Objective. Intervertebral disc degeneration is linked to loss of extracellular matrix (ECM), particularly the early loss of aggrecan. A group of metalloproteinases called aggrecanases are important mediators of aggrecan turnover. The present study was undertaken to investigate the expression of the recognized aggrecanases and their inhibitor, tissue inhibitor of metalloproteinases 3 (TIMP-3), in human intervertebral disc tissue.Methods. Twenty-four nondegenerated and 30 degenerated disc samples were analyzed for absolute messenger RNA (mRNA) copy number of ADAMTS 1, 4, 5, 8, 9, and 15 and TIMP-3 by real-time reverse transcription-polymerase chain reaction. Thirty-six formalin-fixed embedded intervertebral disc samples of varying grades of degeneration were used for immunohistochemical analyses. In addition, samples from 8 subjects were analyzed for the presence of matrix metalloproteinase (MMP)-and aggrecanase-generated aggrecan products.Results. Messenger RNA for all the aggrecanases other than ADAMTS-8 was identified in intervertebral disc tissue, as was mRNA for TIMP-3. Levels of mRNA expression of ADAMTS 1, 4, 5, and 15 were significantly increased in degenerated tissue compared with nondegenerated tissue. All these aggrecanases and TIMP-3 were also detected immunohistochemically in disc tissue, and numbers of nucleus pulposus cells staining positive for ADAMTS 4, 5, 9, and 15 were significantly increased in degenerated tissue compared with nondegenerated tissue. Aggrecan breakdown products generated by MMP and aggrecanase activities were also detected in intervertebral disc tissue.Conclusion. The aggrecanases ADAMTS 1, 4, 5, 9, and 15 may contribute to the changes occurring in the ECM during intervertebral disc degeneration. Targeting these enzymes may be a possible future therapeutic strategy for the prevention of intervertebral disc degeneration and its associated morbidity.Chronic low back pain affects Ͼ70% of people at some point in their lives (1), with ϳ10% being chronically disabled. The causes of low back pain are multifactorial, although ϳ40% of all cases involve degeneration of the intervertebral discs (2). During degeneration, the matrix of the intervertebral disc undergoes structural, mechanical, and molecular changes resulting in a loss of demarcation between the outer annulus fibrosus and the inner nucleus pulposus. Additionally, alterations in collagen type and a decrease in proteoglycan content result in loss of tissue integrity, decreased hydration, and inability to withstand load (3). Importantly, the loss of proteoglycan, predominantly aggrecan, is considered to be an early indicator of intervertebral disc degeneration (4). Aggrecan molecules possess long core proteins with many chondroitin sulfate (CS) and keratan sulfate (KS) glycosaminoglycan (GAG) side chains (3). These GAG side chains are polyanionic due to the high content of carboxyl and sulfate groups, and thus they attract and bind water molecules, hydrating the tissue. Degradation
We report a detailed analysis of heparan sulfate (HS) structure using a model of human colon carcinogenesis. Metabolically radiolabeled HS was isolated from adenoma and carcinoma cells. The chain length of HS was the same in both cell populations (M r 20,000; 45-50 disaccharides), and the chains contained on average of two sulfated domains (S domains), identified by heparinase I scission. This enzyme produced fragments of approximate size 7 kDa, suggesting that the S domains were evenly spaced in the intact HS chain. The degree of polymer sulfation and the patterns of sulfation were strikingly different between the two HS species. When compared with adenoma HS, the iduronic acid 2-O-sulfate content of the carcinoma-derived material was reduced by 33%, and the overall level of N-sulfation was reduced by 20%. However, the level of 6-O-sulfation was increased by 24%, and this was almost entirely attributable to an enhanced level of N-sulfated glucosamine 6-Osulfate, a species whose data implied was mainly located in the mixed sequences of alternating N-sulfated and N-acetylated disaccharides. The results indicate that in the transition to malignancy in human colon adenoma cells, the overall molecular organization of HS is preserved, but there are distinct modifications in both the S domains and their flanking mixed domains that may contribute to the aberrant behavior of the cancer cell.Heparan sulfate is a widespread complex linear polysaccharide that consists of alternate hexuronic acid and N-substituted glucosamine residues. The ability to bind protein