Browsing by Author "Schmidt, Tannin"
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Item Open Access A Proposed Mechanism for Enhanced Titin-Based Force during Ca2+-activation(2017) Powers, Krysta; Herzog, Walter; Pollack, Gerald; Schmidt, Tannin; Syme, Douglas; Nishikawa, Kiisa; Shemanko, Carrie; Federico, SalvatoreWhen an active skeletal muscle sarcomere is stretched, it generates more force after the stretch is completed when compared to a contraction at the same corresponding length. This mechanical property of skeletal muscle defies explanation by any conventional mechanism of contraction. Surmounting evidence indicates that the explanation for enhanced force following active stretch may be provided by the titin protein in the sarcomere, which becomes stiffer during Ca2+-activation. This thesis explores the mechanisms by which titin force is enhanced in actively stretched sarcomeres. Known to stiffen in the presence of Ca2+, the first study quantifies the contribution of Ca2+ to enhanced titin force, showing that an alternative mechanism accounts for the majority of titin force enhancement. The mechanism is further investigated using chemical inhibition of cross-bridges which shows that titin force enhancement is initiated with the development of contractile force. The next set of studies increase support for an interdependence of titin force enhancement and contractile force, showing that titin force enhancement is essentially eliminated and contractile force is decreased in sarcomeres with muscular dystrophy with myositis (mdm), a genetic mutation affecting the titin protein. The final study seeks to determine whether mechanical deficiencies in titin force enhancement are observed in a less reduced, single fiber preparation. Mutant fibers generated comparable contractile force and total force following active stretch beyond filament overlap as control fibers. Titin force enhancement was abolished in some mutant fibers and measured in other mutant fibers suggesting that the mdm mutation differentially affects fibers (and titin) in skeletal muscle. Passive force was increased in mutant fibers, showing that alternative structural components in a fiber can re-establish enhanced active stiffness in the absence of titin force enhancement. Collectively, the findings from this thesis show that titin-based force enhancement is an inherent property of skeletal muscle. The mechanism of titin force enhancement is crucial to sarcomere mechanics; as in its absence, sarcomeres generate less force and alternative structures to titin establish a comparable increase in active stiffness.Item Open Access Biophysical Evidence for a Molecular Interaction between Proteoglycan 4 and Hyaluronan in Solution: Effect of Exposure to a Zwitterionic Detergent on Cartilage Boundary Lubricating Function(2016) Morin, Alyssa; Schmidt, TanninProteoglycan 4 (PRG4) and hyaluronan (HA) are key synovial fluid constituents that contribute synergistically to cartilage boundary lubrication through an undefined mechanism. PRG4 monomers and multimers have previously been separated using size exclusion chromatography with a buffer containing the zwitterionic detergent CHAPS. However, CHAPS may alter the ability of PRG4 to interact with HA and synergistically reduce friction. The objectives of this thesis were to (1) assess the size distribution of PRG4, HA, and PRG4+HA in solution, and (2) determine the effect of exposing PRG4 to CHAPS on the size distribution, cartilage boundary lubricating ability, and cartilage adsorption of PRG4+HA. The size distribution of PRG4+HA was less than PRG4 and HA. Exposure to CHAPS irreversibly altered the size distribution, cartilage boundary lubricating ability, and cartilage adsorption of PRG4+HA. These results suggest a hydrophobic molecular interaction exists between PRG4 and HA, both in solution and at the articular cartilage surface.Item Open Access Boundary Lubricating Ability of Proteoglycan 4 at an Articular Cartilage-Meniscus Biointerface(2015-10-08) Dorosz, Samuel; Schmidt, TanninProteoglycan 4 (PRG4) is a glycoprotein in synovial fluid (SF) and on the articular cartilage surface, where it acts as a boundary lubricant. Hyaluronan (HA) is another SF constituent that acts as a boundary lubricant at the cartilage-cartilage biointerface and interacts with PRG4 to synergistically lower friction. The meniscus is a fibrocartilaginous pad in the knee, on which PRG4 exists, that is critical for joint health. This thesis aimed to develop an in vitro lubrication test to determine the boundary lubricating ability of PRG4 and HA, alone and in combination, at the cartilage-meniscus biointerface, and assess the surface adsorption of recombinant