Versican V1 Research Articles

Use of versican variant V3 and versican antisense expression to engineer cultured human skin containing increased content of insoluble elastin.

Skin substitutes for repair of dermal wounds are deficient in functional elastic fibres. We report that the content of insoluble elastin in the dermis of cultured human skin can be increased though the use of two approaches that enhance elastogenesis by dermal fibroblasts, forced expression of versican variant V3, which lacks glycosaminoglycan (GAG) chains, and forced expression of versican antisense to decrease levels of versican variant V1 with GAG chains. Human dermal fibroblasts transduced with V3 or anti-versican were cultured under standard conditions over a period of 4 weeks to produce dermal sheets, with growth enhanced though multiple seedings for the first 3 weeks. Human keratinocytes, cultured in supplemented media, were added to the 4-week dermal sheets and the skin layer cultured for a further week. At 5 weeks, keratinocytes were multilayered and differentiated, with desmosome junctions thoughout and keratin deposits in the upper squamous layers. The dermal layer was composed of layered fibroblasts surrounded by extracellular matrix of collagen bundles and, in control cultures, small scattered elastin deposits. Forced expression of V3 and versican antisense slowed growth, decreased versican V1 expression, increased tropoelastin expression and/or the deposition of large aggregates of insoluble elastin in the dermal layer, and increased tissue stiffness, as measured by nano-indentation. Skin sheets were also cultured on Endoform Dermal Template™, the biodegradable wound dressing made from the lamina propria of sheep foregut. Skin structure and the enhanced deposition of elastin by forced expression of V3 and anti-versican were preserved on this supportive substrate. Copyright © 2014 John Wiley & Sons, Ltd.

Versican is a critical extracellular matrix protein in lung and brain during embryonic development in the mouse (1046.9)

Versican, a chondroitin sulfate proteoglycan and a component of the extracellular matrix, is associated with fatal cardiac malformations if absent during embryonic development and is increased with injury in adult tissues. Changes in versican gene expression and accumulation have not been examined comprehensively in the lung and brain during mid to late gestation in the mouse. The objective of this work was to determine if versican accumulation and degradation change during lung and brain embryogenesis. Quantitative PCR and immunohistochemistry were used to measure versican expression, accumulation and degradation in the lung, brain, heart and liver of C57Bl/6 mice for five consecutive days during embryonic development. Consistent with previous reports, we found that total versican gene expression was higher in embryonic tissues when compared to adults and the highest total versican gene expression in embryos occurred at embryonic day 13.5 (E13.5). At E15.5, versican gene expression was lowest in the liver, highest in the brain, and intermediate in the heart and lung. There was differential expression of versican isoforms, with V0, V1, and V2 isoforms increased in the brain but only V0 and V1 isoforms increased in the lung. Versican expression in the lung peaked at E13.5, at which time there was strong versican immunoreactivity in the interstitium and airway basement membrane but little accumulation in airway epithelial cells. In the brain, immunoreactivity was strongest in the roof of the hind brain and neopallial cortex and in the deeper white matter tracts, and at later gestational ages there was little accumulation in the developing choroid plexus and periventricular gray matter. During embryonic development of the lung and brain, we found areas of versican expression that colocalized with the expression of DPEAAE, a versican degradation product. These results suggest that versican plays an important role in embryogenesis of the lung and brain.Grant Funding Source: Supported by HL098067, RR030249

TGF-β1 Promotes Osteosarcoma Cell Migration and Invasion Through the miR-143-Versican Pathway

Background & Aims: TGF-β1 is an abundant cytokine present in the tumour microenvironment. It has been shown to trigger versican expression in human osteosarcoma cells, which may account for the metastatic potential of these cells. However, the underlying mechanism of TGF-β1-mediated metastasis remains unclear. The aim of this study was to evaluate the roles of versican in TGF-β1-induced osteosarcoma cell migration and invasion. Methods: Sixty paired osteosarcoma tumour tissues and adjacent normal tissues were obtained, and the relationship between Enneking stage and versican expression was tested by ANOVA analysis. Real-time PCR or Western blot was used to detect versican, Smad and miR-143 expression. Osteosarcoma cell migration and invasion was assessed using Boyden chambers. A luciferase reporter assay was employed to validate the miR-143-versican interaction. Results: Both versican isoforms, V0 and V1, were significantly differentially expressed in tumours at different stages. TGF-β1 promoted osteosarcoma cell migration and invasion in vitro by up-regulating versican. Furthermore, TGF-β1 suppressed miR-143 expression through a Smad 2/3-dependent pathway. miR-143 directly targets the versican 3'-UTR, and anti-miR-143 or versican knockdown blocked the effects of TGF-β1. Conclusion: Our results suggest that TGF-β1 up-regulates versican expression by suppressing miR-143, and this pathway is important for osteosarcoma cell migration and invasion.

