Lubricin Research Articles

Proteoglycan 4 predicts tribological properties of repaired cartilage tissue.

Purpose: One of the essential requirements in maintaining the normal joint motor function is the perfect tribological property of the articular cartilage. Many cartilage regeneration strategies have been developed for treatment in early stages of osteoarthritis, but there is little information on how repaired articular cartilage regains durability. The identification of biomarkers that can predict wear resistant property is critical to advancing the success of cartilage regeneration therapies. Proteoglycan 4 (PRG4) is a macromolecule distributing on the chondrocyte surface that contributes to lubrication. In this study, we investigate if PRG4 expression is associated with tribological properties of regenerated cartilage, and is able to predict its wear resistant status.Methods: Two different strategies including bone marrow enrichment plus microfracture (B/BME-MFX) and microfracture alone (B-MFX) of cartilage repair in sheep were used. PRG4 expression and a series of tribological parameters on regenerated cartilage were rigorously examined and compared.Results: Highly and continuously expression of PRG4 in regenerated cartilage surface was negatively correlated with each tribological parameter (P<0.0001, respectively). Multivariate analysis showed that PRG4 expression was the key predictor that contributed to the promotion of cartilage wear resistance.Conclusion: Higher PRG4 expression in regenerated cartilage is significantly associated with wear resistance improvement. PRG4 may be useful for predicting the wear resistant status of regenerated cartilage and determining the optimal cartilage repair strategy.

Lubricin (PRG4) reduces fouling susceptibility and improves sensitivity of carbon-based electrodes

In order to overcome the problems of reliability and accuracy often associated with electrochemical sensing in bioassays due to electrode surface fouling, we present an innovative antifouling surface coating based on lubricin (LUB). LUB is a large glycoprotein found in the articular joints of mammals with the ability to self-assemble on surfaces of different nature, creating an antiadhesive brush layer. This work demonstrates the size selective and antiadhesive properties of LUB coatings which prevent large biomolecules from adhering onto the electrode surface while simultaneously allowing smaller electroactive species to pass through the layer unimpeded. Electrochemical and surface characterisation experiments were performed using glassy carbon (GC), carbon black SPEs (CB), and reduced graphene oxide SPEs (rGO) in order to evaluate the antifouling capabilities of LUB coatings in solutions containing bovine serum albumin (BSA) and human saliva. The LUB coatings have been shown to exhibit outstanding antiadhesive properties, size selectivity, self-assembly as well as providing excellent electrochemistry in these highly fouling solutions.

Spatially patterned microribbon-based hydrogels induce zonally-organized cartilage regeneration by stem cells in 3D

Regenerating cartilage with biomimetic zonal organization, which is critical for tissue function, remains a great challenge. The objective of this study was to evaluate the potential of spatially-patterned, multi-compositional, macroporous, extracellular matrix-based microribbon (µRB) µRB scaffolds to regenerate cartilage with biochemical, mechanical, and morphological zonal organization by mesenchymal stem cells (MSCs) compared to conventional multi-layer nanoporous hydrogels. MSCs were seeded in either trilayer microribbon (µRB) or hydrogel (HG) scaffolds that were composed of layered biomaterial compositions that had been chosen for their ability to differentiate MSCs into chondrocytes with zonal properties. To mimic the aligned collagen morphology in the superficial layer of native cartilage, an additional experimental group added MSC-laden aligned µRBs to the surface of the superficial layer of a µRB trilayer. Tuning µRB alignment and compositions in different zones led to zonal-specific responses of MSCs to create neocartilage with zonal biochemical, morphological, and mechanical properties, while trilayer HGs led to minimal cartilaginous deposition overall. Trilayer µRBs created neocartilage exhibiting significant increases in compressive modulus (up to 456 kPa) and > 4-fold increase in sGAG production from superficial to deep zones. Aligned gelatin µRBs in the superficial zone further enhanced biomimetic mimicry of the produced neocartilage by leading to robust collagen deposition and superficial zone protein production. Statement of significanceRegenerating cartilage with zonal organization using mesenchymal stem cells (MSCs) remains a great challenge. We developed a spatially-patterned, gradient, macroporous, trilayer microribbon (µRB) scaffold that we used to engineer MSC-based neocartilage with zonal trends that match native cartilage in many aspects, including collagen, sGAG, superficial zone protein, and compressive moduli. This is in direct contrast to conventional trilayer nanoporous hydrogels which led to minimal cartilage deposition and weak mechanical properties. It took only 21 days for MSC-seeded trilayer µRB scaffolds to reach cartilage-mimicking compressive moduli without requiring high cell seeding density, which has never been reported before. While this paper focuses on cartilage zonal organization, gradient µRB scaffolds can be used to repair other tissue interfaces such as osteochondral defects.

