The wound healing response is one of most primitive and conserved physiological responses in the ... more The wound healing response is one of most primitive and conserved physiological responses in the animal kingdom, as restoring tissue integrity/homeostasis can be the difference between life and death. Wound healing in mammals is mediated by immune cells and inflammatory signaling molecules that regulate tissue resident cells, including local progenitor cells, to mediate closure of the wound through formation of a scar. Proteoglycan 4 (PRG4), a protein found throughout the animal kingdom from fish to elephants, is best known as a glycoprotein that reduces friction between articulating surfaces (e.g. cartilage). Previously, PRG4 was also shown to regulate the inflammatory and fibrotic response. Based on this, we asked whether PRG4 plays a role in the wound healing response. Using an ear wound model, topical application of exogenous recombinant human (rh)PRG4 hastened wound closure and enhanced tissue regeneration. Our results also suggest that rhPRG4 may impact the fibrotic response, ...
Proteoglycan 4 (PRG4), also known as lubricin and superficial zone protein, is a mucin-like glyco... more Proteoglycan 4 (PRG4), also known as lubricin and superficial zone protein, is a mucin-like glycoprotein present in synovial fluid (SF) and at the surface of articular cartilage. PRG4 exists in SF as monomeric and disulfidebonded multimeric forms, which are secreted by chondrocytes in bovine cartilage explants in vitro and have been shown to be functionally determinant in cartilage boundary lubricating function. Dynamic shear stimulation of cartilage explant cultures has been shown to increase the quantity of high molecular weight (MW) PRG4 species and up-regulate overall PRG4 secretion by 3-4 times that of unloaded controls. Dynamic compression via oscillation of a ceramic ball over a chondrocyte-seeded scaffold has also been shown to increase PRG4 mRNA expression levels. Collectively, these studies demonstrate that mechanical stimuli regulate PRG4 expression. However, the effect of hydrodynamic shear on PRG4 secretion by chondrocytes in vitro remains to be determined. Therefore, t...
Objective: Local biological and biomechanical-stimuli modulate proteoglycan-4 secretion within sy... more Objective: Local biological and biomechanical-stimuli modulate proteoglycan-4 secretion within synovial joints. For the horse, changes to proteoglycan-4 concentration and function are notable in acute joint injury and osteoarthritis. Proteoglycan-4 (also known as Lubricin) is present in the blood, however the effect of exercise on equine serum levels is unknown. The overall objective of this study was, therefore, to investigate the effect of intense exercise on serum proteoglycan-4 in thoroughbred horses.Methods: Samples of blood were taken from thoroughbreds (n = 12) during a chuckwagon racing event (Alberta, Canada). The chuckwagon race is a sprint racing event where teams of horses pull a combined 1,325 lbs (601 kg) of wagon and driver around a 5/8th mile (1 km) of dirt track, racing at full gallop to the finish. Blood samples were collected 30-min before the race start, and several timepoints post-race: 5-min, 90-min, 3-h, 12-h, and 23-h. Proteoglycan-4 concentrations in serum w...
The synovial fluid glycoprotein lubricin (also known as proteoglycan 4) is a mucin-type O-linked ... more The synovial fluid glycoprotein lubricin (also known as proteoglycan 4) is a mucin-type O-linked glycosylated biological lubricant implicated to be involved in osteoarthritis (OA) development. Lubricin’s ability to reduce friction is related to its glycosylation consisting of sialylated and unsialylated Tn-antigens, core-1 and core-2 structures. The glycans on lubricin have also been suggested to be involved in crosslinking and stabilization of the lubricating superficial layer of cartilage by mediating interaction between lubricin and galectin-3. However, with the spectrum of glycans being found on lubricin, the glycan candidates involved in this interaction were unknown. Here, we confirm that the core-2 O-linked glycans mediate this lubricin-galectin 3 interaction, shown by surface plasmon resonance data indicating that recombinant lubricin (rhPRG4) devoid of core-2 structures did not bind to recombinant galectin-3. Conversely, transfection of Chinese hamster ovary (CHO) cells wit...
