Proteoglycan Depletion Research Articles

Elevated extracellular matrix production and degradation upon bone morphogenetic protein-2 (BMP-2) stimulation point toward a role for BMP-2 in cartilage repair and remodeling

Bone morphogenetic protein-2 (BMP-2) has been proposed as a tool for cartilage repair and as a stimulant of chondrogenesis. In healthy cartilage, BMP-2 is hardly present, whereas it is highly expressed during osteoarthritis. To assess its function in cartilage, BMP-2 was overexpressed in healthy murine knee joints and the effects on proteoglycan (PG) synthesis and degradation were evaluated. Moreover, the contribution of BMP in repairing damage induced by interleukin-1 (IL-1) was investigated. Ad-BMP-2 was injected intra-articularly into murine knee joints, which were isolated 3, 7, and 21 days after injection for histology, immunohistochemistry, and autoradiography. In addition, patellar and tibial cartilage was isolated for RNA isolation or measurement of PG synthesis by means of 35SO4 2- incorporation. To investigate the role for BMP-2 in cartilage repair, cartilage damage was induced by intra-articular injection of IL-1. After 2 days, Ad-BMP-2, Ad-BMP-2 + Ad-gremlin, Ad-gremlin, or a control virus was injected. Whole knee joints were isolated for histology at day 4 or patellae were isolated to measure 35SO42- incorporation. BMP-2 stimulated PG synthesis in patellar cartilage on all days and in tibial cartilage on day 21. Aggrecan mRNA expression had increased on all days in patellar cartilage, with the highest increase on day 7. Collagen type II expression showed a similar expression pattern. In tibial cartilage, collagen type II and aggrecan mRNA expression had increased on days 7 and 21. BMP-2 overexpression also induced increased aggrecan degradation in cartilage. VDIPEN staining (indicating matrix metalloproteinase activity) was elevated on day 3 in tibial cartilage and on days 3 and 7 in patellar cartilage, but no longer was by day 21. Increased NITEGE staining (indicating aggrecanase activity) was found on days 7 and 21. In IL-1-damaged patellar cartilage, BMP-2 boosted PG synthesis. Blocking of BMP activity resulted in a decreased PG synthesis compared with IL-1 alone. This decreased PG synthesis was associated with PG depletion in the cartilage. These data show that BMP-2 boosts matrix turnover in intact and IL-damaged cartilage. Moreover, BMP contributes to the intrinsic repair capacity of damaged cartilage. Increased matrix turnover might be functional in replacing matrix molecules in the repair of a damaged cartilage matrix.

The lonely death: Chondrocyte apoptosis in TNF-induced arthritis

Inflammatory joint disease typically provokes progressive cartilage damage. The proliferative synovial inflammatory tissue directly invades the cartilage and induces the expression and activation of degrading enzymes such as matrix metalloproteases (MMPs) and aggrecanases. However, also chondrocyte apoptosis has been observed in cartilage samples of inflamed joints. It remains unclear whether this is a secondary phenomenon due to cartilage damage or a primary event initiated by the synovial inflammation. To determine the presence or absence of chondrocyte death in experimental arthritis, we longitudinally assessed proteoglycan depletion and chondrocyte apoptosis in paw sections from human tumor necrosis factor transgenic (hTNFtg) mice and wild-type littermates. Whereas, wild-type mice showed no signs of cartilage damage, hTNFtg mice exhibited progressive proteoglycan loss starting at clinical onset of arthritis. However, we already found the first apoptotic chondrocytes well before cartilage matrix breakdown occurred indicating that chondrocyte death can be induced before matrix resorption. Chondrocyte death could constantly be observed until late stages of arthritis causing a continuous increase in the number of empty cartilage lacunae. As apoptotic cells in cartilage cannot be cleared by phagocytes due to their spatial isolation in the avascular lacunae of cartilage, having no contact to professional or amateur phagocytes. The dying cells are compelled to undergo a “lonely death” inevitable ending up in secondary necrosis giving rise to perpetuation of a pro-inflammatory cascade. These data indicate that chondrocyte death may play a primary role in inflammatory arthritis fuelling cartilage inflammation and damage due to secondary necrosis.

