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Lumican is a keratan sulfate proteoglycan that belongs to the small leucine-rich proteoglycan family. Research has lifted the veil on the versatile roles of lumican in the pathogenesis of eye diseases. Lumican has pivotal roles in the maintenance of physiological tissue homogenesis and is often upregulated in pathological conditions, e.g., fibrosis, scar tissue formation in injured tissues, persistent inflammatory responses and immune anomaly, etc. Herein, we will review literature regarding the role of lumican in pathogenesis of inherited congenital and acquired eye diseases, e.g., cornea dystrophy, cataract, glaucoma and chorioretinal diseases, etc., Competing Interests: Declaration of competing interest No conflicting relationship exits for any author., (Copyright © 2023. Published by Elsevier Inc.)

Biomarkers for treatment sensitivity or drug resistance used in precision medicine include prognostic and predictive molecules, critical factors in selecting appropriate treatment protocols and improving survival rates. However, identification of accurate biomarkers remain challenging due to the high risk of false-positive findings and lack of functional validation results for each biomarker. Here, we discovered a mechanical correlation between leucine proline-enriched proteoglycan 1 (LEPRE1) and pelitinib drug sensitivity using in silico statistical methods and confirmed the correlation in acute myeloid leukemia (AML) and A549 lung cancer cells. We determined that high LEPRE1 levels induce protein kinase B activation, overexpression of ATP-binding cassette superfamily G member 2 (ABCG2) and E-cadherin, and cell colonization, resulting in a cancer stem cell-like phenotype. Sensitivity to pelitinib increases in LEPRE1-overexpressing cells due to the reversing effect of ABCG2 upregulation. LEPRE1 silencing induces pelitinib resistance and promotes epithelial-to-mesenchymal transition through actin rearrangement via a series of Src/ERK/cofilin cascades. The in silico results identified a mechanistic relationship between LEPRE1 and pelitinib drug sensitivity, confirmed in two cancer types. This study demonstrates the potential of LEPRE1 as a biomarker in cancer through in-silico prediction and in vitro experiments supporting the clinical development of personalized medicine strategies based on bioinformatics findings., (© 2022. The Author(s).)

Objective: The clinical success of focal metallic resurfacing implants depends largely on the friction between implant and opposing cartilage. Therefore, the present study determines the lubricating ability of the synovial fluid components hyaluronic acid (HA), proteoglycan 4 (PRG4) and a surface-active phospholipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC), on the articulation between cartilage and a Cobalt Chromium Molybdenum (CoCrMo) implant surface, compared with two cartilage surfaces., Methods: A ring-on-disk geometry was used to perform repeated friction measurements at physiologically relevant velocities (6 and 60 mm/s) using lubricants with an increasing number of components present. Shear measurements were performed in order to evaluate the viscosity. To ensure that it is clinically relevant to explore the effect of these components, the presence of PRG4 in synovial fluid obtained from primary and revision knee and hip implant surgeries was examined., Results: PRG4 in the presence of HA was found to significantly reduce the coefficient of friction for both cartilage-cartilage and cartilage-CoCrMo interface. This is relevant, as it was also demonstrated that PRG4 is still present at the time of revision surgeries. The addition of POPC had no effect for either configurations. HA increased the viscosity of the lubricating fluid by one order of magnitude, while PRG4 and POPC had no effect., Conclusion: The present study demonstrates the importance of selecting the appropriate lubrication solution to evaluate implant materials with biotribology tests. Because PRG4 is a key component for reducing friction between cartilage and an opposing surface, developing coatings which bind PRG4 is recommended for cartilage resurfacing implants., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Complex formation and endocytosis of transforming growth factor-β (TGF-β) receptors play important roles in signaling. However, their interdependence remained unexplored. Here, we demonstrate that ALK1, a TGF-β type I receptor prevalent in endothelial cells, forms stable complexes at the cell surface with endoglin and with type III TGF-β receptors (TβRIII). We show that ALK1 undergoes clathrin-mediated endocytosis (CME) faster than ALK5, type II TGF-β receptor (TβRII), endoglin, or TβRIII. These complexes regulate the endocytosis of the TGF-β receptors, with a major effect mediated by ALK1. Thus, ALK1 enhances the endocytosis of TβRIII and endoglin, while ALK5 and TβRII mildly enhance endoglin, but not TβRIII, internalization. Conversely, the slowly endocytosed endoglin has no effect on the endocytosis of either ALK1, ALK5, or TβRII, while TβRIII has a differential effect, slowing the internalization of ALK5 and TβRII, but not ALK1. Such effects may be relevant to signaling, as BMP9-mediated Smad1/5/8 phosphorylation is inhibited by CME blockade in endothelial cells. We propose a model that links TGF-β receptor oligomerization and endocytosis, based on which endocytosis signals are exposed/functional in specific receptor complexes. This has broad implications for signaling, implying that complex formation among various receptors regulates their surface levels and signaling intensities.

Purpose: A previous study found that a lack of SPOCK2 expression was an early event that occurs during the malignant transformation of endometriosis (EMS); however, the role played by SPOCK2 in the pathogenesis of endometriosis and its malignant transformation remains unclear., Materials and Methods: In this study, SPOCK2 expression in human endometrial epithelial cells (hEECs) was downregulated by transfection with shRNA, and the biological behavior of the transfected cells was observed., Results: We found that downregulation of SPOCK2 promoted cell proliferation, adhesion, and invasion., Conclusions: Our data suggest that downregulation of SPOCK2 might participate in the pathogenesis and progression of EMS, as well as its malignant transformation, by promoting the proliferation, adhesion, and invasion of endometrial epithelial cells.

This work analyses the role of proteoglycans on skin ageing, influenced by the presence of glycosylated proteins, which exercise diverse functions on the skin. They are essential components that restore the cells, providing hydration, maintaining hydration of the extracellular matrix, preventing the formation of wrinkles thanks to their ability to combine to other molecules such as collagen or hyaluronic acid and favouring the smoothness of the skin texture. The use of these proteins is a very recent and promising topic, since their application may revolutionize skin ageing therapies. Of the existing proteoglycans, decorin, versican and perlecan are of special note, playing a fundamental role on skin., (© 2020 Society of Cosmetic Scientists and the Société Française de Cosmétologie.)

Loss of popliteal lymphatic vessel (PLV) contractions, which is associated with damage to lymphatic muscle cells (LMCs), is a biomarker of disease progression in mice with inflammatory arthritis. Currently, the nature of LMC progenitors has yet to be formally described. Thus, we aimed to characterize the progenitors of PLV-LMCs during murine development, towards rational therapies that target their proliferation, recruitment, and differentiation onto PLVs. Since LMCs have been described as a hybrid phenotype of striated and vascular smooth muscle cells (VSMCs), we performed lineage tracing studies in mice to further clarify this enigma by investigating LMC progenitor contribution to PLVs in neonatal mice. PLVs from Cre-tdTomato reporter mice specific for progenitors of skeletal myocytes (Pax7 + and MyoD + ) and VSMCs (Prrx1 + and NG2 + ) were analyzed via whole mount immunofluorescent microscopy. The results showed that PLV-LMCs do not derive from skeletal muscle progenitors. Rather, PLV-LMCs originate from Pax7 - /MyoD - /Prrx1 + /NG2 + progenitors similar to VSMCs prior to postnatal day 10 (P10), and from a previously unknown Pax7 - /MyoD - /Prrx1 + /NG2 - muscle progenitor pathway during development after P10. Future studies of these LMC progenitors during maintenance and repair of PLVs, along with their function in other lymphatic beds, are warranted.

