Proteomic analysis of the LPS-induced stress response in rat chondrocytes reveals induction of innate immune response components in articular cartilage

Haglund, Lisbet; Bernier, Suzanne M.; Önnerfjord, Patrik; Recklies, Anneliese D.

doi:10.1016/j.matbio.2007.09.009

Cited by 72 publications

(76 citation statements)

References 55 publications

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“…Subcomponent C1r, factor C3, and factor H have an important role in innate immunity where they are a part of an antigen-independent alternative complement activation pathway. 7,17,18 Factor H was detected in both normal and osteoarthritic cartilage already by Kumar et al, 19 based on a detection of expressed sequence tags, and our study confirms their findings by proteomic analysis of the secretome.…”

supporting

confidence: 88%

“…Proteins containing 13 C 6 labeled peptides, identified in the secretion medium of human articular chondrocytes in explants GAGs secreted by chondrocytes significantly interfered with the 2D gel focusing. 7,15,16 Our approach based on 1D gel protein separation combined with SILAC and LC/ MS-MS has enabled us to identify over 100 different types of proteins produced by chondrocytes during the culture period. To our knowledge, no previous report has shown such a long spectrum of proteins produced by chondrocytes in culture.…”

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confidence: 99%

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Differences in the secretome of cartilage explants and cultured chondrocytes unveiled by SILAC technology

Polacek

Bruun

Johansen

et al. 2010

Journal Orthopaedic Research

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ABSTRACT:The main goal of our study was to analyze and compare the profiles of secreted proteins from adult human articular chondrocytes in monolayers, and cartilage explants in culture, using a de novo protein labeling approach. Stable isotope labeling of proteins in culture was used to differentiate between chondrocyte-derived proteins and other preexisting matrix-derived components, or proteins coming from serum or synovial fluids. Proteins in culture supernatants were resolved by one-dimensional SDS-PAGE electrophoresis, and analyzed in tandem with LC/MS-MS (liquid chromatography/double mass spectrometry). Results from stable isotope labeling with amino acids in cell culture (SILAC) were validated by specific immunoblotting of four relevant proteins identified in the secretion media. After 8-10 days of culture, over 90% of proteins secreted during monolayer growth contained 13 C 6 -Arg and 13 C 6 -Lys. Nonlabeled proteins corresponded mostly to plasma-associated proteins, indicating background contamination of medium with serum remnants. The majority of the secreted proteins in 2D cultures were extracellular matrix components and matrix regulators, along with some inflammatory agents and metabolic enzymes. In explants, only 25%-30% of proteins were labeled with heavy amino acids, corresponding to matrix regulators and carrier molecules. Nonlabeled proteins corresponded primarily to structural matrix components. In qualitative terms, all labeled proteins coming from cartilage explants were also found in chondrocytes supernatants. In summary, our results show differences in the labeling pattern of proteins found in supernatants from explants and monolayers. Most proteins found in the media of explants were subproducts of matrix turnover rather than newly synthesized. To our knowledge, this study is the first one so far applying SILAC technology in the context of cartilage and chondrocytes physiology. ß

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confidence: 88%

mentioning

confidence: 99%

Differences in the secretome of cartilage explants and cultured chondrocytes unveiled by SILAC technology

Polacek

Bruun

Johansen

et al. 2010

Journal Orthopaedic Research

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“…In this part of the review, we focused on proteins identified either directly in the secretome of cartilage explants [12][13][14][15][16][17][18][19][20] or chondrocytes cultures [18,[21][22][23][24][25][26][27][28][29][30][31]), both of which are listed in Table 1. We further complete this list with proteins recently identified by proteomic analysis performed directly on fresh chondrocytes or cartilage tissue [32][33][34][35][36] (sometimes de-cellularized [37]) whereby different locations in the joint were compared, or healthy joint tissues were compared with 6 OA tissues.…”

Section: Chondrocyte Secretomementioning

confidence: 99%

Chondrocyte secretome: a source of novel insights and exploratory biomarkers of osteoarthritis

Sanchez

Bay‐Jensen

Pap

et al. 2017

Osteoarthritis and Cartilage

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The extracellular matrix (ECM) of articular cartilage is comprised of complex networks of proteins and glycoproteins, all of which are expressed by its resident cell, the chondrocyte. Cartilage is a unique tissue given its complexity and ability to resist repeated load and deformation. The mechanisms by which articular cartilage maintains its integrity throughout our lifetime is not fully understood, however there are numerous regulatory pathways known to govern ECM turnover in response to mechanical stimuli. To further our understanding of this field, we envision that proteomic analysis of the secretome will provide information on how the chondrocyte remodels the surrounding ECM in response to load, in addition to providing information on the metabolic state of the cell. In this review, we attempt to summarize the recent mass spectrometry-based proteomic discoveries in healthy and diseased cartilage and chondrocytes, to facilitate the discovery of novel biomarkers linked to degenerative pathologies, such as osteoarthritis (OA).