Multinuclear NMR and microscopic MRI studies of the articular cartilage nanostructure

Studies of the structure of articular cartilage by a number of NMR spectroscopic and imaging techniques are reviewed. Advantage is taken of the fact that the NMR investigations can be done non-invasively on the intact tissue and do not require sectioning, slicing and decalcification as in the case of electron microscopy. The different contributions to 1H T2 relaxation are described and it is pointed out that ignoring the biexponential behavior of the transverse relaxation can lead to serious errors in the proton density measurements and the T2 characterization of the articular cartilage. A way to slow the transverse relaxation and to minimize its angular dependence by the use of dipolar echo is described. 2H double quantum filtered spectroscopic MRI is a powerful technique to follow the orientation and density of the collagen fibers in articular cartilage. Using this technique, it was found that attachment of the cartilage to the bone has a stabilizing effect on the collagen matrix and that the hydroxyapatite in the calcified zone resides near the collagen fibers but does not contribute to their order. In response to mechanical pressure, it was shown that the collagen fibers flatten near the surface and become crimped near the bone. A number of NMR techniques have been described for the measurement of 23Na residual quadrupolar interaction. It was found that this can serve as a very sensitive measure of the depletion of proteoglycans. Finally, a combination of the above techniques was used to study a maturation of articular cartilage in pigs. The increased order and density of the collagen fibers from newborn to adult pigs revealed itself as a shortening of T2 and significant increase of the residual quadrupolar interaction of both 2H and 23Na nuclei.

Oncostatin M induces angiogenesis and cartilage degradation in rheumatoid arthritis synovial tissue and human cartilage cocultures

To investigate the role of oncostatin M (OSM) in cell adhesion, angiogenesis, and matrix degradation in rheumatoid arthritis (RA) synovial tissue and normal human cartilage. Human dermal microvascular endothelial cell (HDMEC) and RA synovial fibroblast (RASF) proliferation and intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expression were assessed by a bromodeoxyuridine proliferation assay and flow cytometry. HDMEC tubule formation and migration were assessed by Matrigel culture and migration assay. Production of matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinases 1 (TIMP-1) in RA synovial explants, and proteoglycan/glycosaminoglycan (GAG) release, vascular endothelial growth factor (VEGF), and angiopoietin 2 production from RASF/normal cartilage cocultures were assessed by enzyme-linked immunosorbent assay and immunohistology. HDMEC/RASF proliferation was induced by OSM and interleukin-1beta (IL-1beta), alone and in combination. OSM enhanced cell surface expression of ICAM-1, but not VCAM-1, on endothelial cells and RASFs. OSM increased endothelial cell tubule formation and migration. In RA synovial explants, OSM induced production of MMP-1 and TIMP-1. When OSM was combined with IL-1beta, however, the MMP-1:TIMP-1 ratio was significantly increased. OSM potentiated IL-1beta-induced MMP-1 and MMP-13 expression in normal human cartilage/RASF cocultures, resulting in a significant increase in the MMP:TIMP ratio. In OSM/IL-1beta- stimulated cocultures, cartilage sections demonstrated significant proteoglycan depletion that was paralleled by a significant increase in GAG release in supernatants. Finally, compared with either cytokine alone, the combination of OSM and IL-1beta significantly induced VEGF production in RASF/cartilage cocultures. These data suggest that OSM promotes angiogenesis and endothelial cell migration and potentiates the effects of IL-1beta in promoting extracellular matrix turnover and human cartilage degradation. Furthermore, the induction of VEGF in cocultures supports the hypothesis of a link between angiogenesis and cartilage degradation.

Differential regulation of matrix degrading enzymes in a TNFα-induced model of nucleus pulposus tissue degeneration

Intervertebral disc degeneration occurs commonly and is linked to persistent back pain and the development of disc herniation. The mechanisms responsible for tissue catabolism have not yet been fully elucidated. Previously we characterized an in vitro model of TNFα-induced nucleus pulposus degeneration, which demonstrates decreased expression of matrix macromolecules, increased expression of matrix degrading enzymes, and the activation of aggrecanase-mediated proteoglycan degradation [Seguin, C.A., Pilliar, R.M., Roughley, P.J., and Kandel, R.A. 2005. Tumor necrosis factor-alpha modulates matrix production and catabolism in nucleus pulposus tissue. Spine 30: 1940–1948]. This study explores the intracellular pathways activated during TNFα-induced matrix degradation. We demonstrate that in nucleus pulposus cells, the p38 and JNK pathways regulate induction of MMP-1 and -3; p38, JNK, and NF-κB regulate the induction of MMP-13; and ERK regulates the up-regulation of MT1-MMP mRNA in response to TNFα. Induction of ADAMTS-4 and -5 mRNA occurred downstream of NF-κB activation. Depletion of tissue proteoglycans was mediated by ERK and NF-κB-dependent “aggrecanase” activity, suggesting MT1-MMP and ADAMTS-4 and -5 as effectors of TNFα-induced tissue catabolism.

