Characterisation of key proteoglycans in the cranial cruciate ligaments (CCLs) from two dog breeds with different predispositions to CCL disease and rupture.

Cranial cruciate ligament disease and rupture (CCLD/R) is one of the most common orthopaedic conditions in dogs, eventually leading to osteoarthritis of the stifle joint. Certain dog breeds such as the Staffordshire bull terrier have an increased risk of developing CCLD/R. Previous studies into CCLD/R have found that glycosaminoglycan levels were elevated in cranial cruciate ligament (CCL) tissue from high-risk breeds when compared to the CCL from a low-risk breed to CCLD/R. Our objective was to determine specific proteoglycans/glycosaminoglycans in the CCL and to see whether their content was altered in dog breeds with differing predispositions to CCLD/R. Disease-free CCLs from Staffordshire bull terriers (moderate/high-risk to CCLD/R) and Greyhounds (low-risk to CCLD/R) were collected and key proteoglycan/glycosaminoglycans were determined by semi-quantitative Western blotting, quantitative biochemistry, quantitative reverse transcription polymerase chain reaction, and immunohistochemistry. Gene expression of fibromodulin (P = 0.03), aggrecan (P = 0.0003), and chondroitin-6-sulphate stubs (P = 0.01) were significantly increased, and for fibromodulin this correlated with an increase in protein content in Staffordshire bull terriers compared to Greyhound CCLs (P = 0.02). Decorin (P = 0.03) and ADAMTS-4 (P = 0.04) gene expression were significantly increased in Greyhounds compared to Staffordshire bull terrier CCLs. The increase of specific proteoglycans and glycosaminoglycans within the Staffordshire bull terrier CCLs may indicate a response to higher compressive loads, potentially altering their risk to traumatic injury. The higher decorin content in the Greyhound CCLs is essential for maintaining collagen fibril strength, while the increase of ADAMTS-4 indicates a higher rate of turnover helping to regulate normal CCL homeostasis in Greyhounds.

Insulin-like growth factor binding protein (IGFBP6) is a cross-species tendon marker.

The main challenge in tendon injury management is suboptimal tissue healing that fails to re-establish original tendon function. Tissue bioengineering is a promising approach for tendon therapy, with potential to improve its functional outcomes. However, evaluation criteria for tissue-engineered tendon are unclear due to the lack of specific markers of differentiated tendon. The study aim was to identify a panel of genes that characterised tendons in comparison to cartilage or muscles and validate those genes, both in human and key species used as models for tendon diseases. Gene expression profiling of rat tendon and cartilage in whole-tissue samples and primary tenocytes and chondrocytes was undertaken using two independent microarray platforms. Genes that demonstrated high expression correlation across two assays were validated by qRT-PCR in rat tendon relative to cartilage and muscle. Five genes demonstrating the highest tendon-related expression in the validation experiment (ASPN, ECM1, IGFBP6, TNMD, THBS4) were further evaluated by qRT-PCR in ovine, equine and human tissue. The group of tendon markers, identified by unbiased transcriptomic analysis of rat musculoskeletal tissues, demonstrated species-dependent profiles of expression. Insulin-like growth factor binding protein 6 (IGFBP6) was identified as the only universal tendon marker. Further investigation in equine tendon showed that IGFBP6 expression was not affected by ageing or tendon function but decreased in anatomical regions subjected to elevated compressive force. IGFBP6 is a robust cross-species marker of tendon phenotype and may find application in evaluation of tendon physiology and guided differentiation of permissive cells towards functional tenocytes.

A systems biology approach to defining regulatory mechanisms for cartilage and tendon cell phenotypes.

Phenotypic plasticity of adult somatic cells has provided emerging avenues for the development of regenerative therapeutics. In musculoskeletal biology the mechanistic regulatory networks of genes governing the phenotypic plasticity of cartilage and tendon cells has not been considered systematically. Additionally, a lack of strategies to effectively reproduce in vitro functional models of cartilage and tendon is retarding progress in this field. De- and redifferentiation represent phenotypic transitions that may contribute to loss of function in ageing musculoskeletal tissues. Applying a systems biology network analysis approach to global gene expression profiles derived from common in vitro culture systems (monolayer and three-dimensional cultures) this study demonstrates common regulatory mechanisms governing de- and redifferentiation transitions in cartilage and tendon cells. Furthermore, evidence of convergence of gene expression profiles during monolayer expansion of cartilage and tendon cells, and the expression of key developmental markers, challenges the physiological relevance of this culture system. The study also suggests that oxidative stress and PI3K signalling pathways are key modulators of in vitro phenotypes for cells of musculoskeletal origin.

