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Recombinant human proteoglycan 4 (rhPRG4) is a macromolecular mucin‐like glycoprotein that is classically studied as a lubricant within eyes and joints. Given that endogenously produced PRG4 is present within atherosclerotic lesions and genetic PRG4 deficiency increases atherosclerosis susceptibility in mice, in the current study we investigated the anti‐atherogenic potential of chronic rhPRG4 treatment. Female low‐density lipoprotein receptor knockout mice were fed an atherogenic Western‐type diet for 6 weeks and injected three times per week intraperitoneally with 0.5 mg rhPRG4 or PBS as control. Treatment with rhPRG4 was associated with a small decrease in plasma‐free cholesterol levels, without a change in cholesteryl ester levels. A marked increase in the number of peritoneal foam cells was detected in response to the peritoneal rhPRG4 administration, which could be attributed to elevated peritoneal leukocyte MSR1 expression levels. However, rhPRG4‐treated mice exhibited significantly smaller aortic root lesions of 278 ± 21 × 103 μm2 compared with 339 ± 15 × 103 μm2 in the aortic root of control mice. The overall decreased atherosclerosis susceptibility coincided with a shift in the monocyte and macrophage polarization states towards the patrolling and anti‐inflammatory M2‐like phenotypes, respectively. Furthermore, rhPRG4 treatment significantly reduced macrophage gene expression levels as well as plasma protein levels of the pro‐inflammatory/pro‐atherogenic cytokine TNF‐alpha. In conclusion, we have shown that peritoneal administration and subsequent systemic exposure to rhPRG4 beneficially impacts the inflammatory state and reduces atherosclerosis susceptibility in mice. Our findings highlight that PRG4 is not only a lubricant but also acts as an anti‐inflammatory agent. Key points: Endogenously produced proteoglycan 4 is found in atherosclerotic lesions and its genetic deficiency in mice is associated with enhanced atherosclerosis susceptibility.In this study we investigated the anti‐atherogenic potential of chronic treatment with recombinant human PRG4 in hypercholesterolaemic female low‐density lipoprotein receptor knockout mice.We show that recombinant human PRG4 stimulates macrophage foam cell formation, but also dampens the pro‐inflammatory state of monocyte/macrophages, eventually leading to a significant reduction in plasma TNF‐alpha levels and a lowered atherosclerosis susceptibility.Our findings highlight that peritoneal recombinant human PRG4 treatment can execute effects both locally and systemically and suggest that it will be of interest to study whether rhPRG4 treatment is also able to inhibit the progression and/or induce regression of previously established atherosclerotic lesions. [ABSTRACT FROM AUTHOR]

Purpose: To search for adaptive response molecules that affect the efficacy of transcatheter arterial chemoembolization (TACE), analyze their clinical correlation with and prognostic value for hepatocellular carcinoma (HCC), and explore their impact on cell biological behavior and their mechanisms of action. Methods: HCC tissue gene sequencing was used to identify differentially expressed genes. The expression of proteoglycan 4 (PRG4) in the serum of 117 patients with HCC who received TACE was detected by enzyme-linked immunosorbent assay. Serum-free medium mimicked TACE-induced nutrient deprivation. Cells with stable knockdown of PRG4 (shPRG4) were constructed to verify the effect and mechanism of PRG4 on the biological behavior of HCC cells in vitro. Results: The expression of PRG4 was significantly elevated under TACEinduced starvation conditions. Low PRG4 expression was associated with worse response to TACE treatment, shorter survival time, and stronger HCC migration ability. Furthermore, in vitro experiments showed that knockdown of PRG4 promoted HCC cell migration by enhancing epithelial-mesenchymal transition (EMT) while did not affect proliferation. When PRG4 expression was low, starvation treatment impaired the migratory ability of HCC cells and reduced the chemosensitivity of HCC cells to epirubicin. Conclusions: PRG4 expression predicts survival and TACE treatment response in patients with HCC. Furthermore, knockdown of PRG4 enhanced EMT, leading to HCC cell migration. PRG4 may serve as a biomarker for HCC patients receiving TACE. [ABSTRACT FROM AUTHOR]

Dry Eye Disease (DED) is a complex pathology affecting millions of people with significant impact on quality of life. Corneal inflammation, including via the nuclear factor kappa B (NFκB) pathway, plays a key etiological role in DED. Recombinant human proteoglycan 4 (rhPRG4) has been shown to be a clinically effective treatment for DED that has anti-inflammatory effects in corneal epithelial cells, but the underlying mechanism is still not understood. Our goal was to understand if rhPRG4 affects tumor necrosis factor α (TNFα)-stimulated inflammatory activity in corneal epithelial cells. We treated hTERT-immortalized corneal epithelial (hTCEpi) cells ± TNFα ± rhPRG4 and performed Western blotting on cell lysate and RNA sequencing. Bioinformatics analysis revealed that rhPRG4 had a significant effect on TNFα-mediated inflammation with potential effects on matricellular homeostasis. rhPRG4 reduced activation of key inflammatory pathways and decreased expression of transcripts for key inflammatory cytokines, interferons, interleukins, and transcription factors. TNFα treatment significantly increased phosphorylation and nuclear translocation of p65, and rhPRG4 significantly reduced both these effects. RNA sequencing identified human leukocyte antigen (HLA)-F adjacent transcript 10 (FAT10), a ubiquitin-like modifier protein which has not been studied in the context of DED, as a key pro-inflammatory transcript increased by TNFα and decreased by rhPRG4. These results were confirmed at the protein level. In summary, rhPRG4 is able to downregulate NFκB activity in hTCEpi cells, suggesting a potential biological mechanism by which it may act as a therapeutic for DED. [ABSTRACT FROM AUTHOR]

Mesenchymal progenitor cells (MPCs) have been recently identified in human and murine epidural fat and have been hypothesized to contribute to the maintenance/repair/regeneration of the dura mater. MPCs can secrete proteoglycan 4 (PRG4/lubricin), and this protein can regulate tissue homeostasis through bio-lubrication and immunomodulatory functions. MPC lineage tracing reporter mice (Hic1) and human epidural fat MPCs were used to determine if PRG4 is expressed by these cells in vivo. PRG4 expression co-localized with Hic1+ MPCs in the dura throughout skeletal maturity and was localized adjacent to sites of dural injury. When Hic1+ MPCs were ablated, PRG4 expression was retained in the dura, yet when Prx1+ MPCs were ablated, PRG4 expression was completely lost. A number of cellular processes were impacted in human epidural fat MPCs treated with rhPRG4, and human MPCs contributed to the formation of epidural fat, and dura tissues were xenotransplanted into mouse dural injuries. We have shown that human and mouse MPCs in the epidural/dura microenvironment produce PRG4 and can contribute to dura homeostasis/repair/regeneration. Overall, these results suggest that these MPCs have biological significance within the dural microenvironment and that the role of PRG4 needs to be further elucidated. [ABSTRACT FROM AUTHOR]