In vitro degradation of articular cartilage: does trypsin treatment produce consistent results?

It is common practice in laboratories to create models of degraded articular cartilage in vitro and use these to study the effects of degeneration on cartilage responses to external stimuli such as mechanical loading. However, there are inconsistencies in the reported action of trypsin, and there is no guide on the concentration of trypsin or the time to which a given sample can be treated so that a specific level of proteoglycan depletion is achieved. This paper argues that before any level of confidence can be established in comparative analysis it is necessary to first obtain samples with similar properties. Consequently, we examine the consistency of the outcome of the artificial modification of cartilage relative to the effects of the common enzyme, trypsin, used in the process of in vitro proteoglycan depletion. The results demonstrate that for a given time and enzyme concentration, the action of trypsin on proteoglycans is highly variable and is dependent on the initial distribution and concentration of proteoglycans at different depths, the intrinsic sample depth, the location in the joint space and the medium type, thereby sounding a note of caution to researchers attempting to model a proteoglycan-based degeneration of articular cartilage in their experimental studies.

Proinflammatory Cytokine Activities, Matrix Metalloproteinase‐3 Activity, and Sulfated Glycosaminoglycan Content in Synovial Fluid of Dogs with Naturally Acquired Cranial Cruciate Ligament Rupture

To measure and compare activities of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and matrix metalloproteinase-3 (MMP-3); as well as sulfated glycosaminoglycan (S-GAG) content in synovial fluid from dogs with cranial cruciate ligament rupture (CCLR) and dogs with clinically normal stifles. To determine whether correlations exist between demographic and disease-related variables and these synovial markers. Prospective clinical study. Dogs with CCLR (n=23) and Beagles with normal stifle joints (n=21). Synovial fluid activities of proinflammatory cytokines (IL-1beta, IL-6, and TNF-alpha) were determined by bioassay. MMP-3 activity was measured using fluorogenic substrate. S-GAG contents were determined by dimethylmethylene blue dye-binding assay. Mann-Whitney U-test was used to compare results from CCLR joints with normal controls. Spearman's rank correlation test was used to evaluate associations between demographic and disease-related markers and synovial markers. Mean values for synovial markers were significantly higher in CCLR joints compared with controls. IL-1beta and MMP-3 were positively correlated with lameness duration. Activities of proinflammatory cytokines, MMP-3 activity and S-GAG contents were significantly elevated in synovial fluid from canine stifle joints with naturally acquired CCLR. These results indicate that there is joint inflammation and increased release of GAGs into synovial fluid, suggesting that these inflammatory changes are associated with depletion of proteoglycan from articular cartilage. Medical and surgical treatments designed to decrease joint inflammation and breakdown of proteoglycans may be of value in the management of CCLR in the dog.

Connective tissue growth factor/CCN2 overexpression in mouse synovial lining results in transient fibrosis and cartilage damage

Characteristics of osteoarthritis (OA) include cartilage damage, fibrosis, and osteophyte formation. Connective tissue growth factor (CTGF; also known as CCN2), is found in high levels in OA chondrocytes and is frequently involved in fibrosis, bone formation, and cartilage repair. The present study was therefore undertaken to investigate the potential role of CTGF in OA pathophysiology. We transfected the synovial lining of mouse knee joints with a recombinant adenovirus expressing human CTGF and measured synovial fibrosis and proteoglycan content in cartilage on days 1, 3, 7, 14, and 28. Messenger RNA (mRNA) expression in synovium and cartilage was measured on days 3, 7, and 21. CTGF induced synovial fibrosis, as indicated by accumulation of extracellular matrix and an increase in procollagen type I-positive cells. The fibrosis reached a maximum on day 7 and had reversed by day 28. Levels of mRNA for matrix metalloproteinase 3 (MMP-3), MMP-13, ADAMTS-4, ADAMTS-5, tissue inhibitor of metalloproteinases 1 (TIMP-1), and transforming growth factor beta were elevated in the fibrotic tissue. TIMP-1 expression was elevated on day 3, while expression of other genes did not increase until day 7 or later. CTGF induced proteoglycan depletion in cartilage as early as day 1. Maximal depletion was observed on days 3-7. Cartilage damage was reduced by day 28. A high level of MMP-3 mRNA expression was found in cartilage. CTGF overexpression did not induce osteophyte formation. CTGF induces transient fibrosis that is reversible within 28 days. Overexpression of CTGF in knee joints results in reversible cartilage damage, induced either by the high CTGF levels or via factors produced by the CTGF-induced fibrotic tissue.