effectors such as growth factors or protease inhibitors (e.g. antithrombin III; Refs. 1 and 2) is strongly influenced by the position and density of sulfate residues that occur most frequently as N-sulfates but are also present as sulfate esters at the 6-O-(or less commonly the 3-O-) position of glucosamine or the 2-O-position of iduronic acid (3). The complexity of HS is achieved through the location of sulfate groups in domains of high and low sulfation (4), the conformational flexibility of iduronate residues (5, 6), and the degree of polymorphism manifest in different tissues (7-9). The strongly anionic zones of HS, 1 the S domains, consist of contiguous glucosamine N-sulfate-containing disaccharides that bear a variable number of O-sulfate moieties. Domains of less sulfated sequences called mixed sequences are believed to flank the S domains, separating them from the unsulfated domains, and these are liberated as tetrasaccharides by low pH nitrous acid, a reagent that cleaves HS at N-sulfated glucosaminecontaining disaccharides (i.e. GlcNSO 3 -␣1-4-hexuronic acid). The ability of HS to act as a growth factor activator (10 -13) and as a component of focal adhesions (14) has focused attention on this molecule as a potential therapeutic target in diseases of aberrant cellular growth or migration such as cancer, diabetic retinopathy, or coronary arterial restenosis. A number of studies have investigated the structural changes in HS in animal models of malignancy w...
IntroductionMucopolysaccharidosis type IH (MPS-IH, Hurler syndrome) is an autosomal recessive disorder resulting from defects in the gene encoding the lysosomal enzyme ␣-L-iduronidase (IDUA). This leads to ineffective degradation of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate. Individuals with very low levels of IDUA present in infancy and early childhood as a consequence of the deleterious accumulation of these GAGs in different organ systems, including the central nervous system, reticuloendothelial system, and the skeleton. Such severely affected patients usually die within the first decade. 1,2 Current therapy for MPS-IH focuses on allogeneic bone marrow transplantation from an unaffected, HLA-compatible donor. This provides normal, enzyme-competent leukocytes that secrete IDUA that can be taken up by enzyme-deficient cells via mannose-6-phosphate receptors. 3 The utility of this approach is significantly limited by the availability of donors and significant toxicity of the intense immunosuppressive conditioning therapy that the recipient requires for donor hemopoiesis to become established without rejection. Even where donor hemopoiesis is fully established (ie, all hemopoietic cells have normal enzyme levels), symptoms (particularly defects in the skeleton and central nervous system) are incompletely and variably corrected. 4,5 Mesenchymal stem cells (MSCs) are multipotent progenitors that can be isolated from bone marrow and are capable of contributing to multiple mesenchymal tissues in vivo. [6][7][8][9][10] In this paper we demonstrate, for the first time, retroviral gene transfer leading to correction of these MSCs in an inherited disorder. Furthermore, there is maintenance of the proliferative and multilineage differentiation potential of these modified cells, and they are able to cross-correct non-gene-modified cells.Numerous studies have demonstrated the presence of donor mesenchymal cells in multiple tissues following transplantation, and MSCs injected into brain are able to differentiate into nerve cells. Taken with these, our data indicate that MSCs may prove a better target than hematopoietic stem cells in the context of gene therapy of multisystem, lysosomal storage disorders. Study design Isolation and culture of MSCsBone marrow samples were obtained from MPS-IH patients and unaffected individuals aged from 0 to 18 years, following informed parental consent and approval from the local research ethics committee. MSCs were isolated and cultured as previously described. 11 For differentiation assays, cells were plated at 5 ϫ 10 3 per well in 6-well plates in growth medium with either osteogenic 12 or adipogenic 13 supplements. For differentiation along the neuronal lineage, cells were preincubated for 24 hours in Dulbecco modified Eagle medium/20% fetal calf serum/1 mM -mercapotethanol and then switched into Dulbecco modified Eagle medium/5 mM -mercapotethanol. 14 Mineralized bone was stained by the von Kossa technique, 15 and adipocytes were stained using oil-red-O. 16 N...
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