human PRG4 (rhPRG4). A novel cartilage-meniscus test demonstrated that (rh)PRG4 and HA, alone and in combination, act as effective boundary lubricants at this biointerface. rhPRG4 adsorbed to the meniscal surface. This work contributes to the understanding of meniscus lubrication and potential development of osteoarthritis biotherapeutic treatments.Item Open Access Characterization of OA Severity in Knee Articular Cartilage in-Vivo Using MR Imaging and Loading Techniques(2017) Dai, Xu; Ronsky, Janet Lenore; Frayne, Richard; Schmidt, Tannin; Boyd, Steven Kyle; Nowicki, Edwin Peter; Holdsworth, David W.Early osteoarthritis (OA) is primarily associated with proteoglycan (PG) loss and changes in collagen structure. MR T2 imaging of knee under in-vivo loading may help to further reveal the differences between healthy and OA cartilage. This study investigated the in-vivo loading effect on MR T2 values of human knee patellar cartilage. The results demonstrated T2 value distributions in patellar cartilage were inhomogeneous. In-vivo loading had a site-specific influence on participants’ T2 values. The in-vivo loading produced a significant difference on T2 values in the middle region of interest (ROI) of patellar cartilage (p=0.004＜0.025), but not at the superior or inferior ROIs. The T2 value variation for the OA group during loading was lower than that of the healthy group (p=0.016＜0.025). The T2 recovery ratio was presented in this study as a new variable. The findings indicated the T2 recovery ratio of the OA group was significantly lower than the healthy group (p=0.042＜0.05) in the patellar cartilage middle ROI. It suggests that the OA cartilage had weaker ability to restore its original status after off-loading than healthy cartilage. This study examined the glycosaminoglycan (GAG) mass% concentration (relating PG) in human cadaveric patellar cartilages using biochemical assay. Results showed that the GAG mass% concentrations in OA lesion positions were lower than that in comparative healthy positions (p＜0.001). MR T2 imaging of healthy and OA cadaver knee joints were performed. Correlations between T2 values and the GAG mass(%) of cadaver patellar cartilage specimens were established. As PG concentration of in-vivo human articular cartilage cannot be directly measured non-invasively, the correlation of cadaveric patellar cartilage may serve as an important bridge between the T2 value and GAG mass% for living human assessment. The findings provide an indirect approach to estimate PG concentration of in-vivo patellar cartilage based on an individual’s cartilage T2 values to evaluate the extent of degradation within cartilage. This subject specific method is especially suitable for longitudinal evaluation of OA. By position-matched comparison of previous and current T2 images, the GAG mass% variation may be estimated to assess OA progression non-invasively.Item Open Access Characterization of proteoglycan 4 and hyaluronan composition and function of ovine synovial fluid following knee surgery(2012) Barton, Kristen; Frank, Cyril B.; Schmidt, TanninItem Open Access Characterization of proteoglycan 4 and hyaluronan composition and function of ovine synovial fluid following knee surgery(2012-08-16) Barton, Kristen; Frank, Cyril; Schmidt, TanninOsteoarthritis is a degenerative joint disease involving the breakdown of articular cartilage, which is common after injury or with aging. Cartilage lubrication is a vital mechanism for the protection and maintenance of joints. Proteoglycan 4 (PRG4), a glycoprotein present in synovial fluid (SF), contributes to the boundary lubrication of cartilage and maintenance of the joint. PRG4 also acts synergistically with hyaluronan (HA), another molecule present in SF, as cartilage boundary lubricants. The objective of this thesis was to determine 1) PRG4 and HA concentration, 2) HA molecular weight (MW) distribution, 3) cartilage lubricating ability, and 4) the isoelectric point (pI) of PRG4 in SF from surgical sham (SHAM), anterior cruciate ligament (ACL)/medial collateral ligament (MCL) transection, and lateral meniscectomy (MEN) in a post-knee injury ovine model at 20 weeks. SHAM (n=5), ACL/MCL transection (n=6), and MEN (n=5) ovine SF (oSF) was collected at euthanization 20 weeks after surgery, with the left joint serving as the non-operative control (CTRL). PRG4 and HA concentration in oSF was measured by a sandwich enzyme-linked immunosorbent assay (ELISA) and HA MW distribution by agarose gel electrophoresis. A cartilage-cartilage friction test under boundary lubrication conditions was used to assess the cartilage lubricating ability of oSF. The pI of PRG4 was determined by twodimensional (2D) gel electrophoresis. PRG4 