The roles of cell division control protein 42 homologue (CDC42) and actin polymerisation in regul... more The roles of cell division control protein 42 homologue (CDC42) and actin polymerisation in regulating the phenotype of superficial-zone chondrocytes (SZCs) have been demonstrated in vitro; however, the signalling pathway(s) downstream have yet to be fully elucidated. The study hypothesis was that Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) act downstream to regulate proteoglycan 4 (PRG4) and tenascin C (TNC). Bovine SZCs grown in monolayer were treated with ML141 (CDC42 inhibitor) or the actin depolymerising agents, latrunculin B and cytochalasin D, to determine the effect on YAP/TAZ. Verteporfin (YAP/TAZ inhibitor) and YAP/TAZ siRNA-mediated knockdown were used to determine their role in regulating PRG4 and TNC. ML141 treatment reduced total YAP/TAZ protein, nuclear TAZ levels and the YAP/TAZ target gene, connective tissue growth factor (CTGF) mRNA levels. Latrunculin B decreased nuclear TAZ, while cytochalasin D treatment trended towards increased nuclear TAZ (p = 0.06), correlating with decreased and increased CTGF mRNA levels, respectively. Verteporfin treatment decreased PRG4 and TNC expression, with no effect on actin polymerisation. siRNA-mediated knockdown of YAP/TAZ revealed that PRG4 was regulated by YAP/TAZ while TNC was regulated by TAZ only. As cytochalasin D can activate myocardin-related transcription factor-A (MRTF-A), siRNA-mediated knockdown was performed to determine the role of MRTF-A in regulating YAP/TAZ. Although nuclear TAZ decreased, no significant changes in total protein levels were observed. Findings suggested that CDC42 and actin polymerisation regulated SZCs through multiple actin-regulated pathways. Understanding the regulation of these chondroprotective molecules may have important implications for prevention/treatment of osteoarthritis.
of this study was to investigate the effect eight weeks of rhythmic aerobic activity on plasma vi... more of this study was to investigate the effect eight weeks of rhythmic aerobic activity on plasma visfatin levels and some metabolic risk-factors in obese females. METHODS: Twenty three obese female college students (age, 25AE4 y), (body mass index, 32AE2), and (waist circumference, 91AE5 cm) were randomly assigned into experimental (n¼13) and control (n¼10) groups. Experimental group completed 8 weeks of rhythmic aerobic exercise, three sessions in each week. Every session lasted for 60 minutes which started with 55% of maximum heart rate at the first week and progressed to 70% of maximum heart rate. Blood samples were collected following 12-h overnight fasting and at the end of 8 weeks training interventions. and were kept at -80C for further assay analysis. Fasting visfatin and insulin concentration were measured by ElISA method. Cholesterol, triglyceride, lipoprotein with high density and, lipo-protein with low density and glucose were measured by enzymatic method and insulin resistance was measured by HOMA equation. RESULTS: Result showed that serum level of visfatin significantly decreased in the experimental trial against control. (P¼0.002). In addition, rhythmic aerobic exercise resulted in reduction in plasma triglyceride concentration (P¼0.04), plasma cholesterol (P¼0.02), plasma LDL concentration (P¼0.01). Also, plasma HDL concentration significantly increased in the experimental group (P¼0.005). However, no significant difference was observed in fasting insulin and glucose concentrations. CONCLUSION: We found that rhythmic aerobic training program effectively improved visfatin level and blood lipid profile, but we failed to report any significant change in insulin response following the training program, which this it might be attributed to lack of monitoring the dietary intake during the study. Therefore, further investigations appear to be necessary to reveal underlying mechanisms.