Composition of the brain extracellular matrix changes in time as maturation proceeds. Chondroitin sulfate proteoglycan 5 (CSPG-5), also known as neuroglycan C, has been previously associated to differentiation since it shapes neurite growth and synapse forming. Here, we show that this proteoglycan persists in the postnatal rat brain, and its expression is higher in cortical regions with plastic properties, including hippocampus and the medial prefrontal cortex at the end of the second postnatal week. Progressively accumulating after birth, CSPG-5 typically concentrates around glutamatergic and GABAergic terminals in twelve-week old rat hippocampus. CSPG-5-containing perisynaptic matrix rings often appear at the peripheral margin of perineuronal nets. Electron microscopy and analysis of synaptosomal fraction showed that CSPG-5 accumulates around, and is associated to synapses, respectively. In vitro analyses suggest that neurons, but less so astrocytes, express CSPG-5 in rat primary neocortical cultures, and CSPG-5 produced by transfected neuroblastoma cells appear at endings and contact points of neurites. In human subjects, CSPG-5 expression shifts in brain areas of the default mode network of suicide victims, which may reflect an impact in the pathogenesis of psychiatric diseases or support diagnostic power., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The endothelial-to-mesenchymal transition (EndoMT) is involved in the complex pathogenesis of renal fibrosis. The soluble proteoglycan endothelial cell-specific molecule 1 (ESM1) is significantly upregulated in many tumor cells and cirrhosis-related disease. The role of ESM1 in renal fibrosis is unknown. This study investigates the role of ESM1 in renal fibrosis, using an in vivo unilateral ureteral obstruction (UUO) mouse model of renal fibrosis and in vitro mouse kidney MES 13 cells overexpressing ESM1. We observed that ESM1 overexpression significantly increased the motility and migration of MES 13 cells, independent of cell viability. In ESM1-overexpressing MES 13 cells, we also observed elevated expression of mesenchymal markers (N-cadherin, vimentin, matrix metallopeptidase 9 (MMP9)) and the fibrosis marker α-smooth muscle actin (α-SMA) and decreased expression of the endothelial marker vascular endothelial cadherin (VE-cadherin) and CD31. In a mouse model of fibrosis induced by unilateral ureter obstruction, we observed time-dependent increases in ESM1, α-SMA, and vimentin expression and renal interstitial collagen fibers in kidney tissue samples. These results suggest that ESM1 may serve as an EndoMT marker of renal fibrosis progression.

In the progression of osteoarthritis, pathological calcification in the affected joint is an important feature. The role of these crystallites in the pathogenesis and progression of osteoarthritis is controversial; it remains unclear whether they act as a disease initiator or are present as a result of joint damage. Recent studies reported that the molecular mechanisms regulating physiological calcification of skeletal tissues are similar to those regulating pathological or ectopic calcification of soft tissues. Pathological calcification takes place when the equilibrium is disrupted. Calcium phosphate crystallites are identified in most affected joints and the presence of these crystallites is closely correlated with the extent of joint destruction. These observations suggest that pathological calcification is most likely to be a disease initiator instead of an outcome of osteoarthritis progression. Inhibiting pathological crystallite deposition within joint tissues therefore represents a potential therapeutic target in the management of osteoarthritis., (© 2020 Cambridge Philosophical Society.)

Objective: Neurodegeneration and hair pigmentation alterations in mice occur consequent to aberrations at the Atrn locus coding for the transmembrane form of attractin. Earlier results pointed to a possible involvement in intracellular trafficking/export of secretory vesicles containing proteoglycan. Here we examined kidney and liver, both heavily dependent upon proteoglycan, of attractin-deficient mice to determine whether abnormalities were observed in these tissues., Results: Histological and histochemical analysis to detect glycosylated protein identified a severe loss in attractin-deficient mice of extracellular proteoglycan between kidney tubules in addition to a loss of glycosylated material within the intratubular brush border. In the liver, extracellular matrix material was significantly depleted between hepatocytes together with swollen sinuses and aberrations in the proteoglycan-dependent space of Disse. These results are consistent with a generalized defect in extracellular proteoglycan deposition in Atrn-mutant mice and support previous reports suggesting a role for attractin in the secretory vesicle pathway.

Hepatitis B virus (HBV), an enveloped partially double-stranded DNA virus, is a widespread human pathogen responsible for more than 250 million chronic infections worldwide. Current therapeutic strategies cannot eradicate HBV due to the persistence of the viral genome in a special DNA structure (covalently closed circular DNA, cccDNA). The identification of sodium taurocholate co-transporting polypeptide (NTCP) as an entry receptor for both HBV and its satellite virus hepatitis delta virus (HDV) has led to great advances in our understanding of the life cycle of HBV, including the early steps of infection in particular. However, the mechanisms of HBV internalization and the host factors involved in this uptake remain unclear. Improvements in our understanding of HBV entry would facilitate the design of new therapeutic approaches targeting this stage and preventing the de novo infection of naïve hepatocytes. In this review, we provide an overview of current knowledge about the process of HBV internalization into cells.

Few studies have investigated the role of extracellular-matrix proteoglycans in the pathogenesis of drug-induced gingival overgrowth (DIGO). SPOCK1 is an extracellular proteoglycan that induces epithelial to mesenchymal transition (EMT) in several cancer cell lines and exhibits protease-inhibitory activity. However, the role of SPOCK1 in non-cancerous diseases such as DIGO has not been well-addressed. We demonstrated that the expression of SPOCK1, TGF-β1, and MMP-9 in calcium channel blocker-induced gingival overgrowth is higher than that in non-overgrowth tissues. Transgenic mice overexpressing Spock1 developed obvious gingival-overgrowth and fibrosis phenotypes, and positively correlated with EMT-like changes. Furthermore, in vitro data indicated a tri-directional interaction between SPOCK1, TGF-β1, and MMP-9 that led to gingival overgrowth. Our study shows that SPOCK1 up-regulation in a noncancerous disease and SPOCK1-induced EMT in gingival overgrowth occurs via cooperation and crosstalk between several potential signaling pathways. Therefore, SPOCK1 is a novel therapeutic target for gingival overgrowth and its expression is a potential risk of EMT induction in cancerous lesions.

Tooth loss can cause a lot of physiological and psychological suffering. And tooth root engineering is a promising way for tooth loss treatment. Two kinds of seed cells are usually adopted for tooth root regeneration. In this study, a practical sandwich structure for tooth root regeneration was developed, which was constituted by only one kind of seed cell: human dental pulp stem cells (hDPSCs) and three kinds of graft materials: Vitamin C (VC) induced hDPSC sheet, human treated dentin matrix (hTDM), and Matrigel. It was found that VC could induce hDPSCs to form a cell sheet with two or three cell layers and promote their collagen type I ( COL1 ) mRNA expression obviously. hDPSCs could attach and grow on hTDM, and the mRNA expression of osteocalcin ( OCN ), dentin sialophosphoprotein ( DSPP ), vascular endothelial growth factor receptor 1 ( VEGFR1 ), and Nestin in hDPSCs was obviously upregulated by hTDM leaching solution. hDPSCs could stretch and proliferate in Matrigel. And when cultured in Matrigel condition medium, they positively expressed CD31, β3-Tubulin, and Nestin proteins, as well as increased the mRNA expression of OCN , ALP , and Nestin. Furthermore, periodontium, dentin, and pulp-like tissues were successfully regenerated after the sandwich structure of hDPSC sheet/TDM/Matrigel was transplanted in nude mice subcutaneously for 3 months. Periodontium-like dense connective tissue was regenerated around the hTDM, and a great mass of predentin was formed on the cavity side of hTDM. Odontoblast-like cells and blood vessel-like structures, even nerve-like fibers, were observed in the pulp cavity. In summary, the above results showed that hDPSCs could be used as seed cells for the whole tooth root regeneration, and the sandwich structure constituted by hDPSC sheet, TDM/hDPSCs, and Matrigel/hDPSCs could be utilized for tooth root regeneration.

Retinal ganglion cell (RGC) axons extend along the inner limiting membrane, which forms the extracellular matrix (ECM) containing laminin, collagen, and proteoglycans. RGC axons express integrin, which is activated by binding to ECM proteins to regulate cytoskeleton. To study the growth of RGC axons in vitro, maintaining the natural environment for them is absolutely necessary. For this purpose, culturing a strip of embryonic chick retina in Matrigel ® is a suitable method. This article describes detailed techniques of the retinal strip culture.

