Influence of beta-aminoproprionitrile (BAPN) on cell growth and elastic fiber formation in cultures of auricular chondrocytes

Auricular chondrocytes isolated from 4-day-old rabbits and grown in vitro for 14 days, proliferated rapidly and produced a conspicuous network of elastic fibers. Beta-aminoproprionitrile (BAPN), which in vivo inhibits cross-linking of elastin, decreased the formation of elastic fibers at a concentration of 10-20 micrograms/ml and prevented formation at 40 micrograms/ml. At a concentration of 5 micrograms/ml only the so-called patches of elastin appeared to be absent. The inhibitory effect of BAPN on cell growth did not exceed 10%, which indicates that BAPN is only slightly harmful to auricular chondrocytes and can safely be used in studies on elastin deposition by these cells in vitro.     

The growth responses of hamster chondrocytes,dermal fibroblasts and embryo cells to whole blood serum and plasma-derived serum

Summary Autoradiographic studies with³H-thymidine demonstrated that the growth responses of hamster chondrocytes, dermal fibroblasts and embryo cells, respectively, differed in media containing whole blood serum (WBS) and plasmaderived serum (PDS). Dermal fibroblasts seemed to require a growth factor from platelets for growth, but chondrocytes did not. Embryo cells showed an intermediate pattern of growth response to this factor.This work was supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture and the Ministry of Health and Welfare of Japan.We thank Miss M. Tanaka and Miss K. Kawana for technical assistance.     

The growth plate’s response to load is partially mediated by mechano-sensing via the chondrocytic primary cilium

Mechanical load plays a significant role in bone and growth-plate development. Chondrocytes sense and respond to mechanical stimulation; however, the mechanisms by which those signals exert their effects are not fully understood. The primary cilium has been identified as a mechano-sensor in several cell types, including renal epithelial cells and endothelium, and accumulating evidence connects it to mechano-transduction in chondrocytes. In the growth plate, the primary cilium is involved in several regulatory pathways, such as the non-canonical Wnt and Indian Hedgehog. Moreover, it mediates cell shape, orientation, growth, and differentiation in the growth plate. In this work, we show that mechanical load enhances ciliogenesis in the growth plate. This leads to alterations in the expression and localization of key members of the Ihh-PTHrP loop resulting in decreased proliferation and an abnormal switch from proliferation to differentiation, together with abnormal chondrocyte morphology and organization. Moreover, we use the chondrogenic cell line ATDC5, a model for growth-plate chondrocytes, to understand the mechanisms mediating the participation of the primary cilium, and in particular KIF3A, in the cell’s response to mechanical stimulation. We show that this key component of the cilium mediates gene expression in response to mechanical stimulation.     

Premiers résultats d'analyse fonctionnelle électro-biochimique sur l'oreillette isolée étalée deScyllium canicula

Summary A new method is described for studying the auricular automatism using quickly reversible inhibitors and dissociators of electrical and mechanical process. By means of carbon dioxide, acetylcholine, adenosine-triphosphoric acid and clupeine-sulfate, it has been found that the valve muscle is more resistant than the auricular muscle.     

Regulation of chondrocyte differentiation by ADAMTS-12 metalloproteinase depends on its enzymatic activity

ADAMTS-12, a metalloproteinase that belongs to ADAMTS family, is strongly upregulated during chondrogenesis and demonstrates prominent expression in the growth plate chondrocytes. ADAMTS-12 potently inhibits chondrocyte differentiation, as revealed by altered expression of both early and later genes critical for chondrogenesis. In addition, ADAMTS-12-mediated inhibition of chondrogenesis depends on its enzymatic activity, since its point mutant lacking enzymatic activity completely loses this activity. Furthermore, the C-terminal four thrombospondin motifs known to bind COMP substrate is necessary for its full proteolytic activity and inhibition of chondrocyte differentiation. Mechanism studies demonstrate that ADAMTS-12 induces PTHrP, whereas it inhibits IHH during chondrogenesis. Furthermore, PTHrP induces ADAMTS-12 and ADAMTS-12 is hardly detectable in PTHrP-/-growth plate chondrocytes. Importantly, knocking down ADAMTS-12 mRNA levels or blocking ADAMTS-12 activity almost abolishes the PTHrP-mediated inhibition of type X collagen expression. Collectively, these findings demonstrate that ADAMTS-12, a downstream molecule of PTHrP signaling, is a novel regulator of chondrogenesis. X. H. Bai, D.W. Wang: These two authors contributed equally to this work.     

