Aggrecan is a critical component of the extracellular matrix of all cartilages. One of the early ... more Aggrecan is a critical component of the extracellular matrix of all cartilages. One of the early hallmarks of osteoarthritis (OA) is the loss of aggrecan from articular cartilage followed by degeneration of the tissue. Mesenchymal progenitor cell (MPC) populations in joints, including those in the synovium, have been hypothesized to play a role in the maintenance and/or repair of cartilage, however, the mechanism by which this may occur is unknown. In the current study, we have uncovered that aggrecan is secreted by synovial MPCs from healthy joints yet accumulates inside synovial MPCs within OA joints. Using human synovial biopsies and a rat model of OA, we established that this observation in aggrecan metabolism also occurs in vivo. Moreover, the loss of the “anti-proteinase” molecule alpha-2 macroglobulin (A2M) inhibits aggrecan secretion in OA synovial MPCs, whereas overexpressing A2M rescues the normal secretion of aggrecan. Using mice models of OA and cartilage repair, we have...
The hypothesis is that mast cell numbers and neuropeptide containing nerve fibres are increased i... more The hypothesis is that mast cell numbers and neuropeptide containing nerve fibres are increased in the elbow joint anterior capsule of patients with post-traumatic contractures when compared to normal capsules. Capsules were obtained from two patients with chronic contractures following radial head fractures and two organ donor elbows free of contractures. Four sections from each capsule were double-labelled with specific antibodies to the mast cell marker chymase and the neuropeptide calcitonin gene-related peptide (CGRP). Species specific secondary fluorescent antibodies were used to detect the marker antibodies and cells were identified with a fluorescent nuclear marker (DAPI). Images were captured using a microscope (200x magnification) and five randomly selected areas were sampled for each section obtained from all joint capsules. Chymase positive cell numbers and numbers of nerve fibers (minimum length fifty micrometres) were gathered. The number of chymase positive mast cells...
Proteoglycan 4 (PRG4/lubricin) is secreted by cells that reside in articular cartilage and line t... more Proteoglycan 4 (PRG4/lubricin) is secreted by cells that reside in articular cartilage and line the synovial joint. Lubricin may play a role in modulating inflammatory responses through interaction with CD44. This led us to examine if lubricin could be playing a larger role in the modulation of inflammation/immunity through interaction with Toll-like receptors (TLRs). Human Embryonic Kidney (HEK) cells overexpressing TLRs 2, 4 or 5 and surface plasmon resonance were employed to determine if full length recombinant human lubricin was able to bind to and activate TLRs. Primary human synovial fibroblasts were also examined using flow cytometry and Luminex multiplex ELISA. A rat destabilization model of osteoarthritis (OA) was used to determine if lubricin injections were able to regulate pain and/or inflammation in vivo. Lubricin can bind to and regulate the activity of TLRs, leading to downstream changes in inflammatory signalling independent of HA. We confirmed these findings in vivo...
Type 2 diabetes adversely affects the properties of native connective tissue. The underlying mech... more Type 2 diabetes adversely affects the properties of native connective tissue. The underlying mechanisms, however, by which diabetes alters connective tissue metabolism, especially tendon, are poorly defined. The aim of this study was to determine the effect of type 2 diabetes on the mechanical, histological, and molecular properties of the intact and healing Achilles tendon. The right Achilles tendon was transected in 11 male diabetic Goto-Kakizaki (GK) and 10 age- and sex-matched Wistar control rats, while the left Achilles tendon was left intact. At 2 wk postinjury the intact and injured tendons were assessed by biomechanical testing and histology. The gene expression of collagen I and III, biglycan, versican, MMP-13, and MMP-3 was measured by quantitative RT-PCR, and their protein distribution was studied by immunohistochemistry. Intact tendons exhibited only small differences between the groups. In injured tendons, however, a significantly smaller transverse area and lower stiff...
