Nasal septum deviation as the consequence of BMP-controlled changes to cartilage properties
ABSTRACT: Nasal septum deviation (NSD) is a common abnormality of the septal cartilage often associated with disordered breathing. The etiology of NSD is unknown. BMP7 neural crest-knockout mice, a well-characterized model for midfacial hypoplasia and nasal airway obstruction, develops a deviated septum by 4 weeks of age. Using comparative gene expression, quantitative proteomics, and immunofluorescence analysis we investigated the septum prior, immediately preceding, and with established deviation. We provide a detailed description of cellular and molecular changes leading to septum deviation, including changes to extracellular matrix organization, cell metabolism, and chondrocyte differentiation. Loss of BMP7 was associated with acquisition of elastic cartilage properties, a switch to glucose metabolism, and molecular characteristics commonly associated with osteoarthritis (OA). Many alterations preceded the deviation establishing that molecular changes to chondrocyte properties predispose to the deviation. Interestingly, NSD was associated with a persistent increase in BMP2. Genetic reduction of BMP2 in BMP7ncko mice restores WNT signalling, energy metabolism, and rescues NSD, showing the importance of balanced BMP signaling for normal cartilage. Many of the cellular and molecular changes have been described for knee osteoarthritis, suggesting that these two pathologies might have a similar underlying etiology.
Project description:Background:In this study, we manufactured a complex of human nasal septal cartilage (hNC) with polycaprolactone (PCL) for transplantation into cartilaginous skeletal defects and evaluated their characteristics. Methods:Nasal septum tissue was obtained from five patients aged ??20 years who were undergoing septoplasty. hNCs were isolated and subcultured for three passages in vitro. To formulate the cell-PCL complex, we used type I collagen as an adhesive between chondrocyte and PCL. Immunofluorescence staining, cell viability and growth in the hNC-PCL complex, and mycoplasma contamination were assessed. Results:hNCs in PCL showed viability ??70% and remained at these levels for 9 h of incubation at 4 °C. Immunostaining of the hNC-PCL complex also showed high expression levels of chondrocyte-specific protein, COL2A1, SOX9, and aggrecan during 24 h of clinically applicable conditions. Conclusion:The hNC-PCL complex may be a valuable therapeutic agent for implantation into injured cartilage tissue, and can be used clinically to repair cartilaginous skeletal defects. From a clinical perspective, it is important to set the short duration of the implantation process to achieve effective functional implantation.
Project description:1. UDP-glucose dehydrogenase has been partially purified from sheep nasal septum cartilage, neonatal rat skin and bovine corneal epithelium. 2. The pH profile, K(m) values for NAD(+) and UDP-glucose, activation energy and molecular weight have been determined for the enzyme from several of the tissues. 3. The sugar nucleotide concentrations in each of the tissues have been related to the spectrum of glycosaminoglycans produced by each tissue. 4. The presence of an allosteric UDP-xylose-binding site distinct from the active site(s) in sheep nasal septum UDP-glucose dehydrogenase has been demonstrated. 5. An active UDP-glucuronic acid nucleotidase has been demonstrated in sheep nasal cartilage. 6. Tissue-space experiments have shown the cell water content of sheep nasal septum cartilage to be 14% of the wet weight. 7. Glucuronic acid 1-phosphate does not occur in measurable amounts in sheep nasal septum cartilage and no UDP-glucuronic acid pyrophosphorylase activity could be detected in this tissue. 8. The inhibition by UDP-xylose with respect to both substrates, UDP-glucose and NAD(+), has been examined, and shown to be allosteric.
Project description:Genetic studies and molecular cloning approaches have been successfully used to identify several transcription factors that regulate the numerous stages of cartilage development. Sex-determining region Y (SRY)-box 9 (Sox9) is an essential transcription factor for the initial stage of cartilage development. Sox5 and Sox6 play an important role in the chondrogenic action of Sox9, presumably by defining its cartilage specificity. Several transcription factors have been identified as transcriptional partners for Sox9 during cartilage development. Runt-related transcription factor 2 (Runx2) and Runx3 are necessary for hypertrophy of chondrocytes. CCAAT/enhancer-binding protein ? (C/EBP?) and activating transcription factor 4 (ATF4) function as co-activators for Runx2 during hypertrophy of chondrocytes. In addition, myocyte-enhancer factor 2C (Mef2C) is required for initiation of chondrocyte hypertrophy, presumably by functioning upstream of Runx2. Importantly, the pathogenic roles of several transcription factors in osteoarthritis have been demonstrated based on the similarity of pathological phenomena seen in osteoarthritis with chondrocyte hypertrophy. We discuss the importance of investigating cellular and molecular properties of articular chondrocytes and degradation mechanisms in osteoarthritis, one of the most common cartilage diseases.
