Human Osteoarthritis Patients Research Articles

Expression of calcitonin gene-related peptide in the infrapatellar fat pad in knee osteoarthritis patients

BackgroundThe infrapatellar fat pad (IPFP) has been implicated as a possible source of osteoarthritis (OA) development and knee pain due to the production of inflammatory mediators and the existence of nerve fibers within this structure. Calcitonin gene-related peptide (CGRP) is a vasodilatory neuropeptide that is localized to joint tissues and has recently been implicated in the development of knee OA and OA pain. To date, however, the expression levels of CGRP in the IPFP of human knee OA patients have not been examined.MethodsIFFP and synovial (SYN) tissues were harvested from 100 individuals with radiographic knee OA (unilateral Kellgren/Lawrence [K/L] grades 2–4) during total knee arthroplasty and subjected to immunohistochemical analysis for CGRP localization. In addition, the messenger RNA (mRNA) expression levels of CGRP and cyclooxygenase-2 (COX-2) in the collected tissues were evaluated and compared using real-time PCR analysis of total RNA extracts. CGRP and COX-2 mRNA expression were also compared among individuals with K/L grades 2–4.ResultsCGRP-positive cells were detected in the capillaries within the IPFP and lining layer of SYN tissue. The expression levels of CGRP in the IPFP were positively correlated with COX-2 and were significantly higher than those in SYN tissue. CGRP expression in tissue from the KL4 group was twofold higher than that from the KL2 group.ConclusionsThe IPFP of knee OA patients produces relatively high levels of CGRP, which may be regulated by COX-2 at the transcriptional level. Further studies are needed to determine if CGRP levels are directly linked to OA pathology.

A Promising New Treatment for Osteoarthritis

At present, there is no definite cure for the underlying causes of osteoarthritis (OA). However, adipose tissuederived stem cells (ASCs) in the form of stromal vascular fraction can offer an alternative at this time. ASCs are one type of mesenchymal stem cells that have been utilized and have demonstrated an ability to regenerate cartilage in human patients with OA. Here, we highlight the possibility of ASCs regenerating cartilage in human OA patients.

Insulin-Like Growth Factor II (IGF-II) Inhibits IL-1β-Induced Cartilage Matrix Loss and Promotes Cartilage Integrity in Experimental Osteoarthritis.

Osteoarthritis (OA) is a widespread chronic joint disease characterized by articular cartilage destruction and accompanied by pain and disability. In this study, we found that the expression of Insulin-like Growth Factor II (IGF-II) was reduced in articular cartilage in human OA patients as well as in the murine experimental OA model of destabilization of the medial meniscus (DMM). In primary human articular chondrocytes, ectopic expression of lentiviral IGF-II inhibited pro-inflammatory cytokine IL-1β-induced NF-κB activation as well as catabolic gene expression. Interestingly, IGF-II did not significantly alter the phosphorylation states of ERK1/2 or Akt, which are kinases typically activated by IGF-I. Instead, it induced the activity of phospholipase C (PLC) and a PLC inhibitor blocked the inhibitory activity of IGF-II against IL-1β, suggesting that this activity is mediated through PLC. Furthermore, IGF-II increased cartilage matrix levels and decreased MMP13 protein expression in explanted human OA cartilage cultures in vitro. In the in vivo DMM model, intraarticular injection of lentiviral IGF-II led to enhanced cartilage matrix levels and decreased MMP13 protein expression, as well as reduced osteophyte formation and subchondral bone sclerosis. Therefore, our results suggest that IGF-II can promote cartilage integrity and halt knee joint destruction in OA.