RNA binding proteins regulate anabolic and catabolic gene expression in chondrocytes.

OBJECTIVE: Regulation of anabolic and catabolic factors is considered essential in maintaining the homoeostasis of healthy articular cartilage. In this study we investigated the influence of RNA binding proteins (RNABPs) in this process. DESIGN: Using small interfering RNA (siRNA), RNABP expression was knocked down in SW1353 chondrosarcoma cells and human articular chondrocytes. Gene expression and messenger RNA (mRNA) decay of anabolic (SOX9, Aggrecan) and catabolic (matrix metalloproteinase (MMP)13) factors were analysed using reverse transcription quantitative polymerase chain reaction (RT-qPCR). RNA-electromobility shift assays (EMSAs) were used to investigate RNABP interactions with the SOX9 mRNA 3' untranslated region (UTR). Immunohistochemical localisation of MMP13 and the RNABP human antigen R (HuR) was performed in E13.5 and E16.5 mouse embryo sections. RESULTS: SOX9 mRNA, mRNA half-life and protein expression were increased with siRNA targeting the RNABP tristetraprolin (TTP) in both HACs and SW1353s. TTP knockdown also stimulated aggrecan mRNA expression but did not affect its stability. RNA-EMSAs demonstrated that adenine uracil (AU)-rich elements in the SOX9 mRNA 3'UTR interacted with chondrocyte proteins with three specific elements interacting with TTP. HuR knockdown significantly increased MMP13 expression and also regulated the expression of a number of known transcriptional repressors of MMP13. HuR was ubiquitously expressed within mouse embryos yet displayed regional down-regulation within developing skeletal structures. CONCLUSION: This study demonstrates for the first time how RNABPs are able to affect the balance of anabolic and catabolic gene expression in human chondrocytes. The post-transcriptional mechanisms controlled by RNABPs present novel avenues of regulation and potential points of intervention for controlling the expression of SOX9 and MMP13 in chondrocytes.

Fat pad-derived mesenchymal stem cells as a potential source for cell-based adipose tissue repair strategies.

BACKGROUND: Mesenchymal stem cells are able to undergo adipogenic differentiation and present a possible alternative cell source for regeneration and replacement of adipose tissue. The human infrapatellar fat pad is a promising source of mesenchymal stem cells with many source advantages over from bone marrow. It is important to determine whether a potential mesenchymal stem-cell exhibits tri-lineage differentiation potential and is able to maintain its proliferation potential and cell-surface characterization on expansion in tissue culture. We have previously shown that mesenchymal stem cells derived from the fat pad can undergo chondrogenic and osteogenic differentiation, and we characterized these cells at early passage. In the study described here, proliferation potential and characterization of fat pad-derived mesenchymal stem cells were assessed at higher passages, and cells were allowed to undergo adipogenic differentiation. MATERIALS AND METHODS: Infrapatellar fat pad tissue was obtained from six patients undergoing total knee replacement. Cells isolated were expanded to passage 18 and proliferation rates were measured. Passage 10 and 18 cells were characterized for cell-surface epitopes using a range of markers. Passage 2 cells were allowed to undergo differentiation in adipogenic medium. RESULTS: The cells maintained their population doubling rates up to passage 18. Cells at passage 10 and passage 18 had cell-surface epitope expression similar to other mesenchymal stem cells previously described. By staining it was revealed that they highly expressed CD13, CD29, CD44, CD90 and CD105, and did not express CD34 or CD56, they were also negative for LNGFR and STRO1. 3G5 positive cells were noted in cells from both passages. These fat pad-derived cells had adipogenic differentiation when assessed using gene expression for peroxisome proliferator-activated receptor γ2 and lipoprotein lipase, and oil red O staining. DISCUSSION: These results indicate that the cells maintained their proliferation rate, and continued expressing mesenchymal stem-cell markers and pericyte marker 3G5 at late passages. These results also show that the cells were capable of adipogenic differentiation and thus could be a promising source for regeneration and replacement of adipose tissue in reconstructive surgery.

Increased blood mRNA expression of inflammatory and anti-fibrotic markers in dogs with congestive heart failure.