In vivo MRI of cartilage pathogenesis in surgical models of osteoarthritis

To examine in vivo time-course changes in macromolecular composition of articular cartilage in two surgical models of osteoarthritis (goat: meniscal transection and cartilage incision; rabbit: medial meniscectomy). Collagen integrity and proteoglycan (PG) content were evaluated in both models by magnetization transfer (MT) and contrast-enhanced MRI, respectively. The MT rate k(m) for the exchange process between the bulk water and water bound to collagen was determined as a marker of the collagen network. Local changes in cartilage fixed charge density, i.e., where PGs are depleted, were derived from T(1) relaxation maps as obtained after an infusion of Gd(DTPA)(2-), a paramagnetic agent. In the goat model, the MT rate constant k(m) was significantly higher at 2 weeks post surgery, a possible sign of cartilage swelling, then decreased below baseline values, most likely indicative of disruption in the collagen framework. Meanwhile, post-Gd(DTPA)(2-) MRI acquisition indicated a significant and sustained loss of PGs. The rabbit model produced milder lesions. Although the difference was non-significant, k(m) steadily decreased in response to the surgical insult while kinetics of Gd(DTPA)(2-) uptake, after reaching a peak level at 6 weeks, were back to normal values after 12 weeks. In the goat model, joint instability and cartilage damage was a permanent trigger for cartilage degeneration producing MRI changes. However, biomechanical stress due to partial medial meniscectomy in knees of mature rabbits produced only mild, focal lesions and PG depletion that was partially reversible. This proof-of-concept study identified MT and T(1) parameters as useful surrogate markers in animal models of osteoarthritis.

Utility of delayed gadolinium-enhanced MRI (dGEMRIC) for qualitative evaluation of articular cartilage of patellofemoral joint

Delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) was used for the measurement of relative proteoglycan depletion of articular cartilage in the patellofemoral (PF) joint following a proprietary protocol, which was compared with the X-ray images, proton density weighted MR images (PDWI) and arthroscopic findings. The study examined 30 knees. The ages ranged from 16 to 74 (average 40.3) years. The Gd-DTPA(2-)containing contrast medium was used in a single dose. The subjects were made to exercise the knee joint for 10 min; and MR images were taken 2 h after intravenous injection of contrast medium. T1-calculated images were produced and the region of interest (ROI) was set as follows. (1) ROI1: entire articular cartilage in a slice through the center of the patella. (2) ROI2: low signal region in T1-calculated images, which were set in a blind fashion by two observers. (3) ROI3: articular cartilage on one side that includes ROI2 where low signal region were detected (medial or lateral). ROI3 was set to examine the contrast of ROI2 with surrounding articular cartilage. The average T1 values of ROI1 was 393.5+/-33.6 ms for radiographic grade 0 and 361.3+/-11.1 ms for grade I, which showed a significant difference (P=0.036). The T1 value of ROI2 was 351.6+/-28.2 ms for grade I, 361.9+/-38.3 ms for grade II, 362.1+/-67.7 ms for grade III, and 297.8+/-54.1 ms for grade IV according to arthroscopic Outerbridge classification. All cases, that demonstrated decrease of T1 values on dGEMRIC (ROI2), showed abnormal arthroscopic or direct viewing findings. The ratio (ROI3/ROI2) in cases of only slight damage classified as Outerbridge grade I (6 cases) was an average of 1.04+/-0.02 and was 1.0 or greater in all cases, thereby indicating well-defined contrast with the surrounding cartilage. The diagnosis of damage in articular cartilage was possible in all 16 cases with radiographic K-L grade I on dGEMRIC, while the intensity changes were not found in 10 of 16 cases on PDWI. The dGEMRIC with a single-dose would be useful on a diagnosis of the area demonstrating early relative proteoglycan depletion in the articular cartilage of the PF joint prior to any discernible changes in the subchondral bone on X-ray images and exceeds to plain MR images for examining deterioration of articular cartilage.

