Anabolic and catabolic gene expression pattern analysis in normal versus osteoarthritic cartilage using complementary DNA–array technology

Objective

To understand changes in gene expression levels that occur during osteoarthritic (OA) cartilage degeneration, using complementary DNA (cDNA)–array technology.

Methods

Nine normal, 6 early degenerated, and 6 late‐stage OA cartilage samples of human knee joints were analyzed using the Human Cancer 1.2 cDNA array and TaqMan analysis.

Results

In addition to a large variability of expression levels between different patients, significant expression patterns were detectable for many genes. Cartilage types II and VI collagen were strongly expressed in late‐stage specimens, reflecting the high matrix‐remodeling activity of advanced OA cartilage. The increase in fibronectin expression in early degeneration suggests that fibronectin is a crucial regulator of matrix turnover activity of chondrocytes during early disease development. Of the matrix metalloproteinases (MMPs), MMP‐3 appeared to be strongly expressed in normal and early degenerative cartilage and down‐regulated in the late stages of disease. This indicates that other degradation pathways might be more important in late stages of cartilage degeneration, involving other enzymes, such as MMP‐2 and MMP‐11, both of which were up‐regulated in late‐stage disease. MMP‐11 was up‐regulated in OA chondrocytes and, interestingly, also in the early‐stage samples. Neither MMP‐1 nor MMP‐8 was detectable, and MMP‐13 and MMP‐2 were significantly detectable only in late‐stage specimens, suggesting that early stages are characterized more by degradation of other matrix components, such as aggrecan and other noncollagenous molecules, than by degradation of type II collagen fibers.

Conclusion

This investigation allowed us to identify gene expression profiles of the disease process and to get new insights into disease mechanisms, for example, to develop a picture of matrix proteinases that are differentially involved in different phases of the disease process.

     

Regulation of anabolic and catabolic gene expression in normal and osteoarthritic adult human articular chondrocytes by osteogenic protein-1

OBJECTIVE: Osteoarthritis is characterized by dramatic changes in chondrocyte metabolism including the overexpression of catabolic enzymes, but also a lack of anabolic activity. In this respect, osteogenic protein 1 (OP-1) appears to be one of the most potent anabolic factors of chondrocytes. In this study, we were interested in: (1) whether recombinant human OP-1 exerts its anabolic effects also on osteoarthritic chondrocytes, (2) whether OP-1 modulates the expression of catabolic genes, and (3) whether the BMP effects are related to the expression levels of its intracellular mediators (R- and I-Smads). METHODS: Chondrocytes were isolated from cartilage of either normal (n = 5) or osteoarthritic (n = 8) human knee joints and cultured in short-term high-density monolayer cultures with and without recombinant OP-1. RNA was isolated and analyzed for mRNA expression levels of anabolic (aggrecan, collagen type II), catabolic (MMP-1, -3, -13, ADAMTS-4), and intracellular signaling mediators (Smad 1, 4, 5, 6, 7, and 8) by quantitative online PCR. RESULTS: After OP-1 stimulation, the anabolic genes were significantly up-regulated in osteoarthritic chondrocytes in comparison to normal chondrocytes. Neither in normal nor osteoarthritic chondrocytes were significant changes observed for the matrix degrading enzymes. Smads were also expressed in both normal and osteoarthritic cells at roughly the same level with and without stimulation with OP-1. CONCLUSION: Osteoarthritic chondrocytes are not hypo-responsive to anabolic stimulation by OP-1. Thus, human recombinant OP-1 could be a suitable anabolic activator of osteoarthritic chondrocytes. This might be of particular interest as chondrocytes themselves showed very low levels of OP-1 expression.     

Suppression of cartilage matrix gene expression in upper zone chondrocytes of osteoarthritic cartilage

Objective. To evaluate the anabolic activity of osteoarthritic chondrocytes in situ by investigating the messenger RNA (mRNA) expression of 3 major cartilage components, type II collagen, aggrecan, and link protein. Methods. In situ hybridization experiments and histochemical analysis for proteoglycan content were performed on parallel sections of normal and osteoarthritic (OA) cartilage specimens. Results. Most chondrocytes in the deeper zones of OA cartilage showed an increase in mRNA expression, in particular, of type II collagen and to a lesser extent, aggrecan, compared with normal specimens. However, chondrocytes of the upper zone were largely negative for aggrecan or type II collagen mRNA. The expression of link protein mRNA was low in normal and OA specimens. Conclusion. These observations suggest that suppression of the anabolic activity of chondrocytes in the upper zones contributes to the metabolic imbalance observed in OA cartilage. Stimulation of matrix anabolism in superficial chondrocytes might be a suitable target for therapeutic intervention.     