Proteoglycans (PGs) are proteins which are vital components located in the extracellular matrix, cell surface or intracellular granules. They are linked to polysaccharides called glycosaminoglycans. There are several aspects associated with PGs, such as cell signaling and organization of the extracellular matrix (ECM), making them pivotal participants in many tissue compositions. In teeth, PGs also play an essential role, as many of its components have elaborate ECM structures. However, lack of information on how PGs constitute the various tissues of the tooth and on their roles makes it difficult to elicit the major importance associated with this class of proteins. This review seeks to detail how proteoglycans are involved in many aspects of tooth organization and development, and as far as we are concerned, this has not been performed yet. We have also exemplified the participation of small leucine-rich proteoglycans, a special class of PGs seen in dental trauma cases., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Brain innate immunity is vital for maintaining normal brain functions. Immune homeostatic imbalances play pivotal roles in the pathogenesis of neurological diseases including Parkinson's disease (PD). However, the molecular and cellular mechanisms underlying the regulation of brain innate immunity and their significance in PD pathogenesis are still largely unknown., Methods: Cre-inducible diphtheria toxin receptor (iDTR) and diphtheria toxin-mediated cell ablation was performed to investigate the impact of neuron-glial antigen 2 (NG2) glia on the brain innate immunity. RNA sequencing analysis was carried out to identify differentially expressed genes in mouse brain with ablated NG2 glia and lipopolysaccharide (LPS) challenge. Neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice were used to evaluate neuroinflammatory response in the presence or absence of NG2 glia. The survival of dopaminergic neurons or glial cell activation was evaluated by immunohistochemistry. Co-cultures of NG2 glia and microglia were used to examine the influence of NG2 glia to microglial activation., Results: We show that NG2 glia are required for the maintenance of immune homeostasis in the brain via transforming growth factor-β2 (TGF-β2)-TGF-β type II receptor (TGFBR2)-CX3C chemokine receptor 1 (CX3CR1) signaling, which suppresses the activation of microglia. We demonstrate that mice with ablated NG2 glia display a profound downregulation of the expression of microglia-specific signature genes and remarkable inflammatory response in the brain following exposure to endotoxin lipopolysaccharides. Gain- or loss-of-function studies show that NG2 glia-derived TGF-β2 and its receptor TGFBR2 in microglia are key regulators of the CX3CR1-modulated immune response. Furthermore, deficiency of NG2 glia contributes to neuroinflammation and nigral dopaminergic neuron loss in MPTP-induced mouse PD model., Conclusions: These findings suggest that NG2 glia play a critical role in modulation of neuroinflammation and provide a compelling rationale for the development of new therapeutics for neurological disorders.

Pancreatic islets are highly vascularized endocrine units. Accordingly, their adequate revascularization is of major importance for successful islet transplantation. The proteoglycan, nerve/glial antigen 2 (NG2) expressed in pericytes is a crucial regulator of angiogenesis. Therefore, we herein analyze whether this surface protein contributes to the revascularization of grafted islets. Islets were isolated from NG2 +/+ (wild-type) and NG2 -/- mice and their cellular composition was analyzed by immunohistochemical detection of insulin, glucagon, somatostatin and CD31. Moreover, insulin secretion was assessed by enzyme-linked immunosorbent assay (ELISA). In addition, isolated islets were transplanted into dorsal skinfold chambers of wild-type mice and their revascularization was determined by intravital fluorescence microscopy and immunohistochemistry. NG2 +/+ and NG2 -/- islets did not differ in their cellular composition and insulin secretion. However, transplanted NG2 -/- islets exhibited a significantly lower functional capillary density and a reduced number of CD31-positive microvessels. These findings demonstrate that the loss of NG2 impairs the revascularization of transplanted islets, underlining the importance of this pericytic proteoglycan for islet engraftment.

The extracellular matrix is a network of secreted macromolecules that provides a harmonious meshwork for the growth and homeostatic development of organisms. It conveys multiple signaling cascades affecting specific surface receptors that impact cell behavior. During cancer growth, this bioactive meshwork is remodeled and enriched in newly formed blood vessels, which provide nutrients and oxygen to the growing tumor cells. Remodeling of the tumor microenvironment leads to the formation of bioactive fragments that may have a distinct function from their parent molecules, and the balance among these factors directly influence cell viability and metastatic progression. Indeed, the matrix acts as a gatekeeper by regulating the access of cancer cells to nutrients. Here, we will critically evaluate the role of selected matrix constituents in regulating tumor angiogenesis and provide up-to-date information concerning their primary mechanisms of action., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Non-muscle myosin II (NMII)-induced multicellular contractility is essential for development, maintenance and remodeling of tissue morphologies. Dysregulation of the cytoskeleton can lead to birth defects or enable cancer progression. We demonstrate that the Matrigel patterning assay, widely used to characterize endothelial cells, is a highly sensitive tool to evaluate cell contractility within a soft extracellular matrix (ECM) environment. We propose a computational model to explore how cell-exerted contractile forces can tear up the cell-Matrigel composite material and gradually remodel it into a network structure. We identify measures that are characteristic for cellular contractility and can be obtained from image analysis of the recorded patterning process. The assay was calibrated by inhibition of NMII activity in A431 epithelial carcinoma cells either directly with blebbistatin or indirectly with Y27632 Rho kinase inhibitor. Using Matrigel patterning as a bioassay, we provide the first functional demonstration that overexpression of S100A4, a calcium-binding protein that is frequently overexpressed in metastatic tumors and inhibits NMIIA activity by inducing filament disassembly, effectively reduces cell contractility., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Explanation of the mechanism of resistance to third-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) and the development of a novel strategy for drug resistance are imperative in third-generation EGFR-TKIs-resistant non-small cell lung cancer (NSCLC). SPOCK1 was found to be abnormally expressed in various tumors including lung cancer, however, there was no study focused on the role of SPOCK1 in third-generation EGFR-TKIs resistant lung cancer cells., Methods and Results: We investigated the roles of SPOCK1 in NSCLC with third-generation EGFR-TKIs resistance. We showed that SPOCK1 was upregulated in the osimertinib-resistant lung cancer cells and knockdown of SPOCK1 inhibits osimertinib-resistant cells growth and overcomes resistance. Furthermore, we demonstrated that the SPOCK1 was higher in clinical NSCLC specimens compared with the normal lung tissues, and the higher expression of SPOCK1 correlated with poor prognosis. In addition, the overexpression of SPOCK1 in NSCLC tissues was positively correlated with MMP11 and TGFβ1., Conclusion: Our study suggested that SPOCK1 could be an independent prognostic factor in NSCLC and would be a candidate for target therapy in osimertinib-resistant lung tumors., (© 2019 Wiley Periodicals, Inc.)

2D electrophysiology is often used to determine the electrical properties of neurons. In the brain however, neurons form extensive 3D networks. Thus, performing electrophysiology in a 3D environment provides a closer situation to the physiological condition and serves as a useful tool for various applications in the field of neuroscience. In this study, 3D electrophysiology is established within a fiber-reinforced matrix to enable fast readouts from transfected cells, which are often used as model systems for 2D electrophysiology. Using melt electrowriting (MEW) of scaffolds to reinforce Matrigel, 3D electrophysiology is performed on a glycine receptor-transfected Ltk-11 mouse fibroblast cell line. The glycine receptor is an inhibitory ion channel associated when mutated with impaired neuromotor behavior. The average thickness of the MEW scaffold is 141.4 ± 5.7 µm, using 9.7 ± 0.2 µm diameter fibers, and square pore spacings of 100, 200, and 400 µm. For the first time, the electrophysiological characterization of glycine receptor-transfected cells is demonstrated with respect to agonist efficacy and potency in a 3D matrix. With the MEW scaffold reinforcement not interfering with the electrophysiological measurement, this approach can now be further adapted and developed for different kinds of neuronal cultures to study and understand pathological mechanisms under disease conditions., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Proteoglycan 4 (PRG4), first identified in synovial fluid, is an extracellular matrix structural protein in the joint implicated in reducing shear at the cartilage surface as well as controlling adhesion-dependent synovial growth and regulating bulk protein deposition onto the cartilage. However, recent evidence suggests that it can bind to and effect downstream signaling of a number of cell surface receptors implicated in regulating the inflammatory response. Therefore, we pose the hypothesis: Does PRG4 regulate the inflammatory response and maintain tissue homeostasis? Based on these novel findings implicating PRG4 as an inflammatory signaling molecule, we will present and discuss several hypotheses regarding potential mechanisms by which PRG4 may be able to regulate inflammation. If future studies can demonstrate that PRG4 is a potent inflammatory mediator, this will change current paradigms in the musculoskeletal and ophthalmological fields regarding the how the inflammatory microenvironment is regulated in these tissues and potentially others., (© 2018 WILEY Periodicals, Inc.)