Osteoarthritis (OA) is a degenerative disease that causes irreversible destruction of articular cartilage for which there is no effective treatment at present. Although articular cartilage lacks intrinsic reparative capacity, numerous studies have confirmed the existence of cartilage-resident stem/progenitor cells (CSPCs) in the superficial zone (SFZ) of articular cartilage. CSPCs are characterized by the expression of mesenchymal stromal cell (MSC)-related surface markers, multilineage differentiation ability, colony formation ability, and migration ability in response to injury. In contrast to MSCs and chondrocytes, CSPCs exhibit extensive proliferative and chondrogenic potential with no signs of hypertrophic differentiation, highlighting them as suitable cell sources for cartilage repair. In this review, we focus on the organizational distribution, markers, cytological features and roles of CSPCs in cartilage development, homeostasis and repair, and the application potential of CSPCs in cartilage repair and OA therapies. [ABSTRACT FROM AUTHOR]

Lubricin, a glycoprotein encoded by the proteoglycan 4 (PRG4) gene, is an essential boundary lubricant that reduces friction between articular cartilage surfaces. The loss of lubricin subsequent to joint injury plays a role in the pathogenesis of posttraumatic osteoarthritis. In this study, we describe the development and evaluation of an adeno-associated virus (AAV)-based PRG4 gene therapy intended to restore lubricin in injured joints. The green fluorescent protein (GFP) gene was inserted the PRG4 gene to facilitate tracing the distribution of the transgene product (AAV-PRG4-GFP) in vivo. Transduction efficiency of AAV-PRG4-GFP was evaluated in joint cells, and the conditioned medium containing secreted PRG4-GFP was used for shear loading/friction and viability tests. In vivo transduction of joint tissues following intra-articular injection of AAV-PRG4-GFP was confirmed in the mouse stifle joint in a surgical model of destabilization of the medial meniscus (DMM), and chondroprotective activity was tested in a rabbit anterior cruciate ligament transection (ACLT) model. In vitro studies showed that PRG4-GFP has lubricin-like cartilage-binding and antifriction properties. Significant cytoprotective effects were seen when cartilage was soaked in PRG4-GFP before cyclic shear loading (n = 3). Polymerase chain reaction and confocal microscopy confirmed the presence of PRG4-GFP DNA and protein, respectively, in a mouse DMM (n = 3 per group). In the rabbit ACLT model, AAV-PRG4-GFP gene therapy enhanced lubricin expression (p = 0.001 vs. AAV-GFP: n = 7–14) and protected the cartilage from degeneration (p = 0.014 vs. AAV-GFP: n = 9–10) when treatments were administered immediately postoperation, but efficacy was lost when treatment was delayed for 2 weeks. AAV-PRG4-GFP gene therapy protected cartilage from degeneration in a rabbit ACLT model; however, data from the ACLT model suggest that early intervention is essential for efficacy. [ABSTRACT FROM AUTHOR]

Osteosarcoma is originated from skeletal system. Recombinant human proteoglycan 4 (rhPRG4) can inhibit cell proliferation and migration in multiple cancers. This research is designed to dig out the role and mechanism of PRG4 in osteosarcoma. Human osteosarcoma cell lines, MG63 and 143B, were transfected with programmed death 1 (PD-L1) overexpression vectors and/or treated with 20, 50, and 100 μg/mL rhPRG4, followed by the determination of cell viability, colony formation, sphere formation, invasion, migration, apoptosis, and the expressions of matrix metalloproteinases (MMPs), PD-L1 and apoptosis-related proteins. Tumor-bearing mouse models were constructed by injection of 143B cells and treatment of anti-PD-L1 antibody and/or adenovirus PRG4 (AdPRG4). Tumor volume was monitored, and immunohistochemical location of Ki67 was performed. Expressions of MMPs, transforming growth factor-β (TGF-β), PD-L1, and epithelial mesenchymal transition (EMT)-related proteins were measured in tumors. RhPRG4 (20, 50, and 100 μg/mL) inhibited the viability, colony formation, sphere formation, invasion, migration, and the expressions of MMP2, MMP9 and Bcl2 in osteosarcoma cells, while promoting cell apoptosis as well as Bax and c-caspase3 expressions, at a dose-dependent manner; by contrast, PD-L1 overexpression reversed the above effects of 100 μg/mL rhPRG4. AdPRG4 or anti-PD-L1 antibody decreased tumor volume, number of pulmonary metastasis nodule, Ki67 location, and expressions of TGF-β, PD-L1, MMP2, MMP9, Vimentin, and Snail, but increased E-cadherin expression in tumor cells. Moreover, anti-PD-L1 antibody and AdPRG4 together functioned more effectively than them alone in reducing tumor burden. PRG4 represses the genesis and metastasis of osteosarcoma via inhibiting PD-L1 expression, and AdPRG4 enhances the effectiveness of anti-PD-L1 therapy. • RhPRG4 repressed malignant phenotypes of osteosarcoma cells. • RhPRG4 restrained PD-L1 expression in osteosarcoma cells. • PD-L1 overexpression overturned the influence of rhPRG4 in osteosarcoma cells. • The combination of Anti-PD-L1 and AdPRG4 reduced tumor burden more than either alone. [ABSTRACT FROM AUTHOR]

PurposeThe objectives of this study were to assess the cartilage boundary lubricating ability of (1) nonreduced (NR) disulfide-bonded proteoglycan 4 (PRG4) multimers versus PRG4 monomers and (2) NR versus reduced and alkylated (R/A) PRG4 monomers and to assess (3) the ability of NR PRG4 multimers versus monomers to adsorb to an articular cartilage surface.Materials and methodsPRG4 was separated into two preparations, PRG4 multimer enriched (PRG4Multi+) and PRG4 multimer deficient (PRG4Multi-), using size exclusion chromatography (SEC) and characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The cartilage boundary lubricating ability of PRG4Multi+ and PRG4Multi- was compared at a physiological concentration (450 μg/mL) and assessed over a range of concentrations (45, 150, and 450 μg/mL). R/A and NR PRG4Multi- were evaluated at 450 μg/mL. Immunohistochemistry with anti-PRG4 antibody 4D6 was performed to visualize the adsorption of PRG4 preparations to the surface of articular cartilage explants.ResultsSeparation into enriched populations of PRG4Multi+ and PRG4Multi- was achieved using SEC and was confirmed by SDS-PAGE. PRG4Multi+ and PRG4Multi- both functioned as effective friction-reducing cartilage boundary lubricants at 450 μg/mL, with PRG4Multi+ being more effective than PRG4Multi-. PRG4Multi+ lubricated in a dose-dependent manner, however, PRG4Multi- did not. R/A PRG4Multi- lubricated similar to NR PRG4Multi-. PRG4-containing solutions showed 4D6 immunoreactivity at the articular surface; the immunoreactive intensity of PRG4Multi+ appeared to be similar to SF, whereas PRG4Multi- appeared to have less intensity.ConclusionsThese results demonstrate that the intermolecular disulfide-bonded multimeric structure of PRG4 is important for its ability to adsorb to a cartilage surface and function as a boundary lubricant. These findings contribute to a greater understanding of the molecular basis of cartilage boundary lubrication of PRG4. Elucidating the PRG4 structure-lubrication function relationship will further contribute to the understanding of PRG4's role in diarthrodial joint homeostasis and disease.

