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Sjogren'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.

Lubricin/proteoglycan 4 (PRG4) is a mucinous glycoprotein secreted by synovial fibroblasts and superficial zone chondrocytes. Recently, we showed that recombinant human PRG4 (rhPRG4) is a putative ligand for CD44 receptor. rhPRG4-CD44 interaction inhibits cytokine-induced rheumatoid arthritis synoviocyte proliferation. The objective of this study is to decipher the autocrine function of PRG4 in regulating osteoarthritic synoviocyte proliferation and expression of catabolic and pro-inflammatory mediators under basal and interleukin-1 beta (IL-1β)-stimulated conditions. Cytosolic and nuclear levels of nuclear factor kappa B (NFκB) p50 and p65 subunits in Prg4 +/+ and Prg4 -/- synoviocytes were studied using western blot. Nuclear translocation of p50 and p65 proteins in osteoarthritis (OA) fibroblast-like synoviocytes (FLS) in response to IL-1β stimulation in the absence or presence of rhPRG4 was studied using DNA binding assays. OA synoviocyte (5000 cells per well) proliferation following IL-1β (20 ng/ml) treatment in the absence or presence of rhPRG4 (50–200 μg/ml) over 48 hours was determined using a colorimetric assay. Gene expression of matrix metalloproteinases (MMPs), tissue inhibitor of metallproteinases-1 (TIMP-1), TIMP-2, IL-1β, IL-6, IL-8, TNF-α, cycloxygenae-2 (COX2) and PRG4 in unstimulated and IL-1β (1 ng/ml)-stimulated OA synoviocytes, in the presence or absence of rhPRG4 (100 and 200 μg/ml), was studied following incubation for 24 hours. Prg4 -/- synoviocytes contained higher nuclear p50 and p65 levels compared to Prg4 +/+ synoviocytes (p

A boundary lubricant attaches and protects sliding bearing surfaces by preventing interlocking asperity-asperity contact. Proteoglycan-4 (PRG4) is a boundary lubricant found in the synovial fluid that provides chondroprotection to articular surfaces. Inflammation of the diarthrodial joint modulates local PRG4 concentration. Thus, we measured the effects of inflammation, with Interleukin-1α (IL-1α) incubation, upon boundary lubrication and PRG4 expression in bovine cartilage explants. We further aimed to determine whether the addition of exogenous human recombinant PRG4 (rhPRG4) could mitigate the effects of inflammation on boundary lubrication and PRG4 expression in vitro. Cartilage explants, following a 7 day incubation with IL-1α, were tested in a disc-on-disc configuration using either rhPRG4 or saline (PBS control) as a lubricant. Following mechanical testing, explants were studied immunohistochemically or underwent RNA extraction for real-time polymerase chain reaction (RT-PCR). We found that static coefficient of friction (COF) significantly decreased to 0.14 ± 0.065 from 0.21 ± 0.059 (p = 0.014) in IL-1α stimulated explants lubricated with rhPRG4, as compared to PBS. PRG4 expression was significantly up regulated from 30.8 ± 19 copies in control explants lubricated with PBS to 3330 ± 1760 copies in control explants lubricated with rhPRG4 (p

Camptodactyly-arthropathy-coxa-vara-pericarditis (CACP) syndrome is an autosomal recessive disorder caused by mutations inConsecutive patients with CACP syndrome were prospectively evaluated at the enrollment and 3 months after the right knee injection with yttrium-90. The outcome variables were patient/parent and physician's global assessment measured by a 3-point scale, right knee swelling and range of motion on a 3-point scale, in addition to magnetic resonance imaging (MRI) assessment of the right knee for bone, cartilage, fluid, synovial hypertrophy and soft tissue changes.Six (three boys, three girls) patients with a mean age of 12 years and mean follow-up duration of 8.5 years completed a single right knee intra-articular yttrium-90 injection with 5 mCi. The procedure was well tolerated without adverse events apart from mild and transient joint pain in two patients. There was a minimal radioisotope leakage to soft tissue in two patients. During the 3-month follow-up interval, there was no improvement in the outcome variables. Patients and parents did not notice favorable therapeutic effects and global physician assessment was unsatisfactory. There was no difference in knee joint swelling or range of motion. Furthermore, MRI findings were unchanged. However, there was a minimal increase in synovial fluid post injection.Yttrium-90 radiosynovectomy seems to be a safe and well tolerated procedure, however, it did not show a beneficial therapeutic effect in arthropathy of CACP syndrome with the given dosage and interval. Studies including a larger number of patients and probably repeated injections are needed to derive satisfactory results about the effectiveness of yttrium-90 in CACP syndrome patients.Amaç: Kamptodaktili-artropati-koksa vara-perikardit (CACP) sendromu eklem ve tendon yüzeylerinin ana lubrikanı olan proteoglikan 4’ü kodlayan PRG4 genindeki mutasyonlara bağlı bir otozomal resesif hastalıktır. Eklemde efüzyon ve sinovyal hipertrofi ile karakterize non-enflamatuvar bir artropatidir. Henüz bu hastalığın etkin bir tedavisi yoktur. Amacımız CACP sendromlu hastaların artropatisinde yttrium-90 radyosinovektominin etkinliğini araştırmaktır. Yöntem: CACP sendromu tanısı alan ardışık hastalar tanı anında ve sağ dize yttrium-90 enjeksiyonu uygulanmasından 3 ay sonra prospektif olarak değerlendirildiler. Sonuç değişkenleri 3-puanlı skala üzerinden hasta/ebeveyn ve doktor genel değerlendirmesi, 3-puanlı skala üzerinden sağ diz ödemi ve hareket aralığı ve kemik, kıkırdak, sıvı, sinovyal hipertrofi ve yumuşak doku değişiklikleri açısından sağ diz manyetik rezonans görüntülemesi (MRG) idi. Bulgular: Ortalama yaşı 12, ortalama takip süreleri 8,5 yıl olan altı hastaya (üç erkek, üç kız) tek sefer sağ dize 5 mCi intra-artiküler yttrium-90 enjeksiyonu uygulandı. İşlem iki hastada hafif ve geçici eklem ağrısı dışında herhangi bir komplikasyon olmadan iyi tolere edildi. İki hastada yumuşak dokuya minimal radyoizotop kaçağı oldu. Üç aylık takip süresince sonuç değerlerinde iyileşme olmadı. Hastalar ve ebeveynleri olumlu terapötik etki fark etmediler ve genel doktor değerlendirmesi tatmin edici değildi. Diz eklemi ödemi ve hareket aralığında değişiklik yoktu. MRG bulguları değişmedi, ancak enjeksiyon sonrası sinovyal sıvıda minimal artış saptandı. Sonuç: Yttrium-90 radyosinovektomi güvenli ve iyi tolere edilen bir işlem olarak görülmektedir, ancak verilen doz ve sürede CACP sendromlu hastaların artropatisinde yararlı bir terapötik etki göstermemiştir. Daha fazla hasta içeren ve muhtemelen tekrarlayan enjeksiyonların uygulandığı çalışmalar yttrium-90’ın CACP sendromlu hastalardaki etkinliği ile ilgili gerekli sonuçları sağlayabilir.

