mature, but not elderly, autistic pair members, HS was reduced compared to their matched TDs. Cellular proliferation (Ki67+) was higher in the autistic individual of the youngest age-matched pair. These preliminary data suggesting that HS may be reduced in young to mature autistic individuals are in agreement with previous findings from the BTBR T+tf/J mouse, an animal model of autism; from mice with genetic modifications reducing HS; and with genetic variants in HS-related genes in autism. They suggest that aberrant extracellular matrix glycosaminoglycan function localized to the subventricular zone of the lateral ventricles may be a biomarker for autism, and potentially involved in the etiology of the disorder.
Subject(s)
Autistic Disorder/metabolism , Heparitin Sulfate/deficiency , Lateral Ventricles/metabolism , Adult , Autistic Disorder/pathology , Cell Proliferation , Child, Preschool , Extracellular Matrix/metabolism , Extracellular Matrix/pathology , Humans , Laminin/metabolism , Lateral Ventricles/cytology , Lateral Ventricles/pathology , Male , Middle Aged , Neurogenesis/physiology , Tissue Banks , Young AdultABSTRACT
The cellular receptor of foamy viruses (FVs) is unknown. The broad spectrum of permissive cells suggests that the cellular receptor is a molecular structure with almost ubiquitous prevalence. Here, we investigated the ability of heparan sulfate (HS), a glycosaminoglycan (GAG) present on the extracellular matrix of many cells, to bind FV particles and to permit prototype FV (PFV) and feline FV (FFV) entry. Permissivity of different cell lines for FV entry correlated with the amount of heparan sulfate present on the cell surface. The resulting 50% cell culture infectious doses (CCID(50)s) were distributed over a range of 4 logs, which means that the most susceptible cell line tested (HT1080) was more than 10,000 times more susceptible for PFV infection than the least susceptible cell line (CRL-2242). HS surface expression varied over a range of 2 logs. HS expression and FV susceptibility were positively correlated (P < 0.001). Enzymatic digestion of heparan sulfate on HT1080 cells diminished permissivity for PFV entry by a factor of at least 500. Using fast protein liquid chromatography (FPLC), we demonstrated binding of FV vector particles to a gel filtration column packed with heparin, a molecule structurally related to heparan sulfate, allowing for the purification of infectious particles. Both PFV and FFV infection were inhibited by soluble heparin. Our results show that FVs bind to HS and that this interaction is a pivotal step for viral entry, suggesting that HS is a cellular attachment factor for FVs.
Subject(s)
Heparitin Sulfate/physiology , Spumavirus/physiology , Virus Attachment , Animals , Cats , Cell Membrane/drug effects , Cell Membrane/physiology , Cell Membrane/virology , Cricetinae , Disease Progression , Heparin/metabolism , Heparin/pharmacology , Heparitin Sulfate/deficiency , Humans , Mice , Receptors, Virus/drug effects , Receptors, Virus/physiology , Retroviridae Infections/prevention & control , Spumavirus/pathogenicity , Virus Attachment/drug effects , Virus Internalization/drug effectsABSTRACT
Heparan sulfate regulates diverse cell-surface signaling events, and its roles in the development of the nervous system recently have been increasingly uncovered by studies using genetic models carrying mutations of genes encoding enzymes for its synthesis. On the other hand, the role of heparan sulfate in the physiological function of the adult brain has been poorly characterized, despite several pieces of evidence suggesting its role in the regulation of synaptic function. To address this issue, we eliminated heparan sulfate from postnatal neurons by conditionally inactivating Ext1, the gene encoding an enzyme essential for heparan sulfate synthesis. Resultant conditional mutant mice show no detectable morphological defects in the cytoarchitecture of the brain. Remarkably, these mutant mice recapitulate almost the full range of autistic symptoms, including impairments in social interaction, expression of stereotyped, repetitive behavior, and impairments in ultrasonic vocalization, as well as some associated features. Mapping of neuronal activation by c-Fos immunohistochemistry demonstrates that neuronal activation in response to social stimulation is attenuated in the amygdala in these mice. Electrophysiology in amygdala pyramidal neurons shows an attenuation of excitatory synaptic transmission, presumably because of the reduction in the level of synaptically localized AMPA-type glutamate receptors. Our results demonstrate that heparan sulfate is critical for normal functioning of glutamatergic synapses and that its deficiency mediates socio-communicative deficits and stereotypies characteristic for autism.