Examining O‐xylosyltransferase in Drosophila

O‐xylosyltransferase (Oxt) is a transmembrane glycosyltransferase that initiates the first step in heperan and chrondroitin sulfate (HS and CS) proteoglycan biosynthesis. HSPGs and CSPGs are abundant in the extracellular environment of many tissues, such as articular cartilage in humans. In this tissue, HSPGs and CSPGs are essential regulation of signaling pathways that maintain tissue homeostasis. If homeostasis is not preserved, joint tissue will degrade and osteoarthritis (OA) can occur. Elevated levels of circulating Oxt have been detected in human OA patients suggesting shed Oxt may have an extracellular function. Little is known about Oxt behavior or function once it is released from the plasma membrane. Cell culture experiments have shown that Oxt, normally located in the Golgi and the endoplasmic reticulum (ER), can be shed by proteolysis activity and move into the media. It is likely that the same result will be observed in vivo. The overall aim of this project is to determine if Oxt released from expressing cells in vivo and what, if any, function extracellular Oxt has in organismal development. The data collected thus far shows localization of Oxt to the Golgi and the ER where it could have an active role in HSPG and CSPG biosynthesis. Movement of Oxt from expressing cells to non‐expressing cells is observed in the imaginal wing disc of Drosophila larva. Extracellular staining shows that Oxt is found predominantly on the apical surface and can be detected in the peripodial space suggesting it is released from expressing cells. Western analysis of Drosophila hemolymph indicated Oxt is released into hemolymph, as observed in humans. The results suggest that cleaved Oxt is found in the hemolymph of Drosophila and future studies will explore the potential function of shed Oxt.

Initial evaluation of nighttime restlessness in a naturally occurring canine model of osteoarthritis pain.

Chronic pain due to osteoarthritis (OA) can lead to significant disruption of sleep and increased restlessness. Our objective was to assess whether naturally occurring canine OA is associated with nighttime restlessness and so has potential as a model of OA-associated sleep disturbance. The study was designed as a two-part prospective masked, placebo-controlled study using client-owned dogs (Part A n = 60; Part B n = 19). Inclusion criteria consisted of OA-associated joint pain and mobility impairment. The primary outcome measure for both parts was nighttime accelerometry. In Part B, quality of sleep was assessed using a clinical metrology instrument (Sleep and Night Time Restlessness Evaluation Score, SNoRE). Part A included dogs receiving two weeks of non-steroidal anti-inflammatory drug (NSAID) preceded with two weeks of no treatment. Part B was a crossover study, with NSAID/placebo administered for two weeks followed by a washout period of one week and another two weeks of NSAID/placebo. Repeated measures analysis of variance was used to assess differences between baseline and treatment. There were no significant changes in accelerometry-measured nighttime activity as a result of NSAID administration. SNoRE measures indicated significant improvements in aspects of the quality of nighttime sleep that did not involve obvious movement. These results reflect the few similar studies in human OA patients. Although accelerometry does not appear to be useful, this model has potential to model the human pain-related nighttime sleep disturbance, and other outcome measures should be explored in this model.

Matrilin-3 Inhibits Chondrocyte Hypertrophy as a Bone Morphogenetic Protein-2 Antagonist

Increased chondrocyte hypertrophy is often associated with cartilage joint degeneration in human osteoarthritis patients. Matrilin-3 knock-out (Matn3 KO) mice exhibit these features. However, the underlying mechanism is unknown. In this study, we sought a molecular explanation for increased chondrocyte hypertrophy in the mice prone to cartilage degeneration. We analyzed the effects of Matn3 on chondrocyte hypertrophy and bone morphogenetic protein (Bmp) signaling by quantifying the hypertrophic marker collagen type X (Col X) gene expression and Smad1 activity in Matn3 KO mice in vivo and in Matn3-overexpressing chondrocytes in vitro. The effect of Matn3 and its specific domains on BMP activity were quantified by Col X promoter activity containing the Bmp-responsive element. Binding of MATN3 with BMP-2 was determined by immunoprecipitation, solid phase binding, and surface plasmon resonance assays. In Matn3 KO mice, Smad1 activity was increased more in growth plate chondrocytes than in wild-type mice. Conversely, Matn3 overexpression in hypertrophic chondrocytes led to inhibition of Bmp-2-stimulated, BMP-responsive element-dependent Col X expression and Smad1 activity. MATN3 bound BMP-2 in a dose-dependent manner. Multiple epidermal growth factor (EGF)-like domains clustered together by the coiled coil of Matn3 is required for Smad1 inhibition. Hence, as a novel BMP-2-binding protein and antagonist in the cartilage extracellular matrix, MATN3 may have the inherent ability to inhibit premature chondrocyte hypertrophy by suppressing BMP-2/Smad1 activity.

Type X collagen levels are elevated in serum from human osteoarthritis patients and associated with biomarkers of cartilage degradation and inflammation.