Objectives: Lubricin, encoded by the proteoglycan 4 (PRG4) gene, is mainly responsible for lubricating joints. However, there is expanding evidence on its involvement in inflammatory pathways. Juvenile idiopathic arthritis (JIA) is a heterogeneous group of chronic arthritides with an unknown origin in children aged below 16 years. It is characterized by chronic joint inflammation, including synovial inflammation, and may result in cartilage destruction. We aimed to determine whether serum lubricin levels are affected in JIA patients. Material and methods: This cross-sectional study included children diagnosed with JIA and 28 healthy controls. The patients were divided into two groups according to the presence of remission at the time of study. Lubricin protein analysis was performed by the enzyme-linked immunosorbent assay method. Serum samples were obtained at the study enrollment, and lubricin levels were measured once, and compared between JIA patients and healthy controls, and between JIA patients with active disease and remission. Results: The study included 52 JIA patients (28 female, 24 male) and 28 healthy controls (18 female, 10 males). The mean age at study enrollment was 11.66 ±4.41 years and 12.72 ±4.52 years in the JIA patient and control groups, respectively. Although median serum lubricin level did not differ between JIA patients (median: 0.66 ng/µl, range: 0.02-3.85 ng/µl) and healthy controls (median: 0.52 ng/µl, range: 0.06-3.84 ng/µl), it was statistically significantly higher in patients with active disease (median: 1.58 ng/µ, range: 0.08-3.85 ng/µl) than both patients in remission (median: 0.57 ng/µl, range: 0.02-3.57 ng/µl) and healthy controls. A low degree positive correlation was also found between serum lubricin levels and erythroid sedimentation rate of the JIA patients (r = 0.383 and p = 0.011). Conclusions: This is the first study investigating serum lubricin levels in JIA patients, and we found elevated serum lubricin levels in JIA patients with active disease. Further studies are needed to clarify our results. [ABSTRACT FROM AUTHOR]

Camptodactyly-arthropathy-coxa vara-pericarditis (CACP) syndrome is a rare familial arthropathy of childhood, commonly misdiagnosed as juvenile idiopathic arthritis. It is characterized by non-inflammatory arthropathy, coxa vara deformity, and sterile pericarditis. We describe two children with CACP syndrome who were referred to the rheumatology clinic for the suspicion of inflammatory arthritis. A literature search was carried out using PubMed/ Medline and Embase databases. English language reports of mutation-proven cases of CACP syndrome reported until 31 March 2020 were retrieved and analysed. Both the children had a delay in diagnosis (age at diagnosis- 12 and 13 years, respectively) and had received immunomodulatory therapy for suspected inflammatory arthritis. Presence of symmetrical arthropathy of large joints, camptodactyly, and normal inflammatory parameters are clues that indicated CACP syndrome. One child with a novel variant in PRG4 also had associated mitral valve prolapse and regurgitation. Both had severe constrictive pericarditis requiring pericardiectomy. On literature review, a total of 98 mutation-proven cases of CACP syndrome have been reported till date. Arthropathy in CACP syndrome mainly involves knees, wrists, ankles, and hips. Pericarditis is usually mild, however, can present rarely with severe symptoms requiring surgical intervention. CACP syndrome can closely mimic inflammatory arthritis and early clinical recognition is important to avoid misdiagnosis. Molecular confirmation is essential for early diagnosis and future genetic counselling for affected families. [ABSTRACT FROM AUTHOR]

The roles of cell division control protein 42 homologue (CDC42) and actin polymerisation in regulating the phenotype of superficial-zone chondrocytes (SZCs) have been demonstrated in vitro; however, the signalling pathway(s) downstream have yet to be fully elucidated. The study hypothesis was that Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) act downstream to regulate proteoglycan 4 (PRG4) and tenascin C (TNC). Bovine SZCs grown in monolayer were treated with ML141 (CDC42 inhibitor) or the actin depolymerising agents, latrunculin B and cytochalasin D, to determine the effect on YAP/TAZ. Verteporfin (YAP/TAZ inhibitor) and YAP/TAZ siRNA-mediated knockdown were used to determine their role in regulating PRG4 and TNC. ML141 treatment reduced total YAP/TAZ protein, nuclear TAZ levels and the YAP/TAZ target gene, connective tissue growth factor (CTGF) mRNA levels. Latrunculin B decreased nuclear TAZ, while cytochalasin D treatment trended towards increased nuclear TAZ (p = 0.06), correlating with decreased and increased CTGF mRNA levels, respectively. Verteporfin treatment decreased PRG4 and TNC expression, with no effect on actin polymerisation. siRNA-mediated knockdown of YAP/TAZ revealed that PRG4 was regulated by YAP/TAZ while TNC was regulated by TAZ only. As cytochalasin D can activate myocardin-related transcription factor-A (MRTF-A), siRNA-mediated knockdown was performed to determine the role of MRTF-A in regulating YAP/TAZ. Although nuclear TAZ decreased, no significant changes in total protein levels were observed. Findings suggested that CDC42 and actin polymerisation regulated SZCs through multiple actin-regulated pathways. Understanding the regulation of these chondroprotective molecules may have important implications for prevention/treatment of osteoarthritis.