Chronic inflammation associated with osteoarthritis (OA) may alter normal vascular responses and ... more Chronic inflammation associated with osteoarthritis (OA) may alter normal vascular responses and contribute to joint degradation. Vascular responses to vasoactive mediators were evaluated in the medial collateral ligament (MCL) of the anterior cruciate ligament (ACL)-deficient knee. Chronic joint instability and progressive OA were induced in rabbit knees by surgical transection of the ACL. Under halothane anesthesia, laser speckle perfusion imaging (LSPI) was used to measure MCL blood flow in unoperated control ( n = 12) and 6-wk ACL-transected knees ( n = 12). ACh, bradykinin, histamine, substance P (SP), and prostaglandin E2 (PGE2) were applied to the MCL vasculature in topical boluses of 100 μl (dose range 10−14 to 10−8 mol). In normal joints, ACh, bradykinin, histamine, and PGE2 evoked a dilatory response. Substance P caused a biphasic response that was dilatory from 10−14 to 10−11 mol and constricting at higher doses. In ACL-deficient knees, ACh, bradykinin, histamine, and SP ...
Proteoglycan 4 (PRG4) is an extracellular matrix protein that maintains homeostasis through its b... more Proteoglycan 4 (PRG4) is an extracellular matrix protein that maintains homeostasis through its boundary lubricating and anti-inflammatory properties. Altered expression and function of PRG4 have been associated with joint inflammatory diseases, including osteoarthritis (OA). We found that mast cell tryptase β cleaves PRG4 in a dose- and time-dependent manner, which was confirmed by silver stain gel electrophoresis and mass spectrometry. Tryptase-treated PRG4 resulted in a reduction of lubrication. Compared to full-length, cleaved PRG4 was shown to further activate NF-κB expression in cells overexpressing TLR2, -4, and − 5. In the destabilization of the medial meniscus (DMM) model of OA in rat, tryptase β and PRG4 colocalized at the site of injury in knee cartilage and were associated with disease severity. Human primary synovial fibroblasts from OA patients or non-OA healthy subjects treated with tryptase β and/or PRG4 were subjected to a quantitative shotgun proteomics and proteom...
Scandinavian journal of medicine & science in sports, 2010
It was hypothesized that mobilization vs immobilization after injury would promote tissue healing... more It was hypothesized that mobilization vs immobilization after injury would promote tissue healing by regulating gene expression for molecules associated with repair. Cast immobilization vs free mobilization was studied after rat Achilles tendon rupture. Reverse transcriptase-polymerase chain reaction was performed at 8 and 17 days post-rupture to assess different growth factors [brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), nerve growth factor (NGF) and insulin-like growth factor-1 (IGF-1)] and inflammatory mediators [cyclooxygenase 1 and 2 (COX 1 and COX 2), inducible nitric oxide synthase and hypoxia-inducible factor-1alpha (HIF-1alpha)] in the healing region. At 8 days post-injury, tendon mRNA levels were comparable in both groups. However, by day 17, the mRNA levels for BDNF, bFGF, COX 1 and HIF-1alpha in the mobilized group had increased significantly. Corresponding mRNA levels in the immobilized group decreased during the same period. There w...
Cartilage degeneration after injury affects a significant percentage of the population, including... more Cartilage degeneration after injury affects a significant percentage of the population, including those that will go on to develop osteoarthritis (OA). Like humans, most mammals, including mice, are incapable of regenerating injured cartilage. Interestingly, it has previously been shown that p21 (Cdkn1a) knockout (p21−/−) mice demonstrate auricular (ear) cartilage regeneration. However, the loss of p21 expression is highly correlated with the development of numerous types of cancer and autoimmune diseases, limiting the therapeutic translation of these findings. Therefore, in this study, we employed a screening approach to identify an inhibitor (17-DMAG) that negatively regulates the expression of p21. We also validated that this compound can induce chondrogenesis in vitro (in adult mesenchymal stem cells) and in vivo (auricular cartilage injury model). Furthermore, our results suggest that 17-DMAG can induce the proliferation of terminally differentiated chondrocytes (in vitro and i...