Tumor-Produced Versican V1 Enhances hCAP18/LL-37 Expression in Macrophages through Activation of TLR2 and Vitamin D3 Signaling to Promote Ovarian Cancer Progression In Vitro

Tumor-associated macrophages have been shown to promote tumor growth. They may have an obligatory function in angiogenesis, invasion, and metastasis through release of inflammatory mediators. Their presence in ovarian cancer has been correlated with poor prognosis in these patients. The human cationic antimicrobial protein-18 (hCAP18)/LL-37 was originally identified as an effector molecule of the innate immune system. It is released by innate immune cells, such as macrophages, to combat microorganisms. Previous studies have characterized the hCAP18/LL-37 as a growth factor that has been shown to promote ovarian tumor progression. However, the role hCAP18/LL-37 has in macrophage-promoted ovarian tumor development and how its expression is controlled in this context remains poorly understood. Here, we demonstrate in co-culture experiments of macrophages and ovarian cancer cells a significant increase in the in vitro proliferation and invasiveness of the tumor cells is observed. These enhanced growth and invasion properties correlated with hCAP18/LL-37 induction. HCAP18/LL-37 expression was diminished by addition of two neutralizing antibodies, TLR2 or TLR6, as well as Cyp27B1 or VDR inhibitors. Furthermore, either the TLR2 or TLR6 antibody reduced vitamin D3 signaling and tumor cell progression in vitro. Addition of Cyp27B1 or VDR inhibitors abrogated TLR2/6 activation-induced expression of hCAP18/LL-37 in macrophages. Knockdown of tumor-produced versican V1 by RNAi in these tumor cells led to a decreased induction of hCAP18/LL-37 in macrophages. Versican V1 knockdown also inhibited TLR2 and vitamin D3 signaling, as well as growth and invasiveness of these tumor cells in the in vitro co-culture. In summary, we have found that versican V1 enhances hCAP18/LL-37 expression in macrophages through activation of TLR2 and subsequent vitamin D-dependent mechanisms which promote ovarian tumor progression in vitro.

Versican V2 isoform enhances angiogenesis by regulating endothelial cell activities and fibronectin expression

Versican is a proteoglycan expressed in the extracellular matrix, where it regulates a variety of cell activities and affects tumor development. With alternative splicing, there are four versican isoforms, denoted V0, V1, V2 and V3. The V2 isoform is highly expressed in the mature brain but its function in the mature brain has not yet been elucidated. Since brain tumors are among the most angiogenic of human tumors, we investigated whether or not the V2 isoform plays a role in angiogenesis and found that the glioblastoma cell line U87 stably transfected with V2 formed tumors containing extensive vasculature. Although the V2-expressing cells grew slowly, they survived well in serum-free medium. They also displayed high adhesive ability to endothelial cells and facilitated tube-like structure formation. Importantly, fibronectin was up-regulated by V2 and mediated V2 function. Thus, versican V2 could be a potential target for intervention of brain tumor angiogenesis.

Versican 3′‐untranslated region (3′‐UTR) functions as a ceRNA in inducing the development of hepatocellular carcinoma by regulating miRNA activity

This study was designed to explore the role of versican in the development of hepatocellular carcinoma (HCC). Ectopic expression of the versican 3'-untranslated region (3'-UTR) was studied as a competitive endogenous RNA for regulating miRNA functions. We used this approach to modulate the expression of versican and its related proteins in 3'-UTR transgenic mice and in the liver cancer cell line HepG2, stably transfected with the 3'-UTR or a control vector. We demonstrated that transgenic mice expressing the versican 3'-UTR developed HCC and increased expression of versican isoforms V0 and V1. HepG2 cells transfected with versican 3'-UTR displayed increased proliferation, survival, migration, invasion, colony formation, and enhanced endothelial cell growth, but decreased apoptosis. We found that versican 3'-UTR could bind to miRNAs miR-133a, miR-199a*, miR-144, and miR-431 and also interacted with CD34 and fibronectin. As a consequence, expression of versican, CD34, and fibronectin was up-regulated by ectopic transfection of the versican 3'-UTR, which was confirmed in HepG2 cells and in transgenic mice as compared with wild-type controls. Transfection with siRNAs targeting the versican 3'-UTR abolished the effects of the 3'-UTR. Taken together, these results demonstrate that versican V0 and V1 isoforms play important roles in HCC development and that versican mRNAs compete with endogenous RNAs in regulating miRNA functions.