Summary Objective We previously identified 3′-phosphoadenosine 5′-phosphosulfate synthase 2 ( PAPSS2 ) as a transcriptional target of transforming growth factor β (TGF-β) in chondrocytes. PAPSS2 is required for proper sulfation of proteoglycans in cartilage. Defective sulfation in the matrix results in alterations in mechanical properties of the cartilage that would be expected to result in degeneration. The objective of this study was to identify factors that regulate PAPSS2 expression and compare to a known TGF-β responsive gene, proteoglycan 4/lubricin ( PRG4 ). In this study, TGF-β-mediated regulation of SOX9 was characterized, and the involvement of SOX9 in regulation of PAPSS2 mRNA was investigated. Design Primary bovine articular chondrocytes grown in micromass culture and ATDC5 cells were used as the model system. Adenoviruses were used to express SOX9 and SMAD3. siRNA was used to knock-down Sox9 and Smad3 . Western blot and real-time quantitative RT-PCR (qPCR) were used to measure changes in protein and mRNA levels in response to treatment. Results Over-expression of SOX9 was sufficient to up-regulate PAPSS2 mRNA. TGF-β treatment of SOX9-expressing cells resulted in enhanced up-regulation of PAPSS2 mRNA, suggesting that SOX9 cooperates with TGF-β signaling. Furthermore, Sox9 was required for full TGF-β-mediated induction of Papss2 . In contrast, PRG4 was regulated by SMAD3 but not SOX9. SOX9 protein levels were increased after treatment with TGF-β, although SOX9 mRNA was not. SOX9 protein was post-translationally stabilized after treatment with TGF-β. Conclusions TGF-β stabilizes SOX9 protein, and SOX9 is sufficient and necessary for TGF-β-mediated regulation of PAPSS2 mRNA, providing a novel mechanism for TGF-β-mediated gene regulation in chondrocytes.

Background: Lubricin, or proteoglycan 4 (PRG4), is a glycoprotein responsible for joint boundary lubrication. PRG4 has been shown previously to be down-regulated after traumatic joint injury such as a meniscal tear. Preliminary evidence suggests that intra-articular injection of PRG4 after injury will reduce cartilage damage in rat models of surgically induced posttraumatic osteoarthritis. Objective: To determine the efficacy of intra-articular injection of full-length recombinant human lubricin (rhPRG4) for reducing cartilage damage after medial meniscal destabilization (DMM) in a preclinical large animal model. Study Design: Controlled laboratory study. Methods: Unilateral DMM was performed in 29 Yucatan minipigs. One week after DMM, animals received 3 weekly intra-articular injections (3 mL per injection): (1) rhPRG4 (1.3 mg/mL; n = 10); (2) rhPRG4+hyaluronan (1.3 mg/mL rhPRG4 and 3 mg/mL hyaluronan [~950 kDA]; n = 10); and (3) phosphate-buffered saline (PBS; n = 9). Hindlimbs were harvested 26 weeks after surgery. Cartilage integrity was evaluated by use of macroscopic (India ink) and microscopic (safranin O–fast green and hematoxylin and eosin) scoring systems. Secondary outcomes evaluated via enzyme-linked immunosorbent assay (ELISA) included PRG4 levels in synovial fluid, carboxy-terminal telepeptide of type II collagen (CTX-II) concentrations in urine and serum, and interleukin 1β (IL-1β) levels in synovial fluid and serum. Results: The rhPRG4 group had significantly less macroscopic cartilage damage in the medial tibial plateau compared with the PBS group ( P = .002). No difference was found between the rhPRG4+hyaluronan and PBS groups ( P = .23). However, no differences in microscopic damage scores were observed between the 3 groups ( P = .70). PRG4 production was elevated in the rhPRG4 group synovial fluid compared with the PBS group ( P = .033). The rhPRG4 group presented significantly lower urinary CTX-II levels, but not serum levels, when compared with the PBS ( P = .013) and rhPRG4+hyaluronan ( P = .011) groups. In serum and synovial fluid, both rhPRG4 ( P = .006; P = .017) and rhPRG4+hyaluronan groups ( P = .009; P = .03) presented decreased IL-1β levels. Conclusion: All groups exhibited significant cartilage degeneration after DMM surgery. However, animals treated with rhPRG4 had the least amount of cartilage damage and less inflammation, providing evidence that intra-articular injections of rhPRG4 may slow the progression of posttraumatic osteoarthritis. Clinical Relevance: Patients with meniscal trauma are at high risk for posttraumatic osteoarthritis. This study demonstrates that an intra-articular injection regimen of rhPRG4 may attenuate cartilage damage after meniscal injury.

Summary 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.

Proteoglycan 4 (or lubricin), a mucin-like glycoprotein, was originally classified as a lubricating substance within diarthrodial joints. More recently, lubricin has been found in other tissues and has been implicated in 2 inflammatory pathways within the cell, via the Toll-like receptors (TLRs) and CD44. Lubricin is an antagonist of TLR2 and TLR4, and appears to enter cells via the CD44 receptor. Because of lubricin's action on these receptors, downstream processes of inflammation are halted, thereby preventing release of cytokines (a hallmark of inflammation and sepsis) from the cell, indicating lubricin's role as a biomarker and possible therapeutic for sepsis.

Background Proteoglycan 4 (PRG4; lubricin) is a member of two gene co-expression network modules associated with human vein graft failure. However, little is known about PRG4 and the vascular system. Therefore, we have investigated the effects of recombinant human PRG4 (rhPRG4) on cell migration and proliferation in human veins. Methods Effects of rhPRG4 on cell migration, proliferation, and neointima formation were determined in human venous tissue and cultured venous smooth muscle cells (SMCs), adventitial cells, and endothelial cells. Expression of PRG4 by cultured human saphenous veins, failed vein grafts, and varicose veins was determined by immunostaining or Western blotting. Results Limited expression of PRG4 in fresh saphenous veins was dramatically increased around medial SMCs after culture ex vivo. rhPRG4 inhibited the migration of cultured SMCs, adventitial cells, and endothelial cells, as well as the proliferation of endothelial cells. rhPRG4 also inhibited the migration of SMCs and adventitial cells from tissue explants, but there was no effect on cell proliferation or neointima formation in ex vivo whole veins. Finally, PRG4 was largely absent in two examples of venous pathology, that is, failed human vein grafts and varicose veins. Conclusions Although rhPRG4 can inhibit the migration of venous SMCs, endothelial cells, and adventitial cells, and the proliferation of endothelial cells, PRG4 was only increased around medial SMCs in veins after ex vivo culture. PRG4 was not observed around medial SMCs in failed human vein grafts and varicose veins, suggesting the possibility that a failure of PRG4 upregulation may promote these pathologies.