Characterization of proteoglycan depletion in articular cartilage using two‐dimensional time domain nuclear magnetic resonance

In vitro proteoglycan (PG) depletion in the 20-40% range (enzymatic PG depletion of normal cartilage in the early osteoarthritis (OA) PG depletion range) was investigated in articular cartilage using 2D time domain NMR relaxation techniques. Spin-lattice relaxation times were measured at low fields (T(1rho)) and at high fields (T(1)) using nonselective and selective excitation pulse sequences. The short relaxation time magnetization components in T(1rho) ( approximately 8% signal) and nonselective T(1) ( approximately 5% signal) experiments were significantly altered with PG degradation. In addition, a magnetization component ( approximately 5% signal) with a "fast " T(1) approximately 7 ms was observed in the T(1) experiment involving selective excitation. This fast T(1) was at least 10 times shorter than the short T(1) in the nonselective experiment and was associated with a strong magnetization exchange mechanism between collagen and PG. The results suggest that T(1rho) and T(1) (nonselective and selective) relaxation based MRI techniques, which focus on the short relaxation time magnetization components, have the potential of detecting molecular abnormalities associated with early OA earlier than single, long relaxation time component approaches.

Products resulting from cleavage of the interglobular domain of aggrecan in samples of synovial fluid collected from dogs with early- and late-stage osteoarthritis

To investigate interglobular domain (IGD) cleavage of aggrecan in dogs with naturally developing osteoarthritis (OA). Samples of synovial fluid (SF) obtained from 3 cubital (elbow) joints and 3 stifle joints of 4 clinically normal dogs, 24 elbow joints of 12 dogs with early-stage OA, 8 stifle joints of 5 dogs with early-stage OA, and 10 stifle joints of 9 dogs with late-stage OA. Fractions of SF were assayed for total glycosaminoglycan (GAG) content and also subjected to Western blot analysis by use of monoclonal antibodies against neoepitopes generated by cleavage of the IGD of the aggrecan protein core by matrix metalloproteinase (MMP; BC-14) and aggrecanase (BC-3). Total GAG content of SF from joints of clinically normal dogs did not differ from that of dogs with early-stage OA. The GAG content of SF from joints of dogs with late-stage OA was significantly lower, compared with GAG content for other SF samples. Aggrecanase-generated fragments were detected in SF from all groups but not in all samples. Matrix metalloproteinase-generated fragments were not detected in any SF samples. In early-stage OA, high-molecular-weight aggrecanase-generated aggrecan catabolites were evident. GAG content of SF obtained from dogs with late-stage OA is significantly decreased, suggesting proteoglycan depletion of cartilage. Aggrecanases, but not MMPs, are the major proteolytic enzymes responsible for IGD cleavage of aggrecan in canine joints. Analyses of SF samples to detect aggrecanase-generated catabolites may provide an early biomarker for discriminating early- and late-stage OA in dogs.

Changes in the amounts of chondroitin sulfate proteoglycans in rat brain after neonatal hypoxia‐ischemia

Chondroitin sulfate proteoglycans have been shown to participate in the pathogenesis of neuronal damages in the injured adult central nervous system (CNS). Upregulated expression of chondroitin sulfate proteoglycans has been reported around the injured sites and depletion of these chondroitin sulfate proteoglycans brings about increased axonal regeneration in the injured adult CNS. To examine if chondroitin sulfate proteoglycans are also involved in the pathologic process of hypoxia-ischemia in the neonatal brain, expressions of three chondroitin sulfate proteoglycans, neurocan, phosphacan, and neuroglycan C, were examined in rat brains after neonatal hypoxia-ischemia. Hypoxic-ischemic rats were produced by ligating the right carotid artery of 7-day-old rats, followed by 8% oxygen exposure. Western blot analysis revealed that in contrast to injured adult CNS, the amount of neurocan was reduced 24 hr after hypoxia in the neonatal hypoxic-ischemic cerebral hemisphere. The amounts of phosphacan and neuroglycan C were also reduced significantly 24 hr after hypoxia at the right injured cortex compared to those at the left cortex. Surprisingly, the immunohistologic staining for phosphacan was conversely intensified both at 24 hr and 8 days after hypoxia at the infarcted area. In addition, the habenula and fascicules retroflexus in the right cerebral hemisphere degenerated and became intensely immunostained with the anti-phosphacan antibody shortly after hypoxia. Hypoxic-ischemic insult may unmask phosphacan epitopes at the injured sites, resulting in intensified immunostaining. Because intensified immunostaining for neurocan and neuroglycan C was not observed, unmasking seems to be specific to phosphacan among these three chondroitin sulfate proteoglycans.

