This study reviewed the occurrence of chondroitin sulfate (CS) motifs 4-C-3, 7-D-4, and 3-B-3(-),... more This study reviewed the occurrence of chondroitin sulfate (CS) motifs 4-C-3, 7-D-4, and 3-B-3(-), which are expressed by progenitor cells in tissues undergoing morphogenesis. These motifs have a transient early expression pattern during tissue development and also appear in mature tissues during pathological remodeling and attempted repair processes by activated adult stem cells. The CS motifs are information and recognition modules, which may regulate cellular behavior and delineate stem cell niches in developmental tissues. One of the difficulties in determining the precise role of stem cells in tissue development and repair processes is their short engraftment period and the lack of specific markers, which differentiate the activated stem cell lineages from the resident cells. The CS sulfation motifs 7-D-4, 4-C-3, and 3-B-3 (-) decorate cell surface proteoglycans on activated stem/progenitor cells and appear to identify these cells in transitional areas of tissue development and in tissue repair and may be applicable to determining a more precise role for stem cells in tissue morphogenesis.
This review highlights the attributes of pentosan polysulfate (PPS) in the promotion of intervert... more This review highlights the attributes of pentosan polysulfate (PPS) in the promotion of intervertebral disc (IVD) repair processes. PPS has been classified as a disease-modifying osteoarthritic drug (DMOAD) and many studies have demonstrated its positive attributes in the countering of degenerative changes occurring in cartilaginous tissues during the development of osteoarthritis (OA). Degenerative changes in the IVD also involve inflammatory cytokines, degradative proteases, and cell signaling pathways similar to those operative in the development of OA in articular cartilage. PPS acts as a heparan sulfate (HS) mimetic to effect its beneficial effects in cartilage. The IVD contains small cell membrane HS proteoglycans (HSPGs) such as syndecan, and glypican and a large multifunctional HS/chondroitin sulfate (CS) hybrid proteoglycan (HSPG2/perlecan), that have important matrix-stabilizing properties and sequester, control, and present growth factors from the FGF, VEGF, PDGF, and BMP families to cellular receptors to promote cell proliferation, differentiation, and matrix synthesis. HSPG2 also has chondrogenic properties and stimulates the synthesis of extracellular matrix (ECM) components and expansion of cartilaginous rudiments, and has roles in matrix stabilization and repair. Perlecan is a perinuclear and nuclear proteoglycan (PG) in IVD cells with roles in chromatin organization and control of transcription factor activity, immunolocalizes to stem cell niches in cartilage, promotes escape of stem cells from quiescent recycling, differentiation and attainment of pluripotency and migratory properties. These participate in tissue development and morphogenesis, ECM remodeling and repair. PPS also localizes in the nucleus of stromal stem cells, promotes development of chondroprogenitor cell lineages, ECM synthesis and repair and discal repair by resident disc cells. The availability of recombinant perlecan and PPS offers new opportunities in repair biology. These multifunctional agents offer welcome new developments in repair strategies for the IVD.
The aim of this study was to assess if the ovine articular cartilage serine proteinase inhibitors... more The aim of this study was to assess if the ovine articular cartilage serine proteinase inhibitors (SPIs) were related to the Kunitz inter--trypsin inhibitor (ITI) family. Ovine articular cartilage was finely diced and extracted in 6M urea and SPIs isolated by sequential anion exchange, HA affinity and Sephadex G100 gel permeation chromatography. Selected samples were also subjected to chymotrypsin and concanavalin-A affinity chromatography. Eluant fractions from these isolation steps were monitored for protein and trypsin inhibitory activity and pooled fractions assessed by affinity blotting using biotinylated trypsin to detect active SPIs and by Western blotting using antibodies to 1-microglobulin, bikunin, TSG-6 and 2-B-6 (+) CS stub epitope generated by chondroitinase-ABC digestion. This identified 2-B-6 (+) positive 220-250,120, 58 and 36 kDa SPIs. The 58 kDa SPI contained 1-microglobulin, bikunin and chondroitin-4-sulphate stub epitope consistent with its identity as the 1-microglobulin-bikunin (AMBP) precursor and was also isolated by concanavalin-A lectin affinity chromatography indicating it had N-glycosylation. Kunitz protease inhibitor (KPI) species of 36, 26, 12 and 6 kDa could be autolytically generated by prolonged storage of the aforementioned 120 and 58 kDa SPIs; chymotrypsin affinity chromatography also generated the 6kDa SPI. KPI domain 1 and 2 SPIs were separated by concanavalin lectin affinity chromatography, domain 1 displayed affinity for this lectin indicating it had N-glycosylation. KPI 1 and 2 both displayed potent inhibitory activity towards trypsin, chymotrypsin, kallikrein, leucocyte elastase and cathepsin G. Localisation of versican, lubricin and HA in the surface regions of articular cartilage represented probable binding sites for the ITI SPs with likely importance in the preservation of joint function.