Adsorption of Amyloidogenic Peptides to Functionalized Surfaces Is Biased by Charge and Hydrophilicity.

Surfaces are abundant in living systems, such as in the form of cellular membranes, and govern many biological processes. In this study, the adsorption of the amyloidogenic model peptides GNNQQNY, NNFGAIL, and VQIVYK as well as the amyloid-forming antimicrobial peptide uperin 3.5 (U3.5) were studied at low concentrations (100 μM) to different surfaces. The technique of a quartz crystal microbalance with dissipation monitoring (QCM-D) was applied as it enables the monitoring of mass binding to sensors at nanogram sensitivity. Gold-coated quartz sensors were used as unmodified gold surfaces or functionalized with self-assembled monolayers (SAMs) of alkanethiols (terminated as methyl, amino, carboxyl, and hydroxyl) resulting in different adsorption affinities of the peptides. Our objective was to evaluate the underlying role of the nature and feature of interfaces in biological systems which could concentrate peptides and impact or trigger peptide aggregation processes. In overall, the largely hydrophobic peptides adsorbed with preference to hydrophobic or countercharged surfaces. Further, the glycoprotein lubricin (LUB) was tested as an antiadhesive coating. Despite its hydrophilicity, the adsorption of peptides to LUB coated sensors was similar to the adsorption to unmodified gold surfaces, which indicates that some peptides diffused through the LUB layer to reach the underlying gold sensor surface. The LUB protein-antiadhesive is thus more effective as a biomaterial coating against larger biomolecules than small peptides under the conditions used here. This study provides directions toward a better understanding of amyloid peptide adsorption to biologically relevant interfaces, such as cellular membranes.

Recombinant human PRG4 (rhPRG4) suppresses breast cancer cell invasion by inhibiting TGFβ-Hyaluronan-CD44 signalling pathway.

Metastasis is the major cause of cancer-related morbidity and mortality. The ability of cancer cells to become invasive and migratory contribute significantly to metastatic growth, which necessitates the identification of novel anti-migratory and anti-invasive therapeutic approaches. Proteoglycan 4 (PRG4), a mucin-like glycoprotein, contributes to joint synovial homeostasis through its friction-reducing and anti-adhesive properties. Adhesion to surrounding extracellular matrix (ECM) components is critical for cancer cells to invade the ECM and eventually become metastatic, raising the question whether PRG4 has an anti-invasive effect on cancer cells. Here, we report that a full-length recombinant human PRG4 (rhPRG4) suppresses the ability of the secreted protein transforming growth factor beta (TGFβ) to induce phenotypic disruption of three-dimensional human breast cancer cell-derived organoids by reducing ligand-induced cell invasion. In mechanistic studies, we find that rhPRG4 suppresses TGFβ-induced invasiveness of cancer cells by inhibiting the downstream hyaluronan (HA)-cell surface cluster of differentiation 44 (CD44) signalling axis. Furthermore, we find that rhPRG4 represses TGFβ-dependent increase in the protein abundance of CD44 and of the enzyme HAS2, which is involved in HA biosynthesis. It is widely accepted that TGFβ has both tumor suppressing and tumor promoting roles in cancer. The novel finding that rhPRG4 opposes HAS2 and CD44 induction by TGFβ has implications for downregulating the tumor promoting roles, while maintaining the tumor suppressive aspects of TGFβ actions. Finally, these findings point to rhPRG4’s potential clinical utility as a therapeutic treatment for invasive and metastatic breast cancer.