and HA concentration in SHAM, ACL/MCL, and MEN oSF was similar in comparison to the contralateral CTRL oSF. The HA MW distribution in the SHAM, ACL/MCL, and MEN oSF for all ranges were similar with respect to the contralateral CTRL oSF. The kinetic coefficient of friction in phosphate buffered saline was significantly higher than all groups, both operated, CTRL oSF, and bovine SF in all cases, which was lower and similar. The pI of PRG4 is ~4.0-4.4 and the pI of PRG4 may have shifted to a higher pI in MEN oSF, in compared to contralateral CTRL oSF. These results suggest that lubricant composition and function was normal 20 weeks post-knee surgery in this model and 2D electrophoresis may provide insight into the structure of PRG4 glycosylations.Item Open Access Characterization of Proteoglycan 4 Supramolecular Structure and its Effect on Lubricating Function(2018-09-14) Martens, Kayla; Schmidt, Tannin; MacCallum, Justin; Derksen, Darren; Sen, ArindomProteoglycan 4 (PRG4) is a mucin-like glycoprotein that is a key synovial fluid constituent, functioning as a cartilage boundary lubricant. PRG4 has been suggested to exist as a supramolecular structure. The objectives of this thesis were to (1) characterize the supramolecular structure of recombinant human PRG4 (rhPRG4) and its dependency on concentration, (2) elucidate the molecular forces underlying the formation of the supramolecular structure, and (3) assess the lubricating ability of the supramolecular structure. rhPRG4’s ability to form a supramolecular structure and reduce friction was concentration dependent. Arginine (0.2M) disrupted the formation of the supramolecular structure of rhPRG4 and diminished its lubricating ability. Conversely, 20 mM calcium enhanced the formation of rhPRG4 supramolecular structure and enhanced its lubricating ability. These results suggest non-covalent interactions mediate rhPRG4’s supramolecular structure, shown here to exist for the first time, which in turn is a functionally (lubrication) determinant property.Item Open Access Effects of Concentration on Synergistic PRG4-Hyaluronan Cartilage Boundary Lubrication(2012) Hunter, Miles; Ludwig, Taryn; Schmidt, TanninItem Open Access Evaluation of in vitro contact lens friction: Effects of recombinant human proteogrlycan 4 and test counterface(2017-12-15) Chan, Amanda; Schmidt, Tannin; Rinker, Kristina; Egberts, PhilipIn vitro contact lenses friction significantly correlates with contact lens discomfort. In this thesis, in vitro friction testing was conducted to evaluate the effects of recombinant human PRG4 (rhPRG4) as an ocular surface boundary lubricant and to evaluate both biological and synthetic test counterfaces. The objectives were to (1) assess the potential effect of different lens types and incubation times on the friction of rhPRG4-incubated contact lenses and (2) evaluate mucin- soaked PDMS as a synthetic test counterface for in vitro friction testing of contact lenses. Results of this thesis work demonstrated that a) rhPRG4 may be useful as a friction reducing lubricant on some, but not all, silicone hydrogel contact lenses; and b) as a synthetic counterface, PDMSmucin can exhibit similar friction coefficients compared to biological counterfaces on certain silicone hydrogel lenses. Overall, results led to an improved understanding of PRG4 and in vitro contact lens friction.Item Open Access Experimental Validation of Finite Element Predicted Bone Strain in the Human Metatarsal(2017) Fung, Anita; Edwards, William Brent; Boyd, Steven Kyle; Schmidt, Tannin; Li, Leping; Wiley, James PrestonThe objective of this study was to verify and validate a finite element modeling routine for the human metatarsal, which is a common location for stress fractures. Experimental strain measurements on 33 human cadaveric metatarsals subject to cantilever bending were compared with strain predictions from finite element (FE) models generated from computed tomography images. For the material property assignment of the FE models, a published density-elasticity relationship was compared with density-elasticity equations developed using optimization techniques. The correlations between the measured and predicted and predicted strains were very high (r2≥0.94) for all of the density-elasticity equations. However, the utilization of an optimized density-elasticity equation improved the accuracy of the finite element models, reducing the maximum error between measured and predicted strains by 10% to 20%. The finite element modeling routine could be used for investigating potential interventions to minimize metatarsal strains and the occurrence of metatarsal stress fractures.Item Open Access Generation of Tissue-Engineered