The aim of this review is to highlight the beneficial attributes of flavonoids, a diverse family ... more The aim of this review is to highlight the beneficial attributes of flavonoids, a diverse family of widely-distributed polyphenolic phytochemicals that have beneficial cell and tissue protective properties. Phytochemicals are widely distributed in plants, herbs and shrubs used in traditional complimentary medical formulations for centuries. The bioactive components that convey beneficial medicinal effects in these complex herbal preparations are now being identified using network pharmacology and molecular docking procedures that identify their molecular targets. Flavonoids have anti-oxidant, anti-inflammatory, antiviral, antibacterial and anti-cancer properties that have inspired the development of potent multifunctional derivatised flavonoids of improved efficacy. The antiviral properties of flavonoids and the emergence of the severe acute respiratory syndrome (SARS-CoV-2) pandemic has resulted in a resurgence of interest in phytochemicals in the search for efficacious compounds that can prevent viral infection or replication, with many promising plant compounds identified. Promising semi-synthetic flavonoid derivatives have also been developed that inhibit multiple pathological neurodegenerative processes; these offer considerable promise in the treatment of diseases of cognitive decline. Clinical trials are currently being undertaken to evaluate the efficacy of dietary supplements rich in flavonoids for the treatment of virally-mediated diseases. Such trials are expected to identify flavonoids with cell and tissue protective properties that can be harnessed in biomedical applications that may serve as supportive adjunctive procedures to conventional anti-viral drug therapies against diseases such as COVID-19.
The aim of this study was to ascertain whether, like many cell types in cartilaginous tissues if ... more The aim of this study was to ascertain whether, like many cell types in cartilaginous tissues if type XI collagen was a pericellular component of annulus fibrosus (AF) cells and chondrocytes. Fine fibrillar networks were visualised which were perlecan, HS (MAb 10E4) and type XI collagen positive. Heparitinase-III pre-digestion abolished the type XI collagen and 10E4 localisation in these fibrillar assemblies demonstrating a putative HS mediated interaction which localised the type XI collagen. Type XI collagen was confirmed to be present in the Heparitinase III treated AF monolayer media samples by immunoblotting. Heparitinase-III generated ΔHS stub epitopes throughout these fibrillar networks strongly visualised by MAb 3-G-10. Monolayers of murine hip articular chondrocytes from C57BL/6 and Hspg2 exon 3 null mice also displayed pericellular perlecan localisations, however type XI collagen was only evident in the Wild type mice. Perlecan was also immunolocalised in control and murine knee articular cartilage from the two mouse genotypes subjected to a medial meniscal destabilisation procedure which induces OA. This resulted in a severe depletion of perlecan levels particularly in the perlecan exon 3 null mice and was consistent with OA representing a disease of the pericellular matrix. A model was prepared to explain these observations between the NPP type XI collagen domain and HS chains of perlecan domain-I in the pericellular matrix of AF cells which likely contributed to cellular communication, tissue stabilization and the regulation of extracellular matrix homeostasis. Keywords Type XI collagen • Intervertebral disc • Annulus fibrosus • Cell-matrix communication • Heparan sulfate • Perlecan Abbreviations IVD Intervertebral disc AF Annulus fibrosus HS Heparan sulfate
Hyaluronan (HA) binding proteins (HABPs) were localized in cartilaginous ovine tissues (articular... more Hyaluronan (HA) binding proteins (HABPs) were localized in cartilaginous ovine tissues (articular cartilage, intervertebral disc) using a biotinylated HA (bHA) oligosaccharide bioaffinity probe. The bHA oligosaccharide probe was prepared by partial digestion of HA with ovine testicular hyaluronidase, and the oligosaccharides were labeled with biotin hydrazide and purified by a combination of aggrecan G1 domain and avidin affinity chromatography. Hyaladherins were prominently visualized in tissue sections using the bHA oligosaccharide probe as pericellular components in hypertrophic epiphyseal and vertebral growth plate chondrocytes and in the enlarged cells of the cartilaginous end plate of the intervertebral disc. Weaker extracellular staining was also evident in the matrix of the ovine newborn hip and knee joint cartilages. The bHA oligosaccharide probe also visualized intracellular HABPs (IHABPs) in the hypertrophic growth plate chondrocytes of the primary ossification centers. Monolayer cultures of ovine chondrocytes rapidly internalized the bHA oligosaccharide affinity probe to discrete cytoplasmic, nuclear, and perinuclear regions, which were visualized by indirect fluorescent microscopy. This bHA oligosaccharide affinity probe may be useful in future investigations designed to characterize these novel cartilage IHABPs and the role that HA endocytosis plays in cellular regulatory processes in cartilage homeostasis.
The aim of this study was to immunolocalize perlecan in human fetal, postnatal, and mature hyalin... more The aim of this study was to immunolocalize perlecan in human fetal, postnatal, and mature hyaline cartilages and to determine information on the structure and function of chondrocyte perlecan. Perlecan is a prominent component of human fetal (12-14 week) finger, toe, knee, and elbow cartilages; it was localized diffusely in the interterritorial extracellular matrix, densely in the pericellular matrix around chondrocytes, and to small blood vessels in the joint capsules and perichondrium. Aggrecan had a more intense distribution in the marginal regions of the joint rudiments and in para-articular structures. Perlecan also had a strong pericellular localization pattern in postnatal (2-7 month) and mature (55-64 year) femoral cartilages, whereas aggrecan had a prominent extracellular matrix distribution in these tissues. Western blotting identified multiple perlecan core protein species in extracts of the postnatal and mature cartilages, some of which were substituted with heparan sulfate and/or chondroitin sulfate and some were devoid of glycosaminoglycan substitution. Some perlecan core proteins were smaller than intact perlecan, suggesting that proteolytic processing or alternative splicing had occurred. Surface plasmon resonance and quartz crystal microbalance with dissipation experiments demonstrated that chondrocyte perlecan bound fibroblast growth factor (FGF)-1 and-9 less efficiently than endothelial cell perlecan. The latter perlecan supported the proliferation of Baf-32 cells transfected with FGFR3c equally well with FGF-1 and-9, whereas chondrocyte perlecan only supported Baf-32 cell proliferation with FGF-9. The function of perlecan therefore may not be universal but may vary with its cellular origin and presumably its structure.