Spatially Patterned Microribbon-Based Hydrogels Induce Zonally-Organized Cartilage Regeneration by Stem Cells in 3D

Regenerating cartilage with biomimetic zonal organization, which is critical for tissue function, remains a great challenge. The objective of this study was to evaluate the potential of spatially-patterned, multi-compositional, macroporous, extracellular matrix-based μRB scaffolds to regenerate cartilage with biochemical, mechanical, and morphological zonal organization by mesenchymal stem cells (MSCs) compared to conventional multi-layer nanoporous hydrogels. MSCs were seeded in either trilayer microribbon (μRB) or hydrogel (HG) scaffolds that were composed of layered biomaterial compositions that had been chosen for their ability to differentiate MSCs into chondrocytes with zonal properties. To mimic the aligned collagen morphology in the superficial layer of native cartilage, an additional experimental group added MSC-laden aligned μRBs to the surface of the superficial layer of a μRB trilayer. Tuning μRB alignment and compositions in different zones led to zonal-specific responses of MSCs to create neocartilage with zonal biochemical, morphological, and mechanical properties, while trilayer HGs led to minimal cartilaginous deposition overall. Trilayer μRBs created neocartilage exhibiting significant increases in compressive modulus (up to 456 kPa) and > 4-fold increase in sGAG production from superficial to deep zones. Aligned gelatin µRBs in the superficial zone further enhanced biomimetic mimicry of the produced neocartilage by leading to robust collagen deposition and superficial zone protein production.

Absence of Proteoglycan 4 (Prg4) Leads to Increased Subchondral Bone Porosity Which Can Be Mitigated Through Intra-Articular Injection of PRG4.

Proteoglycan 4 (PRG4) is a mucin-like glycoprotein important for joint health. Mice lacking Prg4 demonstrate degeneration of the cartilage and altered skeletal morphology. The purpose of this study was to examine if Prg4 deficiency leads to subchondral bone defects and if these defects could be mitigated through intra-articular injection of recombinant human PRG4 (rhPRG4). Mice deficient in Prg4 expression demonstrated increased cartilage thickness and increased subchondral bone porosity compared with C57BL/6 controls. While the porosity of the subchondral bone of Prg4-/- mice decreased over time with maturation, intra-articular injection of rhPRG4 was able to forestall the increase in porosity. In contrast, neither hyaluronan (HA) nor methylprednisolone injections had beneficial effects on the subchondral bone porosity in the Prg4 knockout mice. Bone marrow progenitor cells from Prg4-/- mice demonstrated reduced osteogenic differentiation capacity at 4 weeks of age, but not at 16 weeks of age. While most studies on PRG4/lubricin focus on the health of the cartilage, this study demonstrates that PRG4 plays a role in the maturation of the subchondral bone. Furthermore, increasing joint lubrication/viscosupplementation through injection of HA or controlling joint inflammation through injection of methylprednisolone may help maintain the cartilage surface, but had no positive effect on the subchondral bone in animals lacking Prg4. Therefore, alterations in the subchondral bone in models with absent or diminished Prg4 expression should not be overlooked when investigating changes within the articular cartilage regarding the pathogenesis of osteoarthritis/arthrosis. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:2077-2088, 2019.

Correlation of computed tomography with carotid plaque transcriptomes associates calcification with lesion-stabilization

Background and aimsUnstable carotid atherosclerosis causes stroke, but methods to identify patients and lesions at risk are lacking. We recently found enrichment of genes associated with calcification in carotid plaques from asymptomatic patients. Here, we hypothesized that calcification represents a stabilising feature of plaques and investigated how macro-calcification, as estimated by computed tomography (CT), correlates with gene expression profiles in lesions. MethodsPlaque calcification was measured in pre-operative CT angiographies. Plaques were sorted into high- and low-calcified, profiled with microarrays, followed by bioinformatic analyses. Immunohistochemistry and qPCR were performed to evaluate the findings in plaques and arteries with medial calcification from chronic kidney disease patients. ResultsSmooth muscle cell (SMC) markers were upregulated in high-calcified plaques and calcified plaques from symptomatic patients, whereas macrophage markers were downregulated. The most enriched processes in high-calcified plaques were related to SMCs and extracellular matrix (ECM) organization, while inflammation, lipid transport and chemokine signaling were repressed. These findings were confirmed in arteries with high medial calcification. Proteoglycan 4 (PRG4) was identified as the most upregulated gene in association with plaque calcification and found in the ECM, SMA+ and CD68+/TRAP + cells. ConclusionsMacro-calcification in carotid lesions correlated with a transcriptional profile typical for stable plaques, with altered SMC phenotype and ECM composition and repressed inflammation. PRG4, previously not described in atherosclerosis, was enriched in the calcified ECM and localized to activated macrophages and smooth muscle-like cells. This study strengthens the notion that assessment of calcification may aid evaluation of plaque phenotype and stroke risk.