Project description:While glucocorticoids have been used for over 50 years to treat rheumatoid and osteoarthritis pain, the prescription of glucocorticoids remains controversial because of potentially harmful side effects at the molecular, cellular and tissue levels. One member of the glucocorticoid family, dexamethasone (DEX) has recently been demonstrated to rescue cartilage matrix loss and chondrocyte viability in animal studies and cartilage explant models of tissue injury and post-traumatic osteoarthritis, suggesting the possibility of DEX as a disease-modifying drug if used appropriately. However, the literature on the effects of DEX on cartilage reveals conflicting results on the drug's safety, depending on the dose and duration of DEX exposure as well as the model system used. Overall, DEX has been shown to protect against arthritis-related changes in cartilage structure and function, including matrix loss, inflammation and cartilage viability. These beneficial effects are not always observed in model systems using initially healthy cartilage or isolated chondrocytes, where many studies have reported significant increases in chondrocyte apoptosis. It is crucially important to understand under what conditions DEX may be beneficial or harmful to cartilage and other joint tissues and to determine potential for safe use of this glucocorticoid in the clinic as a disease-modifying drug.
Project description:Osteoarthritis presents as a change in the chondrocyte phenotype and an imbalance between anabolic and catabolic processes. Age affects its onset and progression. Small nucleolar RNAs (SnoRNAs) direct chemical modification of RNA substrates to fine-tune spliceosomal and rRNA function, accommodating changing requirements for splicing and protein synthesis during health and disease. Articular cartilage from young, old and OA knees was used in a microarray study to identify alterations in snoRNA expression. Changes in snoRNAs in osteoarthritis-like conditions were studied in chondrocytes using interleukin-1 and osteoarthritic synovial fluid. SNORD26 and SNORD96A knockdown and overexpression were undertaken using antisense oligonucleotides and overexpression plasmids. We identified panels of snoRNAs differentially expressed due to ageing (including SNORD96A, SNORD44) and osteoarthritis (including SNORD26 and SNORD116). In vitro experiments using osteoarthritis-like conditions affected snoRNA expression. Knockdown or overexpression of SNORD26 or SNORD96A resulted in changes in chondrogenic, hypertrophic, rRNA and osteoarthritis related gene expression. We demonstrate that snoRNA expression changes in cartilage ageing, and osteoarthritis and in osteoarthritis-like conditions, and when the expression of these snoRNAs is altered this affects chondrogenic and hypertrophic gene expression. Thus, we propose an additional dimension in the molecular mechanisms underlying cartilage ageing and osteoarthritis through the dysregulation of snoRNAs.
Project description:Regulation of transcription occurs in a cell type specific manner orchestrated by epigenetic mechanisms including DNA methylation. Methylation changes may also play a key role in lineage specification during stem cell differentiation. To further our understanding of epigenetic regulation in chondrocytes we characterised the DNA methylation changes during chondrogenesis of mesenchymal stem cells (MSCs) by Infinium 450?K methylation array. Significant DNA hypomethylation was identified during chondrogenic differentiation including changes at many key cartilage gene loci. Integration with chondrogenesis gene expression data revealed an enrichment of significant CpGs in upregulated genes, while characterisation of significant CpG loci indicated their predominant localisation to enhancer regions. Comparison with methylation profiles of other tissues, including healthy and diseased adult cartilage, identified chondrocyte-specific regions of hypomethylation and the overlap with differentially methylated CpGs in osteoarthritis. Taken together we have associated DNA methylation levels with the chondrocyte phenotype. The consequences of which has potential to improve cartilage generation for tissue engineering purposes and also to provide context for observed methylation changes in cartilage diseases such as osteoarthritis.