Aggrecan is a critical component of the extracellular matrix of all cartilages. One of the early ... more Aggrecan is a critical component of the extracellular matrix of all cartilages. One of the early hallmarks of osteoarthritis (OA) is the loss of aggrecan from articular cartilage followed by degeneration of the tissue. Mesenchymal progenitor cell (MPC) populations in joints, including those in the synovium, have been hypothesized to play a role in the maintenance and/or repair of cartilage, however, the mechanism by which this may occur is unknown. In the current study, we have uncovered that aggrecan is secreted by synovial MPCs from healthy joints yet accumulates inside synovial MPCs within OA joints. Using human synovial biopsies and a rat model of OA, we established that this observation in aggrecan metabolism also occurs in vivo. Moreover, the loss of the “anti-proteinase” molecule alpha-2 macroglobulin (A2M) inhibits aggrecan secretion in OA synovial MPCs, whereas overexpressing A2M rescues the normal secretion of aggrecan. Using mice models of OA and cartilage repair, we have...
The hypothesis is that mast cell numbers and neuropeptide containing nerve fibres are increased i... more The hypothesis is that mast cell numbers and neuropeptide containing nerve fibres are increased in the elbow joint anterior capsule of patients with post-traumatic contractures when compared to normal capsules. Capsules were obtained from two patients with chronic contractures following radial head fractures and two organ donor elbows free of contractures. Four sections from each capsule were double-labelled with specific antibodies to the mast cell marker chymase and the neuropeptide calcitonin gene-related peptide (CGRP). Species specific secondary fluorescent antibodies were used to detect the marker antibodies and cells were identified with a fluorescent nuclear marker (DAPI). Images were captured using a microscope (200x magnification) and five randomly selected areas were sampled for each section obtained from all joint capsules. Chymase positive cell numbers and numbers of nerve fibers (minimum length fifty micrometres) were gathered. The number of chymase positive mast cells...
Proteoglycan 4 (PRG4/lubricin) is secreted by cells that reside in articular cartilage and line t... more Proteoglycan 4 (PRG4/lubricin) is secreted by cells that reside in articular cartilage and line the synovial joint. Lubricin may play a role in modulating inflammatory responses through interaction with CD44. This led us to examine if lubricin could be playing a larger role in the modulation of inflammation/immunity through interaction with Toll-like receptors (TLRs). Human Embryonic Kidney (HEK) cells overexpressing TLRs 2, 4 or 5 and surface plasmon resonance were employed to determine if full length recombinant human lubricin was able to bind to and activate TLRs. Primary human synovial fibroblasts were also examined using flow cytometry and Luminex multiplex ELISA. A rat destabilization model of osteoarthritis (OA) was used to determine if lubricin injections were able to regulate pain and/or inflammation in vivo. Lubricin can bind to and regulate the activity of TLRs, leading to downstream changes in inflammatory signalling independent of HA. We confirmed these findings in vivo...
Type 2 diabetes adversely affects the properties of native connective tissue. The underlying mech... more Type 2 diabetes adversely affects the properties of native connective tissue. The underlying mechanisms, however, by which diabetes alters connective tissue metabolism, especially tendon, are poorly defined. The aim of this study was to determine the effect of type 2 diabetes on the mechanical, histological, and molecular properties of the intact and healing Achilles tendon. The right Achilles tendon was transected in 11 male diabetic Goto-Kakizaki (GK) and 10 age- and sex-matched Wistar control rats, while the left Achilles tendon was left intact. At 2 wk postinjury the intact and injured tendons were assessed by biomechanical testing and histology. The gene expression of collagen I and III, biglycan, versican, MMP-13, and MMP-3 was measured by quantitative RT-PCR, and their protein distribution was studied by immunohistochemistry. Intact tendons exhibited only small differences between the groups. In injured tendons, however, a significantly smaller transverse area and lower stiff...