Subject(s)
Autistic Disorder/complications , Communication , Heparitin Sulfate/deficiency , Social Behavior , Stereotypic Movement Disorder/complications , Amygdala/pathology , Amygdala/physiopathology , Animals , Autistic Disorder/physiopathology , Heparitin Sulfate/metabolism , Immunohistochemistry , Mice , Mice, Inbred C57BL , Mice, Knockout , N-Acetylglucosaminyltransferases/deficiency , N-Acetylglucosaminyltransferases/metabolism , Nervous System/pathology , Nervous System/physiopathology , Neurons/metabolism , Neurons/pathology , Organ Specificity , Phenotype , Proto-Oncogene Proteins c-fos/metabolism , Stereotypic Movement Disorder/physiopathology , Synaptic Transmission/physiologyABSTRACT
Growth and remodeling of lymphatic vasculature occur during development and during various pathologic states. A major stimulus for this process is the unique lymphatic vascular endothelial growth factor-C (VEGF-C). Other endothelial growth factors, such as fibroblast growth factor-2 (FGF-2) or VEGF-A, may also contribute. Heparan sulfate is a linear sulfated polysaccharide that facilitates binding and action of some vascular growth factors such as FGF-2 and VEGF-A. However, a direct role for heparan sulfate in lymphatic endothelial growth and sprouting responses, including those mediated by VEGF-C, remains to be examined. We demonstrate that VEGF-C binds to heparan sulfate purified from primary lymphatic endothelia, and activation of lymphatic endothelial Erk1/2 in response to VEGF-C is reduced by interference with heparin or pretreatment of cells with heparinase, which destroys heparan sulfate. Such treatment also inhibited phosphorylation of the major VEGF-C receptor VEGFR-3 upon VEGF-C stimulation. Silencing lymphatic heparan sulfate chain biosynthesis inhibited VEGF-C-mediated Erk1/2 activation and abrogated VEGFR-3 receptor-dependent binding of VEGF-C to the lymphatic endothelial surface. These findings prompted targeting of lymphatic N-deacetylase/N-sulfotransferase-1 (Ndst1), a major sulfate-modifying heparan sulfate biosynthetic enzyme. VEGF-C-mediated Erk1/2 phosphorylation was inhibited in Ndst1-silenced lymphatic endothelia, and scratch-assay responses to VEGF-C and FGF-2 were reduced in Ndst1-deficient cells. In addition, lymphatic Ndst1 deficiency abrogated cell-based growth and proliferation responses to VEGF-C. In other studies, lymphatic endothelia cultured ex vivo from Ndst1 gene-targeted mice demonstrated reduced VEGF-C- and FGF-2-mediated sprouting in collagen matrix. Lymphatic heparan sulfate may represent a novel molecular target for therapeutic intervention.
Subject(s)
Lymphangiogenesis , Vascular Endothelial Growth Factor C/physiology , Animals , Endothelium, Lymphatic , Heparitin Sulfate/deficiency , Lymphatic Vessels , Mice , Mitogen-Activated Protein Kinase 3/metabolism , Protein Binding , Sulfotransferases/metabolism , Vascular Endothelial Growth Factor Receptor-3ABSTRACT
Angiogenesis is a hallmark of expanding tissue e.g. during embryogenesis and wound healing in physiology as well as in diseases such as cancer and atherosclerosis. Key steps of the angiogenic process involve growth factor-mediated stimulation of endothelial cell sprouting and tube formation. Heparan sulphate proteoglycans (HSPGs) have been implicated as important co-receptors of several pro-angiogenic proteins. The importance of HSPGs in physiology was underscored by the finding that knockout of the gene encoding HS polymerase, EXT-1, resulted in early embryonic lethality. Here, we describe the establishment of HS-deficient endothelial cells from sprouting aortas as well as from the lungs of EXT-1(flox/flox) mice. Recombination of the loxP-flanked EXT-1 locus by Cre-expressing adenovirus was demonstrated at the mRNA level. Moreover, depletion of HS polysaccharides was verified by flow cytometry and fluorescence microscopy methodology using phage display-derived anti-HS antibodies. In summary, we provide a genetic model to unravel the functional role of HSPGs specifically in primary endothelial cells during early steps of angiogenesis. Our studies are applicable to most loxP-based transgenic mouse strains, and may thus be of general importance in the angiogenesis field.
Subject(s)
Aorta/growth & development , Cell Culture Techniques/methods , Endothelial Cells/metabolism , Heparitin Sulfate/deficiency , Lung/blood supply , N-Acetylglucosaminyltransferases/genetics , Neovascularization, Physiologic , Adenoviridae/genetics , Animals , Cells, Cultured , Endothelial Cells/enzymology , Genetic Vectors/genetics , Heparitin Sulfate/metabolism , Integrases/metabolism , Lung/enzymology , Mice , Mice, Transgenic , N-Acetylglucosaminyltransferases/metabolism , Recombination, Genetic/geneticsABSTRACT
A quantifiable in situ immune fluorescent assay (IFA) was developed to measure bluetongue virus (BTV) binding to mammalian cells. The utility of the assay was demonstrated with both Chinese hamster ovary (CHO) and bovine pulmonary artery endothelial (CPAE) cells. Since heparin sulfate (HS) has been shown to function as a receptor for a number of viruses, its role as a receptor for BTV was evaluated with the in situ IFA. Binding of BTV to both CHO and CPAE cells was inhibited in a dose dependent manner by HS. In addition, HS deficient CHO cells showed greatly diminished binding of BTV when compared to the parental cell line. The IFA protocol will find application, as a non-isotopic, quantifiable technique, to study virus-cell receptor interactions. Information gained from such studies will expand our understanding of the early steps in virus replication.