Synovial fluid (SF) is the complex biofluid that facilitates the exceptional lubrication of articular cartilage in joints. Its primary lubricating macromolecules, the linear polysaccharide hyaluronic acid (HA) and the mucin-like glycoprotein proteoglycan 4 (PRG4 or lubricin), interact synergistically to reduce boundary friction. However, the precise manner in which these molecules influence the rheological properties of SF remains unclear. This study aimed to elucidate this by employing confocal microscopy and multiscale rheometry to examine the microstructure and rheology of solutions containing recombinant human PRG4 (rhPRG4) and HA. Contrary to previous assumptions of an extensive HA-rhPRG4 network, it is discovered that rhPRG4 primarily forms stiff, gel-like aggregates. The properties of these aggregates, including their size and stiffness, are found to be influenced by the viscoelastic characteristics of the surrounding HA matrix. Consequently, the rheology of this system is not governed by a single length scale, but instead responds as a disordered, hierarchical network with solid-like rhPRG4 aggregates distributed throughout the continuous HA phase. These findings provide new insights into the biomechanical function of PRG4 in cartilage lubrication and may have implications in the development of HA-based therapies for joint diseases like osteoarthritis., (© 2023 The Authors. Small published by Wiley‐VCH GmbH.)

Dry Eye Disease (DED) is a complex pathology affecting millions of people with significant impact on quality of life. Corneal inflammation, including via the nuclear factor kappa B (NFκB) pathway, plays a key etiological role in DED. Recombinant human proteoglycan 4 (rhPRG4) has been shown to be a clinically effective treatment for DED that has anti-inflammatory effects in corneal epithelial cells, but the underlying mechanism is still not understood. Our goal was to understand if rhPRG4 affects tumor necrosis factor α (TNFα)-stimulated inflammatory activity in corneal epithelial cells. We treated hTERT-immortalized corneal epithelial (hTCEpi) cells ± TNFα ± rhPRG4 and performed Western blotting on cell lysate and RNA sequencing. Bioinformatics analysis revealed that rhPRG4 had a significant effect on TNFα-mediated inflammation with potential effects on matricellular homeostasis. rhPRG4 reduced activation of key inflammatory pathways and decreased expression of transcripts for key inflammatory cytokines, interferons, interleukins, and transcription factors. TNFα treatment significantly increased phosphorylation and nuclear translocation of p65, and rhPRG4 significantly reduced both these effects. RNA sequencing identified human leukocyte antigen (HLA)-F adjacent transcript 10 (FAT10), a ubiquitin-like modifier protein which has not been studied in the context of DED, as a key pro-inflammatory transcript increased by TNFα and decreased by rhPRG4. These results were confirmed at the protein level. In summary, rhPRG4 is able to downregulate NFκB activity in hTCEpi cells, suggesting a potential biological mechanism by which it may act as a therapeutic for DED.

Osteoarthritis (OA) is a degenerative disease that causes irreversible destruction of articular cartilage for which there is no effective treatment at present. Although articular cartilage lacks intrinsic reparative capacity, numerous studies have confirmed the existence of cartilage-resident stem/progenitor cells (CSPCs) in the superficial zone (SFZ) of articular cartilage. CSPCs are characterized by the expression of mesenchymal stromal cell (MSC)-related surface markers, multilineage differentiation ability, colony formation ability, and migration ability in response to injury. In contrast to MSCs and chondrocytes, CSPCs exhibit extensive proliferative and chondrogenic potential with no signs of hypertrophic differentiation, highlighting them as suitable cell sources for cartilage repair. In this review, we focus on the organizational distribution, markers, cytological features and roles of CSPCs in cartilage development, homeostasis and repair, and the application potential of CSPCs in cartilage repair and OA therapies.

[Display omitted] • Extracellular matrix protein 1 (ECM1) serves as a central regulator of chondrocyte proliferation and hypertrophy, however, its role in OA remains largely unknown. • ECM1 may be a new candidate target for the treatment of osteoarthritis. • Cartilage destruction increased ECM1 expression, which consequently exacerbated OA progression partly by upregulating PRG4 expression through TGF-β/PKA/CREB signaling. Osteoarthritis (OA) is the most common joint disease whose important pathological feature is degeneration of articular cartilage. Although extracellular matrix protein 1 (ECM1) serves as a central regulator of chondrocyte proliferation and hypertrophy, its role in OA remains largely unknown. This study aims to decipher the roles of ECM1 in OA development and therapy in animal models. In the present study, ECM1 expression was examined in clinical OA samples, experimental OA mice and OA cell models. Mice subjected to destabilised medial meniscus (DMM) surgery were intra-articularly injected with adeno-associated virus (AAV) expressing ECM1 (AAV-ECM1) or AAV containing shECM1 (AAV-shECM1). Histological analysis was performed to determine cartilage damage. mRNA sequencing was performed to explore the molecular mechanism. In addition, the downstream signaling was further confirmed by using specific inhibitors. Our data showed that ECM1 was upregulated in the cartilage of patients with OA, OA mice as well as OA cell models. Moreover, ECM1 over-expressing in knee joints by AAV-ECM1 accelerated OA progression, while knockdown of ECM1 by AAV-shECM1 alleviated OA development. Mechanistically, cartilage destruction increased ECM1 expression, which consequently exacerbated OA progression partly by decreasing PRG4 expression in the TGF-β/PKA/CREB-dependent manner. In conclusion, our study revealed the important role of ECM1 in OA progression. Targeted ECM1 inhibition is a potential strategy for OA therapy. [ABSTRACT FROM AUTHOR]

Abstract Background The 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. Methods Confocal 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. Results PRG4 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. Conclusions These 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.

Neuroinflammation and dysfunction of the blood–brain barrier (BBB) are two prominent mechanisms of secondary injury in neurotrauma. It has been suggested that Toll-like receptors (TLRs) play important roles in initiating and propagating neuroinflammation resulting from traumatic brain injury (TBI), but potential beneficial effects of targeting these receptors in TBI have not been broadly studied. Here, we investigated the effect of targeting TLRs with proteoglycan 4 (PRG4) on post-traumatic neuroinflammation and BBB function. PRG4 is a mucinous glycoprotein with strong anti-inflammatory properties, exerting its biological effects by interfering with TLR2/4 signaling. In addition, PRG4 has the ability to inhibit activation of cluster of differentiation 44 (CD44), a cell-surface glycoprotein playing an important role in inflammation. Using the controlled cortical impact model of TBI in rats, we showed a rapid and prolonged upregulation of message for TLR2/4 and CD44 in the injured cortex. In the in vitro model of the BBB, recombinant human PRG4 (rhPRG4) crossed the endothelial monolayers through a high-capacity, saturable transport system. In rats sustaining TBI, PRG4 delivery to the brain was enhanced by post-traumatic increase in BBB permeability. rhPRG4 injected intravenously at 1 h post-TBI potently inhibited post-traumatic activation of nuclear factor kappa B and extracellular signal-regulated kinases 1/2, the two major signal transduction pathways associated with TLR2/4 and CD44, and curtailed the post-traumatic influx of monocytes. In addition, PRG4 restored normal BBB function after TBI by preventing the post-traumatic loss of tight junction protein claudin 5 and reduced neuronal death. Our observations provide support for therapeutic strategies targeting TLRs in TBI. [ABSTRACT FROM AUTHOR]