Enriched Na + channel clustering allows for rapid saltatory conduction at a specialized structure in myelinated axons, the node of Ranvier, where cations are exchanged across the axon membrane. In the extracellular matrix (ECM), highly negatively charged molecules accumulate and wrap around the nodal gaps creating an ECM dome, called the perinodal ECM. The perinodal ECM has different molecular compositions in the central nervous system (CNS) and peripheral nervous system (PNS). Chondroitin sulfate proteoglycans are abundant in the ECM at the CNS nodes, whereas heparan sulfate proteoglycans are abundant at the PNS nodes. The proteoglycans have glycosaminoglycan chains on their core proteins, which makes them electrostatically negative. They associate with other ECM molecules and form a huge stable ECM complex at the nodal gaps. The polyanionic molecular complexes have high affinity to cations and potentially contribute to preventing cation diffusion at the nodes.In this chapter, we describe the molecular composition of the perinodal ECM in the CNS and PNS, and discuss their physiological role at the node of Ranvier.

Inhibins are gonadal hormones that act on pituitary gonadotrope cells to suppress FSH synthesis and secretion. Inhibin A and B are heterodimers of the inhibin ⍺-subunit disulfide-linked to one of two inhibin β-subunits. Homodimers or heterodimers of the inhibin β-subunits form the activins, which stimulate FSH production. Activins signal through complexes of type I and II receptor serine/threonine kinases to increase transcription of the FSHβ subunit gene. According to in vitro observations, inhibins impair FSH synthesis by competitively binding to activin type II receptors, particularly in the presence of the TGFβ type III receptor (TGFBR3, or betaglycan). The role of TGFBR3 in inhibin action in vivo has not been determined. Here, we ablated Tgfbr3 specifically in murine gonadotropes. Conditional knockout females were supra-fertile, exhibiting enhanced folliculogenesis, numbers of ovulated eggs per cycle, and litter sizes relative to control mice. Despite these phenotypes, FSH levels appeared to be unaltered in knockout mice, and the mechanisms underlying their enhanced fertility remain unexplained. Inhibin B is the predominant form of the hormone in males and in females during most stages of the estrous cycle. Remarkably, inhibin A, but not inhibin B, suppression of FSH synthesis was impaired in cultured pituitaries of knockout mice, which may explain the absence of discernible changes in FSH levels in vivo. Collectively, these data challenge current dogma by demonstrating that TGFBR3 (betaglycan) functions as an inhibin A, but not an inhibin B, coreceptor in gonadotrope cells in vivo. Mechanisms of inhibin B action merit further investigation.

The function of ligaments and tendons is to support and transmit loads applied to the musculoskeletal system. These tissues are often able to perform their function for many decades; however, connective tissue disease and injury can compromise ligament and tendon integrity. A range of protein and non-protein constituents, combined in a complex structural hierarchy from the collagen molecule to the tissue and covering nanometer to centimeter length scales, govern tissue function, and impart characteristic non-linear material behavior. This review summarizes the structure of ligaments and tendons, the roles of their constituent components for load transfer across the hierarchy of structure, and the current understanding of how damage occurs in these tissues. Disease and injury can alter the constituent make-up and structural organization of ligaments and tendons, affecting tissue function, while also providing insight to the role and interactions of individual constituents. The studies and techniques presented here have helped to understand the relationship between tissue constituents and the physical mechanisms (e.g., stretching, sliding) that govern material behavior at and between length scales. In recent years, new techniques have been developed to probe ever smaller length scales and may help to elucidate mechanisms of load transfer and damage and the molecular constituents involved in the in the earliest stages of ligament and tendon damage. A detailed understanding of load transfer and damage from the molecular to the tissue level may elucidate targets for the treatment of connective tissue diseases and inform practice to prevent and rehabilitate ligament and tendon injuries. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:3093-3104, 2018., (© 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

The extracellular matrix (ECM) constitutes a highly dynamic three-dimensional structural network comprised of macromolecules, such as proteoglycans/glycosaminoglycans (PGs/GAGs), collagens, laminins, fibronectin, elastin, other glycoproteins and proteinases. In recent years, the field of PGs has expanded rapidly. Due to their high structural complexity and heterogeneity, PGs mediate several homeostatic and pathological processes. PGs consist of a protein core and one or more covalently attached GAG chains, which provide the protein cores with the ability to interact with several proteins. The GAG building blocks of PGs significantly influence the chemical and functional properties of PGs. The primary goal of this comprehensive review is to summarize major achievements and paradigm-shifting discoveries made on the PG/GAG chemistry-biology axis, focusing on structural variability, structure-function relationships, metabolic, molecular, and epigenetic mechanisms underlying their synthesis. Recent insights related to exosome biogenesis, degradation, and cell signaling, their status as diagnostic tools and potential pharmacological targets in diseases as well as current applications in nanotechnology and biotechnology are addressed. Moreover, issues related to docking studies, molecular modeling, GAG/PG interaction networks, and their integration are discussed.

Loss of charged proteoglycans in the knee meniscus, which aid in the support of compressive loads by entraining water, is an effect of degeneration and is often associated with osteoarthritis. In healthy menisci, proteoglycan content is highest in the inner white zone and decreases towards the peripheral red zone. We hypothesized that loss of proteoglycans would reduce both osmotic swelling and compressive stiffness, spatially localized to the avascular white zone of the meniscus. This hypothesis was tested by targeted enzymatic digestion of proteoglycans using hyaluronidase in intact cervine medial menisci. Mechanics were quantified by creep indentation on the femoral surface. Osmotic swelling changes were assessed by measuring collagen fiber crimp period in the radial-axial plane in the lamellar layer along both the tibial and femoral contacting surfaces. All measurements were made in the inner, middle, and outer zones of the anterior, central, and posterior regions. Mechanical measurements showed variation in creep behavior with anatomical location, along with spatially uniform decreases in viscosity (average of 21%) and creep stiffness (average of 15%) with hyaluronidase treatment. Lamellar collagen crimp period was significantly decreased (average of 27%) by hyaluronidase, indicating a decrease in osmotic swelling, with the largest decreases seen in locations with the highest proteoglycan content. Taken together, these results suggest that while proteoglycans have localized effects on meniscus swelling, the resulting effect on compressive properties is distributed throughout the tissue., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The brain extracellular matrix (ECM) plays a crucial role in both the developing and adult brain by providing structural support and mediating cell-cell interactions. In this review, we focus on the major constituents of the ECM and how they function in both normal and injured brain, and summarize the changes in the composition of the ECM as well as how these changes either promote or inhibit recovery of function following traumatic brain injury (TBI). Modulation of ECM composition to facilitates neuronal survival, regeneration and axonal outgrowth is a potential therapeutic target for TBI treatment., (Published 2018. This article is a U.S. Government work and is in the public domain in the USA.)

The intervertebral disc faces high compressive forces during daily activities. Axial compression induces creeping fluid loss and reduction in disc height. With degeneration, disc fluids and height are progressively lost, altering biomechanics. It is assumed that this reduction of fluids is caused by a decline of osmolality within the disc due to proteoglycan depletion. Here we investigate the isolated effect of a reduction in osmosis on the biomechanical properties of the intervertebral disc. Continuous diurnal loading was applied to healthy caprine intervertebral discs in a loaded disc culture system for a total of 6days. We increased testing bath osmolality with two doses of polyethylene-glycol (PEG), thereby reducing the osmotic gradient between the disc and the surrounding fluid. This way we could study the isolated effect of reduced osmosis on axial creep, without damaging the disc. We evaluated: daily creep and recovery, recovery time-constants and compressive stiffness. Additionally, we investigated water content. There was a strong dose-dependent effect of PEG concentration on water content and axial creep behaviour: disc height, amplitude and rate of creep and recovery were all significantly reduced. Axial compressive stiffness of the disc was not affected. Reduction of water content and amplitude of creep and recovery showed similarity to degenerative disc biomechanics. However, the time-constants increased, indicating that the hydraulic permeability was reduced, in contrast to what happens with degeneration. This suggests that besides the osmotic gradient, the permeability of the tissues determines healthy intervertebral disc biomechanics., (Copyright © 2017. Published by Elsevier Ltd.)