Journal of Histochemistry & Cytochemistry, 2020
Inter-α-trypsin inhibitor (IαI) family members are ancient and unique molecules that have evolved... more Inter-α-trypsin inhibitor (IαI) family members are ancient and unique molecules that have evolved over several hundred million years of vertebrate evolution. IαI is a complex containing the proteoglycan bikunin to which heavy chain proteins are covalently attached to the chondroitin sulfate chain. Besides its matrix protective activity through protease inhibitory action, IαI family members interact with extracellular matrix molecules and most notably hyaluronan, inhibit complement, and provide cell regulatory functions. Recent evidence for the diverse roles of the IαI family in both biology and pathology is reviewed and gives insight into their pivotal roles in tissue homeostasis. In addition, the clinical uses of these molecules are explored, such as in the treatment of inflammatory conditions including sepsis and Kawasaki disease, which has recently been associated with severe acute respiratory syndrome coronavirus 2 infection in children:
Veterinary and Comparative Orthopaedics and Traumatology, 1994
SummaryCompositional analyses were undertaken on lumbar (L2L3 - L5L6) and lumbosacral (L6S1) inte... more SummaryCompositional analyses were undertaken on lumbar (L2L3 - L5L6) and lumbosacral (L6S1) intervertebral disc tissues from young adult (2-year-old) merino wethers. The proteoglycan level in the nucleus pulposus of the lumbosacral disc was significantly lower than that found in the nucleus pulposus of lumbar levels (p<0.05). The annulus fibrosus was richer in collagen compared to the nucleus pulposus in all discs examined, and the lumbosacral disc consistently had both higher annulus fibrosus and nucleus pulposus collagen levels than lumbar discs (p<0.05). Aggregation of the high buoyant density proteoglycans, with hyaluronic acid, was generally higher in annulus fibrosus proteogly-cans than nucleus pulposus proteogly-cans irrespective of the spinal level examined. Examination of the inter-vertebral disc proteoglycans, by composite agarose polyacrylamide gel electrophoresis, demonstrated three proteoglycan subpopulations and that the nucleus pulposus generally contained a gr...
Loose bodies are fragments of cartilage or bone present in the synovial fluid. In the present stu... more Loose bodies are fragments of cartilage or bone present in the synovial fluid. In the present study we assessed if loose bodies could be used as a source of autologous human chondrocytes for experimental purposes. Histochemical examination of loose bodies and differential enzymatic digestions were undertaken, the isolated cells were cultured in alginate bead microspheres and immunolocalisations were undertaken for chondrogenic markers such as aggrecan, and type II collagen. Isolated loose body cells had high viability (≥90% viable), expressed chondrogenic markers (aggrecan, type II collagen) but no type I collagen. Loose bodies may be a useful source of autologous chondrocytes of high viability.
Abbreviations used AD Alzheimer's diseas AkT a serine/threonine-specific protein kinase, protein ... more Abbreviations used AD Alzheimer's diseas AkT a serine/threonine-specific protein kinase, protein kinase B ALS Amyotrophic lateral sclerosis AT antithrombin BMP bone morphogenetic protein CCL2 CC Motif Chemokine Ligand 2 CNS/PNS central nervous system/peripheral nervous system Cs chitosan CS chondroitin sulphate DS dermatan sulphate ECM Extracellular matrix ERM condensed term based on the first initial of a family of three highly related cytoskeletal proteins, Ezrin, Radixin, Moeisin, FGF fibroblast growth factor GAG Glycosaminoglycan HA hyaluronic acid, hyaluronan HS heparan sulphate IHH indian hedgehog KS keratan sulphate mAb monoclonal antibody Mef2c MADS box transcription enhancer factor 2. MADS is a conserved motif in a family of MADS box transcription factors. MADS is an acronym derived from the first initial from four founding members of this group, MCM1 from Saccharomyces cerevisiae, AGAMOUS from the thale cress Arabidopsis thaliana, DEFICIENS from the snapdragon Antirrhinum majus and SRF from Homo sapiens. NHERF-1 Na + /H + exchanger regulatory factor 1 PDGF platelet derived growth factor PDZ a term derived from the first initials of post-synaptic density protein (PSD95), Drosophila disc large tumour suppressor protein (Dig1), and zona occludens-1 protein. PEDOT /PSS poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) PG proteoglycan PTEN Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase VEGF vascular endothelial cell growth factor Sox SRY-related high mobility group (HMG)-box genes Wnt a condensation term derived from the terms for the Winged and Int genes Electrostimulation, Glycosaminoglycans, cellular regulation and tissue repair
This narrative review highlights the complexities of the gut microbiome and health-promoting prop... more This narrative review highlights the complexities of the gut microbiome and health-promoting properties of prebiotic xylans metabolized by the gut microbiome. In animal husbandry, prebiotic xylans aid in the maintenance of a healthy gut microbiome. This prevents the colonization of the gut by pathogenic organisms obviating the need for dietary antibiotic supplementation, a practice which has been used to maintain animal productivity but which has led to the emergence of antibiotic resistant bacteria that are passed up the food chain to humans. Seaweed xylan-based animal foodstuffs have been developed to eliminate ruminant green-house gas emissions by gut methanogens in ruminant animals, contributing to atmospheric pollution. Biotransformation of pentosan polysulfate by the gut microbiome converts this semi-synthetic sulfated disease-modifying anti-osteoarthritic heparinoid drug to a prebiotic metabolite that promotes gut health, further extending the therapeutic profile and utility ...