Subject(s)
Bluetongue virus/physiology , Heparitin Sulfate/metabolism , Receptors, Virus/metabolism , Virology/methods , Virus Attachment , Animals , Cattle , Cell Line , Cricetinae , Cricetulus , Female , Fluorescent Antibody Technique, Direct/methods , Heparitin Sulfate/antagonists & inhibitors , Heparitin Sulfate/deficiency , Receptors, Virus/antagonists & inhibitors , Receptors, Virus/deficiencyABSTRACT
The heparan sulfate (HS) is a component of proteoglycans in the extracellular matrix and on cell surfaces, modulating developmental processes. The aim of this study is to investigate whether the defect of HS in the periocular mesenchyme impairs ocular morphogenesis. First, using Protein 0-Cre transgenic mice, we ablated Ext1, which encodes an indispensable enzyme for HS synthesis, in the developing periocular mesenchyme. The expression of Ext1 messenger RNA (mRNA) and HS were observed by RT-PCR and immunohistochemistry, respectively. The phenotypes in the mutant were evaluated by light microscopy and immunohistochemistry for cellular makers. Second, the distribution of the mutant periocular mesenchymal cells was tracked using a Rosa26 Cre-reporter gene. No mutant embryos (Protein 0-Cre;Ext1(flox/flox)) were identified after embryonic day 14.5 (E14.5). RT-PCR showed that an intense band amplified from Ext1 was observed in cDNAs from the control periocular mesenchymal cells at E13.5; however, the band for Ext1 was hardly detectable in cDNA from the mutant embryo, indicating that the mRNA was missing in the mutant periocular mesenchyme at E13.5. The HS expression was disrupted in the periocular mesenchyme of the mutant ocular tissues. The HS deficiency resulted in microphthalmia with reduced axial lengths, lens diameters, and vitreous sizes compared with the littermate eyes. The mutant embryos showed agenesis of the anterior chamber, where cells expressing Cre recombinase were distributed. Moreover, the mutants showed phenotypic alterations in the neural ectoderm including dysgenesis of the presumptive ciliary body and agenesis of the optic nerve head. These findings demonstrate that HS in the periocular mesenchyme plays a critical role in normal ocular morphogenesis, indicating reciprocal interactions between the periocular mesenchyme and the neural ectoderm.
Subject(s)
Ciliary Body/abnormalities , Eye Abnormalities/embryology , Heparitin Sulfate/deficiency , Mesoderm/embryology , Microphthalmos/embryology , Animals , Carbohydrate Epimerases/metabolism , Eye Abnormalities/enzymology , Eye Abnormalities/pathology , Female , Genotype , Immunoenzyme Techniques , Male , Mice , Mice, Transgenic , Microphthalmos/enzymology , Microphthalmos/pathology , Morphogenesis , N-Acetylglucosaminyltransferases/genetics , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Smad Proteins/metabolism , Sulfotransferases/metabolismABSTRACT
The effect of targeted inactivation of the gene encoding N-deacetylase/N-sulfotransferase-1 (Ndst1), a key enzyme involved in the biosynthesis of heparan sulfate (HS) chains, on the inflammatory response associated with allergic inflammation in a murine model of OVA-induced acute airway inflammation was investigated. OVA-exposed Ndst1(f/f)TekCre(+) (mutant) mice deficient in endothelial and leukocyte Ndst1 demonstrated significantly decreased allergen-induced airway hyperresponsiveness and inflammation characterized by a significant reduction in airway recruitment of inflammatory cells (eosinophils, macrophages, neutrophils, and lymphocytes), diminished IL-5, IL-2, TGF-beta1, and eotaxin levels, as well as decreased expression of TGF-beta1 and the angiogenic protein FIZZ1 (found in inflammatory zone 1) in lung tissue compared with OVA-exposed Ndst1(f/f)TekCre(-) wild-type littermates. Furthermore, murine eosinophils demonstrated significantly decreased rolling on lung endothelial cells (ECs) from mutant mice compared with wild-type ECs under conditions of flow in vitro. Treatment of wild-type ECs, but not eosinophils, with anti-HS Abs significantly inhibited eosinophil rolling, mimicking that observed with Ndst1-deficient ECs. In vivo, trafficking of circulating leukocytes in lung microvessels of allergen-challenged Ndst1-deficient mice was significantly lower than that observed in corresponding WT littermates. Endothelial-expressed HS plays an important role in allergic airway inflammation through the regulation of recruitment of inflammatory cells to the airways by mediating interaction of leukocytes with the vascular endothelium. Furthermore, HS may also participate by sequestering and modulating the activity of allergic asthma-relevant mediators such as IL-5, IL-2, and TGF-beta1.