Human Diseased Articular Cartilage Contains a Mesenchymal Stem Cell-Like Population of Chondroprogenitors with Strong Immunomodulatory Responses.

Background: osteoarthritic human articular cartilage (AC)-derived cartilage cells (CCs) with same-donor bone marrow (BMSCs) and adipose tissue (ASCs)-derived mesenchymal stem cells were compared, in terms of stemness features, and secretory and immunomodulatory responses to inflammation. Methods: proteoglycan 4 (PRG4) presence was evaluated in AC and CCs. MSCs and CCs (n = 8) were cultured (P1 to P4) and characterized for clonogenicity, nanog homeobox (NANOG), and POU class 5 homeobox 1 (POU5F1) expression, immunotypification, and tri-lineage differentiation. Their basal and interleukin-1β (IL-1β)-stimulated expression of matrix metalloproteases (MMPs), tissue inhibitors (TIMPs), release of growth factors, and cytokines were analyzed, along with the immunomodulatory ability of CCs. Results: PRG4 was mainly expressed in the intact AC surface, whereas shifted to the intermediate zone in damaged cartilage and increased its expression in CCs upon culture. All cells exhibited a similar phenotype and stemness maintenance over passages. CCs showed highest chondrogenic ability, no adipogenic potential, a superior basal secretion of growth factors and cytokines, the latter further increased after inflammatory stimulation, and an immunomodulatory behavior. All stimulated cells shared an increased MMP expression without a corresponding TIMP production. Conclusion: based on the observed features, CCs obtained from pathological joints may constitute a potential tissue-specific therapeutic target or agent to improve damaged cartilage healing, especially damage caused by inflammatory/immune mediated conditions.

Lubricin on Platinum Electrodes: A Low‐Impedance Protein‐Resistant Surface Towards Biomedical Implantation

AbstractBiofouling on surfaces compromises the function of biomedical devices whose function involves contact with biological fluids. In the context of electrochemical devices, proteins are attracted to the surface via coaction of various forces (hydrogen bond, hydrophobic effect, and other polar interactions) and protein interaction with the surface can significantly alter the surface chemistry. In response to this issue, we have developed an efficient anti‐biofouling surface that employs a glycoprotein, lubricin (LUB), and which generates low impedance layers compatible with electrochemical applications. Herein, we investigate how different LUB densities on platinum (Pt) electrodes affect the surface electrochemistry and its ability to prevent nonspecific adsorption of protein to the surface. Surfaces with higher densities of LUB were more resistant to protein adsorption. The LUB modified Pt electrodes were challenged in artificial perilymph (AP) media under passive and electrically stimulated conditions over 7‐day periods throughout which the LUB layer retained its anti‐biofouling and surface coating stability.

Investigating the effect of proteoglycan 4 on hyaluronan solution properties using confocal fluorescence recovery after photobleaching

BackgroundThe objective of this study was to use confocal fluorescence recovery after photobleaching (FRAP) to examine the specific and dose-dependent effect of proteoglycan 4 (PRG4) on hyaluronan (HA) solutions of different molecular weight; and assess the effect of reduction and alkylation (R/A) of PRG4 on its effects on HA solutions.MethodsConfocal FRAP was used to determine the diffusion coefficient of fluorescein isothiocyanate (FITC)-dextran tracer (Dt) through 1500 kDa and 500 kDa HA solutions (0–3.3 mg/ml) ± PRG4 or a control protein, bovine serum albumin (BSA), at physiological (450 μg/ml) or pathophysiological (45 μg/ml) concentrations. The effect of PRG4 or R/A PRG4 on 1500 kDa HA solutions was also investigated. Empirical constants obtained from fitting data to the universal scaling equation were used to calculate the average distribution of apparent mesh sizes.ResultsPRG4 at both 45 and 450 μg/ml slowed the diffusion of the FITC-dextran tracer for all concentrations of HA and caused a decrease in the apparent mesh size within the HA solution. This effect was specific to PRG4, not observed with BSA, but not dependent on its tertiary/quaternary structure as the effect remained after R/A of PRG4.ConclusionsThese results demonstrate that PRG4 can significantly alter the solution properties of HA; PRG4 essentially reduced the permeability of the HA network. This effect may be due to PRG4 entangling HA molecules through binding and/or HA crowding PRG4 molecules into a self-assembled network. Collectively these findings contribute to the understanding of PRG4 and HA interaction(s) in solution and therefore the function of SF in diarthroidal joints.

