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| 2. |
- Larsson, Staffan, et al.
(author)
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An ARGS-aggrecan assay for analysis in blood and synovial fluid.
- 2014
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In: Osteoarthritis and Cartilage. - : Elsevier BV. - 1063-4584. ; 22:2, s. 242-249
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Journal article (peer-reviewed)abstract
- To validate a modified ligand-binding assay for the detection of aggrecanase generated aggrecan fragments with the ARGS neoepitope in synovial fluid (SF) and blood, and to verify the identity of aggrecan fragments found in blood.
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| 3. |
- Larsson, Staffan, et al.
(author)
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Association between synovial fluid levels of aggrecan ARGS fragments and radiographic progression in knee osteoarthritis.
- 2010
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In: Arthritis Research and Therapy. - : Springer Science and Business Media LLC. - 1478-6362 .- 1478-6354. ; 12:6
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Journal article (peer-reviewed)abstract
- ABSTRACT: INTRODUCTION: Aggrecanase cleavage at the 392Glu-393Ala bond in the interglobular domain (IGD) of aggrecan, releasing N-terminal 393ARGS fragments, is an early key event in arthritis and joint injuries. We determined whether synovial fluid (SF) levels of ARGS-aggrecan distinguish subjects with progressive radiographic knee osteoarthritis (ROA) from those with stable or no ROA. METHODS: We studied 141 subjects who, at examination A, had been given meniscectomies an average of 18 years earlier (range, 15 to 22 years). Seventeen individuals without surgery, and without known injury to the menisci or cruciate ligaments, were used as references. At examinations A and B, with a mean follow-up time of 7.5 years, we obtained SF and standing tibiofemoral and skyline patellofemoral radiographs. SF ARGS-aggrecan was measured with an electrochemiluminescence immunoassay, and we graded radiographs according to the OARSI atlas. The association between SF ARGS levels at examination A and progression of radiographic features of knee OA between examinations A and B was assessed by using logistic regression adjusted for age, gender, body mass index, and time between examinations, and stratified by ROA status at examination A. RESULTS: We found a weak negative association between SF ARGS concentrations and loss of joint space: the likelihood of progression of radiographic joint space narrowing decreased 0.9 times per picomole per milliliter increase in ARGS (odds ratio (OR) 0.89; 95% confidence interval (CI), 0.79 to 0.996). In subjects with and without preexisting ROA at examination A, the association was OR, 0.96; 0.81 to 1.13; and 0.77; 0.62 to 0.95, respectively. Average levels of SF ARGS 18 years after meniscectomy were no different from those of reference subjects and were not correlated to radiographic status at examination A. CONCLUSIONS: In subjects with previous knee meniscectomy but without ROA, levels of SF ARGS-aggrecan were weakly and inversely associated with increased loss of joint space over a period of 7.5 years.
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| 4. |
- Larsson, Staffan, et al.
(author)
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The association between changes in synovial fluid levels of ARGS-aggrecan fragments, progression of radiographic osteoarthritis and self-reported outcomes: a cohort study.
- 2012
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In: Osteoarthritis and Cartilage. - : Elsevier BV. - 1063-4584. ; 20:5, s. 388-395
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Journal article (peer-reviewed)abstract
- OBJECTIVE: To investigate whether change in concentrations over time of aggrecanase generated ARGS-aggrecan in synovial fluid (SF ARGS) associates with progression of radiographic knee Osteoarthritis (OA) and patient-reported outcome in subjects with previous meniscectomy. METHODS: We studied 141 subjects at two time points after meniscectomy. Time point A was on average 18years after meniscectomy, time point B was on average 7.5years later; 74 subjects had SF available from both examinations. We measured SF ARGS by an electrochemiluminescence immunoassay, graded radiographic features of tibiofemoral or patellofemoral OA according to the Osteoarthritis Research Society International (OARSI) atlas, and scored patient-reported outcomes using the Knee Injury and Osteoarthritis Outcome Score (KOOS). Using logistic regression (adjusted for age, gender, body mass index, time between examinations, and SF ARGS at first examination) we assessed associations between change in SF ARGS between first and second examinations and progression of radiographic OA and KOOS. RESULTS: In subjects with decreasing SF ARGS between examinations, the likelihood of loss of joint space and worsening of KOOS pain between examinations was increased 6- and 4-fold respectively compared to those increasing in SF ARGS (OR 5.72; 95% CI 1.53-21.4 and 3.66; 1.01-13.2, respectively). No significant associations were seen between decreasing SF ARGS and progression of osteophytes (OR 0.88; 0.28-2.78), or for patient-reported outcomes other than KOOS pain. CONCLUSION: Having decreasing levels of SF ARGS over time was associated with an increased risk of loss of joint space and pain worsening, but showed no association with other patient-reported outcomes or osteophyte progression.