Chronic inflammation associated with osteoarthritis (OA) may alter normal vascular responses and ... more Chronic inflammation associated with osteoarthritis (OA) may alter normal vascular responses and contribute to joint degradation. Vascular responses to vasoactive mediators were evaluated in the medial collateral ligament (MCL) of the anterior cruciate ligament (ACL)-deficient knee. Chronic joint instability and progressive OA were induced in rabbit knees by surgical transection of the ACL. Under halothane anesthesia, laser speckle perfusion imaging (LSPI) was used to measure MCL blood flow in unoperated control ( n = 12) and 6-wk ACL-transected knees ( n = 12). ACh, bradykinin, histamine, substance P (SP), and prostaglandin E2 (PGE2) were applied to the MCL vasculature in topical boluses of 100 μl (dose range 10−14 to 10−8 mol). In normal joints, ACh, bradykinin, histamine, and PGE2 evoked a dilatory response. Substance P caused a biphasic response that was dilatory from 10−14 to 10−11 mol and constricting at higher doses. In ACL-deficient knees, ACh, bradykinin, histamine, and SP ...
Proteoglycan 4 (PRG4) is an extracellular matrix protein that maintains homeostasis through its b... more Proteoglycan 4 (PRG4) is an extracellular matrix protein that maintains homeostasis through its boundary lubricating and anti-inflammatory properties. Altered expression and function of PRG4 have been associated with joint inflammatory diseases, including osteoarthritis (OA). We found that mast cell tryptase β cleaves PRG4 in a dose- and time-dependent manner, which was confirmed by silver stain gel electrophoresis and mass spectrometry. Tryptase-treated PRG4 resulted in a reduction of lubrication. Compared to full-length, cleaved PRG4 was shown to further activate NF-κB expression in cells overexpressing TLR2, -4, and − 5. In the destabilization of the medial meniscus (DMM) model of OA in rat, tryptase β and PRG4 colocalized at the site of injury in knee cartilage and were associated with disease severity. Human primary synovial fibroblasts from OA patients or non-OA healthy subjects treated with tryptase β and/or PRG4 were subjected to a quantitative shotgun proteomics and proteom...
Scandinavian journal of medicine & science in sports, 2010
It was hypothesized that mobilization vs immobilization after injury would promote tissue healing... more It was hypothesized that mobilization vs immobilization after injury would promote tissue healing by regulating gene expression for molecules associated with repair. Cast immobilization vs free mobilization was studied after rat Achilles tendon rupture. Reverse transcriptase-polymerase chain reaction was performed at 8 and 17 days post-rupture to assess different growth factors [brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor (bFGF), nerve growth factor (NGF) and insulin-like growth factor-1 (IGF-1)] and inflammatory mediators [cyclooxygenase 1 and 2 (COX 1 and COX 2), inducible nitric oxide synthase and hypoxia-inducible factor-1alpha (HIF-1alpha)] in the healing region. At 8 days post-injury, tendon mRNA levels were comparable in both groups. However, by day 17, the mRNA levels for BDNF, bFGF, COX 1 and HIF-1alpha in the mobilized group had increased significantly. Corresponding mRNA levels in the immobilized group decreased during the same period. There w...
Cartilage degeneration after injury affects a significant percentage of the population, including... more Cartilage degeneration after injury affects a significant percentage of the population, including those that will go on to develop osteoarthritis (OA). Like humans, most mammals, including mice, are incapable of regenerating injured cartilage. Interestingly, it has previously been shown that p21 (Cdkn1a) knockout (p21−/−) mice demonstrate auricular (ear) cartilage regeneration. However, the loss of p21 expression is highly correlated with the development of numerous types of cancer and autoimmune diseases, limiting the therapeutic translation of these findings. Therefore, in this study, we employed a screening approach to identify an inhibitor (17-DMAG) that negatively regulates the expression of p21. We also validated that this compound can induce chondrogenesis in vitro (in adult mesenchymal stem cells) and in vivo (auricular cartilage injury model). Furthermore, our results suggest that 17-DMAG can induce the proliferation of terminally differentiated chondrocytes (in vitro and i...
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