BackgroundOsteoarthritis (OA) is the most common degenerative joint disease, of which the pathogenesis is inadequately understood. Hypertrophy-like changes have been observed as part of the progression of OA. The aim of the study was to develop and characterize a novel biomarker of chondrocytes hypertrophy and investigate how this marker was associated with cartilage degradation and inflammation in patients with various degrees of OA.MethodsA competitive ELISA, C-Col10, applying a well-characterized monoclonal antibody was developed as a biomarker of chondrocyte hypertrophy through measurement of type X collagen (ColX). The levels of C-Col10, C2M (matrix metalloproteinase-derived fragments of type II collagen) and hsCRP (high sensitive C-reactive protein) were quantified by ELISAs in serum of 271 OA patients stratified by Kellgren-Lawrence (KL) score 0–4. Associations between serum levels of the three biomarkers (log transformed) were analyzed by Pearson’s correlation and differences in C-Col10 levels between patients with high and low levels of inflammation measured by hsCRP were analyzed by ANOVA.ResultsWe developed a C-Col10 assay measuring the C-terminus of ColX. We found significantly higher levels of ColX in patients with KL score 2 compared to patients with no radiographic evidence of OA (KL0) (p = 0.04). Levels of ColX were significantly elevated in OA patients with above normal hsCRP levels (p < 0.0001), as well as significantly correlated with levels of C2M (r = 0.55, p < 0.0001), which suggested that chondrocyte hypertrophy was associated with inflammation and cartilage degradation. There was no correlation between C2M and hsCRP. Age and BMI adjustment didn’t change the results. Immuno-staining revealed that ColX was predominately located around the hypertrophic chondrocytes and the clustered chondrocytes indicating that C-Col10 measures may be linked to cartilage hypertrophic changes.ConclusionsWe developed a novel assay, C-Col10, for measurement of chondrocyte hypertrophy and found its levels significantly elevated in OA patients with KL score of 2, and also in OA patients with above normal hsCRP levels. Concentration of C-Col10 strongly correlated with levels of C2M, a marker of cartilage destruction. The data suggest that chondrocyte hypertrophy and subsequent collagen X fragmentation seem to be increased in a subset of patients with inflammatory OA.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2474-15-309) contains supplementary material, which is available to authorized users.

Investigation on the role of delta/notch like egf-related receptor in the pathogenesis of osteoarthritis

s / Osteoarthritis and Cartilage 22 (2014) S57–S489 S369 Figure 2. (a) Plot of cartilage thickness and trabecular thickness, wherein each sample specific correlation yields an inverse relationship in sclerotic samples. Individual correlation coefficients are R 1⁄4 0.62, 0.87, 0.88, 0.85. 0.60, 0.77, respectively, (b) Plot of denuded surface and bone volume/total volume, indicating, for each sample, greater bone density in regions of lost cartilage coverage. Individual correlation coefficients are R 1⁄4 0.41, 0.84, 0.74, 0.38, 0.80, 0.41, respectively. The combined correlation coefficients for Cg.Th versus Tb.Th and %DS versus BV/TV, are 0.74 and 0.56, respectively. Conclusions: The results of this work show different relationships between cartilage and bone structures in end stage OA samples displaying bone sclerosis, than for samples with a more osteoporosis-like change. This suggests that identifying the structural cues involved with OA disease progression could allow for a more accurate diagnosis of disease. It must be stated, however, that more osteoporotic-like samples will need to be measured to confirm this result. Specifically, changes associated with sclerotic or osteoporotic-like bone change may require alternative physical therapy or drug regimes. Future work will focus on correlating these structural changes to specific genes and signalling pathways, as well as biomechanical loading and use patterns in the shoulder joint. 648 NKX3.2-BARX1 CROSSTALK IN CHONDROCYTE AND ITS ROLE IN OSTEOARTHRITIS PATHOGENESIS D-W. Kim. Yonsei Univ., Seoul, REPUBLIC OF KOREA Osteoarthritis (OA) is a prevalent disease affecting majority of aged human, yet its effective treatment is currently absent. We have previously shown that Nkx3.2-mediated consitutive activation of NF-kB functions to maintain chondrocyte viability. Besides, Barx1 has been implicated for its role in development of gut, spleen and craniofacial skeletons, but its precise molecular function in chondrocyte has been poorly understood. Here we show that Barx1 can induce programmed cell death in chondrocyte by abrogating Nkx3.2-mediated NF-kB activation. Interestingly, we also observed remarkable decrease and increase in Nkx3.2 and Barx1 expression, respectively, both in mouse OA models and in human OA patients. Consistent with these, in vivo experiments employing intra-articular infection of Lentivirus modifying Nkx3.2 or Barx1 expression revealed that osteoarthritic cartilage destruction can be suppressed by Nkx3.2, whereas Barx1 aggravates it. Furthermore, importantly, we observed that either Nkx3.2 overexpression or Barx1 knockdown is capable of rendering regeneration of damaged cartilage in DMM-OA analyses in mice. Lastly, we demonstrate that OA progression is decelerated and accelerated in Nkx3.2 transgenic and knockout mice, respectively. Thus, these results indicate that Nkx3.2-Barx1 crosstalk plays a role in OA pathogenesis and may serve as promising therapeutic targets for OA modification. 649 INVESTIGATION ON THE ROLE OF DELTA/NOTCH LIKE EGF-RELATED RECEPTOR IN THE PATHOGENESIS OF OSTEOARTHRITIS L. Berninger y, A. Balkenhol y, C. Baier z, U. M€ uller-Ladner y, E. Neumann y, M. Geyer y. y Justus-Liebig-Univ. of Giessen, KerckhoffKlinik, Bad Nauheim, Germany; zUniv. of Regensburg, Regensburg,