Project description:Midface dysgenesis is a feature of more than 200 genetic conditions in which upper airway anomalies frequently cause respiratory distress, but its etiology is poorly understood. Mouse models of Apert and Crouzon craniosynostosis syndromes exhibit midface dysgenesis similar to the human conditions. They carry activating mutations of Fgfr2, which is expressed in multiple craniofacial tissues during development. Magnetic resonance microscopy of three mouse models of Apert and Crouzon syndromes revealed decreased nasal passage volume in all models at birth. Histological analysis suggested overgrowth of the nasal cartilage in the two Apert syndrome mouse models. We used tissue-specific gene expression and transcriptome analysis to further dissect the structural, cellular and molecular alterations underlying midface and upper airway dysgenesis in Apert Fgfr2+/S252W mutants. Cartilage thickened progressively during embryogenesis because of increased chondrocyte proliferation in the presence of Fgf2 Oral epithelium expression of mutant Fgfr2, which resulted in a distinctive nasal septal fusion defect, and premature facial suture fusion contributed to the overall dysmorphology. Midface dysgenesis in Fgfr2-related craniosynostosis is a complex phenotype arising from the combined effects of aberrant signaling in multiple craniofacial tissues.
Project description:Although regeneration of human cartilage is inherently inefficient, age is an important risk factor for Osteoarthritis (OA). Recent reports have provided compelling evidence that juvenile chondrocytes (from donors below 13 years of age) are more efficient at generating articular cartilage as compared to adult chondrocytes. However, the molecular basis for such a superior regenerative capability is not understood. In order to identify the cell-intrinsic differences between juvenile and adult cartilage, we have systematically profiled global gene expression changes between a small cohort of human neonatal/juvenile and adult chondrocytes. No such study is available for human chondrocytes although ‘young’ and ‘old’ bovine and equine cartilage have been recently profiled. Overall design: 3 juvenile chondrocyte samples and 2 adult chondrocyte samples were profiled. Bone-marrow derived mesenchymal cells (MSC) from 2 donors were profiled. We analyzed the changes in gene expression; genes with a fold-change ≥ or ≤1.5, with a difference in intensity of >100 and within the lower 90% confidence bound were selected.
Project description:Although pathways controlling ribosome activity have been described to regulate chondrocyte homeostasis in osteoarthritis, ribosome biogenesis in osteoarthritis is unexplored. We hypothesized that U3 snoRNA, a non-coding RNA involved in ribosomal RNA maturation, is critical for chondrocyte protein translation capacity in osteoarthritis. U3 snoRNA was one of a number of snoRNAs with decreased expression in osteoarthritic cartilage and osteoarthritic chondrocytes. OA synovial fluid impacted U3 snoRNA expression by affecting U3 snoRNA gene promoter activity, while BMP7 was able to increase its expression. Altering U3 snoRNA expression resulted in changes in chondrocyte phenotype. Interference with U3 snoRNA expression led to reduction of rRNA levels and translational capacity, whilst induced expression of U3 snoRNA was accompanied by increased 18S and 28S rRNA levels and elevated protein translation. Whole proteome analysis revealed a global impact of reduced U3 snoRNA expression on protein translational processes and inflammatory pathways. For the first time we demonstrate implications of a snoRNA in osteoarthritis chondrocyte biology and investigated its role in the chondrocyte differentiation status, rRNA levels and protein translational capacity.
Project description:Here, we show the role of melatonin combined with or without exercise as a determinant of multicellular behavior in osteoarthritis. We address the relationship between the molecular components governing local circadian clock and changes in the osteoarthritic musculoskeletal axis. Melatonin was injected subcutaneously in animals with advanced knee osteoarthritis (OA) for 4 weeks. Concurrently, moderate treadmill exercise was applied for 30 min/day. Morphometric, histological, and gene/protein-level analyses were performed in the cartilage, synovium, bone, and gastrocnemius muscle. Primary cultured chondrocytes repeatedly exposed to TNF-? were used in an in vitro study. The symptoms of OA include gait disturbance, osteophyte formation, and abnormal metabolism of the extracellular matrix (ECM) of the cartilage. Low-level expression of clock genes was accompanied by aberrant changes in cartilage specimens. Nanomolar doses of melatonin restored the expression of clock-controlled genes and corrected the abnormal chondrocyte phenotype. Melatonin combined with or without exercise prevented periarticular muscle damage as well as cartilage degeneration. But prolonged melatonin administration promoted the proteolytic cleavage of RANKL protein in the synovium, leading to severe subchondral bone erosion. These musculoskeletal changes apparently occurred via the regulation of molecular clock components, suggesting a role of melatonin as a switch-like regulator for the OA phenotype.