Inflammation and extracellular matrix (ECM) remodeling contribute to the development of congestive heart failure (CHF), but the pathogenesis is still incompletely understood. Therefore, whole blood samples from eight dogs without cardiac disease and eight dogs with CHF were investigated for mRNA expression of IL1ß, IL2, IL4, IL6, IL8, IL10, TNFα, IFNγ, TGFß1-3, MMP1, -2, -3, -9 and TIMP1-4 using quantitative PCR. Dogs with CHF had significantly higher IL1ß (P=0.015), IL2 (P=0.043), MMP1 (P=0.031), TIMP3 (P=0.012) and lower TNFα (P<0.001), TGFß3 (P=0.006), TIMP1 (P=0.015) and TIMP2 (P=0.011) mRNA levels. Increased pro-inflammatory IL1ß and anti-fibrotic MMP1 and reduced pro-fibrotic TGFß and TIMP1 and TIMP2 in dogs with CHF suggest progressive left ventricular remodeling. The reduction of TNFα and increase of immunomodulatory IL2 and TIMP3 might suggest control of the inflammatory response. A better understanding of inflammation and ECM remodeling in cardiac diseases may lead to novel treatment approaches.

Leptin expression in dogs with cardiac disease and congestive heart failure.

BACKGROUND: Leptin belongs to the group of adipokines and has recently attracted attention because of its effects on the cardiovascular system. Increased leptin concentrations are reported in obese dogs but its role in cardiac disease (CD) is not known. Therefore, we investigated leptin expression in blood samples from dogs with congestive heart failure (CHF), and from myocardial samples of dogs with CDs. METHODS: Leptin mRNA was analyzed from blood samples of 8 dogs presented for cardiac screening in which no abnormalities were detected and 8 dogs in CHF. In addition, myocardial samples (interventricular septum, right and left atria, and ventricles) of 10 dogs with no cardiac abnormalities (controls), 7 dogs with acquired and 3 dogs with congenital CDs were investigated using real-time polymerase chain reaction (PCR). RESULTS: Dogs with CHF had significantly higher blood concentrations of leptin mRNA than dogs without CD (P = .013). Myocardial leptin expression was significantly increased in acquired (P = .035) and decreased in congenital CD (P = .016) in comparison to controls. Dogs in heart failure stage D showed higher myocardial leptin concentrations than dogs in stage C3 and B (P = .031). Differences according to myocardial region (P < .05) were detected and higher leptin concentrations were present in the atria in comparison to the ventricles in dogs with CD (P = .005). Comparing male and female dogs with CD revealed higher leptin concentrations in female dogs (P = .001). CONCLUSIONS: These results indicate leptin mRNA concentrations vary with CD, severity of CD, myocardial region, and possibly sex. Therefore, leptin might play a role in canine CD.

Post-transcriptional gene regulation following exposure of osteoarthritic human articular chondrocytes to hyperosmotic conditions.

OBJECTIVE: Osmolarity is a major biophysical regulator of chondrocyte function. Modulation of chondrocytic marker gene expression occurs at the post-transcriptional level following exposure of human articular chondrocytes (HAC) to hyperosmotic conditions. This study aims to further characterise the post-transcriptional response of HAC to hyperosmolarity. METHODS: Gene expression and microRNA (miRNA) levels in freshly isolated HAC after 5h under control or hyperosmotic conditions were measured using microarrays. Regulated genes were checked for the presence of AU rich elements (AREs) in their 3' untranslated regions (3'UTR), whilst gene ontology was examined using Ingenuity Pathway Analysis (IPA). RNA decay rates of candidate ARE-containing genes were determined in HAC using actinomycin D chase experiments and the involvement of the p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathways were investigated using pharmacological inhibitors. RESULTS: Hyperosmolarity led to the regulation of a wide variety of genes. IPA identified enrichment of genes involved with cell stress responses, cell signalling and transforming growth factor ß (TGFß) signalling. Importantly, upregulated genes were over-represented with those containing AREs, and RNA decay analysis demonstrated that many of these were regulated post-transcriptionally by hyperosmolarity in HAC. Analysis of miRNA levels in HAC indicated that they are only modestly regulated by hyperosmotic conditions, whilst inhibitor studies showed that p38 MAPK and ERK1/2 were able to block hyperosmotic induction of many of these genes. CONCLUSION: Through microarray and bioinformatics analysis we have identified genes which are post-transcriptionally regulated in HAC following exposure to hyperosmotic conditions. These genes have a range of functions, and their regulation involves transduction through the p38 MAPK and ERK1/2 pathways. Interestingly, our results suggest that miRNA regulation is not key to the process. Overall, this work illustrates the range of processes regulated in chondrocytes by changes in their osmotic environment, and underlines the importance of post-transcriptional mRNA regulation to chondrocyte function.

Regulation of SOX9 in normal and osteoarthritic equine articular chondrocytes by hyperosmotic loading.