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“…23 The LPS-induced Chi3L1 expression by articular cartilage is a response to TLR activation without significant increase in the production of inflammatory cytokines. 43 Our results show a signal for Chi3L1 10 weeks after ESWT, indicating a reorganization in matrix protein composition connected to cartilage injury and degeneration.…”

Section: Discussionmentioning

confidence: 52%

The effect of high‐energy extracorporeal shock waves on hyaline cartilage of adult rats in vivo

Mayer-Wagner

Ernst

Maier

et al. 2010

Journal Orthopaedic Research

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The aim of this study was to determine if extracorporeal shock wave therapy (ESWT) in vivo affects the structural integrity of articular cartilage. A single bout of ESWT (1500 shock waves of 0.5 mJ/mm 2 ) was applied to femoral heads of 18 adult Sprague-Dawley rats. Two sham-treated animals served as controls. Cartilage of each femoral head was harvested at 1, 4, or 10 weeks after ESWT (n ¼ 6 per treatment group) and scored on safranin-O-stained sections. Expression of tenascin-C and chitinase 3-like protein 1 (Chi3L1) was analyzed by immunohistochemistry. Quantitative real-time polymerase chain reaction (PCR) was used to examine collagen (II)a 1 (COL2A1) expression and chondrocyte morphology was investigated by transmission electron microscopy and no changes in Mankin scores were observed after ESWT. Positive immunostaining for tenascin-C and Chi3L1 was found up to 10 weeks after ESWT in experimental but not in control cartilage. COL2A1 mRNA was increased in samples 1 and 4 weeks after ESWT. Alterations found on the ultrastructural level showed expansion of the rough-surfaced endoplasmatic reticulum, detachment of the cell membrane and necrotic chondrocytes. Extracorporeal shock waves caused alterations of hyaline cartilage on a molecular and ultrastructural level that were distinctly different from control. Similar changes were described before in the very early phase of osteoarthritis (OA). High-energy ESWT might therefore cause degenerative changes in hyaline cartilage as they are found in initial OA. Keywords: shock wave; ESWT; cartilage; degeneration; in vivo Shock waves are transient pressure disturbances that propagate rapidly in three-dimensional space. In clinical applications they are characterized by their energy flux density, frequency, number of impulses and focus. The energy flux density (EFD) of extracorporeal shock wave therapy (ESWT) is an important value that is defined as the energy measured during the time of one impulse within one square millimeter. Although there is no consistent definition on high-and low-energy EFD found in the literature, an EFD of 0.5 mJ/mm 2 is described as high-energy ESWT in most cases. ESWT has been established as common mode of treatment in orthopedic surgery for periarticular affections such as calcifying tendinitis of the shoulder 1 and lateral epicondylitis 2,3 where low-energy treatment is used. Its effectiveness has been shown in a range of diseases by an increasing number of studies. [4][5][6] Osteonecrosis of the femoral head 7 and heterotopic ossifications 8 were treated in contrast by ESWT with high-energy flux density (EFD) in humans. ESWT, of which the exact biological mechanism is not known in detail, has dosedependent effects. 9 Shock waves have been used since 1977 in humans, with the principal clinical application to treat kidney stones. The renal stone fragmentation by extracorporeal shock waves (lithotripsy) has become an established procedure in urology. It was discovered that patients after lithotripsy showed modifications of their bon...

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Proteomic analysis of the LPS-induced stress response in rat chondrocytes reveals induction of innate immune response components in articular cartilage

Cited by 72 publications

References 55 publications

Differences in the secretome of cartilage explants and cultured chondrocytes unveiled by SILAC technology

Differences in the secretome of cartilage explants and cultured chondrocytes unveiled by SILAC technology

Chondrocyte secretome: a source of novel insights and exploratory biomarkers of osteoarthritis

The effect of high‐energy extracorporeal shock waves on hyaline cartilage of adult rats in vivo

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