The aim of this study was to determine the sorption location of full-length recombinant human proteoglycan 4 (rhPRG4) tagged with fluorescein isothiocyanate (FITC) to four silicone hydrogel contact lenses [balafilcon A (PureVision, Bausch + Lomb), senofilcon A (Acuvue Oasys, JohnsonJohnson), comfilcon A (Biofinity, CooperVision), lotrafilcon B (Air Optix, Alcon)] and one conventional hydrogel lens [etafilcon A (Acuvue 2, JohnsonJohnson)], using confocal laser scanning microscopy (CLSM). Lenses (

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

Objective The aim of the study was to determine whether magnetic resonance imaging (MRI) can be used in assessment of biologic activity of intraluminal thrombus (ILT) and proteolytic processes of the abdominal aortic aneurysm wall. Methods Using MRI, 50 patients with asymptomatic infrarenal abdominal aortic aneurysm were analyzed at the maximum aneurysm diameter on T1-weighted images in the arterial phase after administration of contrast material. Relative ILT signal intensity (SI) was determined as the ratio between ILT SI and psoas muscle SI. During surgery, the full thickness of the ILT and the adjacent part of the aneurysm wall were harvested at the maximal diameter for biochemical analysis. The concentrations of matrix metalloproteinase 9 and neutrophil elastase (NE/ELA) were analyzed in harvested thrombi, and the concentrations of collagen type III, elastin, and proteoglycans were analyzed in harvested aneurysm walls. Results A significant positive correlation was found between the NE/ELA concentration of the ILT and the relative SI (ρ = 0.309; P = .029). Furthermore, a negative correlation was observed between the elastin content of the aneurysm wall and the relative SI (ρ = −0.300; P = .034). No correlations were found between relative SI and concentration of matrix metalloproteinase 9, NE/ELA, collagen type III, or proteoglycan 4 in the aneurysm wall. Conclusions These findings indicate a potential novel use of MRI in prediction of thrombus proteolytic enzyme concentrations and the extracellular matrix content of the aneurysm wall, thus providing additional information for the risk of potential aneurysm rupture.

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.

Osteoarthritis, a degenerative disease of the joints, is the most common form of arthritis in the knee. Total joint arthoplasty is a commonly used treatment for joint degeneration and osteoarthritis, and due to these factors, TJA for hip and knee joints is projected to grow by 137% and 601% between 2005 and 2030. Matrix metalloproteases are enzymes found in the extracellular matrix that cleave matrix components. Normally MMPs are downregulated in tissues by Tissue Inhibitors of Metalloproteases, or TIMPs. The relative concentration of TIMPs also may denote some of the activity of the MMPs found in serum. Lubricin (proteoglycan 4) is a molecule found in the synovial fluid that protects joints by dissipating strain energy during locomotion. Lubricin synovial fluid concentration is also diminished in many patients with osteoarthritis, but not all. Given the importance of these three sets of molecules, our lab investigated the correlation between circulating lubricin, MMP levels and TIMPs levels. Blood plasma samples were obtained from de-identified subjects undergoing total joint arthroplasty at Loyola University Medical Center and the University of Utah. Normal blood plasma from pooled healthy individuals served as a control. We analyzed biomarker levels in plasma using ELISA. Our data show that MMP-1 and 9 were increased in TJA patients compared to normal controls, while MMP-2 and 13 were decreased. We also found decreased lubricin and tissue factor in surgical patients relative to controls. These data support the idea that lubricin is vital in protecting the synovial joint and that MMPs play a complex role in the destruction of the joint.

Osteoarthritis (OA) is a common chronic degenerative disease that affects the elderly. Thus far, no pharmacological therapy approved by regulators has shown a convincing effect on OA. Glabridin, a small molecule, is a well-known and powerful natural antioxidant, which has a strong scavenging effect on free radicals. This study attempted to explore the role and underlying mechanisms of Glabridin on OA both in vitro and in vivo. In the in vitro study, Glabridin was found to increase the expression levels of extracellular matrix (ECM) related genes, Collagen II, Aggrecan (ACAN), SRY-box 9 (SOX9) and proteoglycan 4 (PRG4). Moreover, Glabridin was observed to significantly reduce the level of oxidative stress in OA chondrocytes while effectively reducing the apoptosis of chondrocytes. Glabridin was also found to significantly increase the autophagy of human OA chondrocytes. During the in vivo study, intraarticular injection of Glabridin was observed to alleviate OA progression and protect chondrocytes against apoptosis following anterior cruciate ligament transection (ACLT) in rats. Furthermore, the mammalian target of rapamycin (mTOR) mediated autophagy was identified as one of the potential mediators of Glabridin activity. Overall, Glabridin protects articular cartilage from damage in rats with OA by protecting chondrocytes against oxidative stress, apoptosis and promoting mTOR mediated autophagy.

Melatonin receptors (MTNRs) play important roles in regulation of circadian rhythms and seasonal reproduction. However, their origin and evolution in vertebrates have not been investigated. Here, we performed a comprehensive examination by comparative genome mining of MTNRs in vertebrates. We successfully extracted 164 putative encoding sequences for MTNRs (including 57 mtnr1a, 59 mtnr1b and 48 mtnr1c) from 45 high-quality representative genomes. Interestingly, the putative expansions of mtnr1a and mtnr1b in zebrafish were also identified in other Cyprinifomes, but not in other orders of teleost. Using phylogenetic interference, we observed this expansion to be clustered into a primitive position of the Actinopterygii, which may be resulted from teleost-specific genome duplication. The C-terminal extension of MTNR1C, predicted to be proteoglycan 4 (PRG4), originated after the speciation of Monotremata or Marsupialia. Our present genomics survey provides novel insights into the evolution of MTNRs in vertebrates and updates our understanding of these proteins.

Osteoarthritis (OA) is a chronic condition marked by joint pain, inflammation and loss of articular cartilage, that can be treated with total joint arthroplasty (TJA) at end stages. TJA is marked by post-operative inflammation, which directly effects levels of cartilage degradation biomarkers, proteoglycan-4 (PRG4) and matrix metalloproteinase-9 (MMP-9). PRG4 is a protective glycoprotein that is decreased in individuals with OA. MMP-9 is a matrix metalloproteinase that contributes to articular cartilage loss and is elevated in OA patients. It is upregulated by pro-inflammatory markers, such as IL-1, IL-6 and CRP. This study aims to elucidate the immediate post-operative changes in levels of PRG4, MMP-9, IL-6, CRP, and WBC in patients undergoing TJA to clarify the role of inflammation in recovery after surgery and in the overall pathogenesis of OA. Blood was collected at 3 time points (day 0, day 1 post-operatively, and days 5-7 post-operatively), from 63 patients undergoing TJA due to OA, and levels of these biomarkers were quantified. IL-6, CRP, WBC and MMP-9 were lowest at day 0, highest at day 1, and stabilized at an intermediate level at days 5-7. Meanwhile, PRG4 followed the opposite trend. These studies suggest that IL-6, CRP and WBC showed predictable fluctuations, with pro-inflammatory biomarkers upregulating MMP-9 and downregulating PRG4. Measuring these biomarkers may help expose the role of inflammation in the post-surgical recovery of TJA patients and in long-term pathogenesis of OA. These levels may help risk stratify patients pre-operatively and help develop individualized post-surgical plans.