Selective Decline of Synaptic Protein Levels in the Frontal Cortex of Female Mice Deficient in the Extracellular Metalloproteinase ADAMTS1

The chondroitin sulfate-bearing proteoglycans, also known as lecticans, are a major component of the extracellular matrix (ECM) in the central nervous system and regulate neural plasticity. Growing evidence indicates that endogenous, extracellular metalloproteinases that cleave lecticans mediate neural plasticity by altering the structure of ECM aggregates. The bulk of this in vivo data examined the matrix metalloproteinases, but another metalloproteinase family that cleaves lecticans, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), modulates structural plasticity in vitro, although few in vivo studies have tested this concept. Thus, the purpose of this study was to examine the neurological phenotype of a mouse deficient in ADAMTS1. Adamts1 mRNA was absent in the ADAMTS1 null mouse frontal cortex, but there was no change in the abundance or proteolytic processing of the prominent lecticans brevican and versican V2. However, there was a marked increase in the perinatal lectican neurocan in juvenile ADAMTS1 null female frontal cortex. More prominently, there were declines in synaptic protein levels in the ADAMTS1 null female, but not male, frontal cortex beginning at postnatal day 28. These synaptic marker declines did not affect learning or memory in the adult female ADAMTS1 null mice when tested with the radial-arm water maze. These results indicate that in vivo Adamts1 knockout leads to sexual dimorphism in frontal cortex synaptic protein levels. Since changes in lectican abundance and proteolytic processing did not accompany the synaptic protein declines, ADAMTS1 may play a nonproteolytic role in regulating neural plasticity.

Increased Renal Versican Expression Is Associated with Progression of Chronic Kidney Disease

Novel prognostic markers for progression of kidney disease are needed to distinguish patients who might benefit from a more aggressive nephroprotective therapy. Expression of the proteoglycan versican was evaluated in renal transcriptomics profiles and in an independent set of 74 renal biopsies. Versican levels were correlated to histologic damage scores and to renal outcome, and versican expression and regulation was evaluated in vitro. In transcriptomics profiles of renal tissue versican was positively correlated with (i) histological parameters in kidney biopsies, (ii) progressive decline of renal function in proteinuric kidney diseases, and (iii) impaired renal function and histology scores in diabetic nephropathy. In an independent cohort of 74 biopsies of glomerular diseases renal RNA levels of versican isoforms V0 and V1, but not V2 and V3 correlated significantly with creatinine after a mean follow up time of 53 months. Versican isoforms V0 and V1 together with serum creatinine at time of biopsy and the degree of glomerulosclerosis predicted 20% and 24% of the variability of creatinine at follow up, which was significantly more than serum creatinine and histological parameters alone (16%). However, when patients with acute kidney failure at time of biopsy (n = 5) were excluded, the additive predictive value of versican V1 was only marginally higher (35%) than creatinine and glomerulosclerosis alone (34%). Versican isoforms V0 and V1 were primarily expressed in vitro in proximal tubule cells and in fibroblasts. The results in humans were confirmed in three rodent models of kidney disease, in which renal versican expression was significantly upregulated as compared to corresponding controls. These data show for the first time an association of renal versican isoform V0 and V1 expression with progressive renal disease.