SAT0064 The expression of HDAC6 is reduced in rheumatoid arthritis synovial fibroblasts

BackgroundSince rheumatoid synovial cells produce large amounts of various cytokines and proteases, up to 30% of all newly synthesized proteins are unfolded, leading to a cellular stress condition. Histone-deacetylase (HDAC)...

Development and validation of mechanical allodynia as a pain readout in a preclinical model of osteoarthritis

Development and validation of mechanical allodynia as a pain readout in a preclinical model of osteoarthritis

Identification of novel molecular scaffolds for the design of MMP-13 inhibitors: A first round of lead optimization

Osteoarthritis (OA) is the leading cause of joint pain and disability in middle-aged and elderly patients, and is characterized by progressive loss of articular cartilage. Among the various matrix metalloproteinases (MMPs), MMP-13 is specifically expressed in the cartilage of human OA patients and is not present in normal adult cartilage. Thus, MMP-13-selective inhibitors are promising candidates in osteoarthritis therapy. Recently, we designed an N-isopropoxy-arylsulfonamide-based hydroxamate inhibitor, which showed low nanomolar activity and high selectivity for MMP-13. In parallel to further studies aiming to assess the in vivo activity of our compound, we screened the Life Chemicals database through computational docking to seek for novel scaffolds as zinc-chelating non-hydroxamate inhibitors. Experimental evaluation of 20 selected candidate compounds verified five novel leads with IC 50 in the low μM range. These newly discovered inhibitors are structurally unrelated to the ones known so far and provide useful scaffolds to develop compounds with more desirable properties. Finally, a first round of structure-based optimization on lead 1 was accomplished and led to an increase in potency of more than 5 fold.

Downregulation of Protein Kinase CK2 Activity Facilitates Tumor Necrosis Factor-α-Mediated Chondrocyte Death through Apoptosis and Autophagy

Despite the numerous studies of protein kinase CK2, little progress has been made in understanding its function in chondrocyte death. Our previous study first demonstrated that CK2 is involved in apoptosis of rat articular chondrocytes. Recent studies have suggested that CK2 downregulation is associated with aging. Thus examining the involvement of CK2 downregulation in chondrocyte death is an urgently required task. We undertook this study to examine whether CK2 downregulation modulates chondrocyte death. We first measured CK2 activity in articular chondrocytes of 6-, 21- and 30-month-old rats. Noticeably, CK2 activity was downregulated in chondrocytes with advancing age. To build an in vitro experimental system for simulating tumor necrosis factor (TNF)-α-induced cell death in aged chondrocytes with decreased CK2 activity, chondrocytes were co-treated with CK2 inhibitors and TNF-α. Viability assay demonstrated that CK2 inhibitors facilitated TNF-α-mediated chondrocyte death. Pulsed-field gel electrophoresis, nuclear staining, flow cytometry, TUNEL staining, confocal microscopy, western blot and transmission electron microscopy were conducted to assess cell death modes. The results of multiple assays showed that this cell death was mediated by apoptosis. Importantly, autophagy was also involved in this process, as supported by the appearance of a punctuate LC3 pattern and autophagic vacuoles. The inhibition of autophagy by silencing of autophage-related genes 5 and 7 as well as by 3-methyladenine treatment protected chondrocytes against cell death and caspase activation, indicating that autophagy led to the induction of apoptosis. Autophagic cells were observed in cartilage obtained from osteoarthritis (OA) model rats and human OA patients. Our findings indicate that CK2 down regulation facilitates TNF-α-mediated chondrocyte death through apoptosis and autophagy. It should be clarified in the future if autophagy observed is a consequence versus a cause of the degeneration in vivo.