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| 6. |
- Stattin, Eva-Lena, et al.
(author)
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A missense mutation in the aggrecan C-type lectin domain disrupts extracellular matrix interactions and causes dominant familial osteochondritis dissecans
- 2010
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In: American Journal of Human Genetics. - : Elsevier BV. - 0002-9297 .- 1537-6605. ; 86:2, s. 126-137
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Journal article (peer-reviewed)abstract
- Osteochondritis dissecans is a disorder in which fragments of articular cartilage and subchondral bone dislodge from the joint surface. We analyzed a five-generation family in which affected members had autosomal-dominant familial osteochondritis dissecans. A genome-wide linkage analysis identified aggrecan (ACAN) as a prime candidate gene for the disorder. Sequence analysis of ACAN revealed heterozygosity for a missense mutation (c.6907G > A) in affected individuals, resulting in a p.V2303M amino acid substitution in the aggrecan G3 domain C-type lectin, which mediates interactions with other proteins in the cartilage extracellular matrix. Binding studies with recombinant mutated and wild-type G3 proteins showed loss of fibulin-1, fibulin-2, and tenascin-R interactions for the V2303M protein. Mass spectrometric analyses of aggrecan purified from patient cartilage verified that V2303M aggrecan is produced and present in the tissue. Our results provide a molecular mechanism for the etiology of familial osteochondritis dissecans and show the importance of the aggrecan C-type lectin interactions for cartilage function in vivo.
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| 7. |
- Struglics, André, et al.
(author)
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A comparison of different purification methods of aggrecan fragments from human articular cartilage and synovial fluid.
- 2010
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In: Matrix Biology. - : Elsevier BV. - 1569-1802 .- 0945-053X. ; 29, s. 74-83
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Journal article (peer-reviewed)abstract
- In the study of aggrecan fragmentation several methods to extract and purify aggrecan from cartilage and synovial fluid (SF) are used. This work compares and evaluates the effectiveness for purification of aggrecan of the most commonly used methods by the ratio of sulfated glycosaminoglycan (sGAG) to protein and by fragment analysis by Western blot. A novel method for purification of aggrecan fragments from SF by boiling (Boiled SF) is also presented. Of the sGAG extracted from cartilage by guanidinium, 66% was recovered by associative-dissociative cesium chloride density gradient centrifugation (A1D1-D3) with a 9 times higher ratio of sGAG to protein in the A1D1 fraction. Although less enriched in aggrecan, the Western blot aggrecan pattern of the guanidinium extracted sample resembled that of the combined patterns of the A1D1, A1D2 and A1D3 fractions. The recoveries of sGAG from SF purified by anion chromatography and Alcian blue precipitation were around 50%, while the recoveries were over 80% in the associative or dissociative density gradient fractions (A1 and D1) and Boiled SF. The purification compared to neat SF ranged from 9 times in boiled SF to 1800-1900 times in Alcian blue and D1 samples. To obtain reliable results when analyzing synovial fluid aggrecan fragments by Western blot, purification was necessary. The immuno-pattern of anion chromatography purified SF resembled the patterns of A1 and D1, while the pattern of Boiled SF resembled the D1 sample. This work suggests that aggrecan fragments extracted from cartilage by guanidinium need no further purification to be analyzed by Western blot, whereas aggrecan fragments in SF are best analyzed in the A1 and D1 fractions or in the Boiled SF sample.
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| 8. |
- Struglics, André, et al.
(author)
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Aggrecanase cleavage in juvenile idiopathic arthritis patients is minimally detected in the aggrecan interglobular domain but robust at the aggrecan C-terminus.
- 2012
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In: Arthritis and Rheumatism. - : Wiley. - 1529-0131 .- 0004-3591.