Estradiol Protects Dermal Hyaluronan/Versican Matrix during Photoaging by Release of Epidermal Growth Factor from Keratinocytes

Hyaluronan (HA) and versican are key components of the dermis and are responsive to ultraviolet (UV)B-induced remodeling. The aim of this study was to explore the molecular mechanisms mediating the effects of estrogen (E(2)) on HA-rich extracellular matrix during photoaging. Hairless skh-1 mice were irradiated with UVB (three times, 1 minimal erythema dose (80 mJ/cm(2)), weekly) for 10 weeks, and endogenous sex hormone production was abrogated by ovariectomy. Subcutaneous substitution of E(2) by means of controlled-release pellets caused a strong increase in the dermal HA content in both irradiated and nonirradiated skin. The increase in dermal HA correlated with induction of HA synthase HAS3 by E(2). Expression of splice variant 2 of the HA-binding proteoglycan versican was also increased by E(2). In search of candidate mediators of these effects, it was found that E(2) strongly induced the expression of epidermal growth factor (EGF) in UVB-irradiated epidermis in vivo and in keratinocytes in vitro. EGF in turn up-regulated the expression of HAS3 and versican V2 in dermal fibroblasts. HAS3 knockdown by shRNA caused inhibition of fibroblast proliferation. Furthermore, HAS3 and versican V2 induction by E(2) correlated positively with proliferation in vivo. In addition, the accumulation of inflammatory macrophages, expression of inducible cyclooxygenase 2, as well as proinflammatory monocyte chemotactic protein 1 were decreased in response to E(2) in the dermis. Collectively, these data suggest that E(2) treatment increases the amount of dermal HA and versican V2 via paracrine release of EGF, which may be implicated in the pro-proliferative and anti-inflammatory effects of E(2) during photoaging.

Altered Synaptic Marker Abundance in the Hippocampal Stratum Oriens of Ts65Dn Mice is Associated with Exuberant Expression of Versican

DS (Down syndrome), resulting from trisomy of chromosome 21, is the most common cause of genetic mental retardation; however, the molecular mechanisms underlying the cognitive deficits are poorly understood. Growing data indicate that changes in abundance or type of CSPGs (chondroitin sulfate proteoglycans) in the ECM (extracellular matrix) can influence synaptic structure and plasticity. The purpose of this study was to identify changes in synaptic structure in the hippocampus in a model of DS, the Ts65Dn mouse, and to determine the relationship to proteoglycan abundance and/or cleavage and cognitive disability. We measured synaptic proteins by ELISA and changes in lectican expression and processing in the hippocampus of young and old Ts65Dn mice and LMCs (littermate controls). In young (5 months old) Ts65Dn hippocampal extracts, we found a significant increase in the postsynaptic protein PSD-95 (postsynaptic density 95) compared with LMCs. In aged (20 months old) Ts65Dn hippocampus, this increase was localized to hippocampal stratum oriens extracts compared with LMCs. Aged Ts65Dn mice exhibited impaired hippocampal-dependent spatial learning and memory in the RAWM (radial-arm water maze) and a marked increase in levels of the lectican versican V2 in stratum oriens that correlated with the number of errors made in the final RAWM block. Ts65Dn stratum oriens PNNs (perineuronal nets), an extension of the ECM enveloping mostly inhibitory interneurons, were dispersed over a larger area compared with LMC mice. Taken together, these data suggest a possible association with alterations in the ECM and inhibitory neurotransmission in the Ts65Dn hippocampus which could contribute to cognitive deficits.

Fibulin‐1 is required during cardiac ventricular morphogenesis for versican cleavage, suppression of ErbB2 and Erk1/2 activation, and to attenuate trabecular cardiomyocyte proliferation

Trabeculation is an integral component of cardiac ventricular morphogenesis and is dependent on the matrix metalloproteinase, ADAMTS1. A substrate of ADAMTS1 is the proteoglycan versican which is expressed in the developing ventricle and which has been implicated in trabeculation. Fibulin-1 is a versican and ADAMTS1-binding extracellular matrix protein required for ventricular morphogenesis. Here we investigated the involvement of fibulin-1 in ADAMTS1-mediated cleavage of versican in vitro, and the involvement of fibulin-1 in versican cleavage in ventricular morphogenesis. We show that fibulin-1 is a cofactor for ADAMTS1-dependent in vitro cleavage of versican V1, yielding a 70-kDa amino-terminal fragment. Furthermore, fibulin-1-deficiency in mice was found to cause a significant reduction (>90%) in ventricular levels of the 70-kDa versican V1 cleavage product and a 2-fold increase in trabecular cardiomyocyte proliferation. Decreased versican V1 cleavage and augmented trabecular cardiomyocyte proliferation in fibulin-1 null hearts is accompanied by increased ventricular activation of ErbB2 and Erk1/2. By contrast, versican deficiency was found to lead to decreased cardiomyocyte proliferation and reduced ventricular trabeculation. We conclude that fibulin-1 regulates versican-dependent events in ventricular morphogenesis by promoting ADAMTS1 cleavage of versican leading to suppression of trabecular cardiomyocyte proliferation mediated by the ErbB2-Map kinase pathway.