The purpose of this study was to investigate the effects of trauma and subsequent articulation on adult human ankle cartilage subjected to an injurious impact. Trauma was initiated through impaction on talar cartilage explants. Articulation and loading were applied in a joint bioreactor over 5 consecutive days. The early (24 h) effects of impaction included a reduced chondrocytes viability (51% vs. 81% for non-impacted; p = 0.03), increased levels of apoptosis (43% vs. 27%; p = 0.03), and an increase in the histopathology score (4.4 vs. 1.7; p = 0.02) as compared to non-impacted cartilage explants. One of the key findings was that damage also stimulated the PRG4 release (2.2 vs. 1.5 μg/ml). Subsequent articulation for 5 days did not lead to further changes in tissue histopathology and cell viability, neither for injured nor non-injured samples. However, articulation led to an increased apoptosis in the injured samples (p = 0.03 for the interaction term). Articulation also caused a significant increase of PG/GAG release into the culture medium (p = 0.04) for both injured and non-injured samples; however, the synthesis of PG was not affected by articulation (p = 0.45) though the PG synthesis was higher in injured samples (p < 0.01). With regard to the PRG4 release, impacted samples continued to show higher amounts (p = 0.01), adding articulation led to a reduction (p = 0.02). The current study demonstrated that adult human talar cartilage increases both the PRG4 release and biosynthetic activity as an immediate cellular response to injury. Articulation played a less contributing role to biosynthesis and remodeling, behaving mostly neutral, in that no further damage emerged. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:667-676, 2017.

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.

Our aim was to investigate lubricin, cartilage oligomeric matrix protein (COMP), and femoral cartilage deformation in response to different biomechanical loading of the knee joint (running vs cycling). Serum lubricin and COMP concentrations (enzyme-linked immunosorbent assay), and femoral cartilage thickness (suprapatellar transverse ultrasonography) were determined in 11 male runners (age: 40 ± 6 years; weight: 76 ± 8 kg) and 11 male cyclists (35 ± 12 years; 75 ± 5 kg) at baseline, immediately after, and 30 min after vigorous exercise (time trial: 10-km run or 25-km cycle). At baseline, lubricin (runners: 104.0 ± 19.8 ng/ml; cyclists: 119.1 ± 23.9 ng/ml) and COMP (runners: 804.1 ± 87.5 ng/ml; cyclists: 693.0 ± 84.7 ng/ml) did not significantly differ; however, vigorous exercise was accompanied by an increase in lubricin (cyclists: 39.4 %; p

Engineering tissues with a structure and spatial composition mimicking those of native articular cartilage (AC) remains a challenge. This study examined if infrapatellar fat pad-derived stem cells (FPSCs) can be used to engineer cartilage grafts with a bulk composition and a spatial distribution of matrix similar to the native tissue. In an attempt to mimic the oxygen gradients and mechanical environment within AC, FPSC-laden hydrogels (either 2 mm or 4 mm in height) were confined to half of their thickness and/or subjected to dynamic compression (DC). Confining FPSC-laden hydrogels was predicted to accentuate the gradient in oxygen tension through the depth of the constructs (higher in the top and lower in the bottom), leading to enhanced glycosaminoglycan (GAG) and collagen synthesis in 2 mm high tissues. When subjected to DC alone, both GAG and collagen accumulation increased within 2 mm high unconfined constructs. Furthermore, the dynamic modulus of constructs increased from 0.96 MPa to 1.45 MPa following the application of DC. There was no synergistic benefit of coupling confinement and DC on overall levels of matrix accumulation; however in all constructs, irrespective of their height, the combination of these boundary conditions led to the development of engineered tissues that spatially best resembled native AC. The superficial region of these constructs mimicked that of native tissue, staining weakly for GAG, strongly for type II collagen, and in 4 mm high tissues more intensely for proteoglycan 4 (lubricin). This study demonstrated that FPSCs respond to joint-like environmental conditions by producing cartilage tissues mimicking native AC. Copyright © 2016 John Wiley & Sons, Ltd.

The temporomandibular joint (TMJ) is a diarthrodial joint that relies on lubricants for frictionless movement and long-term function. It remains unclear what temporal and causal relationships may exist between compromised lubrication and onset and progression of TMJ disease. Here we report that Proteoglycan 4 (Prg4)-null TMJs exhibit irreversible osteoarthritis-like changes over time and are linked to formation of ectopic mineralized tissues and osteophytes in articular disc, mandibular condyle and glenoid fossa. In the presumptive layer of mutant glenoid fossa’s articulating surface, numerous chondrogenic cells and/or chondrocytes emerged ectopically within the type I collagen-expressing cell population, underwent endochondral bone formation accompanied by enhanced Ihh expression, became entrapped into temporal bone mineralized matrix, and thereby elicited excessive chondroid bone formation. As the osteophytes grew, the roof of the glenoid fossa/eminence became significantly thicker and flatter, resulting in loss of its characteristic concave shape for accommodation of condyle and disc. Concurrently, the condyles became flatter and larger and exhibited ectopic bone along their neck, likely supporting the enlarged condylar heads. Articular discs lost their concave configuration, and ectopic cartilage developed and articulated with osteophytes. In glenoid fossa cells in culture, hedgehog signaling stimulated chondrocyte maturation and mineralization including alkaline phosphatase, while treatment with hedgehog inhibitor HhAntag prevented such maturation process. In sum, our data indicate that Prg4 is needed for TMJ integrity and long-term postnatal function. In its absence, progenitor cells near presumptive articular layer and disc undergo ectopic chondrogenesis and generate ectopic cartilage, possibly driven by aberrant activation of Hh signaling. The data suggest also that the Prg4-null mice represent a useful model to study TMJ osteoarthritis-like degeneration and clarify its pathogenesis.

Enamel matrix derivative (EMD) and collagen membranes (CMs) are simultaneously applied in regenerative periodontal surgery. The aim of this study is to evaluate the ability of two CMs and a collagen matrix to adsorb the activity intrinsic to EMD that provokes transforming growth factor (TGF)-β signaling in oral fibroblasts.Three commercially available collagen products were exposed to EMD or recombinant TGF-β1, followed by vigorous washing. Oral fibroblasts were either seeded directly onto collagen products or were incubated with the respective supernatant. Expression of TGF-β target genes interleukin (IL)-11 and proteoglycan 4 (PRG4) was evaluated by real time polymerase chain reaction. Proteomic analysis was used to study the fraction of EMD proteins binding to collagen.EMD or TGF-β1 provoked a significant increase of IL-11 and PRG4 expression of oral fibroblasts when seeded onto collagen products and when incubated with the respective supernatant. Gene expression was blocked by the TGF-β receptor I kinase inhibitor SB431542. Amelogenin bound most abundantly to gelatin-coated culture dishes. However, incubation of palatal fibroblasts with recombinant amelogenin did not alter expression of IL-11 and PRG4.These in vitro findings suggest that collagen products adsorb a TGF-β receptor I kinase-dependent activity of EMD and make it available for potential target cells.