BMP9 stimulates joint regeneration at digit amputation wounds in mice

A major goal of regenerative medicine is to stimulate tissue regeneration after traumatic injury. We previously discovered that treating digit amputation wounds with BMP2 in neonatal mice stimulates endochondral ossification to regenerate the stump bone. Here we show that treating the amputation wound with BMP9 stimulates regeneration of a synovial joint that forms an articulation with the stump bone. Regenerated structures include a skeletal element lined with articular cartilage and a synovial cavity, and we demonstrate that this response requires the Prg4 gene. Combining BMP2 and BMP9 treatments in sequence stimulates the regeneration of bone and joint. These studies provide evidence that treatment of growth factors can be used to engineer a regeneration response from a non-regenerating amputation wound.

Probing the Molecular Interactions and Lubrication Mechanisms of Purified Full-Length Recombinant Human Proteoglycan 4 (rhPRG4) and Hyaluronic Acid (HA).

Probing the adsorption and lubrication behavior of lubricin, also known as proteoglycan 4 (PRG4), is important for understanding the ultralow friction of cartilage lubrication. Most previous research has focused on native lubricin either purified from synovial fluid or articular cartilage explant culture media. In this work, the adsorption behavior and lubrication mechanism of full-length recombinant human PRG4 (rhPRG4) on mica as well as the effect of adding hyaluronic acid (HA, a polysaccharide) were systematically investigated using a surface forces apparatus (SFA) technique. A low friction coefficient (μ ∼ 0.04) was measured when multilayer rhPRG4 (∼31 nm) was confined in between mica surfaces, even when the load increased to ∼1.2 MPa. Intriguingly, a previously unreported ultralow friction coefficient (μ < 0.005) was observed at a low sliding velocity ( v = 0.14 μm/s) with the applied load P reaching ∼3.6 MPa when a diluted rhPRG4 solution (∼90 μg/mL) was used. The distinct friction behavior is likely due to the smooth and more close-packed lubricin coating, as made evident by the atomic force microscope imaging. Adding HA onto multilayer rhPRG4-coated mica increased the friction coefficient μ to ∼0.1; however, the load bearing property increased, indicating potential synergistic effect between rhPRG4 and HA, which was further demonstrated by the weak adhesion observed when separating rhPRG4-coated mica and HA-coated aminopropyltriethoxysilane-mica (APTES-mica). Alternatively, adding premixed rhPRG4-HA on mica had a friction coefficient (μ ∼ 0.1) close to that of injecting concentrated rhPRG4 (∼450 μg/mL) with lower load sustainability. Our results provide fundamental insights into the adsorption and lubrication behavior of lubricin and its interaction with HA, with useful implications for the underlying mechanism of ultralow friction provided by synovial fluid.

FAMILIAL CAMPTODACTYLY COXA VARA PERICARDITIS SYNDROME MASQUARADING AS JUVUNILE IDIOPATHIC ARTHRITIS

ABSTRACTThe camptodactyly-arthropathy-coxa vara-pericarditis syndrome (CACP) is a rare autosomal recessive disease characterized by congenital or early-onset camptodactyly, childhood-onset non-inflammatory arthropathy associated with synovial hyperplasia, constrictive pericarditis and coxa vara. This rare disease is caused by mutations in PRG4 Gene which encodes for lubricin, a protein present in synovial fluid responsible for lubrication.The case we are reporting is of 9 and half years old boy who presented to us in pediatric Out Patient Department with early onset camptodactyly and non-inflammatory arthropathy. Synovial biopsy shows synovial hyperplasia with numerous CD68 positive multinucleated giant cells. He was discharged and physiotherapy was advised with regular follow ups.KEY WORDS: Coxa Vara (MeSH); Lubricin (MeSH); Joint Diseases (MeSH); Arthropathy (MeSH); Osteoarthritis (MESH); Pericarditis (MeSH); Synovial Membrane (MeSH).