This review highlights the multifunctional properties of perlecan (HSPG2) and its potential roles... more This review highlights the multifunctional properties of perlecan (HSPG2) and its potential roles in repair biology. Perlecan is ubiquitous, occurring in vascular, cartilaginous, adipose, lymphoreticular, bone and bone marrow stroma and in neural tissues. Perlecan has roles in angiogenesis, tissue development and extracellular matrix stabilization in mature weight bearing and tensional tissues. Perlecan contributes to mechanosensory properties in cartilage through pericellular interactions with fibrillin-1, type IV, V, VI and XI collagen and elastin. Perlecan domain I - FGF, PDGF, VEGF and BMP interactions promote embryonic cellular proliferation, differentiation, and tissue development. Perlecan domain II, an LDLR-like domain interacts with lipids, Wnt and Hedgehog morphogens. Perlecan domain III binds FGF-7 and 18 and has roles in the secretion of perlecan. Perlecan domain IV, an immunoglobulin repeat domain, has cell attachment and matrix stabilizing properties. Perlecan domain V...
Osteoarthritis is a disease of multifactorial aetiology characterised by progressive breakdown of... more Osteoarthritis is a disease of multifactorial aetiology characterised by progressive breakdown of articular cartilage. In the early stages of the disease, changes become apparent in the superficial zone of articular cartilage, including fibrillation and fissuring. Normally, a monolayer of lubricating molecules is adsorbed on the surface of cartilage and contributes to the minimal friction and wear properties of synovial joints. Proteoglycan 4 is the lubricating glycoprotein believed to be primarily responsible for this boundary lubrication. Here we have used an established ovine meniscectomy model of osteoarthritis, in which typical degenerative changes are observed in the operated knee joints at three months after surgery, to evaluate alterations in proteoglycan 4 expression and localisation in the early phases of the disease. In normal control joints, proteoglycan 4 was immunolocalised in the superficial zone of cartilage, particularly in those regions of the knee joint covered by...
The aim of this study was to highlight the roles of perlecan in the regulation of the development... more The aim of this study was to highlight the roles of perlecan in the regulation of the development of the rudiment developmental cartilages and growth plate cartilages, and also to show how perlecan maintains permanent articular cartilage homeostasis. Cartilage rudiments are transient developmental templates containing chondroprogenitor cells that undergo proliferation, matrix deposition, and hypertrophic differentiation. Growth plate cartilage also undergoes similar changes leading to endochondral bone formation, whereas permanent cartilage is maintained as an articular structure and does not undergo maturational changes. Pericellular and extracellular perlecan-HS chains interact with growth factors, morphogens, structural matrix glycoproteins, proteases, and inhibitors to promote matrix stabilization and cellular proliferation, ECM remodelling, and tissue expansion. Perlecan has mechanotransductive roles in cartilage that modulate chondrocyte responses in weight-bearing environment...
Cartilage regeneration requires a balance of anabolic and catabolic processes. This study examine... more Cartilage regeneration requires a balance of anabolic and catabolic processes. This study examined the susceptibility of fibromodulin (FMOD) and lumican (LUM) to degradation by MMP-13, ADAMTS-4 and ADAMTS-5, the three major degradative proteinases in articular cartilage in osteoarthritis (OA). Immunolocalisation of FMOD and LUM in foot sections of developmental cartilages demonstrated prominent localisations in metatarsal and phalangeal foetal rudiment cartilages and growth plate. An MMP-13 neoepitope antibody (TsYG11) demonstrated localisation of MMP-13 cleaved FMOD in the hypertrophic chondrocytes of the metatarsal growth plate. FMOD was more prominently localised in the superficial cartilage of normal and fibrillated zones in OA cartilage, TsYG11 positive FMOD was located deeper in the cartilage samples. Ab TsYG11 also identified FMOD fragmentation in Western blots of extracts of normal and fibrillated cartilage and total knee replacement OA cartilage. The C-terminal anti-F...
The aim of the present study was to examine the roles of l-fucose and the glycosaminoglycans (GAG... more The aim of the present study was to examine the roles of l-fucose and the glycosaminoglycans (GAGs) keratan sulfate (KS) and chondroitin sulfate/dermatan sulfate (CS/DS) with selected functional molecules in neural tissues. Cell surface glycans and GAGs have evolved over millions of years to become cellular mediators which regulate fundamental aspects of cellular survival. The glycocalyx, which surrounds all cells, actuates responses to growth factors, cytokines and morphogens at the cellular boundary, silencing or activating downstream signaling pathways and gene expression. In this review, we have focused on interactions mediated by l-fucose, KS and CS/DS in the central and peripheral nervous systems. Fucose makes critical contributions in the area of molecular recognition and information transfer in the blood group substances, cytotoxic immunoglobulins, cell fate-mediated Notch-1 interactions, regulation of selectin-mediated neutrophil extravasation in innate immunity and CD-34-m...