The proteoglycan nerve/glial antigen (NG) 2 is expressed on multiple cell types and mediates cell proliferation and migration. However, little is known about its function in gene regulation. In this study, we demonstrate that in pericytes and glioblastoma cells intercellular adhesion molecule (ICAM)-1, an essential protein for leukocyte adhesion and transmigration, underlies a NG2-dependent expression. As shown by flow cytometry, Western blot analysis and quantitative real-time polymerase chain reaction (qRT-PCR), silencing of NG2 in human placenta-derived pericytes increased the expression of ICAM-1. Pathway analyses revealed that this is mediated by extracellular-regulated-kinases (ERK) 1/2 signaling. Moreover, leukocyte adhesion to NG2 siRNA-treated pericytes was significantly enhanced when compared to scrambled (scr) siRNA-treated control cells. In vivo, we detected increased ICAM-1 protein levels in the retina of mice lacking NG2 expression. To exclude that this novel mechanism is pericyte-specific, we additionally analyzed the expression of ICAM-1 in dependency of NG2 in two glioblastoma cell lines. We found that A1207 and M059K cells exhibit an inverse expression pattern of NG2 and ICAM-1. Finally, downregulation of NG2 in A1207 cells significantly increased ICAM-1 expression. Taken together, these findings indicate that NG2 may represent a promising target for the modulation of ICAM-1-mediated immune responses., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Genetic factors play a large role in cancer, and thus, there is a great desire to understand the effects of different genes in cancer and to also develop gene therapy for better treatments. Therefore, the development of alternative diagnosis and therapy modalities is of utmost importance. The aim of our study was to illuminate the role of ESM1 (endothelial cell-specific molecule-1, also known as Endocan) in proliferation and migration of head and neck cancer, thus helping to pave the way for new treatment modalities and predictive biomarkers., Methods: ESM1 expression was shown with immunofluorescence assay using confocal laser scanning microscope in primary and metastatic head and neck cancer cells. ESM1 expression was knocked down by RNA interference in head and neck cancer cells. Knockdown efficiency was evaluated by quantitative real-time RT-PCR and Western blot. Cell proliferation and migration assays were performed by xCELLigence real-time cell analysis system., Results: Immunofluorescence assay showed nuclear localization and high expression of ESM1 in primary and metastatic head and neck cancer cells. ESM1 mRNA and protein levels were significantly decreased in ESM1-knockdown cells compared to control. ESM1-knockdown cells showed reduced proliferation and migration activity when compared to control cells., Conclusion: These findings suggest that ESM1 has roles on proliferation and migration of head and neck cancer cells., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Many studies have measured the global compressive properties of tissue engineered (TE) cartilage grown on porous scaffolds. Such scaffolds are known to exhibit strain softening due to local buckling under loading. As matrix is deposited onto these scaffolds, the global compressive properties increase. However the relationship between the amount and distribution of matrix in the scaffold and local buckling is unknown. To address this knowledge gap, we studied how local strain and construct buckling in human TE constructs changes over culture times and GAG content. Confocal elastography techniques and digital image correlation (DIC) were used to measure and record buckling modes and local strains. Receiver operating characteristic (ROC) curves were used to quantify construct buckling. The results from the ROC analysis were placed into Kaplan-Meier survival function curves to establish the probability that any point in a construct buckled. These analysis techniques revealed the presence of buckling at early time points, but bending at later time points. An inverse correlation was observed between the probability of buckling and the total GAG content of each construct. This data suggests that increased GAG content prevents the onset of construct buckling and improves the microscale compressive tissue properties. This increase in GAG deposition leads to enhanced global compressive properties by prevention of microscale buckling., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

We used a bone marrow transplantation approach to distinguish the activities of bone marrow-derived macrophages from the activities of central nervous system-resident microglia in phenomena associated with axon demyelination and remyelination. We transplanted wild type or germline NG2 null beta-actin-EGFP expressing bone marrow into irradiated wild type or NG2 null recipient mice, followed by analysis of lysolecithin-induced spinal cord demyelination and remyelination and quantification of Iba-1+/ F4/80+/ EGFP+ macrophages and Iba-1+/ F4/80+/ EGFP- microglia. One week after microinjection of 1% lysolecithin into the spinal cord, wild type recipients receiving NG2 null bone marrow exhibit greatly reduced infiltration of macrophages into lesions, compared to wild type recipients receiving wild type bone marrow. Wild type bone marrow recipients also exhibit larger numbers of demyelinated axons than NG2 null recipients, indicative of macrophage participation in the initial myelin damage. However, wild type bone marrow recipients also exhibit superior myelin repair at 6 weeks post-injury, compared to NG2 null bone marrow recipients, demonstrating the additional importance of macrophages in remyelination. Incompletely repaired lesions in NG2 null bone marrow recipients at 6 weeks post-injury retain elevated numbers of macrophages, in contrast to lower numbers of macrophages in more completely repaired lesions in wild type bone marrow recipients. This suggests that NG2 expression renders macrophages more effective in myelin repair and less likely to promote chronic inflammation. Effective macrophage involvement in myelin repair is due in part to effects on the proliferation and/or recruitment of oligodendrocyte progenitor cells. Reduced numbers of oligodendrocyte progenitors are seen in lesions in NG2 null bone marrow recipients, likely due to deficits in macrophage production of oligodendrocyte progenitor-relevant mitogens and in phagocytosis of inhibitory myelin debris. Microglia also appear to be important for clearance of myelin debris, as indicated by reduced phagocytosis in NG2 null recipients receiving wild type bone marrow.

Chondroitin sulfate proteoglycans (CSPGs) are the main active component of perineuronal nets (PNNs). Digestion of the glycosaminoglycan chains of CSPGs with chondroitinase ABC or transgenic attenuation of PNNs leads to prolongation of object recognition memory and activation of various forms of plasticity in the adult central nervous system. The inhibitory properties of the CSPGs depend on the pattern of sulfation of their glycosaminoglycans, with chondroitin 4-sulfate (C4S) being the most inhibitory form. In this study, we tested a number of candidates for functional blocking of C4S, leading to selection of an antibody, Cat316, which specifically recognizes C4S and blocks its inhibitory effects on axon growth. It also partly blocks binding of semaphorin 3A to PNNs and attenuates PNN formation. We asked whether injection of Cat316 into the perirhinal cortex would have the same effects on memory as chondroitinase ABC treatment. We found that masking C4S with the Cat316 antibody extended long-term object recognition memory in normal wild-type mice to 24 hours, similarly to chondroitinase or transgenic PNN attenuation. We then tested Cat316 for restoration of memory in a neurodegeneration model. Mice expressing tau with the P301S mutation showed profound loss of object recognition memory at 4 months of age. Injection of Cat316 into the perirhinal cortex normalized object recognition at 3 hours in P301S mice. These data indicate that Cat316 binding to C4S in the extracellular matrix can restore plasticity and memory in the same way as chondroitinase ABC digestion. Our results suggest that antibodies to C4S could be a useful therapeutic to restore memory function in neurodegenerative disorders., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objective: Proteoglycans (PGs) are multifunctional biomacromolecules of the extracellular matrix of collagen-based tissues. In teeth, besides a pivotal regulatory role on dentin biomineralization, PGs provide mechanical support to the mineralized tissue and compressive strength to the biosystem. This study assessed enzymatic protocols for selective PGs removal from demineralized dentin to determine the roles of these biomacromolecules in the bulk mechanical properties and biostability of type I collagen., Methods: Selective removal of glycosaminoglycans chains (GAGs) and PGs from demineralized dentin was carried out by enzymatic digestion protocols using chondroitinase ABC (c-ABC) and trypsin (Try). A comprehensive study design included assessment of dentin matrix mass loss, biodegradability of the PGs/GAGs-depleted dentin matrix, ultimate tensile strength (UTS) and energy to fracture tests. Quantitative data was statistically analyzed by two-way and one-way ANOVA followed by the appropriate post hoc tests (α=0.05)., Results: Transmission electron microscopy images show effective GAGs removal by c-ABC and Try and both enzymatic methods released statistically similar amounts of GAGs from the demineralized dentin. Try digestion resulted in about 25% dentin matrix mass loss and increased susceptibility to collagenolytic digestion when compared to c-ABC (p=0.0224) and control (p=0.0901). Moreover, PGs digestion by Try decreased the tensile strengths of dentin. Statistically lower energy to fracture was observed in c-ABC-treated dentin matrix., Conclusions: GAGs plays a pivotal role on tissue mechanics and anisotropy, while the core protein of PGs have a protective role on matrix biostability., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Postpartum effluvium is a well-known clinical fact. However, following some minor research activities in the 1960s, very little has happened on the research front of the subject. It was hypothesized that postpartum hair loss might be a manifestation of a change in the hair growth cycle occurring normally during pregnancy. Recently, new research has been published trying to explain the mechanism of action behind this frequently occurring hair growth disruption, and to develop a functional treatment schedule and regime. Under normal circumstances, postpartum effluvium will disappear by itself as a function of time, and therefore adequate information to the patient is important in order to reduce the anxiety that it will not be a permanent problem. However, in some subjects it can manifest itself for longer stages and even become permanent. At the present time, treatments aim at correcting underlying hormonal imbalances and at improving overall cosmetic appearance. Several treatments in the form of thyroid supplementation, topical progesterone and estradiol lotions, and even oral contraceptive have been studied. All the available studies have significant limitations in their scientific basis, such as small sample size, absence of control group, or highly subjective measurement of treatment response. It is evident from the available studies that no specific treatment has been investigated thoroughly enough to justify recommendation in clinical treatment or to be termed "effective." Without the ability to provide a pathogenic diagnosis or causality criteria, chances are low that a treatment by trial and error will adequately be able to control hair effluvium. Current hair treatment strategies are symptomatic and nonspecific; therefore, future research must aim at developing new and targeted methods with a point of departure in observing concomitant biological mechanisms. Based on the research in the 1960s, current knowledge about the hair follicle and the regulation of the hair cycles, we believe that an anagen inducer in the form of a specific proteoglycan replacement therapy (Nourkrin ® with Marilex ® from Pharma Medico) could be a proper solution to shorten the hair eclipse phenomenon - and give the patient a feeling of control and empowerment., (© 2016 Wiley Periodicals, Inc.)