Cartilage Constructs in Stirred Suspension Bioreactors(2016) Allen, Leah Marie; Sen, Arindom; Hart, David; Matyas, John; Ungrin, Mark; Schmidt, Tannin; Ramirez-Serrano, AlejandroTraumatic injuries to articulating joints, such as the knee and hip, can result in the formation of defects within the articular cartilage contained therein. These defects do not heal spontaneously and can initiate a degenerative process, eventually resulting in osteoarthritis (OA). Current cartilage repair options are limited and do not result in the regeneration of durable cartilage. Mesenchymal stem cells (MSCs) isolated from the synovial fluid within joints have an inherent ability to differentiate towards a chondrogenic lineage. Tissue engineered cartilage constructs (TECs) formed from MSCs have been shown to contribute to cartilage repair when implanted into a defect site, thereby providing a potential approach to prevent the onset or the progression of OA. TECs have traditionally been formed in the wells of static culture plates and serum-containing medium. Unfortunately, TECs made in this manner can exhibit variable characteristics which are likely exacerbated by the use of animal-sourced serum in the medium. Therefore, a need still exists to better optimize the generation of uniform TECs to enhance the clinical translatability of this otherwise promising technology. This work investigated the aggregation and culture of human MSCs within suspension bioreactors and serum-free culture conditions for eventual use in filling articular cartilage defects. It also investigated the impact of low-oxygen tension and chondrogenic medium to enhance the MSC differentiation into a chondrogenic phenotype. Expanding on a body of knowledge, this work demonstrated the ability of suspension bioreactors to create a population of aggregates using serum-free culture conditions and non-osteoarthritic human SF-MSCs. Additionally, it demonstrated that the application of low-oxygen tension and chondrogenic growth factors to the suspension bioreactor system is simple. The culture of MSC aggregates in suspension bioreactors under low-oxygen tension resulted in up-regulated gene expression for aggrecan, significantly more collagen/DNA production, and less necrosis on the inside of the aggregates as compared to ambient oxygen tension. The application of chondrogenic medium to the aggregates in the suspension bioreactors resulted in enhanced collagen type-II gene expression and deposition as compared to the static TECs. Although, the overall amount of ECM quantified and staining for glycosaminoglycans was more prominent in the static TECs.Item Open Access In Vitro Friction of Contact Lenses & Model Contact Lens Biomaterials: Effect of Proteoglycan 4(2017) Samsom, Michael; Schmidt, Tannin; Hart, David; Egberts, PhilipContact lenses are associated with discomfort and dry eye in many users. Friction is currently the only variable known to correlate with contact lens discomfort. Therefore, one approach to improve the performance of contact lenses could be to reduce their friction. Proteoglycan 4 (PRG4) is a mucin-like glycoprotein that is naturally produced at, and lubricates, the ocular surface. Hyaluronan (HA) is a repeating disaccharide produced in the body that has also been shown to function as a boundary lubricant at biological interfaces. It is possible that PRG4 and HA could be used as boundary lubricants for contact lenses. The interaction between these molecules and contact lens materials have not been thoroughly characterized. The motivation of this thesis was to contribute to the development and use of PRG4 and HA for contact lens hydrogels using physiologically relevant in vitro friction testing techniques. Novel and previously characterized methods were used to synthesize model contact lens hydrogels. In vitro friction testing was used to evaluate the boundary lubrication of PRG4 and HA for model contact lens materials (conventional: pHEMA; silicone hydrogel: pHEMA/TRIS, DMAA/TRIS), and commercially available contact lenses. PRG4 solutions were effective lubricants for model and commercial silicone hydrogels. PRG4, immobilized on the surface, was an effective lubricant for pHEMA/TRIS, but not pHEMA. HA incorporated into bulk materials was only effective for pHEMA/TRIS. HA immobilized on the surface of hydrogels did not effect friction. PRG4+HA combinations were effective for DMAA/TRIS and pHEMA/TRIS. There was evidence for PRG4+HA synergistic lubrication for pHEMA/TRIS, both when HA was incorporated and when PRG4+HA were in solution. A novel lid wiper - commercial contact lens in vitro friction test was developed and could resolve differences between lenses tested in