This study reviewed the occurrence of chondroitin sulfate (CS) motifs 4-C-3, 7-D-4, and 3-B-3(-),... more This study reviewed the occurrence of chondroitin sulfate (CS) motifs 4-C-3, 7-D-4, and 3-B-3(-), which are expressed by progenitor cells in tissues undergoing morphogenesis. These motifs have a transient early expression pattern during tissue development and also appear in mature tissues during pathological remodeling and attempted repair processes by activated adult stem cells. The CS motifs are information and recognition modules, which may regulate cellular behavior and delineate stem cell niches in developmental tissues. One of the difficulties in determining the precise role of stem cells in tissue development and repair processes is their short engraftment period and the lack of specific markers, which differentiate the activated stem cell lineages from the resident cells. The CS sulfation motifs 7-D-4, 4-C-3, and 3-B-3 (-) decorate cell surface proteoglycans on activated stem/progenitor cells and appear to identify these cells in transitional areas of tissue development and in tissue repair and may be applicable to determining a more precise role for stem cells in tissue morphogenesis.
This review highlights the attributes of pentosan polysulfate (PPS) in the promotion of intervert... more This review highlights the attributes of pentosan polysulfate (PPS) in the promotion of intervertebral disc (IVD) repair processes. PPS has been classified as a disease-modifying osteoarthritic drug (DMOAD) and many studies have demonstrated its positive attributes in the countering of degenerative changes occurring in cartilaginous tissues during the development of osteoarthritis (OA). Degenerative changes in the IVD also involve inflammatory cytokines, degradative proteases, and cell signaling pathways similar to those operative in the development of OA in articular cartilage. PPS acts as a heparan sulfate (HS) mimetic to effect its beneficial effects in cartilage. The IVD contains small cell membrane HS proteoglycans (HSPGs) such as syndecan, and glypican and a large multifunctional HS/chondroitin sulfate (CS) hybrid proteoglycan (HSPG2/perlecan), that have important matrix-stabilizing properties and sequester, control, and present growth factors from the FGF, VEGF, PDGF, and BMP families to cellular receptors to promote cell proliferation, differentiation, and matrix synthesis. HSPG2 also has chondrogenic properties and stimulates the synthesis of extracellular matrix (ECM) components and expansion of cartilaginous rudiments, and has roles in matrix stabilization and repair. Perlecan is a perinuclear and nuclear proteoglycan (PG) in IVD cells with roles in chromatin organization and control of transcription factor activity, immunolocalizes to stem cell niches in cartilage, promotes escape of stem cells from quiescent recycling, differentiation and attainment of pluripotency and migratory properties. These participate in tissue development and morphogenesis, ECM remodeling and repair. PPS also localizes in the nucleus of stromal stem cells, promotes development of chondroprogenitor cell lineages, ECM synthesis and repair and discal repair by resident disc cells. The availability of recombinant perlecan and PPS offers new opportunities in repair biology. These multifunctional agents offer welcome new developments in repair strategies for the IVD.
The aim of this study was to assess if the ovine articular cartilage serine proteinase inhibitors... more The aim of this study was to assess if the ovine articular cartilage serine proteinase inhibitors (SPIs) were related to the Kunitz inter--trypsin inhibitor (ITI) family. Ovine articular cartilage was finely diced and extracted in 6M urea and SPIs isolated by sequential anion exchange, HA affinity and Sephadex G100 gel permeation chromatography. Selected samples were also subjected to chymotrypsin and concanavalin-A affinity chromatography. Eluant fractions from these isolation steps were monitored for protein and trypsin inhibitory activity and pooled fractions assessed by affinity blotting using biotinylated trypsin to detect active SPIs and by Western blotting using antibodies to 1-microglobulin, bikunin, TSG-6 and 2-B-6 (+) CS stub epitope generated by chondroitinase-ABC digestion. This identified 2-B-6 (+) positive 220-250,120, 58 and 36 kDa SPIs. The 58 kDa SPI contained 1-microglobulin, bikunin and chondroitin-4-sulphate stub epitope consistent with its identity as the 1-microglobulin-bikunin (AMBP) precursor and was also isolated by concanavalin-A lectin affinity chromatography indicating it had N-glycosylation. Kunitz protease inhibitor (KPI) species of 36, 26, 12 and 6 kDa could be autolytically generated by prolonged storage of the aforementioned 120 and 58 kDa SPIs; chymotrypsin affinity chromatography also generated the 6kDa SPI. KPI domain 1 and 2 SPIs were separated by concanavalin lectin affinity chromatography, domain 1 displayed affinity for this lectin indicating it had N-glycosylation. KPI 1 and 2 both displayed potent inhibitory activity towards trypsin, chymotrypsin, kallikrein, leucocyte elastase and cathepsin G. Localisation of versican, lubricin and HA in the surface regions of articular cartilage represented probable binding sites for the ITI SPs with likely importance in the preservation of joint function.