Versican V1 isoform regulates cell‐associated matrix formation and cell behavior differentially from aggrecan in Swarm rat chondrosarcoma cells

Versican, a large chondroitin sulfate proteoglycan that binds hyaluronan and is composed of large extracellular matrix aggregates, has been shown to correlate with tumor progression. No studies have examined the roles of versican in chondrosarcoma nor compared them to those of aggrecan. In clinical specimens of human chondromatous tumors, versican expression was significantly increased in malignant tumors, moreover, as the tumor grade increased. To clarify the roles of versican in chondrosarcoma, versican splicing variant 1, variant 3 or only GFP was stably transfected to Swarm rat chondrosarcoma cells with Trap-In System. Forced expression of versican V1 isoform in Swarm rat chondrosarcoma cells induced a marked increase of cell-associated matrix compared to V3-, GFP- transfected or RCS cells. Versican was immunolocalized in a fashion similar to that of hyaluronan and more diffusively than aggrecan. Anchor-dependent and -independent growth was not affected by versican isoform expression, whereas cell motility and migration were significantly enhanced by V1 isoform transfection. Tumors formed in vivo with V1-transfected cells exhibited more myxomatous area and included more spindle shaped cells. These results support the concept that versican has the capacity to form more extensive cell-associated matrix than aggrecan, and the prominent matrix formation alters the cell behavior of chondrosarcoma more aggressively. These observations suggest that versican expression may serve as a marker of tumor grade determination in chondrosarcoma and possibly help to decide on therapeutic targets in higher grades of chondrosarcoma.

Versican G1 and G3 domains are upregulated and latent transforming growth factor-β binding protein-4 is downregulated in breast cancer stroma

Cancers are supported by a distinct type of stroma, and versican is overexpressed in the stroma of malignant tumors, including breast cancer. Versican interacts with hyaluronan and fibrillin-1 at its amino terminus (G1) and carboxyl terminus (G3), respectively. Fibrillin-1 also associates with latent transforming growth factor-β binding protein (LTBP)-1 and -4. The detailed alteration of these molecules in breast cancer tissues is still unclear. In 18 patients, alteration of versican, fibrillin-1 and LTBP-1 and 4 was elucidated in comparison with matched normal tissues, using real-time reverse transcriptase polymerase chain reaction, slot blotting and immunohistochemistry. The relationship between the protein expression and clinicopathological features was also investigated. In breast cancer tissues, mRNAs for versican V1 and V0 were upregulated, and the extracted protein levels of the versican G1 and G3 domains were increased. Meanwhile, LTBP-4 was decreased, and fibrillin-1 and LTBP-1 remained unchanged. The immunohistochemical observations were consistent with the biochemical findings, and the molecules were localized in the stromal tissue rather than in the cancer cells themselves. The expression of versican G3 and G1 domains was positively related to the Ki67 index of carcinoma cells and tumor size, respectively. The stromal alterations of versican and LTBP-4 might influence the carcinogenesis and progression of breast tumor cells and modulate their biological phenotypes.

Versican V0 and V1 Direct the Growth of Peripheral Axons in the Developing Chick Hindlimb

Peanut agglutinin-binding disaccharides and chondroitin sulfate mark transient mesenchymal barriers to advancing motor and sensory axons innervating the hindlimbs during chick development. Here we show that the vast majority of these carbohydrates are at the critical stage and location attached to the versican splice variants V0 and V1. We reveal that the isolated isoforms of this extracellular matrix proteoglycan suppress axon extension at low concentrations and induce growth cone collapse and rapid retraction at higher levels. Moreover, we demonstrate that versican V0 and/or V1, recombinantly expressed in collagen-I gels or ectopically deposited in the hindlimbs of chicken embryos in ovo, cause untimely defasciculation and axon stalling. Consequently, severe disturbances of nerve patterning are observed in the versican-treated embryos. Our experiments emphasize the inhibitory capacity of versicans V0 and V1 in axonal growth and evidence for their function as basic guidance cues during development of the peripheral nervous system.

Does the Expression of Versican Isoforms Contribute to the Pathogenesis of Neurodegenerative Diseases?

