Quantitative proteomics at different depths in human articular cartilage reveals unique patterns of protein distribution

The articular cartilage of synovial joints ensures friction-free mobility and attenuates mechanical impact on the joint during movement. These functions are mediated by the complex network of extracellular molecules characteristic for articular cartilage. Zonal differences in the extracellular matrix (ECM) are well recognized. However, knowledge about the precise molecular composition in the different zones remains limited. In the present study, we investigated the distribution of ECM molecules along the surface-to-bone axis, using quantitative non-targeted as well as targeted proteomics.\ In a discovery approach, iTRAQ mass spectrometry was used to identify all extractable ECM proteins in the different layers of a human lateral tibial plateau full thickness cartilage sample. A targeted MRM mass spectrometry approach was then applied to verify these findings and to extend the analysis to four medial tibial plateau samples. In the lateral tibial plateau sample, the unique distribution patterns of 70 ECM proteins were identified, revealing groups of proteins with a preferential distribution to the superficial, intermediate or deep regions of articular cartilage. The detailed analysis of selected 29 proteins confirmed these findings and revealed similar distribution patterns in the four medial tibial plateau samples. The results of this study allow, for the first time, an overview of the zonal distribution of a broad range of cartilage ECM proteins and open up further investigations of the functional roles of matrix proteins in the different zones of articular cartilage in health and disease

Quantitative Proteomic Analysis of Eight Cartilaginous Tissues Reveals Characteristic Differences as well as Similarities between Subgroups.

Human synovial joints display a characteristic anatomic distribution of arthritis e.g. rheumatoid arthritis primarily affects the metacarpophalangeal and proximal finger joints, but rarely the distal finger joints, while osteoarthritis occur in the distal and proximal finger joints. Pelvospondylitis has a selective localization to the spine and sacroiliac joints. Is this tropism due to differences between the cartilages at the molecular level? To substantiate this concept the present study provides a background detailed compositional analysis by relative quantification of extracellular matrix proteins in articular cartilages, meniscus, intervertebral disc, rib and tracheal cartilages on samples from 5-6 different individuals using an optimized approach for proteomics. Tissue extraction followed by trypsin digestion and 2D LC-separations coupled to tandem mass spectrometry, relative quantification with isobaric labeling, iTRAQ, was used to compare the relative abundance of about 150 proteins. There were clear differences in protein patterns between different kinds of cartilages. Matrilin-1 and epiphycan were specific for rib and trachea, while asporin was particularly abundant in the meniscus. Interestingly, lubricin was prominent in the intervertebral disc, especially in the nucleus pulposus. Fibromodulin and lumican showed distributions that were mirror images of one other. Analyses of the insoluble residues from guanidine extraction revealed that a fraction of several proteins remained unextracted e.g. asporin, CILP and COMP indicating crosslinking. Distinct differences in protein patterns may relate to different tissue mechanical properties, and to the intriguing tropism in different patterns of joint pathology

Quantitative proteomics analysis of cartilage response to mechanical injury and cytokine treatment

Mechanical damage at the time of joint injury and the ensuing inflammatory response associated with elevated levels of pro-inflammatory cytokines in the synovial fluid, are reported to contribute to the progression to osteoarthritis after injury. In this exploratory study, we used a targeted proteomics approach to follow the progression of matrix degradation in response to mechanical damage and cytokine treatment of human knee cartilage explants, and thereby to study potential molecular biomarkers. This proteomics approach allowed us to unambiguously identify and quantify multiple peptides and proteins in the cartilage medium and explants upon treatment with ±. injurious compression ±. cytokines, treatments that mimic the earliest events in post-traumatic OA. We followed degradation of different protein domains, e.g., G1/G2/G3 of aggrecan, by measuring representative peptides of matrix proteins released into the medium at 7 time points throughout the 21-day culture period. COMP neo-epitopes, which were previously identified in the synovial fluid of knee injury/OA patients, were also released by these human cartilage explants treated with cyt and cyt+inj. The absence of collagen pro-peptides and elevated levels of specific COMP and COL3A1 neo-epitopes after human knee trauma may be relevant as potential biomarkers for post-traumatic OA. This model system thereby enables study of the kinetics of cartilage degradation and the identification of biomarkers within cartilage explants and those released to culture medium. Discovery proteomics revealed that candidate proteases were identified after specific treatment conditions, including MMP1, MMP-3, MMP-10 and MMP-13

Chondrocytes derived from mesenchymal stromal cells and induced pluripotent cells of patients with familial osteochondritis dissecans exhibit an endoplasmic reticulum stress response and defective matrix assembly