Comparison of the in vitro effects of colchicine and its derivative colchiceine on chondrocyte morphology and function

Colchiceine is a colchicine-metabolite which has been reported to inhibit axonal transport although not binding to brain tubulin. In the present study, colchiceine was shown not to depolymerize cytoplasmic microtubules, nor to mimic other effects of colchicine on the ultrastructure of cultured chondrocytes. In addition, the synthesis of proteoglycans was inhibited by colchicine but slightly stimulated by colchiceine. These results support the idea that the disturbances in cultured chondrocytes caused by colchicine are specifically related to a loss of cytoplasmic microtubules.     

Comparison of the in vitro effects of colchicine and its derivative colchiceine on chondrocyte morphology and function

Summary Colchiceine is a colchicine-metabolite which has been reported to inhibit axonal transport although not binding to brain tubulin. In the present study, colchiceine was shown not to depolymerize cytoplasmic microtubules, nor to mimic other effects of colchicine on the ultrastructure of cultured chondrocytes. In addition, the synthesis of proteoglycans was inhibited by colchicine but slightly stimulated by colchiceine. These results support the idea that the disturbances in cultured chondrocytes caused by colchicine are specifically related to a loss of cytoplasmic microtubules.This work was supported by grants from the Swedish Medical Research Council (proj. No. 12X-03355), the King Gustaf V 80th Birthday Fund, the Swedish Society of Medical Sciences, and from the funds of Karolinska Institutet.     

Molecular aspects of pathogenesis in osteoarthritis: the role of inflammation

Arthritic diseases cause enormous burdens in terms of pain, crippling, and disability. Osteoarthritis (OA), the most common form of arthritis, is characterized by a slow progressive degeneration of articular cartilage. The exact etiology of OA is not known, but the degradation of cartilage matrix components is generally agreed to be due to an increased synthesis and activation of extracellular proteinases, mainly matrix metalloproteinases. Insufficient synthesis of new matrix macromolecules is also thought to be involved, possibly as a consequence of deficient stimulation by growth factors. Although OA is defined as a noninflammatory arthropathy, proinflammatory cytokines such as interleukin-1 have been implicated as important mediators in the disease. In response to interleukin-1, chondrocytes upregulate the production of nitric oxide and prostaglandin E₂, two factors that have been shown to induce a number of the cellular changes associated with OA. The generation of these key signal molecules depends on inducible enzymes and can be suppressed by pharmacological inhibitors.     

Molecular basis for differences between human joints

The molecular program of a cell determines responses including induction or inhibition of genes for function and activity, and this is true of the cells within articular cartilage, a major functional component of the joint. While our studies have previously focussed on differences in the molecular programs of the cells within the superficial and deep zones, we have recently begun to focus on relative differences between joints, such as the knee and ankle. In the human, these joints vary greatly in their susceptibility to joint diseases, such as osteoarthritis (OA). We have predicted that there would be a molecular basis for differences between joints that could lead to differences in susceptibility to OA, if inherent pathways locked into the resident cells induce differences in their response to their environment. We have been able to show that there are differences between the matrix components and water content; these properties correspond to a higher equilibrium modulus and dynamic stiffness but lower hydraulic permeability and serve to make the ankle cartilage stiffer, slowing movement of molecules through the cartilage. In addition to these biochemical differences in the cartilage matrix, we have also identified relative differences in the strength of the response to stimulation of chondrocytes from knee and ankle. The stronger response of the knee chondrocytes includes factors that increase damage to the cartilage matrix, such as a depression of matrix synthesis and increased enzyme activity. This response by the knee chondrocytes results in enzyme damage to the matrix that the cells may not be able to repair, while the weaker response of the ankle chondrocytes may allow the cells to repair their matrix damage.     

Demonstration of intracellular release of calcium induced by sodium ions in the auricular trabeculae of the frog

In a Ca-free, Mg-free medium containing EGTA (10-3M) auricular trabecles develop a slow inward current which is a pure sodium current. After 12 min in this medium, it is still possible to obtain a phasic mechanical activity which shows a perfect correlation with the current. This kind of behavior indicates that a mechanism of sodium-induced calcium release is present at the level of some internal sites.     