Background & Aims: Hepatitis C virus (HCV) evades humoral immunity and establishes chronic infections. Virus particles circulate in complex with lipoproteins facilitating antibody escape. Apolipoprotein E (ApoE) is essential for intracellular HCV assembly and for HCV cell entry. We aimed to explore if ApoE released from non-infected cells interacts with and modulates secreted HCV particles., Methods: ApoE secreted from non-infected cells was incubated with HCV from primary human hepatocytes or Huh-7.5 cells. Co-immunoprecipitation, viral infectivity and neutralization experiments were conducted., Results: Physiological levels of secreted ApoE (10-60µg/ml) enhanced the infectivity of HCV up to 8-fold across all genotypes, which indirectly decreased virus neutralization by antibodies targeting E1 or E2 up to 10-fold. Infection enhancement was observed for particles produced in primary human hepatocytes and Huh-7.5 cells. Selective depletion of ApoE ablated infection enhancement. Addition of HA-tagged ApoE to HCV particles permitted co-precipitation of HCV virions. Serum ApoE levels ranged between 10-60µg/ml, which is ca 100-fold higher than in Huh-7.5 conditioned cell culture fluids. Serum-derived HCV particles carried much higher amounts of ApoE than cell culture-derived HCV particles. Serum ApoE levels correlated with efficiency of co-precipitation of HCV upon exogenous addition of HA-ApoE. ApoE-dependent infection enhancement was independent of the hypervariable region 1 and SR-B1, but was dependent on heparan sulfate proteoglycans (HSPGs)., Conclusions: Physiological quantities of secreted ApoE stimulate HCV infection and increase antibody escape, by incorporating into virus particles and enhancing particle interactions with cellular HSPGs. Thus, secreted particles undergo ApoE-dependent maturation to enhance infectivity and to facilitate evasion from neutralizing antibodies. Lay summary: This study shows that HCV particle infectivity is remodeled by secreted ApoE after particle release from cells. Fluctuation of the availability of ApoE likely influences HCV infectivity, antibody escape and transmission., (Copyright © 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Objective: This study investigated the ability of endogenous lubricin secretion to restore joint health following a brief <21 day, postnatal lubricin-null state, in a C57BL/6J Prg4 gene trap (GT) mouse under the control of cre-recombinase. Previously we showed that re-expression of lubricin at 21 days was partly restorative of joint lubrication., Design: The tibio-femoral joints of adult C57BL/6J mice containing lubricin, lacking lubricin, and postnatally lacking lubricin until restoration of lubricin expression at 7 days or 14 days of age were evaluated ex vivo. At 8-weeks of age, whole joint coefficient of friction (COF), and caspase-3 activation were measured and the tibial-femoral joints histologically analyzed for degenerative changes, following progressive cyclic loading. The peroxynitrite content of femoral head cartilage from these mice prior to cyclic loading was measured., Results: Mice that underwent gene recombination at 7 and 14 days of age did not reestablish low COF as joint cycling time increased and were histopathologically indistinguishable from the joints of lubricin-null littermates. However, cartilage from tibio-femoral joints that underwent recombination at 7 and 14 days of age had significantly fewer caspase-3 positive cells and significantly reduced peroxynitrite content compared to lubricin-null littermates., Conclusions: The biological effects of lubricin, which include limiting inflammation via peroxynitrite production and caspase-3 activation, may be achieved without completely restituting low COF. However, fully recapitulating low COF may require undamaged cartilage surfaces or absence of biofouling, which may interfere with the activity of lubricin., (Copyright © 2017 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Tendon's primary function is a mechanical link between muscle and bone. The hierarchical structure of tendon and specific compositional constituents are believed to be critical for proper mechanical function. With increased appreciation for tendon importance and the development of various technological advances, this review paper summarizes recent experimental approaches that have been used to study multiscale tendon mechanics, includes an overview of studies that have evaluated the role of specific tissue constituents, and also proposes challenges/opportunities facing tendon study. Tendon has been demonstrated to have specific structural characteristics (e.g., multi-level hierarchy, crimp pattern, helix) and complex mechanical properties (e.g., non-linearity, anisotropy, viscoelasticity). Physical mechanisms including uncrimping, fiber sliding, and collagen reorganization have been shown to govern tendon mechanical responses under both static and dynamic loading. Several tendon constituents with relatively small quantities have been suggested to play a role in its mechanics, although some results are conflicting. Further research should be performed to understand the interplay and communication of tendon mechanical properties across levels of the hierarchical structure, and further show how each of these components contribute to tendon mechanics. The studies summarized and discussed in this review have helped elucidate important aspects of multiscale tendon mechanics, which is a prerequisite for analyzing stress/strain transfer between multiple scales and identifying key principles of mechanotransduction. This information could further facilitate interpreting the functional diversity of tendons from different species, different locations, and even different developmental stages, and then better understand and identify fundamental concepts related to tendon degeneration, disease, and healing. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1353-1365, 2017., (© 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.)

