The role of the cytoskeleton in articular cartilage chondrocyte homeostasis

Introduction The aetiology of osteoarthritis (OA) is unknown although abnormal loading of the joint is a contributory factor. We have demonstrated previously that a cyclic, compressive load (0.5 MPa, 1Hz) applied to immature articular cartilage induces a significant increase in the expression and activation of MMP‐2 and MMP‐9 ( Blain et al. 2001 ). Using differential RNA display, we identified a mechanically regulated gene –thymosin β₄ ( Blain et al. 2002 ). The primary function of thymosin β₄ is in the sequestration of filamentous actin (F‐actin). Therefore, we hypothesize that the mechanical induction of matrix degradation, i.e. the up‐regulation of MMP gene expression, is initiated via the actin cytoskeleton, whether directly or indirectly remains to be elucidated. Thus, the objective of this study was to determine whether the actin cytoskeleton, in addition to the tubulin and vimentin cytoskeletal networks are involved in the signalling pathways involved in chondrocyte MMP regulation. Materials and methods Primary chondrocytes, isolated from 7‐day‐old bovine calves, were seeded at a density of 1 × 10⁶ cells/ml, and individual cytoskeletal elements were disrupted with 10 µm cytochalasin‐D (for F‐actin), 10 µm colchicine (for tubulin) or 5 mm acrylamide (for vimentin) for 1–7 days. Amounts of sulphated glycosaminoglycan (sGAG) were determined using the DMMB assay, and total collagen content was assessed using the hydroxyproline assay. MMP activity was measured using gelatin substrate zymography and the amounts of their inhibitors, the TIMPs, assessed by reverse zymography. Results We have demonstrated that disruption of the cytoskeletal elements can affect cartilage chondrocyte homeostasis. There was a significant decrease in sGAG release for all three cytoskeletal disruption treatments when compared to untreated controls (P < 0.01). There was a reduction in total collagen released from the cells which was significant after 7 days in actin‐disrupted (P = 0.012) and vimentin‐disrupted cells (P = 0.05). No collagen was detected in tubulin‐disrupted cells at any time point, which may be due to a decrease in cell viability. Interestingly, at days 3 and 7, both tubulin and vimentin disruption abrogated synthesis of pro‐MMP‐2 and significantly reduced the amount of MMP‐2 activation (P < 0.01). Actin disruption significantly enhanced both synthesis and activation of MMP‐2 (P < 0.02). In comparison, TIMP‐1 expression was also abrogated in cells without a functioning tubulin or vimentin network, whereas in actin‐disrupted cells, there was a reduction in TIMP‐1 compared to untreated controls (P < 0.001). Discussion Clearly disruption of the cytoskeletal networks can affect cartilage chondrocyte homeostasis. Thymosin β₄ has been shown to induce MMP activity in our cell culture system which may be directly attributable to F‐actin depolymerization. Addition of cytochalasin‐D to chondrocytes revealed an increase in MMP‐2 synthesis/activation and reduced TIMP‐1 expression implicating the actin cytoskeleton in this process, whether directly or indirectly remains to be determined. We are currently using antibodies that recognize key signalling intermediates, i.e. FAK¹²⁵, p38 kinase and ERK 1/2 to assess the involvement of these molecules in events proceeding cytoskeletal disruption and prior to the mediation of MMP expression. We are also starting to investigate the mechanisms involved in abrogation of MMP synthesis after tubulin and vimentin disruption in chondrocytes. Elucidation of the role that the three cytoskeletal elements play in cartilage homeostasis will enable us to fully appreciate their functions in cartilage tissue turnover and dysregulation in disease. This work is supported by the EU 5th Framework and ARC (Grant No. D0600).     

The biomechanical role of the chondrocyte pericellular matrix in articular cartilage

The pericellular matrix (PCM) is a narrow tissue region that surrounds chondrocytes in articular cartilage. Previous parametric studies of cell-matrix interactions suggest that the mechanical properties of the PCM relative to those of the extracellular matrix (ECM) can significantly affect the micromechanical environment of the chondrocyte. The goal of this study was to use recently quantified mechanical properties of the PCM in a biphasic finite element model of the cell-PCM-ECM structure to determine the potential influence of the PCM on the mechanical environment of the chondrocyte under normal and osteoarthritic conditions. Our findings suggest that the mismatch between the Young's moduli of PCM and ECM amplifies chondrocyte compressive strains and exhibits a significant stress shielding effect in a zone-dependent manner. Furthermore, the lower permeability of PCM relative to the ECM inhibits fluid flux near the cell by a factor of 30, and thus may have a significant effect on convective transport to and from the chondrocyte. Osteoarthritic changes in the PCM and ECM properties significantly altered the mechanical environment of the chondrocyte, leading to approximately 66% higher compressive strains and higher fluid flux near the cell. These findings provide further support for a potential biomechanical role for the chondrocyte PCM, and suggest that changes in the properties of the PCM with osteoarthritis may alter the stress-strain and fluid flow environment of the chondrocytes.     

The effect of chondrocyte growth factor on membrane transport by articular chondrocytes in monolayer culture.