saline, ATS, and worn vs. fresh lenses. These results demonstrate that PRG4 and HA lubrication of contact lens materials is dependent on the composition of surfaces, and is effective for certain hydrogels. Additionally, a physiologically relevant lid wiper - contact lens test was developed and characterized. This work provides a foundation and framework for use of these natural lubricants for current and future contact lens materials.Item Open Access Investigating the effect of proteoglycan 4 on hyaluronan solution properties using confocal fluorescence recovery after photobleaching(2014-08-26) Bloom, Adam; Schmidt, TanninHyaluronan (HA) and proteoglycan 4 (PRG4) are vital macromolecular constituents of synovial fluid (SF) that contribute synergistically to its biophysical properties. The objective of this thesis was to use confocal fluorescence recovery after photobleaching (FRAP) to determine whether PRG4 will decrease the permeability of simple HA solutions due to specific interactions between PRG4 and HA polymers in solution. Bovine and recombinant human PRG4 caused HA solutions of all concentrations to become less permeable to tracer molecules. This effect, specific to PRG4 and not seen with a control protein, was not dependent on protein structure as determined by reducing and alkylating PRG4. Additionally, confocal FRAP was successfully employed to study human SF samples of interest. These results demonstrate PRG4 can alter solution properties of HA, possibly due to entanglement through binding and/or HA crowding PRG4 into a self-assembled network. These results contribute to the understanding of PRG4’s role in SF.Item Open Access Investigation of Depolarization as the Trigger for Lubricin and Hyaluronan Secretion from Non-Excitable Human Synovial Fibroblasts(2017) Narsingani, Karim; Giles, Wayne; Schmidt, Tannin; Benseler, Susanne; Braun, AndrewHyaluronan (HA) and Lubricin are major components of synovial fluid that are produced by synovial fibroblasts. Their secretion pathway remains unclear. However, calcium has been shown to play a major role. Depolarization may increase intracellular calcium. This study hypothesized that depolarization may lead to an increase in HA and Lubricin. Human synovial fibroblasts (HSF) from two donors were depolarized with 100mM extracellular K+. The media was analyzed for HA and Lubricin using and HA ELISA and AlphaLISA, respectively. Calcium fluorescence determined intracellular calcium concentration ([Ca2+]i). A generalized estimating equation (GEE) was used for statistical analysis. In the high K+ group HA concentration decreased (p=0.000); however, the Lubricin concentration did not change (p=1.000) and [Ca2+]i decreased. Depolarization with 100mM K+ for 12h did not increase [Ca2+]i, Lubricin levels and decreased HA levels in the HSF conditioned media. Results may indicate K+ is not the ion responsible for the resting membrane potential.Item Open Access Lubricin Binds to and Regulates Toll-like Receptor (TLR) Activity(2015-10-01) Iqbal, Shah; Krawetz, Roman; Schmidt, TanninIn this study, lubricin, a protein involved in the lubrication of joints, was discovered to bind to and activate toll-like receptors (TLR). Since TLRs are a major component of the innate immune system, this suggests that lubricin may also play a role in the regulation of the inflammatory response. We attempted to elucidate whether lubricin has a pro- or anti-inflammatory role in the joint following TLR binding. To determine if lubricin-TLR interaction could be playing a role in Osteoarthritis (OA), we examined the cytokine secretions of OA fibroblasts treated with lubricin and demonstrated a potentially anti-inflammatory effect. Furthermore, in vivo studies demonstrated lubricin’s ability to mitigate pain, joint damage, and inflammation following an injury in a rat OA model. The experiments presented in this thesis suggest that lubricin could have anti-inflammatory effects when binding to TLRs at the cellular and tissue level.Item Open Access Prescription of Specialized Footwear for Individuals with Knee Osteoarthritis(2016-02-02) Lewinson, Ryan; Stefanyshyn, Darren; Worobets, Jay; Herzog, Walter; Wiley, Preston; Schmidt, Tannin; Hildebrand, Kevin; Messier, StephenWedged footwear insoles can reduce peak knee adduction moments during gait, which are associated with knee osteoarthritis development and progression; however, randomized trials have been mixed in terms of wedged insole clinical efficacy. To address methodological and technical limitations of past studies, the purposes of this thesis were to (1) identify the most suitable footwear control condition, from a biomechanical perspective, (2) identify a method that can predict the