Biomimetic Bottlebrush Polymer Coatings for Fabrication of Ultralow Fouling Surfaces.

Demand for long-lasting antifouling surfaces has steered the development of accessible, novel, biocompatible and environmentally friendly materials. Inspired by lubricin (LUB), a component of mammalian synovial fluid with excellent antifouling properties, three block polymers offering stability, efficacy, and ease of use were designed. The bottlebrush-structured polymers adsorbed strongly on silica surfaces in less than 10 minutes by a simple drop casting or online exposure method and were extremely stable in high-salinity solutions and across a wide pH range. Antifouling properties against proteins and bacteria were evaluated with different techniques and ultralow fouling properties demonstrated. With serum albumin and lysozyme adsorption <0.2 ng cm-2 , the polymers were 50 and 25 times more effective than LUB and known ultralow fouling coatings. The antifouling properties were also tested under MPa compression pressures by direct force measurements using surface forces apparatus. The findings suggest that these polymers are among the most robust and efficient antifouling agents currently known.

Biomimetic Bottlebrush Polymer Coatings for Fabrication of Ultralow Fouling Surfaces

AbstractDemand for long‐lasting antifouling surfaces has steered the development of accessible, novel, biocompatible and environmentally friendly materials. Inspired by lubricin (LUB), a component of mammalian synovial fluid with excellent antifouling properties, three block polymers offering stability, efficacy, and ease of use were designed. The bottlebrush‐structured polymers adsorbed strongly on silica surfaces in less than 10 minutes by a simple drop casting or online exposure method and were extremely stable in high‐salinity solutions and across a wide pH range. Antifouling properties against proteins and bacteria were evaluated with different techniques and ultralow fouling properties demonstrated. With serum albumin and lysozyme adsorption &lt;0.2 ng cm−2, the polymers were 50 and 25 times more effective than LUB and known ultralow fouling coatings. The antifouling properties were also tested under MPa compression pressures by direct force measurements using surface forces apparatus. The findings suggest that these polymers are among the most robust and efficient antifouling agents currently known.

Proteoglycan 4: From Mere Lubricant to Regulator of Tissue Homeostasis and Inflammation: Does proteoglycan 4 have the ability to buffer the inflammatory response?

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.

Recombinant human proteoglycan-4 reduces phagocytosis of urate crystals and downstream nuclear factor kappa B and inflammasome activation and production of cytokines and chemokines in human and murine macrophages

BackgroundGout is an inflammatory arthritis caused by monosodium urate monohydrate (MSU) crystals’ joint deposition. MSU phagocytosis by resident macrophages is a key step in gout pathogenesis. MSU phagocytosis triggers nuclear factor kappa B (NFκB) activation and production of cytokines and chemokines. Proteoglycan-4 (PRG4) is a glycoprotein produced by synovial fibroblasts and exerts an anti-inflammatory effect in the joint mediated by its interaction with cell surface receptor CD44. PRG4 also binds and antagonizes TLR2 and TLR4. The objective of this study is to evaluate the efficacy of recombinant human PRG4 (rhPRG4) in suppressing MSU-induced inflammation and mechanical allodynia in vitro and in vivo.MethodsTHP-1 macrophages were incubated with MSU crystals ± rhPRG4 or bovine submaxillary mucin (BSM), and crystal phagocytosis, cytokines and chemokines expression and production were determined. NFκB p65 subunit nuclear translocation, NLRP3 induction, caspase-1 activation and conversion of proIL-1β to mature IL-1β were studied. MSU phagocytosis by Prg4+/+ and Prg4−/− peritoneal macrophages was determined in the absence or presence of rhPRG4, BSM, anti-CD44, anti-TLR2, anti-TLR4 and isotype control antibodies. Rhodamine-labeled rhPRG4 was incubated with murine macrophages and receptor colocalization studies were performed. Lewis rats underwent intra-articular injection of MSU crystals followed by intra-articular treatment with PBS or rhPRG4. Weight bearing and SF myeloperoxidase activities were determined.ResultsrhPRG4 reduced MSU crystal phagocytosis at 4 h (p < 0.01) and IL-1β, TNF-α, IL-8 and MCP-1 expression and production at 6 h (p < 0.05). BSM did not alter MSU phagocytosis or IL-1β production in human and murine macrophages. rhPRG4 treatment reduced NFκB nuclear translocation, NLRP3 expression, caspase-1 activation and generation of mature IL-1β (p < 0.05). MSU-stimulated IL-1β production was higher in Prg4−/− macrophages compared to Prg4+/+ macrophages (p < 0.001). rhPRG4, anti-CD44, anti-TLR2 and anti-TLR4 antibody treatments reduced MSU phagocytosis and IL-1β production in murine macrophages (p < 0.05). rhPRG4 preferentially colocalized with CD44 on Prg4−/− peritoneal macrophages compared to TLR2 or TLR4 (p < 0.01). rhPRG4 normalized weight bearing and reduced SF myeloperoxidase activity compared to PBS in vivo.ConclusionrhPRG4 inhibits MSU crystal phagocytosis and exhibits an anti-inflammatory and anti-nociceptive activity in vitro and in vivo. rhPRG4’s anti-inflammatory mechanism may be due to targeting CD44 on macrophages.