CD44 and integrin matrix receptors participate in cartilage homeostasis

Articular chondrocytes express the matrix receptors CD44 and integrins. Both of these receptors exhibit interactions with adjacent extracellular matrix macromolecules. In addition, both integrins and CD44 have the capacity for signal transduction as well as modulated interactions with the actin cytoskeleton. As such, both receptor families provide the chondrocytes a means to detect changes in matrix composition or to function as mechanotransducers. Disruption of CD44 or integrin-mediated cell-matrix interactions, either experimentally induced or when present in osteoarthritis, have profound effects on cartilage metabolism. Thus, CD44 and integrin receptors play a critical role in maintaining cartilage homeostasis.     

Cathétérisme intracardiaque en clinique

Summary Cardiac catheterization, initially conducted byForssmann in 1929, has become a qualified clinical method. A fine rubber catheter is introduced into the antecubital vein and mechanically reaches the right heart chambers; auricular and ventricular, as well as the pulmonary artery. In some exceptional cases the left auricle and ventricle likewise lend themselves to exploration.     

Quantitative studies on in vitro transformation of hamster chondrocytes.

Hamster chondrocytes could be transformed in a quantitative assay system which used X-irradiated feeder layer cells. Morphological transformation occurred on addition of, 4NQO, but not in control cultures. Differentiation was classified into 3 types (good, poor and none); normal and transformed colonies contained similar proportions of the 3 types.     

Differentiation of L6 myoblastic cells into chondrocytes

Under the influence of demineralized bone pieces L6 cells differentiate into chondrocytes. The cartilage formed is identifiable histologically. The results demonstrate that these myoblastic cells, which are committed to produce muscle, may still be influenced to express another potentiality of their genome.     

Quantitative studies on in vitro transformation of hamster chondrocytes

Summary Hamster chondrocytes could be transformed in a quantitative assay system which used X-irradiated feeder layer cells. Morphological transformation occurred on addition of, 4NQO, but not in control cultures. Differentiation was classified into 3 types (good, poor and none); normal and transformed colonies contained similar proportions of the 3 types.This work was supported by a grant from the Ministry of Education, Science and Culture, Japan.We thank Miss M. Tanaka for her technical assistance.     

Differentiation of L6 myoblastic cells into chondrocytes

Summary Under the influence of demineralized bone pieces L6 cells differentiate into chondrocytes. The cartilage formed is identifiable histologically. The results demonstrate that these myoblastic cells, which are committed to produce muscle, may still be influenced to express another potentiality of their genome.     

Annexin V interactions with collagen

Annexin V was originally identified as a collagen-binding protein called anchorin CII and was isolated from chondrocyte membranes by affinity chromatography on native type II collagen. The binding of annexin V to native collagen type II is stable at physiological ionic strength when annexin V is reconstituted in liposomes. The binding to native collagen types II and X, and to some extent to type I as well, was confirmed using recombinant annexin V. A physiological role for annexin V interactions with extracellular collagen is consistent with the localization of annexin V on the outer cell surface of chondrocytes, microvilli of hypertrophic chondrocytes, fibroblasts and osteoblasts. A breakthrough in our understanding of the function of annexin V was made with the discovery of its calcium channel activity. At least one of several putative functions of annexin V became obvious from studies on matrix vesicles derived from calcifying cartilage. It was found that calcium uptake by matrix vesicles depend on collagen type II and type X binding to annexin V in the vesicles and was lost when collagens were digested with collagenase; calcium influx was reconstituted after adding back native collagen II or V. These findings indicate that annexin V plays a major role in matrix vesicle-initiated cartilage calcification as a collagen-regulated calcium channel.     

川芎嗪对IL-1β诱导的兔原代软骨细胞增殖和凋亡影响

目的 观察川芎嗪对白细胞介素-1β诱导的软骨细胞的增殖及凋亡的影响.方法 兔原代软骨细胞培养及鉴定,用IL-1β10ng/ml和/或不同浓度川芎嗪共培养兔原代软骨细胞48h后,利用流式细胞仪检测各组软骨细胞的周期及凋亡率;利用MTT法检测软骨细胞的生长状态.结果 与对照组比较,IL-1β诱导下软骨细胞的凋亡率显著增加,差异具有显著统计学意义(P＜0.01);加入川芎嗪能明显降低IL-1β诱导的软骨细胞凋亡率,有显著统计学意义(P＜0.01);与对照组比较,IL-1β诱导下软骨细胞被明显阻滞在G1期(P＜0.01);加入川芎嗪能明显降低IL-1β对软骨细胞G1期的阻滞作用,细胞增殖指数明显增加(P＜0.05或P＜0.01).结论 川芎嗪对IL-1β诱导的兔原代软骨细胞抑制凋亡并促进生长.