OBJECTIVES: SOX9 is a transcription factor that is essential for cartilage extracellular matrix (ECM) formation. Osteoarthritis (OA) is characterised by a loss of cartilage ECM. In chondrocytes SOX9 gene expression is regulated by osmotic loading. Here we characterise SOX9 mRNA regulation through static and cyclical application of hyperosmotic conditions in normal and OA monolayer equine chondrocytes. Furthermore, we investigate whether extracellular signal-regulated protein kinase (ERK)1/2 mitogen-activated protein kinases (MAPK) pathways have a role in this regulation of SOX9. METHODS: Equine chondrocytes harvested from normal or OA joints were subjected to different osmotic loading patterns as either primary (P0) or passaged (P2) cells. The involvement of MEK-ERK signalling was demonstrated by using pharmacological inhibitors. In addition SOX9 gene stability was determined. Levels of transcripts encoding SOX9, Col2A1 and aggrecan were measured using qRT-PCR. De novo glycosaminoglycan synthesis of explants was determined with (35)S sulphate during static hyperosmolar loading. RESULTS: MEK-ERK signalling increases glycosaminoglycans (GAG) synthesis in explants. Static hyperosmotic conditions significantly reduced SOX9 mRNA in normal P2 and OA P0 but not normal P0 chondrocytes. SOX9 mRNA was stabilised by hyperosmotic conditions. Cyclical loading of normal P2 and OA P0 but not normal P0 cells led to an increase in SOX9 gene expression and this was prevented by MEK1/2 inhibition. CONCLUSIONS: The response to osmotic loading of SOX9 mRNA is dependent on the nature of the osmotic stimulation and the chondrocyte phenotype. This variation may be important in disease progression.

The epitope characterisation and the osteogenic differentiation potential of human fat pad-derived stem cells is maintained with ageing in later life.

Some clinical settings are deficient in osteogenic progenitors, e.g. atrophic nonunited fractures, large bone defects, and regions of scarring and osteonecrosis. These benefit from the additional use of bone marrow-derived mesenchymal stem cells, but these cells exhibit an age-related decline in lifespan, proliferation and osteogenic potential. Therapeutic approaches for the repair of bone could be optimised by the identification of a stem cell source that does not show age-related changes. Fat pad-derived stem cells are capable of osteogenesis, but a detailed study of the effect of ageing on their epitope profile and osteogenic potential has so far not been performed. Fat pad-derived cells were isolated from 2 groups of 5 patients with a mean age of 57 years (S.D. 3 years) and 86 years (S.D. 3 years). The proliferation, epitope profile and osteogenic differentiation potential of cells from the 2 groups were compared. Cells isolated from the fat pad of both groups showed similar proliferation rates and exhibited a cell surface epitope profile similar but not identical to that of bone marrow-derived stem cells. The cells from both groups cultured in osteogenic medium exhibited osteogenesis as shown by a significant upregulation of alkaline phosphatase and osteocalcin genes, and significantly greater alkaline phosphatase enzyme activity compared to cells cultured in the control medium. The cells cultured in the osteogenic medium also showed greater calcium phosphate deposition on alizarin red staining. There was no significant difference between the osteogenic potential of the two age groups for any of the parameters studied. The fat pad is a consistent and homogenous source of stem cells that exhibits osteogenic differentiation potential with no evidence of any decline with ageing in later life. This has many potential therapeutic tissue engineering applications for the repair of bone defects in an increasingly ageing population.

The reactions of articular cartilage to experimental wounding: role of apoptosis.

OBJECTIVE: To determine the cellular and matrix responses to experimental wounding of articular cartilage. METHODS: Immature and mature bovine articular cartilage was used as an in vitro model system to study the cellular responses to cartilage wounding. Explant cultures were wounded centrally with a trephine and maintained for up to 10 days. TUNEL labeling together with ultrastructural analyses were used to assess the nature of the observed cell death. In vitro labeling with 3H-thymidine was used to detect cell proliferation, and 2 antibodies (COL2-3/4M and BC-13) were used to detect changes in matrix turnover. RESULTS: Cell death was observed as a response to wounding and was considered to be a combination of necrosis and apoptosis. In immature tissue, cell death was more pronounced, particularly in the articular surface region. Within the area of cell death, many cells that did not die subsequently underwent proliferation. The collagenous network showed evidence of denaturation in the area of the wound, but "aggrecanase" activity was not detected. CONCLUSION: There are 2 contrasting, but related, responses to cartilage wounding--apoptosis and proliferation. In order to improve cartilage repair, future studies need to elucidate the regulatory mechanisms that determine these responses.