Objective: The superficial zone (SZ) of articular cartilage is responsible for distributing shear forces for optimal cartilage loading and contributes to joint lubrication through the production of proteoglycan 4 (PRG4). PRG4 plays a critical role in joint homeostasis and is chondroprotective. Normal PRG4 production is affected by inflammation and irregular mechanical loading in post-traumatic osteoarthritis (PTOA). THe SZ chondrocyte (SZC) phenotype, including PRG4 expression, is regulated by the actin cytoskeleton in vitro . There remains a limited understanding of the regulation of PRG4 by the actin cytoskeleton in native articular chondrocytes. The filamentous (F)-actin cytoskeleton is a potential node in crosstalk between mechanical stimulation and cytokine activation and the regulation of PRG4 in SZCs, therefore developing insights in the regulation of PRG4 by actin may identify molecular targets for novel PTOA therapies., Materials and Methods: A comprehensive literature search on PRG4 and the regulation of the SZC phenotype by actin organization was performed., Results: PRG4 is strongly regulated by the actin cytoskeleton in isolated SZCs in vitro . Biochemical and mechanical stimuli have been characterized to regulate PRG4 and may converge upon actin cytoskeleton signaling., Conclusion: Actin-based regulation of PRG4 in native SZCs is not fully understood and requires further elucidation. Understanding the regulation of PRG4 by actin in SZCs requires an in vivo context to further potential of leveraging actin arrangement to arthritic therapeutics., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Schmidt and Jay have interest stakes in Lubris, LLC.

Objective: The in vivo mechanoresponsive and lubricating changes of the mandibular condylar cartilage (MCC) associated with mandibular lateral shift (MLS) and recovery are poorly understood. Using growing rats, we investigated whether the expression of mechanoresponsive factors, including proteoglycan-4 (PRG4), Indian hedgehog (Ihh) and transforming growth factor-β1 (TGF-β1), would be affected by MLS. We also investigated whether these changes could recover to the control level after a 2-week treatment reversal (TR). Materials and methods: The MLS appliances were placed for 2 or 4 weeks in 5-week-old rats and removed from 7-week-old rats in the TR group. The MCC was analysed histomorphometrically by toluidine blue staining. Reverse transcription-polymerase chain reaction and immunohistochemistry were performed to evaluate the expression of PRG4, Ihh, PTHrP (parathyroid hormone-related protein), TGF-β1, Matrix metallopeptidase 13 (MMP-13) and a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS-5). Results: A thickened superficial layer and an enhanced expression of PRG4 were detected in MLS groups. PTHrP-Ihh expression correlated positively with the up-regulation of PRG4. TGF-β1 expression decreased in the early stage of MLS but recovered to the control level in the TR group. A significantly enhanced expression of MMP-13 in MLS groups was detected. Conclusion: MLS treatment, which acted on the growth stage of rats, affected the morphology and expression of lubrication factor in the MCC. Elimination of this mechanical stimulus may help MCC recover to normal conditions. Clinical relevance: Our study supports that the adaptive changes of MCC, which are caused by mandibular functional deviation, could be largely recovered by early treatment. [ABSTRACT FROM AUTHOR]

Objective: Lubricin is increasingly being evaluated as an outcome measure in studies investigating post-traumatic and naturally occurring osteoarthritis. However, there are discrepancies in results, making it unclear as to whether lubricin is increased, decreased or unchanged in osteoarthritis. The purpose of this study was to review all papers that measured lubricin in joint injury or osteoarthritis in order to draw conclusions about lubricin regulation in joint disease.Design: A systematic search of the Pubmed, Web of Knowledge, and EBSCOhost databases for papers was performed. Inclusion criteria were in vivo studies that measured lubricin in humans or animals with joint injury, that investigated lubricin supplementation in osteoarthritic joints, or that described the phenotype of a lubricin knock-out model. A methodological assessment was performed.Results: Sixty-two studies were included, of which thirty-eight measured endogenous lubricin in joint injury or osteoarthritis. Nineteen papers found an increase or no change in lubricin and nineteen reported a decrease. Papers that reported a decrease in lubricin were cited four times more often than those that reported an increase. Fifteen papers described lubricin supplementation, and all reported a beneficial effect. Eleven papers described lubricin knock-out models.Conclusions: The human literature reveals similar distributions of papers reporting increased lubricin as compared to decreased lubricin in osteoarthritis. The animal literature is dominated by reports of decreased lubricin in the rat anterior cruciate ligament transection model, whereas studies in large animal models report increased lubricin. Intra-articular lubricin supplementation may be beneficial regardless of whether lubricin increases or decreases in OA. [ABSTRACT FROM AUTHOR]

The objectives of this study were to evaluate temporal changes in lubricin, hyaluronan (HA), and HA molecular weight (MW) distributions in three distinct models of equine joint injury affecting the carpal (wrist), tarsal (ankle), and femoropatellar (knee) joints. To establish ranges for lubricin, HA, and HA MW distributions across multiple joints, we first evaluated clinically healthy, high‐motion equine joints. Synovial fluid was collected from high‐motion joints in horses without clinical signs of joint disease (n = 11 horses, 102 joints) and from research horses undergoing carpal osteochondral fragmentation (n = 8), talar cartilage impact injury (n = 7), and femoral trochlear ridge full‐thickness cartilage injury (n = 22) prior to and following arthroscopically induced joint injury. Lubricin and HA concentrations were measured via enzyme‐linked immunosorbent assays, and gel electrophoresis was performed to evaluate HA MW distributions. Synovial fluid parameters were analyzed via linear regression models, revealing that lubricin and HA concentrations were conserved across healthy, high‐motion joints. Lubricin concentrations increased post‐injury in all osteoarthritis models (carpal fragmentation P =.001; talar impact P <.001; femoral trochlear ridge cartilage defect P =.03). Sustained loss of HA was noted post‐arthroscopy following carpal osteochondral fragmentation (P <.0001) and talar impact injury (P <.001). Lubricin may be elevated to compensate for the loss of HA and to protect cartilage post‐injury. Further investigation into the mechanisms regulating lubricin and HA following joint injury and their effects on joint homeostasis is warranted, including whether lubricin has value as a biomarker for post‐traumatic osteoarthritis. [ABSTRACT FROM AUTHOR]

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 [ABSTRACT FROM AUTHOR]