This study investigated whether ovariohysterectomy (OVH) surgery to induce menopause resulted in changes to modulus, failure strain and lubricin/proteoglycan 4 (PRG4) gene expression in rabbit medial collateral ligaments (MCLs), similar to aging (Thornton et al., 2015a). The MCLs from adult rabbits that underwent OVH surgery as adolescents (15-week-old) and adults (1-year-old) were compared by evaluating mechanical behaviour (adolescent OVH, n=8; adult OVH, n=7; normal, n=7), gene expression (adolescent OVH, n=9; adult OVH, n=8; normal, n=8), and collagen and glycosaminoglycan (adolescent OVH, n=9; adult OVH, n=8; normal, n=8) and water (adolescent OVH, n=9; adult OVH, n=8; normal, n=8) content. Mechanical behaviour evaluated cyclic, static and total creep strain, and ultimate tensile strength, modulus and failure strain. The RT-qPCR assessed mRNA levels for matrix regulatory genes. Adult OVH MCLs exhibited increased cyclic creep and failure strain, and decreased modulus with increased mRNA levels for lubricin/PRG4 and collagen I compared with normal MCLs. Adolescent OVH MCLs exhibited increased cyclic, static and total creep strain with decreased mRNA levels for the progesterone receptor. Lubricin/PRG4 plays a role in the lubrication of collagen fascicles which is likely related to the decreased modulus and increased failure strain observed in ligaments from adult OVH rabbits. Progesterone and its receptor are thought to play a role in the stretching of ligaments in pelvic organ prolapse and pregnancy which is likely related to the increase in creep strain observed in ligaments from adolescent OVH rabbits. Ovariohysterectomy in adult rabbits resulted in changes that were consistent with the aging MCL.

Neuron glia antigen-2 ((NG2), also known as chondroitin sulphate proteoglycan 4, or melanoma-associated chondroitin sulfate proteoglycan) is a type-1 membrane protein expressed by many central nervous system (CNS) cells during development and differentiation and plays a critical role in proliferation and angiogenesis. ‘NG2’ often references either the protein itself or the highly proliferative and undifferentiated glial cells expressing high levels of NG2 protein. NG2 glia represent the fourth major type of neuroglia in the mammalian nervous system and are classified as oligodendrocyte progenitor cells by virtue of their committed oligodendrocyte generation in developing and adult brain. Here, we discuss NG2 glial cells as well as NG2 protein and its expression and role with regards to CNS neoplasms as well as its potential as a therapeutic target for treating childhood CNS cancers.

Extracorporeal irradiation sterilizes resected tumor bone used as autografts in reconstruction surgery. Therapeutic irradiation is a standard technique in head and neck cancer therapy that aims to preserve organ function. Bone irradiation has a complex, mostly inhibitory, effect on remodeling and regeneration, although the underlying mechanisms are still not fully understood. It remains unclear if extracorporeal irradiation affects the paracrine-like activity of the corresponding autografts. We recently reported that bone-conditioned medium from autogenous bone chips contains a number of factors that might affect cell activity. In the present study, we investigated the effects of extracorporeal irradiation of porcine cortical bone chips on the activity of the corresponding bone-conditioned medium. The effects of bone-conditioned medium on the expressions of transforming growth factor-beta (TGF-β) target genes in oral fibroblasts were assessed. Bone-conditioned medium from bone chips exposed to a total radiation dose up to 120 Gy did not affect expressions of TGF-β target genes, including adrenomedullin, BTB/POZ domain-containing protein 11, proteoglycan 4, NADPH oxidase 4, and interleukin 11, in oral fibroblasts. In conclusion, bone irradiation does not alter the capability of the corresponding bone-conditioned medium to provoke a robust fibroblastic cell response in vitro. (J Oral Sci 58, 325-331, 2016).

Background: Neuron glial antigen 2 or chondroitin sulphate proteoglycan 4 (NG2/CSPG4) is expressed by immature precursors/progenitor cells and is possibly involved in malignant cell transformation. The aim of this study was to investigate its role on the progression and survival of sixty-one adult gliomas and nine glioblastoma (GB)-derived cell lines. Methods: NG2/CSPG4 protein expression was assessed by immunohistochemistry and immunofluorescence. Genetic and epigenetic alterations were detected by molecular genetic techniques. Results: NG2/CSPG4 was frequently expressed in IDH-mutant/1p19q-codel oligodendrogliomas (59.1%) and IDH-wild type GBs (40%) and rarely expressed in IDH-mutant or IDH-wild type astrocytomas (14.3%). Besides tumor cells, NG2/CSPG4 immunoreactivity was found in the cytoplasm and/or cell membranes of reactive astrocytes and vascular pericytes/endothelial cells. In GB-derived neurospheres, it was variably detected according to the number of passages of the in vitro culture. In GB-derived adherent cells, a diffuse positivity was found in most cells. NG2/CSPG4 expression was significantly associated with EGFR gene amplification (p = 0.0005) and poor prognosis (p = 0.016) in astrocytic tumors. Conclusion: The immunoreactivity of NG2/CSPG4 provides information on the timing of the neoplastic transformation and could have prognostic and therapeutic relevance as a promising tumor-associated antigen for antibody-based immunotherapy in patients with malignant gliomas.

Supplemental Digital Content is available in the text., Objectives: Sepsis is a leading cause of death in the United States. Putative targets to prevent systemic inflammatory response syndrome include antagonism of toll-like receptors 2 and 4 and CD44 receptors in vascular endothelial cells. Proteoglycan-4 is a mucinous glycoprotein that interacts with CD44 and toll-like receptor 4 resulting in a blockade of the NOD-like receptor pyrin domain-containing-3 pathway. We hypothesized that endothelial cells induced into a sepsis phenotype would have less interleukin-6 expression after recombinant human proteoglycan 4 treatment in vitro. Design: Enzyme-linked immunosorbent assay and reverse transcriptase-quantitative polymerase chain reaction to measure interleukin-6 protein and gene expression. Setting: Research laboratory. Subjects: Human umbilical vascular endothelial cells, human lung microvascular endothelial cells, and transgenic mouse (wild type) (Cd44+/+/Prg4+/+), Cd44–/– (Cd44tm1HbgPrg4+/+), Prg4GT/GT (Cd44+/+ Prg4tm2Mawa/J), and double knockout (Cd44tm1Hbg Prg4tm2Mawa/J) lung microvascular endothelial cells. Interventions: Cells were treated with 100 or 250 ng/mL lipopolysaccharide-Escherichia coli K12 and subsequently treated with recombinant human proteoglycan 4 after 30 minutes. Interleukin-6 levels in conditioned media were measured via enzyme-linked immunosorbent assay and gene expression was measured via reverse transcriptase-quantitative polymerase chain reaction with ΔΔ–Ct analysis. Additionally, human umbilical vascular endothelial cells and human lung microvascular endothelial cells were treated with 1:10 diluted plasma from 15 patients with sepsis in culture media. After 30 minutes, either 50 or 100 µg/mL recombinant human proteoglycan 4 was administered. Interleukin-6 protein and gene expression were assayed. Proteoglycan 4 levels were also compared between control and sepsis patient plasma. Measurements and Main Results: Human umbilical vascular endothelial cell, human lung microvascular endothelial cell, and mouse lung microvascular endothelial cell treated with lipopolysaccharide had significantly increased interleukin-6 protein compared with controls. Recombinant human proteoglycan-4 significantly reduced interleukin-6 in human and mouse endothelial cells. Interleukin-6 gene expression was significantly increased after lipopolysaccharide treatment compared with controls. This response was reversed by 50 or 100 µg/mL recombinant human proteoglycan-4 in 80% of sepsis samples in human umbilical vascular endothelial cells and in 60–73% in human lung microvascular endothelial cells. In Cd44–/– genotypes of the mouse lung microvascular endothelial cells, recombinant human proteoglycan-4 significantly reduced interleukin-6 protein levels after lipopolysaccharide treatment, indicating that Cd44 is not needed for recombinant human proteoglycan-4 to have an effect in a toll-like receptor 4 agonist inflammation model. Patient sepsis samples had higher plasma levels of native proteoglycan-4 than controls. Interpretation and Conclusions: Recombinant human proteoglycan-4 is a potential adjunct therapy for sepsis patients and warrants future in vivo model studies.