Blocking of Interleukin-17 during Reactivation of Experimental Arthritis Prevents Joint Inflammation and Bone Erosion by Decreasing RANKL and Interleukin-1

Rheumatoid arthritis is characterized by an intermittent course of disease with alternate periods of remission and relapse. T cells, and in particular the T-cell cytokine interleukin-17 (IL-17), are expected to be involved in arthritic flares. Here, we report that neutralizing endogenous IL-17 during reactivation of antigen-induced arthritis prevents joint inflammation and bone erosion. Synovial IL-17 mRNA expression was clearly up-regulated during primary arthritis and was further enhanced after antigen rechallenge. Neutralization of IL-17 significantly prevented joint swelling at day 1 of flare and significantly suppressed joint inflammation and cartilage proteoglycan depletion at day 4, as assessed by histology. Blocking IL-17 also clearly reduced bone erosions. Cathepsin K, a marker of osteoclast-like activity, and synovial RANKL mRNA expression were both suppressed. The degree of bone erosions strongly correlated with the severity of joint inflammation, suggesting that anti-IL-17 treatment reduced bone erosion by suppressing joint inflammation. Interestingly, blocking IL-17 suppressed synovial expression of both IL-1beta and tumor necrosis factor-alpha, whereas blocking IL-1 did not affect tumor necrosis factor-alpha levels. These data indicate that IL-17 is an important upstream mediator in joint pathology during flare-up of experimental arthritis.

Intrajoint comparisons of gene expression patterns in human osteoarthritis suggest a change in chondrocyte phenotype

Osteoarthritis (OA) is a degenerative cartilage disease with varying degrees of severity within a given joint. The purpose of this study was to define a sampling procedure for comparing human minimal and advanced OA cartilage in the same patient and to determine basic patterns of gene expression in these regions. A specific hypothesis under study was that the expression level of Bcl-2 would correlate with Sox9 and aggrecan mRNA expression in vivo as has been demonstrated in vitro. Femoral condylar advanced OA cartilage was located within 1 cm of overt lesions, and minimal cartilage was taken from areas with no obvious surface defects. Histological sections were scored for disease severity and chondroitin sulfate and hydroxyproline content was determined. The expression level of nine specific genes (aggrecan, collagen type II, Bcl-2, Sox9, Link protein, osteopontin, and MMP-13, -3, and -9) was determined by quantitative real time PCR. The scores for fibrillation, chondrocyte cloning, and proteoglycan depletion were significantly different between advanced and minimal OA cartilage. The advanced OA cartilage had significantly less chondroitin sulfate than the minimal OA cartilage. Osteopontin mRNA expression showed a 3.6-fold increase in advanced compared to minimal OA cartilage. In contrast, the level of mRNA coding for aggrecan, link protein, Bcl-2, Sox9 and MMP-3, -9, -13 were all decreased in advanced compared to minimal cartilage in the majority of the patients studied. Collagen type II mRNA expression displayed a wide-range of variation. A statistically significant correlation was observed both between Bcl-2 and Sox9 mRNA level, and between Bcl-2 and aggrecan mRNA expression. The patient matched comparison of minimal and advanced OA cartilage revealed differences in cellular and tissue characteristics, and changes in gene expression that may be involved in OA progression. In addition, Bcl-2 may also play a role in regulating the expression of aggrecan through Sox9 in vivo as well as in vitro.

Association of articular cartilage degradation and loss of boundary‐lubricating ability of synovial fluid following injury and inflammatory arthritis

To study the relationship between the boundary-lubricating ability of synovial fluid (SF) and articular cartilage damage in a rabbit knee injury model, to correlate collagen markers of such damage with SF boundary-lubricating ability and elastase activity, and to examine the lubricating ability of SF, together with collagen markers of articular cartilage damage, under the inflammatory conditions of knee joint synovitis (KJS) and rheumatoid arthritis (RA). SF was aspirated weekly from the affected knee joints of 10 adult rabbits following transection of the anterior and posterior cruciate ligaments. The boundary-lubricating ability of SF was determined in vitro using a previously described friction apparatus. Lubricin concentrations and type II collagen (CII) peptides were quantified by sandwich enzyme-linked immunosorbent assays (ELISAs). Levels of the C-terminal neoepitope 9A4 (derived from collagenase degradation of CI, CII, and CIII) and of epitope 5-D-4 of keratan sulfate (a marker of proteoglycan depletion) were quantified by inhibition ELISAs. Elastase activity was measured spectrophotometrically. The sensitivity of purified human lubricin to digestion by neutrophil elastase (NE) was examined by Western blotting. The lubricating ability of SF from injured rabbit knees was significantly decreased at weeks 2 and 3 compared with week 1 after injury. Lubricin concentrations were significantly higher at week 1 than at weeks 2 and 3. CII peptide concentrations increased significantly at weeks 2 and 3 compared with week 1, while 9A4 neoepitope concentrations increased significantly at week 3 compared with weeks 1 and 2. There were no significant differences in epitope 5-D-4 concentrations among the 3 weeks. Elastase activity in SF increased significantly at weeks 2 and 3 compared with week 1. Elastase activity correlated significantly with diminishing lubrication at weeks 1, 2, and 3. SF from patients with KJS or RA exhibited deficient lubrication and elevated levels of CII peptides compared with SF from normal controls. NE was shown to completely degrade purified human lubricin in vitro. Loss of boundary-lubricating ability of SF after injury is associated with damage to the articular cartilage matrix. This can be attributed to inflammatory processes resulting from the injury, particularly in the early phases. This association also exists in patients with acute knee injuries or progressive chronic inflammatory arthritis.