Apoptosis in normal and osteoarthritic human articular cartilage

OBJECTIVES: To investigate whether apoptosis occurs in osteoarthritis (OA), and if this phenomenon is modulated by human recombinant interleukin 1beta (hrIL1beta). METHODS: Human articular cartilage samples were obtained at the time of hip arthroplasty because of femoral neck fracture (normal cartilage) (n=4) or advanced coxarthrosis (OA cartilage) (n=14). Apoptotic chondrocytes, isolated by collagenase digestion and cultivated for 24 hours, or present in situ in frozen cartilage sections, were quantified by fluorescent microscopy using two apoptosis markers: the TUNEL reaction, which detects nuclear DNA fragmentation, and Annexin-V-fluos, which labels at the membrane level the externalisation of phosphatidylserine. RESULTS: In OA cartilage 18-21% of chondrocytes showed apoptotic features, compared with 2-5% in normal cartilage. The results were similar for the two comparative studies (in situ and in vitro) and for both apoptosis markers. Moreover, hrIL1beta increased the apoptosis rate in vitro in a dose dependent manner in OA and normal chondrocytes. CONCLUSION: These results suggest that apoptosis may be an important factor in the evolution of OA and may be a new target for treatment of OA.     

Immunohistological analysis of cytokine expression in human osteoarthritic and healthy cartilage

OBJECTIVE: To investigate osteoarthritic cartilage in comparison to normal cartilage in humans for the presence of the most relevant cytokines/growth factors known to be important for degradation and formation of new cartilage. METHODS: Cartilage from knee or hip joints was obtained from 10 patients with osteoarthritis (OA) and from 7 age matched control patients with intact cartilage. Additionally, normal cartilage from 2 young patients (12 and 17 years old) was obtained after knee traumas. Immunohistological staining of cartilage sections was performed using antibodies for the following cytokines/growth factors: tumor necrosis factor alpha (TNF-alpha), interleukin 1alpha (IL-1alpha), IL-1beta, interferon-gamma, IL-6, IL-4, IL-10, transforming growth factor beta1 (TGF-beta1), insulin-like growth factor I (IGF-I), IGF-II, platelet derived growth factor AA (PDGF-AA), and PDGF-BB. RESULTS: Immunohistochemical stainings were positive for all cytokines in OA cartilage, while only a faint or no staining was found in healthy cartilage. Activated chondrocytes expressing most of the cytokines were located in the middle and partly in the lower layer of cartilage, with the exception of IGF-I, which was expressed exclusively in the upper cartilage layer close to the surface. More chondrocytes stained positive for TNF-alpha than for IL-1, and expression of the degrading cytokine TNF-alpha was inversely correlated to the expression of the regulatory cytokines IL-4, IL-10, and TGF-beta. CONCLUSION: The most relevant cytokines known to be involved in cartilage metabolism are produced by chondrocytes themselves. They are upregulated in OA cartilage, suggesting that they serve some regulatory function and could be a target for future treatment.     

Characterisation of human articular cartilage link proteins from normal and osteoarthritic cartilage.

Proteoglycan link proteins were isolated from human articular cartilage obtained from normal and osteoarthritic femoral heads and purified to homogeneity employing a method previously described by this laboratory. The link proteins were analysed for amino acid composition, molecular weight on sodium dodecyl sulphate polyacrylamide gels, and ability to stabilise proteoglycan aggregates. The results of these studies were compared with those obtained with bovine link proteins. Two link proteins were identified in the purified fraction from normal and osteoarthritic human cartilage with apparent molecular weights of 54 000 (link 1) and 48 000 (link 2). Functionally the link proteins, isolated from osteoarthritic and normal cartilage, were indistinguishable as measured by their ability to stabilise aggregate. The amino acid compositions of normal and osteoarthritic link proteins were also found to be similar to each other but significantly different from the amino acid composition reported for the bovine link proteins. The quantities of these proteins in extracts from normal and diseased tissue were similar, as was the ratio of link protein 1 to link protein 2.     

Metabolism of a cartilage matrix glycoprotein in normal and osteoarthritic canine articular cartilage

We have recently described a 550,000-dalton noncollagenous cartilage matrix glycoprotein (CMGP), with subunits of 130,000, which is present in hyaline cartilage and fibrocartilage. Biosynthetic studies indicated that CMGP was synthesized by short-term organ cultures of normal canine articular cartilage, representing approximately 9% of the total 3H-leucine incorporated into protein in 24-hour cultures. There was no incorporation of 35S-sulfate or 3H-mannose into CMGP under these conditions, but the protein did incorporate 32P-phosphate. The majority of the 3H-leucine-labeled CMGP was removed after 24 hours of chase with unlabeled leucine, and only a small amount remained at 72 hours, which suggests that there was rapid metabolism of the protein. CMGP was not detected in cartilage after addition of cycloheximide to the culture medium; this confirms its short half-life. Cultures of osteoarthritic cartilage obtained from dogs 8-10 weeks after anterior cruciate ligament transection revealed no difference in the metabolism of CMGP in this tissue compared with that found in cultures of normal articular cartilage.     