Despite emerging targeted and immunotherapy treatments, no monoclonal antibodies or antibody-drug conjugates (ADCs) directly targeting tumor cells are currently approved for melanoma therapy. The tumor-associated antigen chondroitin sulphate proteoglycan 4 (CSPG4), a neural crest glycoprotein over-expressed on 70% of melanomas, contributes to proliferative signaling pathways, but despite highly tumor-selective expression it has not yet been targeted using ADCs. We developed a novel ADC comprising an anti-CSPG4 antibody linked to a DNA minor groove-binding agent belonging to the novel pyrridinobenzodiazepine (PDD) class. Unlike conventional DNA-interactive pyrrolobenzodiazepine (PBD) dimer payloads that cross-link DNA, PDD-based payloads are mono-alkylating agents but have similar efficacy and substantially enhanced tolerability profiles compared to PBD-based cross-linkers. We investigated the anti-tumor activity and safety of the anti-CSPG4-(PDD) ADC in vitro and in human melanoma xenografts. Anti-CSPG4-(PDD) inhibited CSPG4-expressing melanoma cell growth and colony formation and triggered apoptosis in vitro at low nanomolar to picomolar concentrations without off-target Fab-mediated or Fc-mediated toxicity. Anti-CSPG4-(PDD) restricted xenograft growth in vivo at 2 mg/kg doses. One 5 mg/kg injection triggered tumor regression in the absence of overt toxic effects or of acquired residual tumor cell resistance. This anti-CSPG4-(PDD) can deliver a highly cytotoxic DNA mono-alkylating payload to CSPG4-expressing tumors at doses tolerated in vivo.

Proteoglycan 4 (Prg4) is a mediator in inflammatory processes, either directly through interactions with CD44 [1] or toll-like receptors [2], or indirectly as a growth factor for hematopoietic cell lineages [3]. In line with this, in our article “Proteoglycan 4 regulates macrophage function without altering atherosclerotic lesion formation in a murine bone marrow-specific deletion model” published in Atherosclerosis, we showed that Prg4 deficiency attenuated the LPS-induced pro-inflammatory response in macrophages [4].

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.

Toxin-producing strains of Clostridioides difficile and Clostridium perfringens cause infections of the gastrointestinal tract in humans and ruminants, with the toxins being major virulence factors, essential for the infection, and responsible for the onset of severe symptoms. C. difficile toxin A (TcdA) and toxin B (TcdB), and the large cytotoxin (TpeL) from C. perfringens are single chain bacterial protein toxins with an AB-like toxin structure. The C-terminal delivery domain mediates cell entry of the N-terminal glycosyltransferase domain by receptor-mediated endocytosis. Several cell surface proteins have been proposed to serve as toxin receptors, including chondroitin-sulfate proteoglycan 4 (CSPG4), poliovirus receptor-like 3 (PVRL3), and frizzled-1/2/7 (FZD1/2/7) for TcdB and LDL-receptor-related protein-1 (LRP1) for TpeL. The expression of the TcdB receptors was investigated in human intestinal organoids (HIOs) and in cultured cell lines. HIOs from four human donors exhibited a comparable profile of receptor expression, with PVRL3, LRP1, and FZD7 being expressed and CSPG4 and FZD2 not being expressed. In human epithelial Caco-2 cells and HT29 cells as well as in immortalized murine fibroblasts, either receptor FZD2/7, CSPG4, PVRL3, and LRP1 was expressed. The question whether the toxins take advantage of the normal turnover of their receptors (i.e., constitutive endocytosis and recycling) from the cell surface or whether the toxins activity induce the internalization of their receptors has not yet been addressed. For the analysis of receptor internalization, temperature-induced uptake of biotinylated toxin receptors into immortalized mouse embryonic fibroblasts (MEFs) and Caco-2 cells was exploited. Solely LRP1 exhibited constitutive endocytosis from the plasma membrane to the endosome, which might be abused by TpeL (and possibly TcdB as well) for cell entry. Furthermore, internalization of CSPG4, PVRL3, FZD2, and FZD7 was observed neither in MEFs nor in Caco-2 cells. FZD2/7, CSPG4, and PVRL3 did thus exhibit no constitutive recycling. The presence of TcdB and the p38 activation induced by anisomycin were not able to induce or enhance CSPG4 or PVRL3 uptake in MEFs. In conclusion, FZD2/7, CSPG4, and PVRL3 seem to serve as cell surface binding receptors rather than internalizing receptors of TcdB.

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.

Despite the effort to optimize soft contact lens performance, almost half of the 140 million contact lens wearers worldwide experience symptoms of ocular dryness and discomfort, especially towards the end of the day. These symptoms are attributed to reduced compatibility between the contact lens and the ocular surface and are the main reason for contact lens discontinuation. As the interactions of the contact lens-eye interface are dynamic, the surface properties play a key role in improving ocular compatibility, comfort and overall performance of contact lenses. One promising method to reduce adverse interfacial interactions between the contact lens and the ocular surface is to modify the contact lens surface with a biomimetic layer inspired by the ocular surface and the tear film. Hyaluronic acid (HA) is a non-sulfated glycosaminoglycan naturally found in the ocular environment providing ocular hydration and lubrication. Proteoglycan 4 (PRG4), a mucin-like glycoprotein naturally produced at the ocular surface contributes to natural lubrication during blinking and to tear film stability. Surface modification with HA or PRG4 has been shown to result in improved wetting, lubricating and antifouling properties. Moreover, HA and PRG4 have been previously found to interact and synergistically reduce friction further. In the current work, novel HA and PRG4-grafted soft contact lens surfaces were prepared, and the impact of the surface tethered layer on important contact lens properties was assessed. Furthermore, the potential synergistic effect between HA and rhPRG4 on the examined properties was evaluated. Surface immobilization of HA on model conventional (pHEMA) and silicone (pHEMA-co-TRIS) hydrogel contact lenses was achieved by thiol-ene “click” chemistry, while full-length recombinant human PRG4 (rhPRG4) was surface grafted via carbonyldiimidazole (CDI) linking chemistry respectively. The chemical structure after each modification step was determined by attenuated total reflectance FTIR (FTIR-ATR) and X-ray photoelectron spectroscopy (XPS) analyses. HA-grafted model soft contact lenses were characterized by improved surface wettability, antifouling and water retentive properties, while a decreasing trend in boundary friction was observed but only for the HA-grafted pHEMA-co-TRIS materials. Surface-tethering of rhPRG4 was found to effectively enhance the surface wettability and boundary lubricating properties of pHEMA-co-TRIS hydrogels only, whereas both rhPRG4-grafted pHEMA and pHEMA-co-TRIS materials exhibited lower protein sorption and dehydration rate. Overall, the surface immobilization processes followed herein did not alter the optical transparency of the model soft contact lenses or their in vitro compatibility with human corneal epithelial cells. Finally, there was evidence that HA and rhPRG4 synergistically interacted, further improving the contact lens properties. However, the degree of HA/rhPRG4 synergy was found to be dependent on the configuration of the formed HA/rhPRG4 complex as well as the composition of the substrate hydrogel material, with the noted improvement being more significant for the model silicone hydrogels. This is the first study to examine surface grafted full-length rhPRG4 and the effect of this modification on contact lens properties. Moreover, the study is the first to investigate the interactions between covalently tethered rhPRG4 and solutions containing HA. The results of this thesis demonstrate that HA and rhPRG4 are good candidates for the development of novel biomimetic surfaces, especially for silicone hydrogel contact lenses. The potential for using these compounds in synergy was also demonstrated, with wetting solutions of HA showing promise for modifying rhPRG4 modified materials to improve symptoms of discomfort. These naturally occurring ocular agents have the potential to improve the management of ocular dryness and discomfort, thus optimizing the overall soft contact lens performance. Thesis Doctor of Philosophy (PhD)