Classical neurodegenerative diseases such as Alzheimer's, Parkinson's, and Huntington's are most commonly seen in older persons. The incidence rate increases as life expectancy increases. Even though neuronal loss, neuronal death and accumulated toxic proteins are well investigated, the mechanism(s) of neurodegenerative disorders is not yet fully understood. Versican is a large extracellular matrix proteoglycan. Its isoforms are aberrantly expressed in central nervous system injuries. Diverse lines of evidence suggest that versican isoforms play a vital role in regulating neuronal differentiation, maturation, neurite outgrowth, and synaptic transmission. Some toxic proteins may be increased and less sensitive to degeneration due to the chondroitin sulfate (CS) chains of versicans. We propose that the patterns of versican V1 and V2 isoforms act as a fine-tuned mechanism for guiding the change of neural microenvironment, and the unbalanced expression of V1 and V2 isoforms may contribute to the pathogenesis of neurodegenerative diseases. The emergence of versican isoforms indicates that it may explain the pathogenesis of the common sporadic forms of complex diseases.

A Large Chondroitin Sulfate Proteoglycan, Versican, in Porcine Predentin

Proteoglycans and their constituent glycosaminoglycan (GAG) have been proposed to be involved in the inhibition of mineralization in unmineralized tissue, predentin. Among the proteoglycans secreted by odontoblasts, we focused on the large chondroitin sulfate proteoglycan, versican, for its large binding capacity for calcium ions. The aims of this study were the determination of the full-length sequence and splicing variants of the porcine versican, and the detection of versican in the porcine predentin. The complete coding sequence of the porcine versican mRNA was cloned to be 11,775 nucleotides long and encode 3,924 amino acids, and four splicing variants, V0, V1, V2 and V3, were characterized in the isolated porcine cartilage cells. The number of potential GAG attachment sites was 15 in the V0 variant, 13 in the V1 variant, 2 in the V2 variant and 0 in the V3 variant. They were deposited in DDBJ. The V1 variant was determined by RT-PCR in the odontoblasts, dental papilla cells, dental follicle cells, periodontal ligament cells, dental pulp cells, and gingival cells of pigs, although a small amount of the V0 valiant was found in the dental papilla cells. The predentin was prepared from developing porcine permanent incisor tooth germs and its soluble proteins were extracted in order to be partially characterized by protein and proteinase profiles. The versican V1 cleavage products were detected in the predentin extract by Western blotting analysis. These results suggested that the versican splice variant V1 implicates both the control of the mineralization and the activities of the predentin metalloproteinases, because it has 13 GAG chains that bind a large amount of calcium.

A Large Chondroitin Sulfate Proteoglycan, Versican, in Porcine Predentin

Proteoglycans and their constituent glycosaminoglycan (GAG) have been proposed to be involved in the inhibition of mineralization in unmineralized tissue, predentin. Among the proteoglycans secreted by odontoblasts, we focused on the large chondroitin sulfate proteoglycan, versican, for its large binding capacity for calcium ions. The aims of this study were the determination of the full-length sequence and splicing variants of the porcine versican, and the detection of versican in the porcine predentin. The complete coding sequence of the porcine versican mRNA was cloned to be 11,775 nucleotides long and encode 3,924 amino acids, and four splicing variants, V0, V1, V2 and V3, were characterized in the isolated porcine cartilage cells. The number of potential GAG attachment sites was 15 in the V0 variant, 13 in the V1 variant, 2 in the V2 variant and 0 in the V3 variant. They were deposited in DDBJ. The V1 variant was determined by RT-PCR in the odontoblasts, dental papilla cells, dental follicle cells, periodontal ligament cells, dental pulp cells, and gingival cells of pigs, although a small amount of the V0 valiant was found in the dental papilla cells. The predentin was prepared from developing porcine permanent incisor tooth germs and its soluble proteins were extracted in order to be partially characterized by protein and proteinase profiles. The versican V1 cleavage products were detected in the predentin extract by Western blotting analysis. These results suggested that the versican splice variant V1 implicates both the control of the mineralization and the activities of the predentin metalloproteinases, because it has 13 GAG chains that bind a large amount of calcium.