Objective: The role of Snorc, a novel cartilage specific transmembrane proteoglycan, was studied during skeletal development using two Snorc knockout mouse models. Hypothesizing that Snorc, like the other transmembrane proteoglycans, may be a coreceptor, we also studied its interaction with growth factors., Methods: Skeletal development was studied in wild type (WT) and Snorc knockout mice during postnatal development by whole mount staining, X-ray imaging, histomorphometry, immunohistochemistry and qRT-PCR. Snorc promoter activity was studied by applying the LacZ reporter expressed by the targeting construct. Slot blot binding and cell proliferation assays were used to study the interaction of Snorc with several growth factors., Results: Snorc expression was localized in the knee epiphyses especially to the prehypertrophic chondrocytes delineating the cartilage canals and secondary ossification center (SOC). Snorc was demonstrated to have a glycosaminoglycan independent affinity to FGF2 and it inhibited FGF2 dependent cell growth of C3H101/2 cells. In Snorc deficient mice, SOCs in knee epiphyses were smaller, and growth plate (GP) maturation was disturbed, but total bone length was not affected. Central proliferative and hypertrophic zones were enlarged with higher extracellular matrix (ECM) volume and rounded chondrocyte morphology at postnatal days P10 and P22. Increased levels of Ihh and Col10a1, and reduced Mmp13 mRNA expression were observed at P10., Conclusions: These findings suggest a role of Snorc in regulation of chondrocyte maturation and postnatal endochondral ossification. The interaction identified between recombinant Snorc core protein and FGF2 suggest functions related to FGF signaling., (Copyright © 2017 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Negative outcomes of mild traumatic brain injury (mTBI) can be exacerbated by repeated insult. Animal models of repeated closed-head mTBI provide the opportunity to define acute pathological mechanisms as the number of mTBI increases. Furthermore, little is known about the effects of mTBI impact site, and how this may affect brain function. We use a closed head, weight drop model of mTBI that allows head movement following impact, in adult female rats to determine the role of the number and location of mTBI on brain pathology and behaviour. Biomechanical assessment of two anatomically well-defined mTBI impact sites were used, anterior (bregma) and posterior (lambda). Location of the impact had no significant effect on impact forces (450 N), and the weight impact locations were on average 5.4 mm from the desired impact site. No between location vertical linear head kinematic differences were observed immediately following impact, however, in the 300 ms post-impact, significantly higher mean vertical head displacement and velocity were observed in the mTBI lambda trials. Breaches of the blood brain barrier were observed with three mTBI over bregma, associated with immunohistochemical indicators of damage. However, an increased incidence of hairline fractures of the skull and macroscopic haemorrhaging made bregma an unsuitable impact location to model repeated mTBI. Repeated mTBI over lambda did not cause skull fractures and were examined more comprehensively, with outcomes following one, two or three mTBI or sham, delivered at 1 day intervals, assessed on days 1-4. We observe a mild behavioural phenotype, with subtle deficits in cognitive function, associated with no identifiable neuroanatomical or inflammatory changes. However, an increase in lipid peroxidation in a subset of cortical neurons following two mTBI indicates increasing oxidative damage with repeated injury in female rats, supported by increased amyloid precursor protein immunoreactivity with three mTBI. This study of acute events following closed head mTBI identifies lipid peroxidation in neurons at the same time as cognitive deficits. Our study adds to existing literature, providing biomechanics data and demonstrating mild cognitive disturbances associated with diffuse injury, predominantly to grey matter, acutely following repeated mTBI.

Background: Lubricin/proteoglycan 4 (PRG4) is a mucinous glycoprotein secreted by synovial fibroblasts and superficial zone chondrocytes. Recently, we showed that recombinant human PRG4 (rhPRG4) is a putative ligand for CD44 receptor. rhPRG4-CD44 interaction inhibits cytokine-induced rheumatoid arthritis synoviocyte proliferation. The objective of this study is to decipher the autocrine function of PRG4 in regulating osteoarthritic synoviocyte proliferation and expression of catabolic and pro-inflammatory mediators under basal and interleukin-1 beta (IL-1β)-stimulated conditions., Methods: Cytosolic and nuclear levels of nuclear factor kappa B (NFκB) p50 and p65 subunits in Prg4 +/+ and Prg4 -/- synoviocytes were studied using western blot. Nuclear translocation of p50 and p65 proteins in osteoarthritis (OA) fibroblast-like synoviocytes (FLS) in response to IL-1β stimulation in the absence or presence of rhPRG4 was studied using DNA binding assays. OA synoviocyte (5000 cells per well) proliferation following IL-1β (20 ng/ml) treatment in the absence or presence of rhPRG4 (50-200 μg/ml) over 48 hours was determined using a colorimetric assay. Gene expression of matrix metalloproteinases (MMPs), tissue inhibitor of metallproteinases-1 (TIMP-1), TIMP-2, IL-1β, IL-6, IL-8, TNF-α, cycloxygenae-2 (COX2) and PRG4 in unstimulated and IL-1β (1 ng/ml)-stimulated OA synoviocytes, in the presence or absence of rhPRG4 (100 and 200 μg/ml), was studied following incubation for 24 hours., Results: Prg4 -/- synoviocytes contained higher nuclear p50 and p65 levels compared to Prg4 +/+ synoviocytes (p < 0.05). rhPRG4 (100 μg/ml) reduced p50 and p65 nuclear levels in Prg4 +/+ and Prg4 -/- synoviocytes (p < 0.001). Similarly, rhPRG4 (200 μg/ml) inhibited NFκB translocation and cell proliferation in OA synoviocytes in a CD44-dependent manner (p < 0.001) via inhibition of IκBα phosphorylation. IL-1β reduced PRG4 expression in OA synoviocytes and rhPRG4 (100 μg/ml) treatment reversed this effect (p < 0.001). rhPRG4 (200 μg/ml) reduced basal gene expression of MMP-1, MMP-3, MMP-13, IL-6, IL-8, and PRG4 in OA synoviocytes, while increasing TIMP-2 and cycloxygenase-2 (COX2) expression (p < 0.001). rhPRG4 (200 μg/ml) reduced IL-1β induction of MMP-1, MMP-3, MMP-9, MMP-13, IL-6, IL-8, and COX2 expression in a CD44-dependent manner (p < 0.001)., Conclusion: PRG4 plays an important anti-inflammatory role in regulating OA synoviocyte proliferation and reduces basal and IL-1β-stimulated expression of catabolic mediators. Exogenous rhPRG4 autoregulates native PRG4 expression in OA synoviocytes.

Biomechanical properties of human meniscus have been shown to be site-specific. However, it is not known which meniscus constituents at different depths and locations contribute to biomechanical properties obtained from indentation testing. Therefore, we investigated the composition and structure of human meniscus in a site- and depth-dependent manner and their relationships with tissue site-specific biomechanical properties. Elastic and poroelastic properties were analyzed from experimental stress-relaxation and sinusoidal indentation measurements with fibril reinforced poroelastic finite element modeling. Proteoglycan (PG) and collagen contents, as well as the collagen orientation angle, were determined as a function of tissue depth using microscopic and spectroscopic methods, and they were compared with biomechanical properties. For all the measurement sites (anterior, middle and posterior) of lateral and medial menisci (n=26), PG content and collagen orientation angle increased as a function of tissue depth while the collagen content had an initial sharp increase followed by a decrease across tissue depth. The highest values (p<0.05) of elastic parameters (equilibrium and instantaneous moduli) and strain-dependent biomechanical parameters (strain-dependent fibril network modulus and permeability) were observed in the anterior horn of the medial meniscus. This location had also higher (p<0.05) PG content in the deep meniscus, higher (p<0.05) collagen content in the entire tissue depth, and lower (p<0.05) collagen orientation angle at the superficial tissue, as compared to many other locations. On the other hand, in certain comparisons (such as anterior vs. middle sites of the medial meniscus) significantly higher (p<0.05) collagen content and lower orientation angle, without any difference in the PG content, were consistent with increased meniscus modulus and/or nonlinear permeability. This study suggests that nonlinear biomechanical properties of meniscus, caused by the collagen network and fluid, may be strongly influenced by tissue osmotic swelling from the deep meniscus caused by the increased PG content, leading to increased collagen fibril tension. These nonlinear biomechanical properties are suggested to be further amplified by higher collagen content at all tissue depths and superficial collagen fibril orientation. However, these structure-function relationships are suggested to be highly site-specific., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Activated protein C/endothelial protein C receptor (APC/EPCR) axis is physiologically involved in anticoagulant and cytoprotective activities in endothelial cells. Emerging evidence indicates that EPCR also plays a role in breast stemness and human tumorigenesis. Yet, its contribution to breast cancer progression and metastasis has not been elucidated., Methods: Transcriptomic status of EPCR was examined in a cohort of 286 breast cancer patients. Cell growth kinetics was evaluated in control and EPCR and SPARC/osteonectin, Cwcv, and kazal-like domains proteoglycan (SPOCK1/testican 1) silenced breast cancer cells in 2D, 3D, and in co-culture conditions. Orthotopic tumor growth and lung and osseous metastases were evaluated in several human and murine xenograft breast cancer models. Tumor-stroma interactions were further studied in vivo by immunohistochemistry and flow cytometry. An EPCR-induced gene signature was identified by microarray analysis., Results: Analysis of a cohort of breast cancer patients revealed an association of high EPCR levels with adverse clinical outcome. Interestingly, EPCR knockdown did not affect cell growth kinetics in 2D but significantly reduced cell growth in 3D cultures. Using several human and murine xenograft breast cancer models, we showed that EPCR silencing reduced primary tumor growth and secondary outgrowths at metastatic sites, including the skeleton and the lungs. Interestingly, these effects were independent of APC ligand stimulation in vitro and in vivo. Transcriptomic analysis of EPCR-silenced tumors unveiled an effect mediated by matricellular secreted proteoglycan SPOCK1/testican 1. Interestingly, SPOCK1 silencing suppressed in vitro 3D growth. Moreover, SPOCK1 ablation severely decreased orthotopic tumor growth and reduced bone metastatic osteolytic tumors. High SPOCK1 levels were also associated with poor clinical outcome in a subset breast cancer patients. Our results suggest that EPCR through SPOCK1 confers a cell growth advantage in 3D promoting breast tumorigenesis and metastasis., Conclusions: EPCR represents a clinically relevant factor associated with poor outcome and a novel vulnerability to develop combination therapies for breast cancer patients.