Familial osteochondritis dissecans (FOCD) is an inherited skeletal defect characterized by the development of large cartilage lesions in multiple joints, short stature, and early onset of severe osteoarthritis. It is associated with a heterozygous mutation in the ACAN gene, resulting in a Val-Met replacement in the C-type lectin domain of aggrecan. To understand the cellular pathogenesis of this condition, we studied the chondrogenic differentiation of patient bone marrow mesenchymal stromal cells (BM-MSCs). We also looked at cartilage derived from induced pluripotent stem cells (iPSCs) generated from patient fibroblasts. Our results revealed several characteristics of the differentiated chondrocytes that help to explain the disease phenotype and susceptibility to cartilage injury. First, patient chondrogenic pellets had poor structural integrity but were rich in glycosaminoglycan. Second, it was evident that large amounts of aggrecan accumulated within the endoplasmic reticulum of chondrocytes differentiated from both BM-MSCs and iPSCs. In turn, there was a marked absence of aggrecan in the extracellular matrix. Third, it was evident that matrix synthesis and assembly were globally dysregulated. These results highlight some of the abnormal aspects of chondrogenesis in these patient cells and help to explain the underlying cellular pathology. The results suggest that FOCD is a chondrocyte aggrecanosis with associated matrix dysregulation. The work provides a new in vitro model of osteoarthritis and cartilage degeneration based on the use of iPSCs and highlights how insights into disease phenotype and pathogenesis can be uncovered by studying differentiation of patient stem cells.We sincerely thank the family members who participated in the study and Dr. Yelverton Tegner, Luleå Technical University, Luleå, Sweden, for providing skin samples. We acknowledge the facilities and technical assistance of the Flow Cytometry Facility at the National University of Ireland Galway, a facility that is funded by National University of Ireland Galway and the Irish Government's Programme for Research in Third Level Institutions, Cycle5, National Development Plan 2007–2013. We thank Pierce Lalor, Dr. Kerry Thompson, and the Centre for Microscopy and Imaging (http://www.imaging.nuigalway.ie) for assistance with TEM and confocal imaging, and Jingqiu Zhang (A*STAR Institute of Medical Biology, Singapore) for assistance with the iPSC reprogramming technique. We also thank Maggie Hall for helpful editing of the manuscript. This study was funded by Science Foundation Ireland Grant SFI 09/SRC.B1794 and Irish Research Council Grant GOIPG/2014/96. This project has also received funding from the European Union's Seventh Framework Programme for Research, Technological Development and Demonstration under grant agreement no. 223298.peer-reviewe

Monovalent Interactions of Galectin-1

Galectin-1, beta-galactoside binding lectin involved in immunoregulation and cancer, binds natural and many synthetic multivalent glycoconjugates with an apparent glycoside cluster effect, that is, affinity above and beyond what would be expected from the concentration of the determinant sugar. Here we have analyzed the mechanism of such cluster effects in solution at physiological concentration using a fluorescence anisotropy assay with a novel fluorescent high-affinity galectin-1 binding probe. The interaction of native dimeric and monomeric mutants of rat and human galectin-1 with mono- and divalent small molecules, fetuin, asialofetuin, and human serum glycoproteins was analyzed. Surprisingly, high-affinity binding did not depend much on the dimeric state of galectin-1 and thus is due mainly to monomeric interactions of a single carbohydrate recognition domain. The mechanism for this is unknown, but one possibility includes additional interactions that high-affinity ligands make with an extended binding site on the carbohydrate recognition domain, It follows that such weak additional interactions must be important for the biological function of galectin-1 and also for the design of galectin-1 inhibitors

Elucidating the Molecular Composition of Cartilage by Proteomics.

Articular cartilage consists of chondrocytes and two major components, a collagen-rich framework and highly abundant proteoglycans. Most prior studies defining the zonal distribution of cartilage have extracted proteins with guanidine-HCl. However, an unextracted collagen-rich residual is left after extraction. In addition, the high abundance of anionic polysaccharide molecules extracted from cartilage adversely affects the chromatographic separation. In this study, we established a method for removing chondrocytes from cartilage sections with minimal extracellular matrix protein loss. The addition of surfactant to guanidine-HCl extraction buffer improved protein solubility. Ultrafiltration removed interference from polysaccharides and salts. Almost four-times more collagen peptides were extracted by the in situ trypsin digestion method. However, as expected, proteoglycans were more abundant within the guanidine-HCl extraction. These different methods were used to extract cartilage sections from different cartilage layers (superficial, intermediate, and deep), joint types (knee and hip), and disease states (healthy and osteoarthritic), and the extractions were evaluated by quantitative and qualitative proteomic analyses. The results of this study led to the identifications of the potential biomarkers of osteoarthritis (OA), OA progression, and the joint specific biomarkers