The distribution of YKL-40 in osteoarthritic and normal human articular cartilage.

YKL-40, also called human cartilage glycoprotein-39, is a major secretory protein of human chondrocytes in cell culture. YKL-40 mRNA is expressed by cartilage from patients with rheumatoid arthritis, but is not detectable in normal human cartilage. The aim was to investigate the distribution of YKL-40 in osteoarthritic (n=9) and macroscopically normal (n=5) human articular cartilage, collected from 12 pre-selected areas of the femoral head, to discover a potential role for YKL-40 in cartilage remodelling in osteoarthritis. Immunohistochemical analysis showed that YKL-40 staining was found in chondrocytes of osteoarthritic cartilage mainly in the superficial and middle zone of the cartilage rather than the deep zone. There was a tendency for high number of YKL-40 positive chondrocytes in areas of the femoral head with a considerable biomechanical load. The number of chondrocytes with a positive staining for YKL-40 was in general low in normal cartilage. The present findings, together with previous observations, suggests that YKL-40 may be of importance in cartilage remodelling/degradation of osteoarthritic joints.     

Biochemical and metabolic abnormalities in normal and osteoarthritic human articular cartilage

Incorporation of radioactive precursors into macromolecules was studied with human normal and osteoarthritic articular cartilage organ culture. Analysis of the salt extracted matrix components separated by cesium chloride buoyant density gradient centrifugation showed an increase in the specific activities of all gradient fractions prepared from the osteoarthritic cartilage. Further analysis of these fractions showed the osteoarthritic cartilage contained 5 times as much sulfate incorporated into proteoglycans, and an even greater amount of ³H-glucosamine incorporated into material sedimenting to the middle of the gradient. Greater than half of this radioactive middle fraction appears to be hyaluronate, as judged by the position it elutes from a DEAE column and its susceptibility to hyaluronidase digestion. This study supports earlier findings showing increased rates of macromolecular synthesis in osteoarthritis, and in addition, an even greater synthetic rate for hyaluronic acid is demonstrated.     

Physiopathology of osteoarthritis. From normal cartilage to osteoarthritic cartilage: risk factors and inflammatory mechanisms

INTRODUCTION: osteoarthritis is the most prevalent form of arthritis in the world. With the progressive ageing of the population, it becomes a major problem of public health. Osteoarthritis is a degenerative affection characterized by many disorders leading to a structural and functional defect of one or several joints. CURRENT KNOWLEDGE AND KEY POINTS: In this review, we focus on the main inflammatory mechanisms occurring in cartilage during primary osteoarthritis. We also describe some well established risk factors involved in the pathogenesis of this disease such as age, overload and genetic factors. Indeed, osteoarthritis is the result of an imbalance between the processes of degradation and the attempts of repair by the chondrocyte which is the exclusive cell type in cartilage. Degradation is induced by several chemical substances such as proteolytic enzymes (metalloproteinases) and pro-inflammatory cytokines especially interleukin 1beta. To face these events, the chondrocyte starts attempts of repair by secreting growth factors (Transforming Growth Factor and Insulin Growth Factor) or anti-inflammatory cytokines (interleukins 4 and 10) unsuccessfully. All these events will lead to the structural modifications observed in the osteoarthritic cartilage. PROSPECTS: A better comprehension of the physiopathology of osteoarthritis will allow an improvement of therapeutic strategies of this common and invalidating disease.     

Human osteoarthritic cartilage is synthetically more active but in culture less vital than normal cartilage.

The proteoglycan turnover of human osteoarthritic (OA) cartilage was compared to that of normal (N) cartilage. The cartilage was obtained postmortem from human femoral knee condyles. Short term cultures were compared to longterm cultures, and proteoglycan synthesis rate, content and release determined. Proteoglycan synthesis rate, determined shortly after collection of the cartilage, was higher in OA cartilage than in N cartilage. After longterm culture, the initially higher proteoglycan synthesis rate of OA cartilage became lower than that of N cartilage. An increased percentage release of proteoglycans from OA cartilage compared to N cartilage was apparent both shortly after collection of the cartilage and after culture. Thus, although OA cartilage in vivo is synthetically more active, in vitro it has an increased catabolic and a decreased anabolic activity, from which we conclude that OA cartilage is less vital than N cartilage.     

Upregulation of insulin-like growth factor I gene expression in the lesions of osteoarthritic human articular cartilage.