Calcification is a prominent feature of late-stage atherosclerosis, but the mechanisms driving this process are unclear. Using a biobank of carotid endarterectomies, we recently showed that Proteoglycan 4 (PRG4) is a key molecular signature of calcified plaques, expressed in smooth muscle cell (SMC) rich regions. Here, we aimed to unravel the PRG4 role in vascular remodeling and intimal calcification. PRG4 expression in human carotid endarterectomies correlated with calcification assessed by preoperative computed tomographies. PRG4 localized to SMCs in early intimal thickening, while in advanced lesions it was found in the extracellular matrix, surrounding macro-calcifications. In experimental models, Prg4 was upregulated in SMCs from partially ligated ApoE−/− mice and rat carotid intimal hyperplasia, correlating with osteogenic markers and TGFb1. Furthermore, PRG4 was enriched in cells positive for chondrogenic marker SOX9 and around plaque calcifications in ApoE−/− mice on warfarin. In vitro, PRG4 was induced in SMCs by IFNg, TGFb1 and calcifying medium, while SMC markers were repressed under calcifying conditions. Silencing experiments showed that PRG4 expression was driven by transcription factors SMAD3 and SOX9. Functionally, the addition of recombinant human PRG4 increased ectopic SMC calcification, while arresting cell migration and proliferation. Mechanistically, it suppressed endogenous PRG4, SMAD3 and SOX9, and restored SMC markers’ expression. PRG4 modulates SMC function and osteogenic phenotype during intimal remodeling and macro-calcification in response to TGFb1 signaling, SMAD3 and SOX9 activation. The effects of PRG4 on SMC phenotype and calcification suggest its role in atherosclerotic plaque stability, warranting further investigations.

Purpose: To investigate the anti-adhesive mechanisms of PXL01 in sodium hyaluronate (HA) by using the rabbit lactoferrin peptide, rabPXL01 in HA, in a rabbit model of healing tendons and tendon sheaths. The mechanism of action for PXL01 in HA is interesting since a recent clinical study of the human lactoferrin peptide PXL01 in HA administered around repaired tendons in the hand showed improved digit mobility. Materials and methods: On days 1, 3, and 6 after tendon injury and surgical repair, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was used to assess mRNA expression levels for genes encoding the mucinous glycoprotein PRG4 (also called lubricin) and a subset of matrix proteins, cytokines, and growth factors involved in flexor tendon repair. RabPXL01 in HA was administered locally around the repaired tendons, and mRNA expression was compared with untreated repaired tendons and tendon sheaths. Results: We observed, at all time points, increased expression of PRG4 mRNA in tendons treated with rabPXL01 in HA, but not in tendon sheaths. In addition, treatment with rabPXL01 in HA led to repression of the mRNA levels for the pro-inflammatory mediators interleukin (IL)-1β, IL-6, and IL-8 in tendon sheaths. Conclusions: RabPXL01 in HA increased lubricin mRNA production while diminishing mRNA levels of inflammatory mediators, which in turn reduced the gliding resistance and inhibited the adhesion formation after flexor tendon repair.

Osteoarthritis (OA) is a degenerative disease of synovial joints characterized by progressive loss of articular cartilage, subchondral bone remodeling, and intra-articular inflammation with synovitis that results in chronic pain and motor impairment. Despite the economic and health impacts, current medical therapies are targeted at symptomatic relief of OA and fail to alter its progression. Given the complexity of OA pathogenesis, we hypothesized that a combinatorial gene therapy approach, designed to inhibit inflammation with interleukin-1 receptor antagonist (IL-1Ra) while promoting chondroprotection using lubricin (PRG4), would improve preservation of the joint compared to monotherapy alone. Employing two surgical techniques to model mild, moderate and severe posttraumatic OA, we found that combined delivery of helper-dependent adenoviruses (HDVs), expressing IL-1Ra and PRG4, preserved articular cartilage better than either monotherapy in both models as demonstrated by preservation of articular cartilage volume and surface area. This improved protection was associated with increased expression of proanabolic and cartilage matrix genes together with decreased expression of catabolic genes and inflammatory mediators. In addition to improvements in joint tissues, this combinatorial gene therapy prolonged protection against thermal hyperalgesia compared to either monotherapy. Taken together, our results show that a combinatorial strategy is superior to monotherapeutic approaches for treatment of posttraumatic OA. [ABSTRACT FROM AUTHOR]

The objective of this study was to determine the effect of different sliding interface materials (counterface) on the cartilage lubricating ability of proteoglycan 4 (PRG4) and hyaluronan (HA) by measuring the kinetic coefficient of friction on cartilage‐glass and cartilage–cartilage interfaces over a wide range of sliding velocities. The lubrication properties of PRG4 and HA were assessed at cartilage‐glass and cartilage–cartilage interfaces using a previously described test setup with a stationary area of contact. Samples were articulated at varying effective sliding velocities of 10, 3, 1, 0.3, 0.1, and 0.01 mm/s. The response of PRG4 and HA as effective friction‐reducing cartilage boundary lubricants was varied and was dependent primarily on the test counterface. At a physiological cartilage–cartilage interface both HA and PRG4 effectively reduced friction compared to PBS at slower speeds while at higher speeds PRG4 was similar to PBS, and HA similar to SF. Conversely, at a cartilage‐glass interface HA demonstrated no friction reducing ability compared to PBS, and PRG4 appeared just as effective as SF. Cartilage‐glass friction coefficients were also significantly greater than cartilage–cartilage friction coefficients. These results indicate the in vitro friction coefficient of putative cartilage boundary lubricants can be affected by the test counterface and suggest that use of synthetic surfaces in studying cartilage boundary lubrication may not always be appropriate for all molecules of interest. As such, care should be taken when interpreting such data, specifically when comparing to in vitro data obtained at a cartilage–cartilage interface, and especially when extrapolating to in vivo situations. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2923–2931, 2018. [ABSTRACT FROM AUTHOR]

Aortic valve stenosis (AVS), a consequence of increased fibrosis and calcification of the aortic valve leaflets, causes progressive narrowing of the aortic valve. Proteoglycans, structural components of the aortic valve, accumulate in regions with fibrosis and moderate calcification. Particularly, proteoglycan 4 (PRG4) has been identified in fibrotic parts of aortic valves. However, the role of PRG4 in the context of AVS and aortic valve calcification has not yet been determined. Here, transcriptomics, histology, and immunohistochemistry were performed in human aortic valves from patients undergoing aortic valve replacement. Human valve interstitial cells (VICs) were used for calcification experiments and RNA expression analysis. PRG4 was significantly upregulated in thickened and calcified regions of aortic valves compared with healthy regions. In addition, mRNA levels of PRG4 positively associated with mRNA for proteins involved in cardiovascular calcification. Treatment of VICs with recombinant human PRG4 enhanced phosphate-induced calcification and increased the mRNA expression of bone morphogenetic protein 2 and the runt-related transcription factor 2. In summary, PRG4 was upregulated in the development of AVS and promoted VIC osteogenic differentiation and calcification. These results suggest that an altered valve leaflet proteoglycan composition may play a role in the progression of AVS.