The aim of this review is to highlight the beneficial attributes of flavonoids, a diverse family ... more The aim of this review is to highlight the beneficial attributes of flavonoids, a diverse family of widely-distributed polyphenolic phytochemicals that have beneficial cell and tissue protective properties. Phytochemicals are widely distributed in plants, herbs and shrubs used in traditional complimentary medical formulations for centuries. The bioactive components that convey beneficial medicinal effects in these complex herbal preparations are now being identified using network pharmacology and molecular docking procedures that identify their molecular targets. Flavonoids have anti-oxidant, anti-inflammatory, antiviral, antibacterial and anti-cancer properties that have inspired the development of potent multifunctional derivatised flavonoids of improved efficacy. The antiviral properties of flavonoids and the emergence of the severe acute respiratory syndrome (SARS-CoV-2) pandemic has resulted in a resurgence of interest in phytochemicals in the search for efficacious compounds that can prevent viral infection or replication, with many promising plant compounds identified. Promising semi-synthetic flavonoid derivatives have also been developed that inhibit multiple pathological neurodegenerative processes; these offer considerable promise in the treatment of diseases of cognitive decline. Clinical trials are currently being undertaken to evaluate the efficacy of dietary supplements rich in flavonoids for the treatment of virally-mediated diseases. Such trials are expected to identify flavonoids with cell and tissue protective properties that can be harnessed in biomedical applications that may serve as supportive adjunctive procedures to conventional anti-viral drug therapies against diseases such as COVID-19.
The aim of this study was to ascertain whether, like many cell types in cartilaginous tissues if ... more The aim of this study was to ascertain whether, like many cell types in cartilaginous tissues if type XI collagen was a pericellular component of annulus fibrosus (AF) cells and chondrocytes. Fine fibrillar networks were visualised which were perlecan, HS (MAb 10E4) and type XI collagen positive. Heparitinase-III pre-digestion abolished the type XI collagen and 10E4 localisation in these fibrillar assemblies demonstrating a putative HS mediated interaction which localised the type XI collagen. Type XI collagen was confirmed to be present in the Heparitinase III treated AF monolayer media samples by immunoblotting. Heparitinase-III generated ΔHS stub epitopes throughout these fibrillar networks strongly visualised by MAb 3-G-10. Monolayers of murine hip articular chondrocytes from C57BL/6 and Hspg2 exon 3 null mice also displayed pericellular perlecan localisations, however type XI collagen was only evident in the Wild type mice. Perlecan was also immunolocalised in control and murine knee articular cartilage from the two mouse genotypes subjected to a medial meniscal destabilisation procedure which induces OA. This resulted in a severe depletion of perlecan levels particularly in the perlecan exon 3 null mice and was consistent with OA representing a disease of the pericellular matrix. A model was prepared to explain these observations between the NPP type XI collagen domain and HS chains of perlecan domain-I in the pericellular matrix of AF cells which likely contributed to cellular communication, tissue stabilization and the regulation of extracellular matrix homeostasis. Keywords Type XI collagen • Intervertebral disc • Annulus fibrosus • Cell-matrix communication • Heparan sulfate • Perlecan Abbreviations IVD Intervertebral disc AF Annulus fibrosus HS Heparan sulfate
Hyaluronan (HA) binding proteins (HABPs) were localized in cartilaginous ovine tissues (articular... more Hyaluronan (HA) binding proteins (HABPs) were localized in cartilaginous ovine tissues (articular cartilage, intervertebral disc) using a biotinylated HA (bHA) oligosaccharide bioaffinity probe. The bHA oligosaccharide probe was prepared by partial digestion of HA with ovine testicular hyaluronidase, and the oligosaccharides were labeled with biotin hydrazide and purified by a combination of aggrecan G1 domain and avidin affinity chromatography. Hyaladherins were prominently visualized in tissue sections using the bHA oligosaccharide probe as pericellular components in hypertrophic epiphyseal and vertebral growth plate chondrocytes and in the enlarged cells of the cartilaginous end plate of the intervertebral disc. Weaker extracellular staining was also evident in the matrix of the ovine newborn hip and knee joint cartilages. The bHA oligosaccharide probe also visualized intracellular HABPs (IHABPs) in the hypertrophic growth plate chondrocytes of the primary ossification centers. Monolayer cultures of ovine chondrocytes rapidly internalized the bHA oligosaccharide affinity probe to discrete cytoplasmic, nuclear, and perinuclear regions, which were visualized by indirect fluorescent microscopy. This bHA oligosaccharide affinity probe may be useful in future investigations designed to characterize these novel cartilage IHABPs and the role that HA endocytosis plays in cellular regulatory processes in cartilage homeostasis.
The aim of this study was to immunolocalize perlecan in human fetal, postnatal, and mature hyalin... more The aim of this study was to immunolocalize perlecan in human fetal, postnatal, and mature hyaline cartilages and to determine information on the structure and function of chondrocyte perlecan. Perlecan is a prominent component of human fetal (12-14 week) finger, toe, knee, and elbow cartilages; it was localized diffusely in the interterritorial extracellular matrix, densely in the pericellular matrix around chondrocytes, and to small blood vessels in the joint capsules and perichondrium. Aggrecan had a more intense distribution in the marginal regions of the joint rudiments and in para-articular structures. Perlecan also had a strong pericellular localization pattern in postnatal (2-7 month) and mature (55-64 year) femoral cartilages, whereas aggrecan had a prominent extracellular matrix distribution in these tissues. Western blotting identified multiple perlecan core protein species in extracts of the postnatal and mature cartilages, some of which were substituted with heparan sulfate and/or chondroitin sulfate and some were devoid of glycosaminoglycan substitution. Some perlecan core proteins were smaller than intact perlecan, suggesting that proteolytic processing or alternative splicing had occurred. Surface plasmon resonance and quartz crystal microbalance with dissipation experiments demonstrated that chondrocyte perlecan bound fibroblast growth factor (FGF)-1 and-9 less efficiently than endothelial cell perlecan. The latter perlecan supported the proliferation of Baf-32 cells transfected with FGFR3c equally well with FGF-1 and-9, whereas chondrocyte perlecan only supported Baf-32 cell proliferation with FGF-9. The function of perlecan therefore may not be universal but may vary with its cellular origin and presumably its structure.