ABSTRACTSynovial fluid lubricin (proteoglycan 4) is a mucin-type O-linked glycosylated (60% of th... more ABSTRACTSynovial fluid lubricin (proteoglycan 4) is a mucin-type O-linked glycosylated (60% of the mass) biological lubricant involved in osteoarthritis (OA) development. Lubricin has been reported to be cross-linked by synovial galectin-3 on the lubricating articular surface. Here, we confirm that binding to galectin-3 depended on core-2 O-linked glycans, where surface plasmon resonance of a recombinant lubricin (rhPRG4) devoid of core-2 structures lacked binding capacity to recombinant galectin-3. Both galectin-3 levels and interactions with synovial lubricin were found to be decreased in late-stage OA patients coinciding with an increase of truncated and less sialylated core 1 O-glycans. These data suggest a defect cross-linking of surface active molecules in OA and provides novel insights into OA molecular pathology.
Metastasis is the major cause of cancer-related morbidity and mortality. The ability of cancer ce... more Metastasis is the major cause of cancer-related morbidity and mortality. The ability of cancer cells to become invasive and migratory contribute significantly to metastatic growth, which necessitates the identification of novel anti-migratory and anti-invasive therapeutic approaches. Proteoglycan 4 (PRG4), a mucin-like glycoprotein, contributes to joint synovial homeostasis through its friction-reducing and anti-adhesive properties. Adhesion to surrounding extracellular matrix (ECM) components is critical for cancer cells to invade the ECM and eventually become metastatic, raising the question whether PRG4 has an antiinvasive effect on cancer cells. Here, we report that a full-length recombinant human PRG4 (rhPRG4) suppresses the ability of the secreted protein transforming growth factor beta (TGFβ) to induce phenotypic disruption of three-dimensional human breast cancer cellderived organoids by reducing ligand-induced cell invasion. In mechanistic studies, we find that rhPRG4 suppresses TGFβ-induced invasiveness of cancer cells by inhibiting the downstream hyaluronan (HA)-cell surface cluster of differentiation 44 (CD44) signalling axis. Furthermore, we find that rhPRG4 represses TGFβ-dependent increase in the protein abundance of CD44 and of the enzyme HAS2, which is involved in HA biosynthesis. It is widely accepted that TGFβ has both tumor suppressing and tumor promoting roles in cancer. The novel finding that rhPRG4 opposes HAS2 and CD44 induction by TGFβ has implications for downregulating the tumor promoting roles, while maintaining the tumor suppressive aspects of TGFβ actions. Finally, these findings point to rhPRG4's potential clinical utility as a therapeutic treatment for invasive and metastatic breast cancer.
Background: The objective of this study was to use confocal fluorescence recovery after photoblea... more 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 (D t ) 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.
Journal of orthopaedic research : official publication of the Orthopaedic Research Society, 2018
Generating the best possible bioengineered cartilage from passaged chondrocytes requires culture ... more Generating the best possible bioengineered cartilage from passaged chondrocytes requires culture condition optimization. In this study, the use of adherent agarose mold (adAM) cultures to support redifferentiation of passaged twice (P2) chondrocytes and serve as a scalable platform to assess the effect of growth factor combinations on proteoglycan accumulation by cells was examined. By 2 days in adAM culture, bovine P2 cells were partially redifferentiated as demonstrated by regression of actin-based dedifferentiation signalling and fibroblast matrix and contractile gene expression. By day 10, aggrecan and type II collagen gene expression were significantly increased in adAM cultured cells. At day 20, a continuous layer of cartilage tissue was observed. There was no evidence of tissue contraction by P2 cells in adAM cultures. The matrix properties of the resultant tissue as well as proteoglycan 4 (PRG4) secreted by the cells were dependent on the initial cell seeding density. AdAM c...