effect of wedged insoles on resultant knee adduction moments, and (3) evaluate the effects of a reduced knee adduction moment intervention on clinical outcomes for individuals with knee osteoarthritis. In fifteen healthy individuals, three sham footwear conditions were compared against participant’s own footwear to determine if sham footwear were biomechanically inert. It was found that all three sham footwear conditions significantly altered biomechanical variables during gait, including the knee adduction moment, and thus participant’s own footwear was recommended as the best control condition, from a biomechanical perspective, for future clinical studies. In fifteen healthy and nineteen knee osteoarthritis participants, a method was discovered that utilizes two dimensional video data from a single stepping motion to predict the change in knee adduction moment induced by wedged insoles during walking. When compared to actual walking data, the method successfully identified the correct insole intervention for 12/15 healthy and 17/19 knee osteoarthritis individuals. It is hoped this method may be implemented into clinical settings for improved footwear prescription capability for individuals with knee osteoarthritis. Finally, a three month randomized trial was conducted. Biomechanical and clinical data were collected from 48 individuals with knee osteoarthritis at baseline, and participants were randomized to either an insole (knee adduction moment reduction) group, or a waitlist control group. At follow-up, no significant differences were noted between groups in terms of change in pain. Knee adduction moment reduction was not associated with reduced pain for the insole group. These data suggest that reduction of knee adduction moments do not confer a clinical benefit in the short term, but do not rule out the possibility that reduced moments are beneficial from a prevention standpoint, or for long term management.Item Open Access Proteolytic Regulation of Proteoglycan 4 in Inflammation(2023-05-05) Das, Nabangshu Shekhar; Dufour, Antoine; Schmidt, Tannin; Krawetz, Roman; Rezansoff, AlexanderProteoglycan 4 (PRG4) is an extracellular macromolecule that is synthesized and secreted by cells lining surfaces of multiple tissues. While it has traditionally been studied and described as a boundary lubricant, recent evidence suggests that it can bind to and affect downstream signaling of several cell surface receptors, including toll-like receptors (TLRs) that are involved in regulating inflammatory responses. Although previous studies have shown that proteolysis of PRG4 reduces its boundary lubricating ability in vitro compared to intact PRG4, the effect of proteolysis on inflammatory signaling remained uncharacterized. Furthermore, while differential levels of PRG4 expression have been associated with various inflammatory conditions, such as osteoarthritis, the role of PRG4 in maintaining inflammatory signaling during normal aging under homeostatic conditions is still unclear. The objectives of this thesis were to 1- Determine expression of PRG4 in global proteomes during inflammation, and 2- Characterize of proteolytic processing of PRG4 and the mechanism and effects in OA and 3- Examine the effects of PRG4 on the global proteomes during age-related joint inflammation. Global proteome analysis revealed PRG4 expression levels changed locally during inflammation as demonstrated by a reduction in the PRG4 expression in the tears while an increase in the saliva of Sjogren’s syndrome patients comparing to healthy individuals. These differential levels of PRG4 expression were associated with differential expression profiles of different signaling molecules (i.e., proteases) associated with multiple inflammatory pathways including neutrophil degranulation both of the tissue environment. With respect to proteolytic susceptibility of PRG4 during inflammation, this study revealed that the proinflammatory serine protease, tryptase β, cleaves PRG4 altering its functional properties with respect to lubrication and inflammation: it reduces the ability to provide boundary lubrication and increases the ability to activate NF-κB-mediated inflammation through the TLR pathway. In a destabilization of medial meniscus (DMM) model of osteoarthritis (OA) in rat knee joint, differential colocalization of tryptase β and PRG4 in knee joints was associated with the development of OA and disease progression. Treatment with an intra-articular injection of exogenous PRG4 was able to resolve joint inflammation and OA phenotypes in the post-DMM rat knee by restoring the expression of PRG4 on articular cartilage and tryptase β by cartilage chondrocytes. In the absence Prg4, chondrocytes