Oligodendrocyte Myelin Glycoprotein Does Not Influence Node of Ranvier Structure or Assembly

Oligodendrocyte myelin glycoprotein (OMgp) is expressed by both neurons and oligodendrocytes in the CNS. It has been implicated in growth cone collapse and neurite outgrowth inhibition by signaling through the Nogo receptor and paired Ig-like receptor B (PirB). OMgp was also reported to be an extracellular matrix (ECM) protein surrounding CNS nodes of Ranvier and proposed to function as (1) an inhibitor of nodal collateral sprouting and (2) an important contributor to proper nodal and paranodal architecture. However, we show here that the anti-OMgp antiserum used in previous studies to define the functions of OMgp at nodes is not specific. Among all reported nodal ECM components, the antiserum exhibited strong cross-reactivity against versican V2 isoform, a chondroitin sulfate proteoglycan. Furthermore, the OMgp antiserum labeled OMgp-null nodes, but not nodes from versican V2-deficient mice, and preadsorption of the OMgp antiserum with recombinant versican V2 blocked nodal labeling. Analysis of CNS nodes in OMgp-null mice failed to reveal any nodal or paranodal defects, or increased nodal collateral sprouting, indicating that OMgp does not participate in CNS node of Ranvier assembly or maintenance. We successfully identified a highly specific anti-OMgp antibody and observed OMgp staining in white matter only after initiation of myelination. OMgp immunoreactivity decorated the surface of mature myelinated axons, but was excluded from compact myelin and nodes. Together, our results strongly argue against the nodal localization of OMgp and its proposed functions at nodes, and reveal OMgp's authentic localization relative to nodes and myelin.

Differentiation of cardiomyocytes from human embryonic stem cells is accompanied by changes in the extracellular matrix production of versican and hyaluronan

Proteoglycans and hyaluronan play critical roles in heart development. In this study, human embryonic stem cells (hESC) were used as a model to quantify the synthesis of proteoglycans and hyaluronan in hESC in the early stages of differentiation, and after directed differentiation into cardiomyocytes. We demonstrated that both hESC and cardiomyocyte cultures synthesize an extracellular matrix (ECM) enriched in proteoglycans and hyaluronan. During cardiomyocyte differentiation, total proteoglycan and hyaluronan decreased and the proportion of proteoglycans bearing heparan sulfate chains was reduced. Versican, a chondroitin sulfate proteoglycan, accumulated in hESC and cardiomyocyte cultures. Furthermore, versican synthesized by hESC contained more N- and O-linked oligosaccharide than versican from cardiomyocytes. Transcripts for the versican variants, V0, V1, V2, and V3, increased in cardiomyocytes compared to hESC, with V1 most abundant. Hyaluronan in hESC had lower molecular weight than hyaluronan from cardiomyocyte cultures. These changes were accompanied by an increase in HAS-1 and HAS-2 mRNA in cardiomyocyte cultures, with HAS-2 most abundant. Interestingly, HAS-3 was absent from the cardiomyocyte cultures, but expressed by hESC. These results indicate that human cardiomyocyte differentiation is accompanied by specific changes in the expression and accumulation of ECM components and suggest a role for versican and hyaluronan in this process.

Bral1: Its Role in Diffusion Barrier Formation and Conduction Velocity in the CNS

At the nodes of Ranvier, excitable axon membranes are exposed directly to the extracellular fluid. Cations are accumulated and depleted in the local extracellular nodal region during action potential propagation, but the impact of the extranodal micromilieu on signal propagation still remains unclear. Brain-specific hyaluronan-binding link protein, Bral1, colocalizes and forms complexes with negatively charged extracellular matrix (ECM) proteins, such as versican V2 and brevican, at the nodes of Ranvier in the myelinated white matter. The link protein family, including Bral1, appears to be the linchpin of these hyaluronan-bound ECM complexes. Here we report that the hyaluronan-associated ECM no longer shows a nodal pattern and that CNS nerve conduction is markedly decreased in Bral1-deficient mice even though there were no differences between wild-type and mutant mice in the clustering or transition of ion channels at the nodes or in the tissue morphology around the nodes of Ranvier. However, changes in the extracellular space diffusion parameters, measured by the real-time iontophoretic method and diffusion-weighted magnetic resonance imaging (MRI), suggest a reduction in the diffusion hindrances in the white matter of mutant mice. These findings provide a better understanding of the mechanisms underlying the accumulation of cations due to diffusion barriers around the nodes during saltatory conduction, which further implies the importance of the Bral1-based extramilieu for neuronal conductivity.