Journal of Histochemistry & Cytochemistry, 2020
Inter-α-trypsin inhibitor (IαI) family members are ancient and unique molecules that have evolved... more Inter-α-trypsin inhibitor (IαI) family members are ancient and unique molecules that have evolved over several hundred million years of vertebrate evolution. IαI is a complex containing the proteoglycan bikunin to which heavy chain proteins are covalently attached to the chondroitin sulfate chain. Besides its matrix protective activity through protease inhibitory action, IαI family members interact with extracellular matrix molecules and most notably hyaluronan, inhibit complement, and provide cell regulatory functions. Recent evidence for the diverse roles of the IαI family in both biology and pathology is reviewed and gives insight into their pivotal roles in tissue homeostasis. In addition, the clinical uses of these molecules are explored, such as in the treatment of inflammatory conditions including sepsis and Kawasaki disease, which has recently been associated with severe acute respiratory syndrome coronavirus 2 infection in children:
Veterinary and Comparative Orthopaedics and Traumatology, 1994
SummaryCompositional analyses were undertaken on lumbar (L2L3 - L5L6) and lumbosacral (L6S1) inte... more SummaryCompositional analyses were undertaken on lumbar (L2L3 - L5L6) and lumbosacral (L6S1) intervertebral disc tissues from young adult (2-year-old) merino wethers. The proteoglycan level in the nucleus pulposus of the lumbosacral disc was significantly lower than that found in the nucleus pulposus of lumbar levels (p<0.05). The annulus fibrosus was richer in collagen compared to the nucleus pulposus in all discs examined, and the lumbosacral disc consistently had both higher annulus fibrosus and nucleus pulposus collagen levels than lumbar discs (p<0.05). Aggregation of the high buoyant density proteoglycans, with hyaluronic acid, was generally higher in annulus fibrosus proteogly-cans than nucleus pulposus proteogly-cans irrespective of the spinal level examined. Examination of the inter-vertebral disc proteoglycans, by composite agarose polyacrylamide gel electrophoresis, demonstrated three proteoglycan subpopulations and that the nucleus pulposus generally contained a gr...
Loose bodies are fragments of cartilage or bone present in the synovial fluid. In the present stu... more Loose bodies are fragments of cartilage or bone present in the synovial fluid. In the present study we assessed if loose bodies could be used as a source of autologous human chondrocytes for experimental purposes. Histochemical examination of loose bodies and differential enzymatic digestions were undertaken, the isolated cells were cultured in alginate bead microspheres and immunolocalisations were undertaken for chondrogenic markers such as aggrecan, and type II collagen. Isolated loose body cells had high viability (≥90% viable), expressed chondrogenic markers (aggrecan, type II collagen) but no type I collagen. Loose bodies may be a useful source of autologous chondrocytes of high viability.
Abbreviations used AD Alzheimer's diseas AkT a serine/threonine-specific protein kinase, protein ... more Abbreviations used AD Alzheimer's diseas AkT a serine/threonine-specific protein kinase, protein kinase B ALS Amyotrophic lateral sclerosis AT antithrombin BMP bone morphogenetic protein CCL2 CC Motif Chemokine Ligand 2 CNS/PNS central nervous system/peripheral nervous system Cs chitosan CS chondroitin sulphate DS dermatan sulphate ECM Extracellular matrix ERM condensed term based on the first initial of a family of three highly related cytoskeletal proteins, Ezrin, Radixin, Moeisin, FGF fibroblast growth factor GAG Glycosaminoglycan HA hyaluronic acid, hyaluronan HS heparan sulphate IHH indian hedgehog KS keratan sulphate mAb monoclonal antibody Mef2c MADS box transcription enhancer factor 2. MADS is a conserved motif in a family of MADS box transcription factors. MADS is an acronym derived from the first initial from four founding members of this group, MCM1 from Saccharomyces cerevisiae, AGAMOUS from the thale cress Arabidopsis thaliana, DEFICIENS from the snapdragon Antirrhinum majus and SRF from Homo sapiens. NHERF-1 Na + /H + exchanger regulatory factor 1 PDGF platelet derived growth factor PDZ a term derived from the first initials of post-synaptic density protein (PSD95), Drosophila disc large tumour suppressor protein (Dig1), and zona occludens-1 protein. PEDOT /PSS poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) PG proteoglycan PTEN Phosphatidylinositol 3,4,5-trisphosphate 3-phosphatase and dual-specificity protein phosphatase VEGF vascular endothelial cell growth factor Sox SRY-related high mobility group (HMG)-box genes Wnt a condensation term derived from the terms for the Winged and Int genes Electrostimulation, Glycosaminoglycans, cellular regulation and tissue repair
This narrative review highlights the complexities of the gut microbiome and health-promoting prop... more This narrative review highlights the complexities of the gut microbiome and health-promoting properties of prebiotic xylans metabolized by the gut microbiome. In animal husbandry, prebiotic xylans aid in the maintenance of a healthy gut microbiome. This prevents the colonization of the gut by pathogenic organisms obviating the need for dietary antibiotic supplementation, a practice which has been used to maintain animal productivity but which has led to the emergence of antibiotic resistant bacteria that are passed up the food chain to humans. Seaweed xylan-based animal foodstuffs have been developed to eliminate ruminant green-house gas emissions by gut methanogens in ruminant animals, contributing to atmospheric pollution. Biotransformation of pentosan polysulfate by the gut microbiome converts this semi-synthetic sulfated disease-modifying anti-osteoarthritic heparinoid drug to a prebiotic metabolite that promotes gut health, further extending the therapeutic profile and utility ...