ABSTRACT: Osteoarthritis (OA) is a degenerative disease that initially manifests as loss of the superficial zone (SZ) of articular cartilage. SZ chondrocytes (SZC) differ in morphology from other chondrocytes as they are elongated and oriented parallel to the tissue surface. Proteoglycan 4 (PRG4) and tenascin C (TNC) are molecules expressed by SZC, which have been shown to be chondroprotective. Identification of the signalling pathway(s) regulating expression of SZ molecules may lead to a therapeutic target that can be used to delay or prevent the onset of OA. The hypothesis of this study is that expression of SZ molecules are regulated in part, by the CDC42‐actin‐myocardin‐related transcription factor‐A (MRTF‐A) signaling pathway. SZC from bovine metacarpal‐phalangeal joints were isolated and grown in monolayer culture. Each target in the CDC42‐actin‐MRTF‐A pathway was inhibited and the effect on cell shape, actin cytoskeleton status, and expression of PRG4 and TNC were determined. Treatment with the CDC42 inhibitor ML141 decreased PRG4 and TNC expression, and correlated with increased cell circularity and G‐/F‐actin ratio. PRG4 and TNC expression were differentially regulated by actin depolymerizing agents, latrunculin B and cytochalasin D. Chemical inhibition of MRTF‐A resulted in decreased expression of both PRG4 and TNC; however, specific knockdown by small interfering RNA only decreased expression of TNC indicating that TNC, but not PRG4, is regulated by MRTF‐A. Although PRG4 and TNC expression are both regulated by CDC42 and actin, it appears to occur through different downstream signaling pathways. Further study is required to elucidate the pathway regulating PRG4. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:2421–2430, 2018. [ABSTRACT FROM AUTHOR]

Background and aims Proteoglycan 4 (Prg4) has a high structural similarity with the established atherosclerosis-modulating proteoglycan versican, but its role in atherogenesis is still unknown. Therefore, the impact of Prg4 deficiency on macrophage function in vitro and atherosclerosis susceptibility in vivo was investigated. Methods The presence and localization of Prg4 was studied in atherosclerotic lesions. Furthermore, the effect of Prg4 deficiency on macrophage foam cell formation, cholesterol efflux and lipopolysaccharide (LPS) response was determined. Finally, susceptibility for atherosclerotic lesion formation was investigated in bone marrow-specific Prg4 knockout (KO) mice. Results Prg4 mRNA expression was induced 91-fold ( p <0.001) in murine initial atherosclerotic lesions and Prg4 protein co-localized with human lesional macrophages. Murine Prg4 KO macrophages showed increased foam cell formation (+2.1-fold, p <0.01). In parallel, the expression of the cholesterol efflux genes ATP-binding cassette transporter A1 and scavenger receptor type B1 was lower (−35%, p <0.05;-40%, p <0.05) in Prg4 KO macrophages. This translated into an impaired cholesterol efflux to high-density lipoprotein (−13%, p <0.001) and apolipoprotein A1 (−8%, p <0.05). Furthermore, Prg4 KO macrophages showed an impaired LPS-induced rise in TNFα secretion as compared to wild-type controls (−31%, p <0.001), indicating a reduced inflammatory response. Combined, these pro- and anti-atherogenic effects did not translate into a significant difference in atherosclerotic lesion formation upon bone marrow-specific deletion of Prg4 in low-density lipoprotein receptor KO mice. Conclusions Prg4 is present in macrophages in both murine and human atherosclerotic lesions and critically influences macrophage function, but deletion of Prg4 in bone marrow-derived cells does not affect atherosclerotic lesion development. [ABSTRACT FROM AUTHOR]

Abstract: Contact lens friction significantly correlates with subjective comfort. Hyaluronan (HA) and proteoglycan 4 (PRG4) are natural boundary lubricants present in the body. The objective of this study was to assess the effect of crosslinked HA into the bulk of model contact lens materials pHEMA, pHEMA/TRIS, and DMAA/TRIS on surface wettability, protein sorption, and boundary lubricating properties at a material–cornea biointerface, both alone and synergistically with PRG4 in solution. Surface wettability was assessed by water contact angle measurement, protein sorption by lysozyme sorption assay, and boundary lubricating properties using an in vitro friction test method. HA incorporation (HAinc) increased the surface wettability of all materials, and reduced protein sorption for pHEMA and DMAA/TRIS. HAinc increased friction for pHEMA, and DMAA/TRIS, whereas a decrease was observed for pHEMA/TRIS. A combination of HAinc and PRG4sol had a synergistic effect of reducing friction only for pHEMA/TRIS. This combination had similar friction reduction compared with PRG4sol alone for DMAA/TRIS. These results indicate HA incorporation could be an effective internal wetting agent, antiadhesive, and boundary lubricant for pHEMA/TRIS silicone hydrogels. In conclusion, HA incorporation can reduce friction of hydrogels alone and in combination with PRG4 in solution, though in a hydrogel composition‐dependent (e.g., TRIS) manner. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1818–1826, 2018. [ABSTRACT FROM AUTHOR]

Abstract: Clinical data show that in vitro contact lens friction is related to in vivo comfort. Solutions of biological lubricants hyaluronan (HA) and proteoglycan 4 (PRG4, also known as lubricin) reduce friction at a cornea–polydimethylsiloxane (PDMS) interface. The purpose of this study was to (1) determine if PRG4 can sorb to and lubricate model contact lens materials and (2) assess the boundary lubricating ability of PRG4 and HA compared to saline on model contact lens materials. PRG4 was obtained from bovine cartilage culture and suspended in saline at 300 µg/mL. N,N‐Dimethylacrylamidetris (trimethylsiloxy) silane, (DMAA/TRIS) and methacryloxypropyltris (trimethylsiloxy) silane (pHEMA/TRIS) silicone hydrogels were prepared. A previously described in vitro eyelid–hydrogel and cornea–hydrogel biomechanical friction test was used to determine boundary lubricant effect. PRG4 sorption to the hydrogels was assessed using a soak‐rinse protocol and western blotting. PRG4 effectively lubricated both silicone hydrogel materials and HA effectively lubricated pHEMA/TRIS, as indicated by a statistically significant reduction in friction compared to the saline control lubricant. An HA and PRG4 combination showed a synergistic effect for pHEMA/TRIS and effectively lubricated DMAA/TRIS. Biological boundary lubricants HA and PRG4 were shown to effectively lubricate silicone hydrogels when in solution. Additionally, HA and PRG4 showed synergistic lubrication for pHEMA/TRIS. The purpose of this study was not to replicate the friction coefficients of contact lenses, but rather to investigate lubricant–surface interactions for common contact lens constituents. These findings contribute to the potential development of biomolecule based lubricant drops for contact lens wearers. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1329–1338, 2018. [ABSTRACT FROM AUTHOR]