The Journal of thoracic and cardiovascular surgery, Jan 26, 2018
Intrapericardial fibrous adhesions increase the risk of sternal reentry. Proteoglycan 4/lubricin ... more Intrapericardial fibrous adhesions increase the risk of sternal reentry. Proteoglycan 4/lubricin (PRG4) is a mucin-like glycoprotein that lubricates tissue compartments and prevents inflammation. We characterized PRG4 expression in human pericardium and examined its effects in vitro on human cardiac myofibroblast fibrotic activity and in vivo as a measure of its therapeutic potential to prevent adhesions. Full-length PRG4 expression was determined using Western blot analysis and amplified luminescent proximity homogeneous assay in human pericardial tissues obtained at cardiotomy. The in vitro effects of PRG4 were investigated on human cardiac myofibroblasts for cell adhesion, collagen gel contraction, and cell-mediated extracellular matrix remodeling. The influence of PRG4 on pericardial homeostasis was determined in a chronic porcine animal model. PRG4 is expressed in human pericardial fluid and colocalized with pericardial mesothelial cells. Recombinant human PRG4 prevented human ...
BACKGROUND: "Ultra-fast track" and "fast track" cardiac anesthesia seek to improve outcomes by ac... more BACKGROUND: "Ultra-fast track" and "fast track" cardiac anesthesia seek to improve outcomes by achieving earlier extubation after cardiac surgery, either within the operating room (OR) itself or within 6-12 hours of intensive care unit (ICU) admission, respectively. Previous reports have described benefits of early extubation on length of stay (LOS) and complication rates after cardiac surgery, but its impact on the occurrence of postoperative delirium (PoD) remains unclear. We sought to determine the effect of earlier extubation on PoD rates after coronary artery bypass grafting (CABG). METHODS AND RESULTS: A single-centre retrospective review of all consecutive patients undergoing isolated CABG from January 1, 2010 to December 31, 2015 was conducted. Baseline demographics, preoperative comorbidities, intraoperative data and postoperative data including major adverse events (death, stroke, myocardial infarction, and requirement for dialysis) were collected for all patients. Patients were grouped according to extubation time and a logistic regression was performed with a priori adjustments made for age, sex, and Euro-SCORE II in order to investigate the association between earlier extubation and PoD (determined by the confusion assessment method). Logistic regression was limited to extubation within the first 24 hours of ICU admission in order to exclude relatively sicker, likely non-candidates for early extubation. In the 2784 patients included in the study, the OR and 24-hour extubation rates were 33.0% and 92.1%, respectively. The postoperative intubation duration was associated with a higher PoD incidence following adjustment (hourly odds ratio 1.05; 95% CI, 1.03e1.07; P < 0.001). This effect was pronounced in patients that exceeded the 12-hour time point. Earlier extubation was associated with reductions in hospital LOS, ICU LOS, and risk of major adverse events (Figure ). CONCLUSION: To date, this is one of the largest reports of outcomes following earlier extubation after cardiac surgery. In addition to known benefits of earlier extubation on LOS and adverse event rates, our findings suggest that the risk of PoD increases for every hour that a patient is kept intubated in the ICU. This study provides the basis for consideration of the appropriate selection of earlier extubation to minimize PoD in the cardiac surgery patient.
Sjögren's syndrome (SS) is an autoimmune disease affecting the lacrimal and salivary glands w... more Sjögren's syndrome (SS) is an autoimmune disease affecting the lacrimal and salivary glands with hallmark clinical symptoms of dry eye and dry mouth. Recently, markedly increased cathepsin S (CTSS) activity has been observed in the tears of SS patients. Proteoglycan 4 (PRG4), also known as lubricin, is an effective boundary lubricant that is naturally present on the ocular surface. While PRG4 is susceptible to proteolytic digestion, the potential effect of CTSS on PRG4 remains unknown. The objective of this study was to assess the ability of CTSS to enzymatically degrade purified PRG4, and PRG4 naturally present in human tears, and alter ocular surface boundary lubricating properties. To assess the potential time course and dose-dependency of PRG4 digestion by CTSS, full-length recombinant human PRG4 (rhPRG4) was incubated at 37 °C with or without CTSS in an enzymatic digestion buffer. Digestion of PRG4 by CTSS was also examined within normal human tear samples, both with and wi...
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Papers by Suresh Regmi