Three-dimensional (3D) cell cultures on reconstituted basement membrane (rBM) enable the study of complex interactions between extracellular matrix (ECM) components and epithelial cells, which are crucial for the establishment of cell polarity and functional development of epithelia. 3D cultures of mammary epithelial cells (MECs) on Matrigel (a laminin-rich ECM derived from the Engelbreth-Holm-Swarm (EHS) murine tumor) promote interactions of MECs with the matrix via integrins, leading to formation of spherical monolayers of polarized cells surrounding a hollow lumen (acini). Acini closely resemble mammary alveoli found in the mammary gland. Thus, it is possible to study ECM-cell interactions and signalling pathways that regulate formation and maintenance of tissue-specific shape and functional differentiation of MECs in 3D under in vitro conditions. Here we present experimental protocols used to investigate the role of mitogen-activated protein kinase phosphatases (MKPs) during development of the alveoli-like structures by primary mouse mammary epithelial cells (PMMEC) cultured on Matrigel. We present detailed protocols for PMMEC isolation, and establishment of 3D cultures using an "on top" method, use of specific kinase and phosphatases inhibitors (PD98059 and pervanadate, respectively) administered at different stages of acinus development, and give examples of analyses carried out post-culture (Western blot, immunofluorescence staining, and confocal imaging).

Branching morphogenesis is a fundamental process in the development of diverse epithelial organs such as the lung, kidney, liver, pancreas, prostate, salivary, lacrimal and mammary glands. A unifying theme during organogenesis is the importance of epithelial cell interactions with the extracellular matrix (ECM) and growth factors (GFs). The diverse developmental mechanisms giving rise to these epithelial organs involve many organ-specific GFs, but a unifying paradigm during organogenesis is the regulation of GF activity by heparan sulfates (HS) on the cell surface and in the ECM. This primarily involves the interactions of GFs with the sulfated side-chains of HS proteoglycans. HS is one of the most diverse biopolymers and modulates GF binding and signaling at the cell surface and in the ECM of all tissues. Here, we review what is known about how HS regulates branching morphogenesis of epithelial organs with emphasis on the developing salivary gland, which is a classic model to investigate epithelial-ECM interactions. We also address the structure, biosynthesis, turnover and function of HS during organogenesis. Understanding the regulatory mechanisms that control HS dynamics may aid in the development of therapeutic interventions for diseases and novel strategies for tissue engineering and regenerative medicine., (Published by Elsevier B.V.)

In addition to contraction, myoepithelia have diverse paracrine effects, including a tumor suppression effect. However, certain myoepithelial markers have been shown to contribute to tumor progression. Transforming growth factor-β (TGF-β) is involved in the transdifferentiation of fibroblasts to contractile myofibroblasts. We investigated whether TGF-β can upregulate potential myoepithelial markers, which may have functional and clinicopathological significance in breast cancer. We found that TGF-β induced SPOCK1 expression in MCF10A, MCF12A, and M10 breast cells and demonstrated SPOCK1 as a novel myoepithelial marker that was immunolocalized within or beneath myoepithelia lining ductolobular units. A functional study showed that overexpression of SPOCK1 enhanced invasiveness in mammary immortalized and cancer cells. To further determine the biological significance of SPOCK1 in breast cancer, we investigated the expression of SPOCK1 in 478 invasive ductal carcinoma (IDC) cases through immunohistochemistry and correlated the expression with clinicopathological characteristics. SPOCK1 expression was significantly correlated with high pathological tumor size (P = 0.012), high histological grade (P = 0.013), the triple-negative phenotype (P = 0.022), and the basal-like phenotype (P = 0.026) and was correlated with a significantly poorer overall survival on univariate analysis (P = 0.001, log-rank test). Multivariate Cox regression analysis demonstrated that SPOCK1 expression maintained an independent poor prognostic factor of overall survival. Analysis of SPOCK1 expression on various non-IDC carcinoma subtypes showed an enrichment of SPOCK1 expression in metaplastic carcinoma, which is pathogenetically closely related to epithelial-mesenchymal transition (EMT). In conclusion, we identified SPOCK1 as a novel TGF-β-induced myoepithelial marker and further demonstrated that SPOCK1 enhanced invasion in breast cancer cells and correlated with poor prognosis in breast cancer clinical samples. The enrichment of SPOCK1 expression in metaplastic carcinoma and the correlation between SPOCK1 expression and high histological grading and basal-like phenotypes in IDC evidence an association between SPOCK1 and EMT., Competing Interests: The authors have declared that no competing interests exist.

Decorin, a prototype small leucine-rich proteoglycan, regulates a vast array of cellular processes including collagen fibrillogenesis, wound repair, angiostasis, tumor growth, and autophagy. This functional versatility arises from a wide array of decorin/protein interactions also including interactions with its single glycosaminoglycan side chain. The decorin-binding partners encompass numerous categories ranging from extracellular matrix molecules to cell surface receptors to growth factors and enzymes. Despite the diversity of the decorin interacting network, two main roles emerge as prominent themes in decorin function: maintenance of cellular structure and outside-in signaling, culminating in anti-tumorigenic effects. Here we present contemporary knowledge regarding the decorin interacting network and discuss in detail the biological relevance of these pleiotropic interactions, some of which could be targeted by therapeutic interventions., (Copyright © 2016. Published by Elsevier B.V.)

Rationale: Lymphatic vessel growth is mediated by major prolymphangiogenic factors, such as vascular endothelial growth factor (VEGF-C) and VEGF-D, among other endothelial effectors. Heparan sulfate is a linear polysaccharide expressed on proteoglycan core proteins on cell membranes and matrix, playing roles in angiogenesis, although little is known about any function(s) in lymphatic remodeling in vivo., Objective: To explore the genetic basis and mechanisms, whereby heparan sulfate proteoglycans mediate pathological lymphatic remodeling., Methods and Results: Lymphatic endothelial deficiency in the major heparan sulfate biosynthetic enzyme N-deacetylase/N-sulfotransferase-1 (Ndst1; involved in glycan-chain sulfation) was associated with reduced lymphangiogenesis in pathological models, including spontaneous neoplasia. Mouse mutants demonstrated tumor-associated lymphatic vessels with apoptotic nuclei. Mutant lymphatic endothelia demonstrated impaired mitogen (Erk) and survival (Akt) pathway signaling and reduced VEGF-C-mediated protection from starvation-induced apoptosis. Lymphatic endothelial-specific Ndst1 deficiency (in Ndst1(f/f)Prox1(+/CreERT2) mice) was sufficient to inhibit VEGF-C-dependent lymphangiogenesis. Lymphatic heparan sulfate deficiency reduced phosphorylation of the major lymphatic growth receptor VEGF receptor-3 in response to multiple VEGF-C species. Syndecan-4 was the dominantly expressed heparan sulfate proteoglycan in mouse lymphatic endothelia, and pathological lymphangiogenesis was impaired in Sdc4((-/-)) mice. On the lymphatic cell surface, VEGF-C induced robust association between syndecan-4 and VEGF receptor-3, which was sensitive to glycan disruption. Moreover, VEGF receptor-3 mitogen and survival signaling was reduced in the setting of Ndst1 or Sdc4 deficiency., Conclusions: These findings demonstrate the genetic importance of heparan sulfate and the major lymphatic proteoglycan syndecan-4 in pathological lymphatic remodeling. This may introduce novel future strategies to alter pathological lymphatic-vascular remodeling., (© 2016 The Authors.)