Sjögren's syndrome (SS) is an autoimmune disease affecting the lacrimal and salivary glands with hallmark clinical symptoms of dry eye and dry mouth. Recently, markedly increased cathepsin S (CTSS) activity has been observed in the tears of SS patients. Proteoglycan 4 (PRG4), also known as lubricin, is an effective boundary lubricant that is naturally present on the ocular surface. While PRG4 is susceptible to proteolytic digestion, the potential effect of CTSS on PRG4 remains unknown. The objective of this study was to assess the ability of CTSS to enzymatically degrade purified PRG4, and PRG4 naturally present in human tears, and alter ocular surface boundary lubricating properties. To assess the potential time course and dose-dependency of PRG4 digestion by CTSS, full-length recombinant human PRG4 (rhPRG4) was incubated at 37 °C with or without CTSS in an enzymatic digestion buffer. Digestion of PRG4 by CTSS was also examined within normal human tear samples, both with and without supplementation by rhPRG4. Finally, digestion of endogenous PRG4 by CTSS, and the effect of a CTSS inhibitor, was examined in SS tears on Schirmer strips. Digestion products were separated on 3–8% SDS-PAGE and visualized by protein staining and western blotting. The boundary lubricating ability of rhPRG4 samples was assessed using an in vitro human eyelid-cornea friction test. Finally, SDS-PAGE protein stain bands resulting from rhPRG4 digestion were submitted for tandem mass spectrometry analysis to confirm their identity as PRG4 and identify non-tryptic cleavage sites. CTSS digested rhPRG4 in a time and dose dependent manner. CTSS digestion of rhPRG4 at 1% (where % is the mass ratio of CTSS to rhPRG4) resulted in a time dependent decrease in the full-length, ∼460 kDa, monomeric rhPRG4 band, and an appearance of lower MW fragments. After 20 h, no full-length rhPRG4 was observed. Furthermore, with an increased relative enzyme concentration of 3%, no protein bands were observed after 2 h, indicating complete digestion of rhPRG4. Western blotting demonstrated PRG4 is present in normal human tears, and that rhPRG4, tears, and tears supplemented with rhPRG4 incubated with 3–9% CTSS demonstrated decreased intensity of high MW PRG4 bands, indicative of partial degradation by CTSS. Similarly, western blotting of PRG4 in SS tears incubated with CTSS demonstrated decreased intensity of high MW PRG4 bands, which was reversed in the presence of the CTSS inhibitor. CTSS treatment of rhPRG4 resulted in an increased friction coefficient, compared to untreated controls. Lastly, the lower MW bands were confirmed to be PRG4 fragments by tandem mass spectrometry, and 6 non-tryptic cleavage sites were identified. rhPRG4 is susceptible to proteolytic digestion by CTSS, both alone and in human tears, which results in diminished ocular surface boundary lubricating ability. Moreover, endogenous PRG4 is susceptible to proteolytic digestion by CTSS, both in normal and SS tears. Given the elevated activity of CTSS in SS tears, and the role intact PRG4 plays in ocular surface health and lubrication, degradation of PRG4 by CTSS is a potential mechanism for diminished ocular surface lubrication in SS. Collectively these results suggest that tear supplementation of PRG4 may be beneficial for SS patients. [ABSTRACT FROM AUTHOR]

Purpose:To investigate the anti-adhesive mechanisms of PXL01 in sodium hyaluronate (HA) by using the rabbit lactoferrin peptide, rabPXL01 in HA, in a rabbit model of healing tendons and tendon sheaths. The mechanism of action for PXL01 in HA is interesting since a recent clinical study of the human lactoferrin peptide PXL01 in HA administered around repaired tendons in the hand showed improved digit mobility. Materials and methods:On days 1, 3, and 6 after tendon injury and surgical repair, reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) was used to assess mRNA expression levels for genes encoding the mucinous glycoprotein PRG4 (also called lubricin) and a subset of matrix proteins, cytokines, and growth factors involved in flexor tendon repair. RabPXL01 in HA was administered locally around the repaired tendons, and mRNA expression was compared with untreated repaired tendons and tendon sheaths. Results:We observed, at all time points, increased expression ofPRG4mRNA in tendons treated with rabPXL01 in HA, but not in tendon sheaths. In addition, treatment with rabPXL01 in HA led to repression of the mRNA levels for the pro-inflammatory mediators interleukin (IL)-1β, IL-6, and IL-8 in tendon sheaths. Conclusions:RabPXL01 in HA increased lubricin mRNA production while diminishing mRNA levels of inflammatory mediators, which in turn reduced the gliding resistance and inhibited the adhesion formation after flexor tendon repair. [ABSTRACT FROM AUTHOR]

Objective: The goals of this study were (1) to quantify proteoglycan 4 (PRG4) gene expression; (2) to assess lubricin immunostaining; and (3) to measure synovial fluid lubricin concentrations in clinical and experimental models of equine carpal osteoarthritis (OA).Design: Lubricin synovial fluid concentrations and cartilage and synovial membrane PRG4 expression were analyzed in research horses undergoing experimental OA induction (n = 8) and in equine clinical patients with carpal OA (n = 58). Lubricin concentrations were measured using a custom sandwich enzyme-linked immunosorbent assay, and PRG4 expression was quantified using qRT-PCR. Lubricin immunostaining was assessed in synovial membrane and osteochondral sections in the experimental model.Results: Lubricin concentrations increased in synovial fluid following induction of OA, peaking at 21 days post-operatively in OA joints vs sham-operated controls (331 ± 69 μg/mL vs 110 ± 19 μg/mL, P = 0.001). Lubricin concentrations also increased in horses with naturally occurring OA as compared to control joints (152 ± 32 μg/mL vs 68 ± 4 μg/mL, P = 0.003). Synovial membrane PRG4 expression increased nearly 2-fold in naturally occurring OA (P = 0.003), whereas cartilage PRG4 expression decreased 2.5-fold (P = 0.025). Lubricin immunostaining was more pronounced in synovial membrane from OA joints as compared to controls, with intense lubricin localization to sites of cartilage damage.Conclusions: Although PRG4 gene expression decreases in OA cartilage, synovial membrane PRG4 expression, synovial fluid lubricin concentrations and lubricin immunostaining all increase in an equine OA model. Lubricin may be elevated to protect joints from post-traumatic OA. [ABSTRACT FROM AUTHOR]