Chondrocytes derived from mesenchymal stromal cells and induced pluripotent cells of patients with familial osteochondritis dissecans exhibit an endoplasmic reticulum stress response and defective matrix assembly

Familial osteochondritis dissecans (FOCD) is an inherited skeletal defect characterized by the development of large cartilage lesions in multiple joints, short stature, and early onset of severe osteoarthritis. It is associated with a heterozygous mutation in the ACAN gene, resulting in a Val-Met replacement in the C-type lectin domain of aggrecan. To understand the cellular pathogenesis of this condition, we studied the chondrogenic differentiation of patient bone marrow mesenchymal stromal cells (BM-MSCs). We also looked at cartilage derived from induced pluripotent stem cells (iPSCs) generated from patient fibroblasts. Our results revealed several characteristics of the differentiated chondrocytes that help to explain the disease phenotype and susceptibility to cartilage injury. First, patient chondrogenic pellets had poor structural integrity but were rich in glycosaminoglycan. Second, it was evident that large amounts of aggrecan accumulated within the endoplasmic reticulum of chondrocytes differentiated from both BM-MSCs and iPSCs. In turn, there was a marked absence of aggrecan in the extracellular matrix. Third, it was evident that matrix synthesis and assembly were globally dysregulated. These results highlight some of the abnormal aspects of chondrogenesis in these patient cells and help to explain the underlying cellular pathology. The results suggest that FOCD is a chondrocyte aggrecanosis with associated matrix dysregulation. The work provides a new in vitro model of osteoarthritis and cartilage degeneration based on the use of iPSCs and highlights how insights into disease phenotype and pathogenesis can be uncovered by studying differentiation of patient stem cells

Chondrocytes derived from mesenchymal stromal cells and induced pluripotent cells of patients with familial osteochondritis dissecans exhibit an endoplasmic reticulum stress response and defective matrix assembly

Familial osteochondritis dissecans (FOCD) is an inherited skeletal defect characterized by the development of large cartilage lesions in multiple joints, short stature, and early onset of severe osteoarthritis. It is associated with a heterozygous mutation in the ACAN gene, resulting in a Val-Met replacement in the C-type lectin domain of aggrecan. To understand the cellular pathogenesis of this condition, we studied the chondrogenic differentiation of patient bone marrow mesenchymal stromal cells (BM-MSCs). We also looked at cartilage derived from induced pluripotent stem cells (iPSCs) generated from patient fibroblasts. Our results revealed several characteristics of the differentiated chondrocytes that help to explain the disease phenotype and susceptibility to cartilage injury. First, patient chondrogenic pellets had poor structural integrity but were rich in glycosaminoglycan. Second, it was evident that large amounts of aggrecan accumulated within the endoplasmic reticulum of chondrocytes differentiated from both BM-MSCs and iPSCs. In turn, there was a marked absence of aggrecan in the extracellular matrix. Third, it was evident that matrix synthesis and assembly were globally dysregulated. These results highlight some of the abnormal aspects of chondrogenesis in these patient cells and help to explain the underlying cellular pathology. The results suggest that FOCD is a chondrocyte aggrecanosis with associated matrix dysregulation. The work provides a new in vitro model of osteoarthritis and cartilage degeneration based on the use of iPSCs and highlights how insights into disease phenotype and pathogenesis can be uncovered by studying differentiation of patient stem cells.We sincerely thank the family members who participated in the study and Dr. Yelverton Tegner, Luleå Technical University, Luleå, Sweden, for providing skin samples. We acknowledge the facilities and technical assistance of the Flow Cytometry Facility at the National University of Ireland Galway, a facility that is funded by National University of Ireland Galway and the Irish Government\u27s Programme for Research in Third Level Institutions, Cycle5, National Development Plan 2007–2013. We thank Pierce Lalor, Dr. Kerry Thompson, and the Centre for Microscopy and Imaging (http://www.imaging.nuigalway.ie) for assistance with TEM and confocal imaging, and Jingqiu Zhang (A*STAR Institute of Medical Biology, Singapore) for assistance with the iPSC reprogramming technique. We also thank Maggie Hall for helpful editing of the manuscript. This study was funded by Science Foundation Ireland Grant SFI 09/SRC.B1794 and Irish Research Council Grant GOIPG/2014/96. This project has also received funding from the European Union\u27s Seventh Framework Programme for Research, Technological Development and Demonstration under grant agreement no. 223298