S100A8 and S100A9 induce a catabolic shift in chondrocytes from human osteoarthritis patients

Background and objectiveS100A8 and S100A9 are damage associated molecular patterns that are associated with inflammation and cartilage and bone erosion during human rheumatoid arthritis. They are produced in large amounts...

New Hope for the Treatment of Osteoarthritis Through Selective Inhibition of MMP-13

Osteoarthritis (OA) is the leading cause of joint pain and disability in middle-aged and elderly patients, and is characterized by progressive loss of articular cartilage that eventually leads to a complex process involving degradation of various components of the cartilage matrix, chief among them are the cartilage-specific type II collagen (CII) and aggrecan. While the loss of aggrecan is thought to be an early and reversible process, degradation of CII is considered to be irreversible and a key step in the loss of structural and functional integrity of cartilage. Among the various matrix metalloproteinases (MMPs), MMP-13 is specifically expressed in the cartilage of human OA patients and is not present in normal adult cartilage. It is the major collagenase in OA cartilage and has the highest activity against CII. However, the clinical utility of broad-spectrum MMP inhibitors developed for treatment of OA has been restricted by dose- and duration-dependent musculoskeletal side effects in humans. Consequently, selectively inhibiting the MMP-13 would seem to be an attractive therapeutic objective. This review mainly focuses on selective MMP-13 inhibitors development in terms of OA since the late 90s, in terms of synthetic compounds of low molecular mass incorporating specific zinc-binding groups, non-zinc-binding groups. In addition, dual inhibitors of MMP-13 and aggrecanase are also reviewed. Special emphasis is placed on logistic concerns for lead compound search as well as the structure-activity relationship (SAR) in this field. Through these methods, new hope is emerging for the treatment of OA through selective inhibition of MMP-13.

Periodic knee injections of BMP‐7 delay cartilage degeneration induced by excessive running in rats

Strenuous running of rats enhances mechanical stress on the knee, thereby inducing degeneration of articular cartilage. Bone morphogenetic protein-7 (BMP-7) has an inhibitory effect on cartilage degeneration, suggesting its usefulness for human osteoarthritis patients. However, its mode of administration should be investigated. We examined whether weekly knee injections of BMP-7 delayed the progression of cartilage degeneration. Wistar rats were forced to run 30 km in 6 weeks on a rodent treadmill, and BMP-7 was injected weekly into the knee. Macroscopically and histologically, this strenuous running regimen induced cartilage degeneration. Weekly injections of 250 ng BMP-7 delayed the progression of cartilage degeneration. Immunohistochemically, in the control knee, type II collagen expression decreased, while BMP-7 expression in chondrocytes slightly increased. Interestingly, weekly injection of BMP-7 increased BMP-7 expression even 9 days after the final injection. Disulfate disaccharide keratan sulfate in serum transiently increased in the control group, while it remained at a low level in the BMP-7 group. Weekly BMP-7 injection increased BMP-7 expression in chondrocytes and its effect seemed to last more than 7 days. The effect of BMP-7 could be monitored by serum keratan sulfate concentration. Periodical injections of BMP-7 delayed progression of cartilage degeneration induced by excessive running in rats.

Changes in gait after bilateral meniscectomy in sheep: effect of two hyaluronan preparations

BackgroundThis study examined the effect of bilateral meniscectomy on ground reaction forces (GRFs) in sheep, and the therapeutic effect of two hyaluronan (HA) preparations. MethodsEighteen sheep were subjected to bilateral lateral meniscectomy and were treated from 16 to 20 weeks post-operatively with intraarticular Hyalgan (Fidia Farmaceutici), HYADD4-G (a novel amide derivative; Fidia Farmaceutici), or saline placebo (n = 6 per group). GRFs were assessed at baseline and 6, 12, 16, 22, and 26 weeks postoperatively. Rheological parameters and HA content of synovial fluid samples were assessed using micro-Fourier rheometry. ResultsMeniscectomy significantly reduced GRF and abolished the normal two-peak vector. GRF deficits were partially ameliorated by both HA preparations: Hyalgan increased peak vertical forces at 6 weeks post-treatment (week 22), while HYADD4-G increased vertical impulse post-treatment. Both HA treatments, but not saline placebo, restored a two-peak composite force vector at 6 weeks post-treatment. Neither HA preparation significantly modulated osteoarthritis (OA) severity, or synovial fluid parameters. ConclusionsThis study showed that GRF responses to bilateral meniscectomy in sheep mimic available data for human meniscectomy and OA patients. However, this time course suggests that gait deficits are temporally unrelated to observed cartilage or synovial fluid changes. The bilateral ovine meniscectomy model demonstrates modest but quantifiable changes in GRF that mimic human OA and are amenable to modification by known OA therapies such as HA.