Induction of cartilage damage by overexpression of T cell interleukin‐17A in experimental arthritis in mice deficient in interleukin‐1

To examine the capacity of T cell interleukin-17A (IL-17A; referred to hereinafter as IL-17) to induce cartilage damage during experimental arthritis in the absence of IL-1. Local IL-17 gene transfer was performed in the knee joint of IL-1-deficient mice and wild-type controls during streptococcal cell wall (SCW)-induced arthritis. Knee joints were isolated at various time points for histologic analysis of cartilage proteoglycan (PG) depletion. Expression of messenger RNA for inducible nitric oxide synthase, matrix metalloproteinases (MMPs) 3, 9, and 13, and ADAMTS-4 was determined by quantitative polymerase chain reaction analysis. VDIPEN staining was analyzed to study MMP-mediated cartilage damage. In addition, systemic anti-IL-1alpha/beta antibody treatment was performed in mice immunized with type II collagen and injected locally with an adenoviral vector expressing IL-17 or with control adenovirus. Knee joints were isolated and analyzed for cartilage PG depletion, chondrocyte death, and cartilage surface erosion. During SCW-induced arthritis, local T cell IL-17 gene transfer turned this acute, macrophage-driven joint inflammation into a severe, chronic arthritis accompanied by aggravated cartilage damage. Of high interest, the IL-1 dependency of cartilage PG depletion was fully abrogated when IL-17 was locally overexpressed in the joint. Moreover, local IL-17 gene transfer increased MMP expression without the need for IL-1, although IL-1 remained essential for part of the cartilage VDIPEN expression. Furthermore, when IL-17 was overexpressed in the knee joints of mice with collagen-induced arthritis, anti-IL-1 treatment did not reduce the degree of chondrocyte death or cartilage surface erosion. These data show the capacity of IL-17 to replace the catabolic function of IL-1 in cartilage damage during experimental arthritis.

NADPH-oxidase-driven oxygen radical production determines chondrocyte death and partly regulates metalloproteinase-mediated cartilage matrix degradation during interferon-γ-stimulated immune complex arthritis

In previous studies we have found that FcγRI determines chondrocyte death and matrix metalloproteinase (MMP)-mediated cartilage destruction during IFN-γ-regulated immune complex arthritis (ICA). Binding of immune complexes (ICs) to FcγRI leads to the prominent production of oxygen radicals. In the present study we investigated the contribution of NADPH-oxidase-driven oxygen radicals to cartilage destruction by using p47phox-/- mice lacking a functional NADPH oxidase complex. Induction of a passive ICA in the knee joints of p47phox-/- mice resulted in a significant elevation of joint inflammation at day 3 when compared with wild-type (WT) controls as studied by histology. However, when IFN-γ was overexpressed by injection of adenoviral IFN-γ in the knee joint before ICA induction, a similar influx of inflammatory cells was found at days 3 and 7, comprising mainly macrophages in both mouse strains. Proteoglycan depletion from the cartilage layers of the knee joints in both groups was similar at days 3 and 7. Aggrecan breakdown in cartilage caused by MMPs was further studied by immunolocalisation of MMP-mediated neoepitopes (VDIPEN). VDIPEN expression in the cartilage layers of arthritic knee joints was markedly lower (between 30 and 60%) in IFN-γ-stimulated arthritic p47phox-/- mice at day 7 than in WT controls, despite significant upregulation of mRNA levels of various MMPs such as MMP-3, MMP-9, MMP-12 and MMP-13 in synovia and MMP-13 in cartilage layers as measured with quantitative RT-PCR. The latter observation suggests that oxygen radicals are involved in the activation of latent MMPs. Chondrocyte death, determined as the percentage of empty lacunae in articular cartilage, ranged between 20 and 60% at day 3 and between 30 and 80% at day 7 in WT mice, and was completely blocked in p47phox-/- mice at both time points. FcγRI mRNA expression was significantly lower, and FcγRII and FcγRIII were higher, in p47phox-/- mice than in controls. NADPH-oxidase-driven oxygen radical production determines chondrocyte death and aggravates MMP-mediated cartilage destruction during IFN-γ-stimulated IC-mediated arthritis. Upregulation of FcγRI by oxygen radicals may contribute to cartilage destruction.

