PubMed Health⌕ Search

Biomedical subjects

Peter Roughley

Publications and source records attributed to Peter Roughley.

9 recordsLinked to original sources
PubMed2006

Age-related accumulation of pentosidine in aggrecan and collagen from normal and degenerate human intervertebral discs.

During aging and degeneration, many changes occur in the structure and composition of human cartilaginous tissues, which include the accumulation of the AGE (advanced glycation end-product), pentosidine, in long-lived proteins. In the present study, we investigated the accumulation of pentosidine in constituents of the human IVD (intervertebral disc), i.e. collagen, aggrecan-derived PG (proteoglycan) (A1) and its fractions (A1D1-A1D6) in health and pathology. We found that, after maturity, pentosidine accumulates with age. Over the age range studied, a linear 6-fold increase was observed in pentosidine accumulation for A1 and collagen with respective rates of 0.12 and 0.66 nmol x (g of protein)(-1) x year(-1). Using previously reported protein turnover rate constants (k(T)) obtained from measurements of the D-isomer of aspartic residue in collagen and aggrecan of human IVD, we could calculate the pentosidine formation rate constants (k(F)) for these constituents [Sivan, Tsitron, Wachtel, Roughley, Sakkee, van der Ham, DeGroot, Roberts and Maroudas (2006) J. Biol. Chem. 281, 13009-13014; Tsitron (2006) MSc Thesis, Technion-Israel Institute of Technology, Haifa, Israel]. In spite of the comparable formation rate constants obtained for A1D1 and collagen [1.81+/-0.25 compared with 3.71+/-0.26 micromol of pentosidine x (mol of lysine)(-1) x year(-1) respectively], the higher pentosidine accumulation in collagen is consistent with its slower turnover (0.005 year(-1) compared with 0.134 year(-1) for A1D1). Pentosidine accumulation increased with decreasing buoyant density and decreasing turnover of the proteins from the most glycosaminoglycan-rich PG components (A1D1) to the least (A1D6), with respective k(F) values of 1.81+/-0.25 and 3.18+/-0.37 micromol of pentosidine.(mol of lysine)(-1) x year(-1). We concluded that protein turnover is an important determinant of pentosidine accumulation in aggrecan and collagen of human IVD, as was found for articular cartilage. Correlation of pentosidine accumulation with protein half-life in both normal and degenerate discs further supports this finding.

Adult↗
PubMed2006

The structure, location, and function of perlecan, a prominent pericellular proteoglycan of fetal, postnatal, and mature hyaline cartilages.

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.

Age Factors↗
PubMed2006

Degradation of small leucine-rich repeat proteoglycans by matrix metalloprotease-13: identification of a new biglycan cleavage site.

A major and early feature of cartilage degeneration is proteoglycan breakdown. Matrix metalloprotease (MMP)-13 plays an important role in cartilage degradation in osteoarthritis (OA). This MMP, in addition to initiating collagen fibre cleavage, acts on several proteoglycans. One of the proteoglycan families, termed small leucine-rich proteoglycans (SLRPs), was found to be involved in collagen fibril formation/interaction, with some members playing a role in the OA process. We investigated the ability of MMP-13 to cleave members of two classes of SLRPs: biglycan and decorin; and fibromodulin and lumican. SLRPs were isolated from human normal and OA cartilage using guanidinium chloride (4 mol/l) extraction. Digestion products were examined using Western blotting. The identities of the MMP-13 degradation products of biglycan and decorin (using specific substrates) were determined following electrophoresis and microsequencing. We found that the SLRPs studied were cleaved to differing extents by human MMP-13. Although only minimal cleavage of decorin and lumican was observed, cleavage of fibromodulin and biglycan was extensive, suggesting that both molecules are preferential substrates. In contrast to biglycan, decorin and lumican, which yielded a degradation pattern similar for both normal and OA cartilage, fibromodulin had a higher level of degradation with increased cartilage damage. Microsequencing revealed a novel major cleavage site (... G177/V178) for biglycan and a potential cleavage site for decorin upon exposure to MMP-13. We showed, for the first time, that MMP-13 can degrade members from two classes of the SLRP family, and identified the site at which biglycan is cleaved by MMP-13. MMP-13 induced SLRP degradation may represent an early critical event, which may in turn affect the collagen network by exposing the MMP-13 cleavage site in this macromolecule. Awareness of SLRP degradation products, especially those of biglycan and fibromodulin, may assist in early detection of OA cartilage degradation.

Adult↗
PubMed2006

Limitations of using aggrecan and type X collagen as markers of chondrogenesis in mesenchymal stem cell differentiation.

The study was initially designed to differentiate human bone marrow-derived mesenchymal stem cells (MSC) into chondrocyte-like cells, for use in tissue engineering. We cultured MSCs in defined chondrogenic medium as pellet cultures supplemented with transforming growth factor (TGF)-beta1 or -beta3 and dexamethazone, as they are commonly used to promote in vitro chondrogenesis. Markers of chondrogenesis used were type II collagen and aggrecan, with type X collagen being used as a marker of late-stage chondrocyte hypertrophy (associated with endochondral ossification). Our results show that aggrecan is constitutively expressed by MSCs and that type X collagen is expressed as an early event. Furthermore, we found that type X collagen was expressed before type II collagen in some cases. This is surprising because it is understood that stem cells have to be differentiated into chondrocytes before they can become hypertrophic. Thus, caution must be exercised when using aggrecan and type X collagen as markers for chondrogenesis and chondrocyte hypertrophy, respectively, in association with stem cell differentiation from this source.

Aggrecans↗
PubMed2005

The potential of chitosan-based gels containing intervertebral disc cells for nucleus pulposus supplementation.