36Proteoglycan 4 (Prg4) is highly expressed in metabolic tissues. The proteoglycan is a target gene for the nuclear receptor peroxisome proliferator receptor gamma which acts as metabolic switch. So, our aim was to investigate the function of Prg4 in metabolism.Male Prg4 knock-out (KO) mice and wild-type (WT) littermates were challenged with a obesogenic high fat diet (45% kcal lard fat) for 16 weeks. To stimulate the development of a diabetic phenotype, 10% fructose water was provided. Mice were subjected to an oral glucose tolerance test to investigate glucose handling. Flux studies with glycerol [3H]oleate-labeled VLDL-like particles were performed to determine the uptake of triglyceride-derived fatty acids in metabolically active organs.No significant difference in body weight was observed between both groups. However, both plasma triglyceride levels (-16%; phepatic steatosis in Prg4 KO mice is most likely driven by decreased de novo lipogenesis, as judged by the significantly reduced expression of lipogenic genes ACC and SCD1 (-21% and -38%; pfatty acids. White adipose tissue of Prg4 KO mice showed decreased uptake of triglyceride-derived fatty acids (-46%; pCD68 and MCP1 was lower in Prg4 KO adipose tissue as compared to that of WT controls (2.9- and 5.1-fold decrease; poral glucose tolerance test (AUC: -29%; pPrg4 KO and WT mice have a similar obesogenic potential, however Prg4 KO mice are protected from high fat diet-induced metabolic disruptions.

Osteoarthritis (OA) is characterized by synovitis and synovial fibrosis. Synoviocytes are fibroblast-like resident cells of the synovium that are activated by transforming growth factor (TGF)-β to proliferate, migrate, and produce extracellular matrix. Synoviocytes secrete hyaluronan (HA) and proteoglycan-4 (PRG4). HA reduces synovial fibrosis in vivo, and the Prg4−/−mouse exhibits synovial hyperplasia. We investigated the antifibrotic effects of increased intracellular cAMP in TGF-β-stimulated human OA synoviocytes. TGF-β1 stimulated collagen I (COL1A1), α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase (TIMP)-1, and procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) expression, and procollagen I, α-SMA, HA, and PRG4 production, migration, and proliferation of OA synoviocytes were measured. Treatment of OA synoviocytes with forskolin (10 μM) increased intracellular cAMP levels and reduced TGF-β1-stimulated COL1A1, α-SMA, and TIMP-1 expression, with no change in PLOD2 expression. Forskolin also reduced TGF-β1-stimulated procollagen I and α-SMA content as well as synoviocyte migration and proliferation. Forskolin (10 μM) increased HA secretion and PRG4 expression and production. A cell-permeant cAMP analog reduced COL1A1 and α-SMA expression and enhanced HA and PRG4 secretion by OA synoviocytes. HA and PRG4 reduced α-SMA expression and content, and PRG4 reduced COL1A1 expression and procollagen I content in OA synoviocytes. Prg4−/−synovium exhibited increased α-SMA, COL1A1, and TIMP-1 expression compared with Prg4+/+synovium. Prg4−/−synoviocytes demonstrated strong α-SMA and collagen type I staining, whereas these were undetected in Prg4+/+synoviocytes and were reduced with PRG4 treatment. We conclude that increasing intracellular cAMP levels in synoviocytes mitigates synovial fibrosis through enhanced production of HA and PRG4, possibly representing a novel approach for treatment of OA synovial fibrosis.

ObjectiveProteoglycan 4 (Prg4) has emerged from human association studies as a possible factor contributing to weight gain, dyslipidemia and insulin resistance. In the current study, we investigated the causal role of Prg4 in controlling lipid and glucose metabolism in mice.MethodsPrg4 knockout (KO) mice and wild-type (WT) littermates were challenged with an obesogenic high-fat diet (45% of total calories as fat) for 16 weeks. To further stimulate the development of metabolic alterations, 10% fructose water was provided starting from week 13.ResultsPrg4 deficiency only tended to reduce diet-induced body weight gain, but significantly improved glucose handling (AUC: −29%; p glycolysis (Gck: −30%; p lipogenesis (Acc: −21%; p triglyceride levels (−56%; p WT mice. Prg4 KO mice likely had lower glucose utilization by skeletal muscle as compared to WT mice, judged by a significant reduction in the genes Glut4 (−29%; p white adipose tissue phenotype with lower uptake of triglyceride-derived fatty acids (−46%; p Cd68, Mcp1 and Tnfα (−65%, −81% and −63%, respectively; p ConclusionPrg4 KO mice are protected from high-fat diet-induced glucose intolerance and fatty liver disease.

Chimeric antigen receptor (CAR)-T cells have been used successfully for cancer immunotherapy. While substantial tumor regression was observed in leukaemia and lymphoma, CAR therapy of solid tumors needs further improvement. A major obstacle to the efficiency of engineered T cells is posed by triggering of inhibitory receptors, for example programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4), leading to an impaired antitumor activity. To boost CAR-T-cell function, we co-electroporated T cells with both, mRNA encoding a CAR specific for chondroitin sulphate proteoglycan 4 (CSPG4) and small-interfering RNAs (siRNAs) to downregulate PD-1 (siPD-1) and CTLA-4 (siCTLA-4). Flow cytometry revealed that activation-induced upregulation of both PD-1 and CTLA-4 was suppressed when compared to CAR-T cells electroporated with negative control siRNA. The siRNA transfection showed no influence on CAR expression of engineered T cells. Functionality assays were performed using PD-L1- and CD80-transfected melanoma cells endogenously expressing CSPG4. CAR-T cells transfected with siPD-1 alone showed improvement in cytokine secretion. Additionally, CAR-T cells transfected with either siPD-1 alone or together with siCTLA-4 exhibited a significantly increased cytotoxicity. No or only little effects were observed when CAR-T cells were co-transfected with siCTLA-4 only. Taken together, it is feasible to optimize CAR-T cells by co-transfection of CAR-encoding mRNA and siRNAs to downregulate inhibitory receptors. Our in vitro data indicate an improvement of the functionality of these CAR-T cells, suggesting that this strategy could represent a novel method to enhance CAR-T-cell immunotherapy of cancer.