Reduced transforming growth factor-beta signaling in cartilage of old mice: role in impaired repair capacity

Osteoarthritis (OA) is a common joint disease, mainly effecting the elderly population. The cause of OA seems to be an imbalance in catabolic and anabolic factors that develops with age. IL-1 is a catabolic factor known to induce cartilage damage, and transforming growth factor (TGF)-beta is an anabolic factor that can counteract many IL-1-induced effects. In old mice, we observed reduced responsiveness to TGF-beta-induced IL-1 counteraction. We investigated whether expression of TGF-beta and its signaling molecules altered with age. To mimic the TGF-beta deprived conditions in aged mice, we assessed the functional consequence of TGF-beta blocking. We isolated knee joints of mice aged 5 months or 2 years, half of which were exposed to IL-1 by intra-articular injection 24 h prior to knee joint isolation. Immunohistochemistry was performed, staining for TGF-beta1, -2 or -3, TGF-betaRI or -RII, Smad2, -3, -4, -6 and -7 and Smad-2P. The percentage of cells staining positive was determined in tibial cartilage. To mimic the lack of TGF-beta signaling in old mice, young mice were injected with IL-1 and after 2 days Ad-LAP (TGF-beta inhibitor) or a control virus were injected. Proteoglycan (PG) synthesis (35S-sulfate incorporation) and PG content of the cartilage were determined. Our experiments revealed that TGF-beta2 and -3 expression decreased with age, as did the TGF-beta receptors. Although the number of cells positive for the Smad proteins was not altered, the number of cells expressing Smad2P strongly dropped in old mice. IL-1 did not alter the expression patterns. We mimicked the lack of TGF-beta signaling in old mice by TGF-beta inhibition with LAP. This resulted in a reduced level of PG synthesis and aggravation of PG depletion. The limited response of old mice to TGF-beta induced-IL-1 counteraction is not due to a diminished level of intracellular signaling molecules or an upregulation of intracellular inhibitors, but is likely due to an intrinsic absence of sufficient TGF-beta receptor expression. Blocking TGF-beta distorted the natural repair response after IL-1 injection. In conclusion, TGF-beta appears to play an important role in repair of cartilage and a lack of TGF-beta responsiveness in old mice might be at the root of OA development.

Stimulation of proteoglycan synthesis by glucuronosyltransferase-I gene delivery: A strategy to promote cartilage repair

Osteoarthritis is a degenerative joint disease characterized by a progressive loss of articular cartilage components, mainly proteoglycans (PGs), leading to destruction of the tissue. We investigate a therapeutic strategy based on stimulation of PG synthesis by gene transfer of the glycosaminoglycan (GAG)-synthesizing enzyme, beta1,3-glucuronosyltransferase-I (GlcAT-I) to promote cartilage repair. We previously reported that IL-1beta down-regulated the expression and activity of GlcAT-I in primary rat chondrocytes. Here, by using antisense oligonucleotides, we demonstrate that GlcAT-I inhibition impaired PG synthesis and deposition in articular cartilage explants, emphasizing the crucial role of this enzyme in PG anabolism. Thus, primary chondrocytes and cartilage explants were engineered by lipid-mediated gene delivery to efficiently overexpress a human GlcAT-I cDNA. Interestingly, GlcAT-I overexpression significantly enhanced GAG synthesis and deposition as evidenced by (35)S-sulfate incorporation, histology, estimation of GAG content, and fluorophore-assisted carbohydrate electrophoresis analysis. Metabolic labeling and Western blot analyses further suggested that GlcAT-I expression led to an increase in the abundance rather than in the length of GAG chains. Importantly, GlcAT-I delivery was able to overcome IL-1beta-induced PG depletion and maintain the anabolic activity of chondrocytes. Moreover, GlcAT-I also restored PG synthesis to a normal level in cartilage explants previously depleted from endogenous PGs by IL-1beta-treatment. In concert, our investigations strongly indicated that GlcAT-I was able to control and reverse articular cartilage defects in terms of PG anabolism and GAG content associated with IL-1beta. This study provides a basis for a gene therapy approach to promote cartilage repair in degenerative joint diseases.