Premature osteoarthritis in the Disproportionate micromelia ( Dmm) mouse

Degeneration of articular cartilage leads to the development of osteoarthritis (OA), but the molecular pathology of the disease is poorly understood. The Disproportionate micromelia (Dmm) mouse has a deletion mutation in the C-propeptide encoding region of Col2a1, which leads to a defective cartilage matrix. The objective of this study was to determine whether heterozygous (Dmm/+) mice develop premature OA, and could therefore serve as an animal model for studying the molecular pathways leading to OA. Histological analysis was utilized to determine the state of articular cartilage degeneration in Dmm/+ mice at 3, 6, 9, 12, 15, and 22 months of age. Severity of OA was quantified with a modified Mankin scoring system. In addition, articular cartilage thickness, cell density, and the extracellular matrix (ECM) fraction of articular cartilage were quantified. Articular cartilage erosion was significantly more severe in Dmm/+ than in wild-type (+/+) mice beginning at 9 months, and modified Mankin scoring revealed Dmm/+ articular cartilage to be in a more severe osteoarthritic state as early as 3 months. In addition, Dmm/+ articular cartilage was thinner than +/+ cartilage and showed increased cell density and decreased matrix fraction compared with +/+ from the earliest time points measured. The present study demonstrates that Dmm/+ mice develop premature OA. The observed degenerative changes of Dmm/+ articular cartilage closely resemble those of human OA patients, with or without Col2a1 mutations, suggesting that Dmm/+ mice are a useful model for investigating mechanisms involved in OA.

Expression of mouse HtrA1 serine protease in normal bone and cartilage and its upregulation in joint cartilage damaged by experimental arthritis

Levels of HtrA1 protein in cartilage have been reported to elevate in joints of human osteoarthritis patients. To understand roles of HtrA1 in normal osteogenesis as well as in pathogenesis of arthritis, we examine HtrA1 expression pattern during bone and cartilage development and in articular cartilage affected by experimental arthritis. HtrA1 is not expressed in mesenchymal or cartilage condensations before initiation of ossification. When ossification begins in the condensations, the expression of HtrA1 starts in chondrocytes undergoing hypertrophic differentiation near the ossification center. Hypertrophic chondrocytes found in adult articular cartilage and epiphyseal growth plates also express HtrA1. When arthritis is induced by injection of anti-collagen antibodies and lipopolysaccharide, resting chondrocytes proceed to terminal hypertrophic differentiation and start expressing HtrA1. These data suggest that hypertrophic change induces HtrA1 expression in chondrocytes both in normal and pathological conditions. HtrA1 has been reported to inhibit TGF-β signaling. We show that HtrA1 digests major components of cartilage, such as aggrecan, decorin, fibromodulin, and soluble type II collagen. HtrA1 may, therefore, promote degeneration of cartilage by inducing terminal hypertrophic chondrocyte differentiation and by digesting cartilage matrix though its TGF-β inhibitory activity and protease activity, respectively. In bone, active cuboidal osteoblasts barely express HtrA1, but osteoblasts which flatten and adhere to the bone matrix and osteocytes embedded in bone are strongly positive for HtrA1 production. The bone matrix shows a high level of HtrA1 protein deposition akin to that of TGF-β, suggesting a close functional interaction between TGF-β and HtrA1.