in murine knee cartilage express elevated level of proteases, particularly tryptase β and Htra-1, along with concurrent development of neutrophil-like phenotypes during aging. The neutrophil-like phenotype of chondrocytes is associated with loss of joint integrity in Prg4-/- mice. Overall, this thesis demonstrates the importance of expression levels and structural integrity of PRG4 in maintaining homeostasis through regulating inflammation, provides greater insights into the complex interplay between PRG4, proteases and inflammation, and provide the foundation and motivation for the development of new treatments for inflammatory diseases.Item Open Access The autocrine role of proteoglycan-4 (PRG4) in modulating osteoarthritic synoviocyte proliferation and expression of matrix degrading enzymes(2017-05-08) Alquraini, Ali; Jamal, Maha; Zhang, Ling; Schmidt, Tannin; Jay, Gregory D; Elsaid, Khaled AAbstract Background Lubricin/proteoglycan 4 (PRG4) is a mucinous glycoprotein secreted by synovial fibroblasts and superficial zone chondrocytes. Recently, we showed that recombinant human PRG4 (rhPRG4) is a putative ligand for CD44 receptor. rhPRG4-CD44 interaction inhibits cytokine-induced rheumatoid arthritis synoviocyte proliferation. The objective of this study is to decipher the autocrine function of PRG4 in regulating osteoarthritic synoviocyte proliferation and expression of catabolic and pro-inflammatory mediators under basal and interleukin-1 beta (IL-1β)-stimulated conditions. Methods Cytosolic and nuclear levels of nuclear factor kappa B (NFκB) p50 and p65 subunits in Prg4 +/+ and Prg4 -/- synoviocytes were studied using western blot. Nuclear translocation of p50 and p65 proteins in osteoarthritis (OA) fibroblast-like synoviocytes (FLS) in response to IL-1β stimulation in the absence or presence of rhPRG4 was studied using DNA binding assays. OA synoviocyte (5000 cells per well) proliferation following IL-1β (20 ng/ml) treatment in the absence or presence of rhPRG4 (50–200 μg/ml) over 48 hours was determined using a colorimetric assay. Gene expression of matrix metalloproteinases (MMPs), tissue inhibitor of metallproteinases-1 (TIMP-1), TIMP-2, IL-1β, IL-6, IL-8, TNF-α, cycloxygenae-2 (COX2) and PRG4 in unstimulated and IL-1β (1 ng/ml)-stimulated OA synoviocytes, in the presence or absence of rhPRG4 (100 and 200 μg/ml), was studied following incubation for 24 hours. Results Prg4 -/- synoviocytes contained higher nuclear p50 and p65 levels compared to Prg4 +/+ synoviocytes (p < 0.05). rhPRG4 (100 μg/ml) reduced p50 and p65 nuclear levels in Prg4 +/+ and Prg4 -/- synoviocytes (p < 0.001). Similarly, rhPRG4 (200 μg/ml) inhibited NFκB translocation and cell proliferation in OA synoviocytes in a CD44-dependent manner (p < 0.001) via inhibition of IκBα phosphorylation. IL-1β reduced PRG4 expression in OA synoviocytes and rhPRG4 (100 μg/ml) treatment reversed this effect (p < 0.001). rhPRG4 (200 μg/ml) reduced basal gene expression of MMP-1, MMP-3, MMP-13, IL-6, IL-8, and PRG4 in OA synoviocytes, while increasing TIMP-2 and cycloxygenase-2 (COX2) expression (p < 0.001). rhPRG4 (200 μg/ml) reduced IL-1β induction of MMP-1, MMP-3, MMP-9, MMP-13, IL-6, IL-8, and COX2 expression in a CD44-dependent manner (p < 0.001). Conclusion PRG4 plays an important anti-inflammatory role in regulating OA synoviocyte proliferation and reduces basal and IL-1β-stimulated expression of catabolic mediators. Exogenous rhPRG4 autoregulates native PRG4 expression in OA synoviocytes.Item Open Access The Effects Of Increased Stimulation On Sarcomere Number(2016) Engel, Maria; Herzog, Walter; Joumaa, Venus; Syme, Douglas; Schmidt, Tannin; Butterfield, TimothyTabary et al. (1981) accelerated serial sarcomere loss by applying electrical stimulation, resulting in a 25% decrease in sarcomere number (SN) in the guinea pig soleus muscle within 12 hours. A duplication of these results was attempted in our laboratory. Rabbit lower hind limb muscles were stimulated for 10 hours continuously. As a result, muscles lost approximately 25% of sarcomeres in series. During laser diffraction analysis, it was observed that the first order diffraction band was wider in experimental compared to control muscles, implying some form of non-uniformity within the fascicle. Therefore, sarcomere length non-uniformity and structural disorganization was analyzed using light microscopy. Electrical stimulation indeed caused an increase in SL nonuniformity. In an attempt to examine the possibility of sarcomere loss in a more clinically relevant environment, muscle morphology and SN was analyzed in the genetically spastic mouse. The gastrocnemius muscle was shorter, but SN was generally unchanged.