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Journal article (peer-reviewed)abstract
- OBJECTIVE: To understand aggrecan degradation in juvenile idiopathic arthritis (JIA), the pattern and abundance of aggrecan fragments in synovial fluid aspirates from JIA patients were analysed and compared with aggrecan fragments in synovial fluids from patients with other arthritides, juvenile knee injury and a knee-healthy reference group. METHODS: The concentration of sulphated glycosaminoglycans in synovial fluid was measured by the Alcian blue precipitation assay. Aggrecan fragments were purified by dissociative CsCl density gradient centrifugation, deglycosylated and analysed by Western blot using antibodies specific for either aggrecanase-derived ARGS, SELE and KEEE neoepitopes, or the aggrecan G3-domain. RESULTS: The concentration of sulphated glycosaminoglycans in JIA synovial fluids was significantly lower compared with the levels in fluids from OA (P<0.001), juvenile knee injury (P=0.006) and knee-healthy reference (P=0.022) groups. Western blot analysis detected KEEE, SELE, and G3 fragments generated by aggrecanase cleavage in the chondroitin sulphate-rich region of JIA aggrecan. The pattern of JIA aggrecan fragments was not identical to that in synovial fluids pooled from OA patients, although there were notable similarities. Surprisingly, aggrecanase-derived ARGS fragments were barely detectable in the JIA synovial fluids, in marked contrast to the levels of ARGS fragments in OA synovial fluids. CONCLUSIONS: Aggrecanases appear to cleave minimally in the interglobular domain of aggrecan in JIA patients despite robust levels of cleavage in aggrecan's chondroitin-sulphate rich region. The results suggest that unlike other arthritides, aggrecanase cleavage in the aggrecan interglobular domain might not be a major pathogenic event in JIA. © 2012 American College of Rheumatology.
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| 9. |
- Struglics, André, et al.
(author)
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Calpain is involved in C-terminal truncation of human aggrecan
- 2010
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In: Biochemical Journal. - 0264-6021. ; 18, s. 10-10
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Journal article (peer-reviewed)abstract
- Mature aggrecan is generally C-terminally truncated at several sites in the CS (chondroitin sulfate) region. Aggrecanases and MMPs (matrix metalloproteinases) have been suggested to be responsible for this digestion. To identify whether calpain, a common intracellular protease, has a specific role in the proteolysis of aggrecan we developed neoepitope antibodies (anti-PGVA, anti-GDLS and anti-EDLS) against calpain cleavage sites and used Western blot analysis to identify alpain-generated fragments in normal and OA (osteoarthritis) knee cartilage and SF (synovial fluid) samples. Our results showed that human aggrecan contains six calpain cleavage sites: one in the IGD (interglobular domain), one in the KS (keratan sulfate) region, two in the CS1 and two in the CS2 region. Kinetic studies of calpain proteolysis against aggrecan showed that the aggrecan molecule was cleaved in a specific order where cuts in CS1 was the most preferred and cuts in KS region was the second most preferred cleavage. OA and normal cartilage contained low amounts of a calpain-generated G1-PGVA fragment (0.5-2%) compared with aggrecanase-generated G1-TEGE (71-76%) and MMP-generated G1-IPEN (23-29%) fragments. Significant amounts of calpain-generated GDLS and EDLS fragments were found in OA and normal cartilage, and a ARGS-EDLS fragment was detected in arthritic SF samples. The results of the present study indicate that calpains are involved in the C-terminal truncation of aggrecan and might have a minor role in arthritic diseases.
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| 10. |
- Struglics, André, et al.
(author)
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Human aggrecanase generated synovial fluid fragment levels are elevated directly after knee injuries due to proteolysis both in the inter globular and chondroitin sulfate domains.
- 2011
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In: Osteoarthritis and Cartilage. - : Elsevier BV. - 1063-4584. ; 19, s. 1047-1057
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Journal article (peer-reviewed)abstract
- OBJECTIVE: To examine different aggrecanase generated fragments in synovial fluid (SF) from patients with acute and chronic knee injuries and from knee healthy subjects. METHODS: We prepared SF-D1 samples from acute (n=35) and chronic (n=35) knee injury patients and knee healthy subjects (n=10). Aggrecan fragments were analyzed in the SF-D1 samples by quantitative (G1, ARGS, KEEE and G3 antibodies) and non-quantitative (GRGT and AGEG antibodies) Western blot. RESULTS: ARGS-SELE, ARGS-chondroitin sulfate (CS)1, GRGT-, GLGS- and AGEG-G3 fragments were the main ARGS and G3 fragments in injured and reference samples. In the acute injury samples the concentrations of these fragments were increased compared to the reference, and the level of the ARGS-SELE remained elevated for at least 2 years after the joint injury. Both SF ARGS fragments and aggrecanase generated G3 fragments had high sensitivity and specificity as biomarkers in distinguishing injured from healthy knee joints, although the ARGS fragments had higher area under the receiver operating characteristic curve (AUC) values for injuries (74-86%) than the G3 fragments (AUC values 63-68%). CONCLUSION: Our results suggest that during the acute phase after knee injury there is an increased aggrecanase activity against both the interglobular domain (IGD) and the CS2 cleavage sites of joint cartilage aggrecan. This increase in SF aggrecanolytic fragments is present for several years after the injury. SF ARGS fragments are better biomarkers than the aggrecanase generated G3-fragments in distinguishing injured from healthy knee joints.
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