Expression of insulin-like growth factor I (IGF-I)mRNA and IGF-I protein was studied in human osteoarthritic and young articular cartilage by in situ hybridisation and immunohistochemistry. In situ hybridisation showed that relatively low amounts of IGF-I mRNA signal were present in anatomically normal regions of osteoarthritic and young cartilage. In fibrillated osteoarthritic cartilage, however, the signal intensity was significantly higher than in non-fibrillated cartilage. Particularly high levels of IGF-I mRNA were present in the surface cell clones of more advanced lesions, the amount of signal being about four to five times greater than in anatomically normal cartilage. The amount of message varied with cartilage depth. In young cartilage there was less IGF-I mRNA in the superficial zone than in the middle and deep zones. In fibrillated regions of osteoarthritic joints the amount of message in surface cells was greater than in deeper regions. A specific human IGF-I antibody was used to show the presence intracellularly of IGF-I protein in osteoarthritic and young cartilage. Raised levels of IGF-I message in osteoarthritic chondrocytes may represent an attempt at increased matrix repair, operating by an autocrine/paracrine mechanism.     

Organization of ground substance proteoglycans in normal and osteoarthritic knee cartilage

A study of the organization of proteoglycans in articular cartilage indicates that nonaggregated proteoglycans existed in larger numbers in osteoarthritic than in normal cartilage and that proteoglycan aggregates in arthritic cartilage were smaller than normal. After dissociation from hyaluronic acid and tissue glycoproteins, no difference in hydrodynamic size of disaggregated proteoglycans was noted, but chondroitin sulfate chains of those from diseased cartilage were shorter than normal. The data suggest that there is a defect in proteoglycan aggregation in osteoarthritic cartilage which could be of pathogenetic significance.     

Identification of mesenchymal progenitor cells in normal and osteoarthritic human articular cartilage

OBJECTIVE: To determine the presence of mesenchymal progenitor cells (MPCs) in human articular cartilage. METHODS: Primary cell cultures established from normal and osteoarthritic (OA) human knee articular cartilage were analyzed for the expression of CD105 and CD166, cell surface markers whose coexpression defines mesenchymal stem cells (MSCs) in bone marrow and perichondrium. The potential of cartilage cells to differentiate to adipogenic, osteogenic, and chondrogenic lineages was analyzed after immunomagnetic selection for CD105+/CD166+ cells and was compared with bone marrow-derived MSCs (BM-MSCs). RESULTS: Up to 95% of isolated cartilage cells were CD105+ and approximately 5% were CD166+. The mean +/- SEM percentage of CD105+/CD166+ cells in normal cartilage was 3.49 +/- 1.93%. Primary cell cultures from OA cartilage contained significantly increased numbers of CD105+/CD166+ cells. Confocal microscopy confirmed the coexpression of both markers in the majority of BM-MSCs and a subpopulation of cartilage cells. Differentiation to adipocytes occurred in cartilage-derived cell cultures, as indicated by characteristic cell morphology and oil red O staining of lipid vacuoles. Osteogenesis was observed in isolated CD105+/CD166+ cells as well as in primary chondrocytes cultured in the presence of osteogenic supplements. Purified cartilage-derived CD105+/CD166+ cells did not express markers of differentiated chondrocytes. However, the cells were capable of chondrocytic differentiation and formed cartilage tissue in micromass pellet cultures. CONCLUSION: These findings indicate that multipotential MPCs are present in adult human articular cartilage and that their frequency is increased in OA cartilage. This observation has implications for understanding the intrinsic repair capacity of articular cartilage and raises the possibility that these progenitor cells might be involved in the pathogenesis of arthritis.     

基因芯片技术对哮喘病人相关基因的筛选和分析

目的 利用基因芯片技术寻找哮喘病患者与正常人外周血单核细胞之间差异表达基因,拟为哮喘的早期诊断及预防提供分子标记.方法 用淋巴细胞分离液分别提取16例哮喘病患者与16例正常人外周血单核细胞,用QIAGEN Rneasy Kit提取纯化样本总RNA,并合成用荧光标记的cRNA,分别与含有41 000条基因序列的全基因芯片杂交,以基因表达倍数值≥2.0和基因表达倍数值≤-2.0为阈值来确定差异表达基因,然后用Genespring软件利用生物信息学方法对差异表达基因进行功能分类分析.结果 按P＜0.05差异显著性标准,从34 183条表达基因谱中,筛选出哮喘患者与正常对照差异表达2倍以上的基因有4177条,差异表达2倍以上已知与哮喘相关的基因有19条.经代谢途径分析发现这些差异基因主要涉及到炎症反应、免疫反应、防御反应、创伤反应、外部刺激反应等8大功能分类.结论 哮喘的发生涉及众多基因表达的改变,芯片技术可以有效地筛选出哮喘患者与正常对照的差异表达基因,对进一步探索哮喘的发病机制、有效的干预或逆转哮喘具有重要意义.