Aging is a main risk factor for osteoarthritis (OA). FoxO transcription factors protect against cellular and organismal aging and FoxO expression in cartilage is reduced with aging and in OA. To investigate FoxO in cartilage function, single and triple FoxO1/3/4 floxed mice were crossed with Col2-Cre mice to generate Col2Cre-FoxO1, 3, and 4 single knockout (KO) and triple KO mice (Col2Cre-TKO). Articular cartilage in Col2Cre-TKO and Col2Cre-FoxO1 KO mice was significantly thicker than in control mice at 1 or 2 months. This was associated with increased proliferation of chondrocytes of Col2Cre-TKO mice in vivo and in vitro. OA-like changes developed in cartilage, synovium and subchondral bone between 4 and 6 months of age in Col2Cre-TKO and Col2Cre-FoxO1 KO mice. Col2Cre-FoxO3 and FoxO4 KO mice showed no cartilage abnormalities until 18 months when Col2Cre-FoxO3 KO mice had more severe spontaneous OA. Autophagy and anti-oxidant defense genes were reduced in Col2Cre-TKO mice. Deletion of triple FoxO1/3/4 in mature mice using Aggrecan(Acan)-CreERT2 (AcanCreERT-TKO) also led to spontaneous cartilage degradation and increased severity of OA induced by surgical knee instability or treadmill running. The superficial zone of Col2Cre-TKO and AcanCreERT-TKO mice exhibited reduced cell density and markedly decreased Prg4. In vitro, ectopic FoxO1 expression increased Prg4 and synergized with TGFβ stimulation. In OA chondrocytes, overexpression of FoxO1 reduced inflammatory mediators, cartilage-degrading enzymes, increased protective genes and antagonized IL-1β effects. Our observations suggest that FoxOs play a key role in postnatal cartilage development, maturation and homeostasis and protects against OA-associated cartilage damage.

Osteoarthritis (OA), a degenerative joint disease characterized by progressive cartilage degeneration, is one of the leading causes of disability worldwide owing to the limited regenerative capacity of adult articular cartilage. Currently, there are no disease-modifying pharmacological or surgical therapies for OA. Fetal mammals, in contrast to adults, are capable of regenerating injured cartilage in the first two trimesters of gestation. A deeper understanding of the properties intrinsic to the response of fetal tissue to injury would allow us to modulate the way in which adult tissue responds to injury. In this study, we employed secretome proteomics to compare fetal and adult protein regulation in response to cartilage injury using an ovine cartilage defect model. The most relevant events comprised proteins associated with the immune response and inflammation, proteins specific for cartilage tissue and cartilage development, and proteins involved in cell growth and proliferation. Alarmins S100A8, S100A9 and S100A12 and coiled-coil domain containing 88A (CCDC88A), which are associated with inflammatory processes, were found to be significantly upregulated following injury in adult, but not in fetal animals. By contrast, cartilage-specific proteins like proteoglycan 4 were upregulated in response to injury only in fetal sheep postinjury. Our results demonstrate the power and relevance of the ovine fetal cartilage regeneration model presented here for the first time. The identification of previously unrecognized modulatory proteins that plausibly affect the healing process holds great promise for potential therapeutic interventions., Summary: Secretome proteomics identifies differential regulation of inflammation modulators during fetal and adult articular cartilage defect healing, offering novel strategies for therapy.

Neuron glial antigen 2 (NG2) is a chondroitin sulphate proteoglycan 4 (CSPG4) that occurs in developing and adult central nervous systems (CNSs) as a marker of oligodendrocyte precursor cells (OPCs) together with platelet-derived growth factor receptor α (PDGFRα). It behaves variably in different pathological conditions, and is possibly involved in the origin and progression of human gliomas. In the latter, NG2/CSPG4 induces cell proliferation and migration, is highly expressed in pericytes, and plays a role in neoangiogenesis. NG2/CSPG4 expression has been demonstrated in oligodendrogliomas, astrocytomas, and glioblastomas (GB), and it correlates with malignancy. In rat tumors transplacentally induced by N-ethyl-N-nitrosourea (ENU), NG2/CSPG4 expression correlates with PDGFRα, Olig2, Sox10, and Nkx2.2, and with new vessel formation. In this review, we attempt to summarize the normal and pathogenic functions of NG2/CSPG4, as well as its potential as a therapeutic target.

Background and aim: Chronic Obstructive Pulmonary Disease (COPD) is characterized by exacerbation and stable periods. Early diagnosis in the exacerbation period of COPD can reduce morbidity and mortality rates. The aim of this study is to investigate the relationship between Proteoglycan 4 (PRG4) and other parameters, including Procalcitonin (PCT) and C-reactive protein (CRP) levels and White Blood Cell (WBC) count, in the exacerbation period. Materials and methods: This preliminary study was conducted on patients admitted to the Emergency Department and Chest Diseases Department in University Medical Faculty Hospital. Patient demographics, spirometric measurements, clinical scoring used for COPD classification and laboratory test results were recorded for each patient. The final diagnosis of COPD was made by pulmonary physicians. Results: A total of 78 patients (38 in exacerbation and 40 in stable period) with COPD were included in the study. PRG4 levels of patients in the exacerbation period (183.7 ± 9.4 ng/ml) were significantly higher than those of patients in the stable period (177.1 ± 15.9 ng/ml; p=0.034). The sensitivity of PRG4 (88.9%) was equal to that of PCT (88.9%) and higher than that of CRP (86.1%) and WBC (80.6%); the specificity of PRG4 (50%) was higher than that of the other parameters (33.3%, 43.0%, and 40.0%, respectively). Conclusion: PRG4 can be used for discriminating between the exacerbation and stable periods of COPD and is superior in diagnosing the exacerbation period compared with other laboratory parameters. © 2018, Scientific Publishers of India. All rights reserved.

Purpose : The nutritional intervention program “DiOGenes” focuses on how obesity can be prevented and treated from a dietary perspective. We generated differential plasma proteome profiles in the DiOGenes cohort to identify proteins associated with weight loss and maintenance and explore their relation to body mass index, fat mass, insulin resistance and sensitivity. Experimental Design : Relative protein quantification was obtained at baseline and after combined weight loss/maintenance phases using isobaric tagging and MS/MS. A Welch t-test determined proteins differentially present after intervention. Protein relationships with clinical variables were explored using univariate linear models, considering collection center, gender and age as confounding factors. Results : 473 subjects were measured at baseline and end of the intervention; 39 proteins were longitudinally differential. Proteins with largest changes were sex hormone-binding globulin, adiponectin, C-reactive protein, calprotectin, serum amyloid A, and proteoglycan 4 (PRG4), whose association with obesity and weight loss is known. We identified new putative biomarkers for weight loss/maintenance. Correlation between PRG4 and proline-rich acidic protein 1 (PRAP1) variation and Matsuda insulin sensitivity increment was showed. Conclusions and Clinical Relevance : MS-based proteomic analysis of a large cohort of non-diabetic overweight and obese individuals concomitantly identified known and novel proteins associated with weight loss and maintenance. This article is protected by copyright. All rights reserved