This review highlights the multifunctional properties of perlecan (HSPG2) and its potential roles... more This review highlights the multifunctional properties of perlecan (HSPG2) and its potential roles in repair biology. Perlecan is ubiquitous, occurring in vascular, cartilaginous, adipose, lymphoreticular, bone and bone marrow stroma and in neural tissues. Perlecan has roles in angiogenesis, tissue development and extracellular matrix stabilization in mature weight bearing and tensional tissues. Perlecan contributes to mechanosensory properties in cartilage through pericellular interactions with fibrillin-1, type IV, V, VI and XI collagen and elastin. Perlecan domain I - FGF, PDGF, VEGF and BMP interactions promote embryonic cellular proliferation, differentiation, and tissue development. Perlecan domain II, an LDLR-like domain interacts with lipids, Wnt and Hedgehog morphogens. Perlecan domain III binds FGF-7 and 18 and has roles in the secretion of perlecan. Perlecan domain IV, an immunoglobulin repeat domain, has cell attachment and matrix stabilizing properties. Perlecan domain V...
Osteoarthritis is a disease of multifactorial aetiology characterised by progressive breakdown of... more Osteoarthritis is a disease of multifactorial aetiology characterised by progressive breakdown of articular cartilage. In the early stages of the disease, changes become apparent in the superficial zone of articular cartilage, including fibrillation and fissuring. Normally, a monolayer of lubricating molecules is adsorbed on the surface of cartilage and contributes to the minimal friction and wear properties of synovial joints. Proteoglycan 4 is the lubricating glycoprotein believed to be primarily responsible for this boundary lubrication. Here we have used an established ovine meniscectomy model of osteoarthritis, in which typical degenerative changes are observed in the operated knee joints at three months after surgery, to evaluate alterations in proteoglycan 4 expression and localisation in the early phases of the disease. In normal control joints, proteoglycan 4 was immunolocalised in the superficial zone of cartilage, particularly in those regions of the knee joint covered by...
The aim of this study was to highlight the roles of perlecan in the regulation of the development... more The aim of this study was to highlight the roles of perlecan in the regulation of the development of the rudiment developmental cartilages and growth plate cartilages, and also to show how perlecan maintains permanent articular cartilage homeostasis. Cartilage rudiments are transient developmental templates containing chondroprogenitor cells that undergo proliferation, matrix deposition, and hypertrophic differentiation. Growth plate cartilage also undergoes similar changes leading to endochondral bone formation, whereas permanent cartilage is maintained as an articular structure and does not undergo maturational changes. Pericellular and extracellular perlecan-HS chains interact with growth factors, morphogens, structural matrix glycoproteins, proteases, and inhibitors to promote matrix stabilization and cellular proliferation, ECM remodelling, and tissue expansion. Perlecan has mechanotransductive roles in cartilage that modulate chondrocyte responses in weight-bearing environment...
Cartilage regeneration requires a balance of anabolic and catabolic processes. This study examine... more Cartilage regeneration requires a balance of anabolic and catabolic processes. This study examined the susceptibility of fibromodulin (FMOD) and lumican (LUM) to degradation by MMP-13, ADAMTS-4 and ADAMTS-5, the three major degradative proteinases in articular cartilage in osteoarthritis (OA). Immunolocalisation of FMOD and LUM in foot sections of developmental cartilages demonstrated prominent localisations in metatarsal and phalangeal foetal rudiment cartilages and growth plate. An MMP-13 neoepitope antibody (TsYG11) demonstrated localisation of MMP-13 cleaved FMOD in the hypertrophic chondrocytes of the metatarsal growth plate. FMOD was more prominently localised in the superficial cartilage of normal and fibrillated zones in OA cartilage, TsYG11 positive FMOD was located deeper in the cartilage samples. Ab TsYG11 also identified FMOD fragmentation in Western blots of extracts of normal and fibrillated cartilage and total knee replacement OA cartilage. The C-terminal anti-F...
The aim of the present study was to examine the roles of l-fucose and the glycosaminoglycans (GAG... more The aim of the present study was to examine the roles of l-fucose and the glycosaminoglycans (GAGs) keratan sulfate (KS) and chondroitin sulfate/dermatan sulfate (CS/DS) with selected functional molecules in neural tissues. Cell surface glycans and GAGs have evolved over millions of years to become cellular mediators which regulate fundamental aspects of cellular survival. The glycocalyx, which surrounds all cells, actuates responses to growth factors, cytokines and morphogens at the cellular boundary, silencing or activating downstream signaling pathways and gene expression. In this review, we have focused on interactions mediated by l-fucose, KS and CS/DS in the central and peripheral nervous systems. Fucose makes critical contributions in the area of molecular recognition and information transfer in the blood group substances, cytotoxic immunoglobulins, cell fate-mediated Notch-1 interactions, regulation of selectin-mediated neutrophil extravasation in innate immunity and CD-34-m...
Uploads
Papers by James Melrose