Catabolic stress induces expression of hypoxia-inducible factor (HIF)-1α in articular chondrocytes: involvement of HIF-1α in the pathogenesis of osteoarthritis

Transcription factor hypoxia-inducible factor (HIF)-1 protein accumulates and activates the transcription of genes that are of fundamental importance for oxygen homeostasis – including genes involved in energy metabolism, angiogenesis, vasomotor control, apoptosis, proliferation, and matrix production – under hypoxic conditions. We speculated that HIF-1α may have an important role in chondrocyte viability as a cell survival factor during the progression of osteoarthritis (OA). The expression of HIF-1α mRNA in human OA cartilage samples was analyzed by real-time PCR. We analyzed whether or not the catabolic factors IL-1β and H2O2 induce the expression of HIF-1α in OA chondrocytes under normoxic and hypoxic conditions (O2 <6%). We investigated the levels of energy generation, cartilage matrix production, and apoptosis induction in HIF-1α-deficient chondrocytes under normoxic and hypoxic conditions. In articular cartilages from human OA patients, the expression of HIF-1α mRNA was higher in the degenerated regions than in the intact regions. Both IL-1β and H2O2 accelerated mRNA and protein levels of HIF-1α in cultured chondrocytes. Inhibitors for phosphatidylinositol 3-kinase and p38 kinase caused a significant decrease in catabolic-factor-induced HIF-1α expression. HIF-1α-deficient chondrocytes did not maintain energy generation and cartilage matrix production under both normoxic and hypoxic conditions. Also, HIF-1α-deficient chondrocytes showed an acceleration of catabolic stress-induced apoptosis in vitro. Our findings in human OA cartilage show that HIF-1α expression in OA cartilage is associated with the progression of articular cartilage degeneration. Catabolic-stresses, IL-1β, and oxidative stress induce the expression of HIF-1α in chondrocytes. Our results suggest an important role of stress-induced HIF-1α in the maintenance of chondrocyte viability in OA articular cartilage.

Intra-articular hyaluronan: A disease modifying therapy?

Hyaluronan (HA) is a macromolecular glycosaminoglycan which is an important constituent of both synovial fluid and the extracellular matrix of cartilage. Research into the functions of HA have suggested a number of different biophysical, biochemical and cell regulatory roles in joint synovial tissues. Intra-articular HA therapy is now widely used in various countries for the treatment of osteoarthritis (OA) in both humans and animals. Numerous high purity pharmaceutical preparations are commercially available which have a variety of forms and molecular weights ranging from less than 1.0 × 106 Da (generally regarded as low molecular weight) up to 3–6.0 × 106 Da (high molecular weight). This range of molecular weights in the commercially available preparations is a direct reflection of the variation in results derived from both in vitro and in vivo studies of the use of HA in various experimental models. Intra-articular HA therapy has been registered by some companies as a device rather than a drug, which reflects both the initial theory that HA exerted its action through viscosupplementation of deficient synovial fluid, and the current scientific debate as to the specific therapeutic effects of HA on synovial tissues. Significant research is ongoing to further define the likely multiple actions of HA on joint tissues. The discovery that HA is rapidly cleared from joints after intra-articular injection, coupled with the realization that the symptomatic relief experienced by human OA patients treated by intra-articular HA, together suggest that viscosupplementation is not the only mode of action of HA. Studies in various animal models of inflammation and pain have supported an anti-inflammatory action of HA in joint disease. In vivo animal studies using models of OA suggest that low molecular weight HA provides better protection of cartilage than high molecular weight preparations. This is in direct contrast to the results of in vitro studies using chondrocyte or cartilage explant cultures in which high molecular weight HA preparations were found to be more effective at protecting cartilage from degradation than low molecular weight preparations. Evidence that low molecular weight forms of HA penetrate the joint tissues more deeply is a partial explanation for this, as it is likely that the low molecular weight HA is able to achieve higher concentrations of the drug in the pericellular environment. However, recent research has provided additional evidence of specific interaction between HA, various cell receptors such as CD44. This, together with other studies in cell biology which have demonstrated that signal transduction and gene expression arise from HA receptor binding suggest an immunoregulatory role for HA. Our present knowledge of interactions of HA at the cellular and molecular level is still inadequate, but there is strong evidence from animal model experiments as well as from studies on human synovial biopsies that the positive effects exhibited by HA in OA joints are largely mediated by its anti-inflammatory, cytoprotective and immunoregulatory properties. There is significant scientific evidence that intra-articular HA therapy can provide cartilage protection. However, this probably does not arise from a direct effect of HA on chondrocytes, but rather is secondary to the down regulation of cytokine, free radical and proteolytic activities in the synovial fluid and synovium, all of which can adversely affect chondrocyte metabolism.