A functional polypeptide N-acetylgalactosaminyltransferase (PGANT) initiates O-glycosylation in cultured silkworm BmN4 cells.

Mucin-type O-glycosylation is initiated by UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts or PGANTs), attaching GalNAc to serine or threonine residue of a protein substrate. In the insect model from Lepidoptera, silkworm (Bombyx mori), however, O-glycosylation pathway is totally unexplored and remains largely unknown. In this study, as the first report regarding protein O-glycosylation analysis in silkworms, we verified the O-glycan profile that a common core 1 Gal (β1-3) GalNAc disaccharide branch without terminally sialylated structure is mainly formed for a baculovirus-produced human proteoglycan 4 (PRG4) protein. Intriguingly, functional screenings in cultured silkworm BmN4 cells for nine Bmpgants reveal that Bmpgant2 is the solo functional BmPGANT for PRG4, implying that Bmpgants may have unique cell/tissue or protein substrate preferences. Furthermore, a recombinant BmPGANT2 protein was successfully purified from silkworm-BEVS and exhibited a high ability to transfer GalNAc for both peptide and protein substrates. Taken together, the present results clarified the functional BmPGANT2 in cultured silkworm cells, providing crucial fundamental insights for future studies dissecting the detailed silkworm O-glycosylation pathways and productions of glycoproteins with O-glycans.

Surface-Functionalized Model Contact Lenses with a Bioinspired Proteoglycan 4 (PRG4)-Grafted Layer.

Ocular dryness and discomfort are the primary reasons for the discontinuation of contact lens wear. This is mainly due to poorly hydrated contact lens surfaces and increased friction, particularly at the end of the day and can potentially cause reduced vision or even inflammation. Proteoglycan 4 (PRG4) is a mucinous glycoprotein with boundary lubricating properties, naturally found in the eye, able to prevent tear film evaporation and protect the ocular surface during blinking. Aiming to improve the interfacial interactions between the ocular surface and the contact lens, the synthesis and characterization of surface-modified model contact lenses with PRG4 is described. Full-length recombinant human PRG4 (rhPRG4) was successfully grafted onto the surface of model conventional and silicone hydrogel (SiHy) contact lenses via its somatomedin B-like end-domain using N, N'-carbonyldiimidazole linking chemistry. Grafting was assessed by Fourier transform infrared spectroscopy-attenuated total reflectance, X-ray photoelectron spectroscopy, and radioactive (I131) labeling. Surface immobilization of rhPRG4 led to model conventional and SiHy materials with improved antifouling properties, without impacting optical transparency or causing any toxic effects to human corneal epithelial cells in vitro. The surface wettability and the boundary friction against human corneal tissue were found to be substrate-dependent, with only the rhPRG4-grafted model SiHy exhibiting a reduced contact angle and kinetic friction coefficient compared to the unmodified surfaces. Hence, clinical grade rhPRG4 can be an attractive candidate for the development of novel bioinspired SiHy contact lenses, providing improved comfort and overall lens performance.