The suitability of chitosan-based hydrogels as scaffolds for the encapsulation of intervertebral disc (IVD) cells and the accumulation of a functional extracellular matrix mimicking that of the nucleus pulposus (NP) was investigated. The specific hypothesis under study was that the cationic chitosan would form an ideal environment in which large quantities of newly synthesized anionic proteoglycan could be entrapped. Indeed, all the formulations of cell-seeded chitosan hydrogels, studied under in vitro culture conditions, showed that the majority of proteoglycan produced by encapsulated NP cells was retained within the gel rather than released into the culture medium. This was not always the case when annulus fibrosus cells were encapsulated, as unlike the nucleus cells the annulus cells often did not survive when cultured in chitosan. The results support the concept that chitosan may be a suitable scaffold for cell-based supplementation to help restore the function of the NP during the early stages of IVD degeneration.

Animals↗
PubMed2005

Airway proteoglycans are differentially altered in fatal asthma.

It has been suggested that airway remodelling is responsible for the persistent airway obstruction and decline in lung function observed in some asthmatic patients. The small airways are thought to contribute significantly to this functional impairment. Proteoglycans (PGs) are important components of the extracellular matrix (ECM) in the lungs. Besides controlling biophysical properties of the ECM, they play important roles in the regulation of some cytokines. Increased subepithelial PG deposition in the airways of mild asthmatics has been reported. However, there are no data on the PG content in small airways in asthma. This study has compared the content and distribution of PGs in large and small airways of patients who died of asthma with those in control lungs. Immunohistochemistry and image analysis were used to determine the content of lumican, decorin, biglycan, and versican in large (internal perimeter >6 mm) and small (internal perimeter < or =6 mm) airways of 18 patients who had died of asthma (A) and ten controls (C). The results were expressed as PG area (microm2)/epithelial basement membrane length (microm). The main differences between asthmatics and controls were observed in the small airways. There was a significant decrease in decorin and lumican contents in the external area of small airways in asthmatics (decorin: A = 1.05 +/- 0.27 microm, C = 3.97 +/- 1.17 microm, p = 0.042; lumican: A = 1.97 +/- 0.37 microm, C = 5.66 +/- 0.99 microm, p = 0.002). A significant increase in versican content in the internal area of small and large airways in asthmatics was also observed (small: A = 7.48 +/- 0.84 microm, C = 5.16 +/- 0.61 microm, p = 0.045; large: A = 18.38 +/- 1.94 microm, C = 11.90 +/- 2.86 microm, p = 0.028). The results show that PGs are differentially expressed in the airways of fatal asthma and may contribute to airway remodelling. These data reinforce the importance of the small airways in airway remodelling in asthma.

Adolescent↗
PubMed2003

A synthetic peptide of link protein stimulates the biosynthesis of collagens II, IX and proteoglycan by cells of the intervertebral disc.

To date, there have been no reports on the effect on disc cells of the intervertebral disc (IVD) of the amino terminal peptide of link protein (DHLSDNYTLDHDRAIH) (link N) which is generated by the cleavage of human link protein by stromelysins 1 and 2, gelatinase A and B, and collagenase between His(16) and Ile(17). However, link N has been shown to act as a growth factor and stimulate synthesis of proteoglycans and collagen by chondrocytes of human articular cartilage. There are also no studies on the effect of link N on type IX collagen in any tissue. In the studies reported here, a serum-free pellet culture system has been used to examine whether link N can play a role in maintaining the integrity of disc matrix, specifically at the level of matrix assembly by cells of the IVD. Using this culture system, we determined the capacity of link N to stimulate accumulation of these matrix proteins in the annulus fibrosus (AF) and nucleus pulposus (NP). Gross inspection of separate AF and NP pellet cultures in the absence of link N revealed a progressive increase in size and a transition from "spherical" to "polygonal" pellets after centrifugation. Addition of 10 ng/ml link N resulted in increased pellet sizes for both AF and NP pellet cultures. Link N increased proteoglycan, type II and type IX collagen contents with an increase in DNA content over time. This study demonstrates that link N can act directly on disc cells to stimulate matrix production, which involves increased accumulation of proteoglycan, and types II and IX collagens. This study also identifies the value of pellet cultures for studies of the IVD cells in a serum-free chemically defined medium, in which pellets can continue growing in size in response to growth factors with minimal cell loss. Link N may have value in stimulating the growth and regeneration of the damaged IVD.

Amino Acid Sequence↗
PubMedDate not supplied

Biological evaluation of chitosan salts cross-linked to genipin as a cell scaffold for disk tissue engineering.

Degenerative disc disease has been implicated as a major component of spine pathology. However, although biological repair of the degenerate disk would be the ideal treatment, there is no universally accepted scaffold for tissue engineering of the intervertebral disk. To help remedy this, we investigated the gelation kinetics of various concentrations (2.5 to 10%) of two water-soluble chitosan chlorides (low molecular weight Protasan UP CL113 and high molecular weight Protasan UP CL213) and two chitosan glutamates (low molecular weight Protasan UP G113 and high molecular weight Protasan UP G213). Various concentrations (5 to 20%) of genipin, a naturally occurring cross-linking reagent used in herbal medicine and in the fabrication of food dyes, were used to prepare cross-linked chitosan hydrogels. The results show that 2.5% Protasan UP G213 cross-linked to 5% genipin was the best candidate. This formulation gelled fastest at 37 degrees C, and maintained 95% viability of encapsulated cultured disk cells. The gel did not produce an inflammatory reaction when injected subcutaneously into C57BL/6 mice and is therefore biocompatible. Most importantly, when injected into the degenerated nucleus pulposus of human cadaveric intervertebral disk, the gel flowed into the clefts without leakage. This study demonstrates that 2.5% Protasan UP G213 cross-linked to 5% genipin might be a promising scaffold for disk tissue engineering.

Aged↗