Chondrocyte growth factor (CGF), a contaminant of pituitary glycoprotein hormones, stimulates growth of cultured lapine articular chondrocytes while depressing SO4-proteoglycan synthesis. To study its effect on membrane transport, NIH-bTSH and two other preparations with comparable CGF activity were employed. In early log phase (36 hr) cultures CGF (64 microgram/ml) did not alter thymidine (dThd) uptake during the first 5 min. By 15 min however, TCA-precipitable dThd was 4-fold greater than in controls while the TCA-soluble fraction remained the same. CGF increased deoxy-glucose (DG) uptake in 36-hr old cultures. At 66 hr, CGF reduced DG transport. The transport of cycloleucine (CL) and aminoisobutyric acid (AIB) was reduced by CGF in 36 and 66-hr old cultures. There was a dose dependency between CGF concentration, the lowered uptake of DG, CL and AIB, and cell protein content. The effect of CGF on DG transport and dThd incorporation into DNA was not immediate but required prior exposure of the cells to CGF. CGF did not alter DG transport in rabbit or mouse fibrocytes or Chang liver cells. This and the reported finding that pituitary fibroblast growth factor (FGF), increases amino acid transport in other cells suggests that the biological specificity of CGF may not be identical to that of FGF.     

Characteristics of a scaffold-free articular chondrocyte plate grown in rotational culture

We investigated whether articular chondrocytes could form three-dimensional tissue-engineered cartilage in a rotational culture system without a scaffold. A suspension of chondrocytes derived from Japanese white rabbits was inoculated into a mold. Eight hours later, the cell suspension in the mold showed cell aggregation, forming a chondrocyte plate. The mold was removed, and the plate was cultured under static conditions. After 7 days of primary static culture, the plate was cultured under dynamic conditions, using rotational culture. After 2-3 weeks of rotational culture, the chondrocyte plate maintained a constant form and was considered stable enough to be handled with surgical pincers. Conversely, after 3 weeks of static culture, the plate gradually changed into an arch over that time. Histological and immunohistochemical evaluations indicated that the plate had cartilaginous qualities in terms of cell distribution and organization and the production of glycosaminoglycans and type II collagen in rotational cultures. Chondron units were detected with scanning electron microscopy. In contrast, a plate cultivated in static culture for 3 weeks was irregular in shape, and histological analysis indicated irregularly accumulated glycosaminoglycans. TUNEL-positive cells had increased significantly in the central region in 3-week static cultures, compared with those in 3-week rotational cultures. In this study, cartilaginous tissue in a scaffold-free environment has been produced. Significantly rotational cultures produce a construct, which is stable enough to be handled with surgical forceps after only 2 weeks of rotational culture. This system should be useful for implantation in the future.     

The aggregation of pig articular chondrocyte and synthesis of extracellular matrix by a lactose-modified chitosan

A reductive amination reaction (N-alkylation) obtained exploiting the aldheyde group of lactose and the amino group of the glucosamine residues of chitosan (d.a. 89%) afforded a highly soluble engineered polysaccharide (chitlac) for a potential application in the repair of the articular cartilage. Chitosan derivatives with 9% and 64% of side chain groups introduced have been prepared and characterized by means of potentiometric titration, (1)H-NMR and intrinsic viscosity. Both polymers, with respect to the unmodified chitosan, induce cell aggregation when in contact with a primary culture of pig chondrocytes, leading to the formation of nodules of considerable dimensions (up to 0.5-1 mm in diameter). The nodules obtained from chondrocytes treated with chitlac with the higher degree of substitution have been studied by means of optical and electron microscopy (SEM, TEM) and the production of glycosaminoglycans (GAGs) and collagen has been measured by means of colorimetric assays. The chondro-specificity of GAG and collagen was determined by RT-PCR. The results show that the lactose-modified chitosan is non-toxic and stimulates the production of aggrecan and type II collagen.     

The role of the neutral amino acid transporter SNAT2 in cell volume regulation

Sodium-dependent neutral amino acid transporter-2 (SNAT2), the ubiquitous member of SLC38 family, accounts for the activity of transport system A for neutral amino acids in most mammalian tissues. As the transport process performed by SNAT2 is highly energized, system A substrates, such as glutamine, glycine, proline and alanine, reach high transmembrane gradients and constitute major components of the intracellular amino acid pool. Moreover, through a complex array of exchange fluxes, involving other amino acid transporters, and of metabolic reactions, such as the synthesis of glutamate from glutamine, SNAT2 activity influences the cell content of most amino acids, thus determining the overall size and the composition of the intracellular amino acid pool. As amino acids represent a large fraction of cell organic osmolytes, changes of SNAT2 activity are followed by modifications in both cell amino acids and cell volume. This mechanism is utilized by many cell types to perform an effective regulatory volume increase (RVI) upon hypertonic exposure. Under these conditions, the expression of SNAT2 gene is induced and newly synthesized SNAT2 proteins are preferentially targeted to the cell membrane, leading to a significant increase of system A transport Vmax. In cultured human fibroblasts incubated under hypertonic conditions, the specific silencing of SNAT2 expression, obtained with anti-SNAT2 siRNAs, prevents the increase in system A transport activity, hinders the expansion of intracellular amino acid pool, and significantly delays cell volume recovery. These results demonstrate the pivotal role played by SNAT2 induction in the short-term hypertonic RVI and suggest that neutral amino acids behave as compatible osmolytes in hypertonically stressed cells.     

The role of acetylcholine in the regulation of ion transport by rat colon mucosa

1. Acetylcholine increases the potential difference across rat proximal colon both in vivo and in vitro.2. There is a sigmoid relationship between the change in potential difference and the logarithm of the dose of acetylcholine. The dose-response curve is shifted to the left by neostigmine and to the right by atropine, suggesting that the action of acetylcholine is mediated by a muscarinic type of receptor.3. The dose-response curve for acetylcholine in vivo is not altered by the ganglion-blocking agents hexamethonium and pentolinium, suggesting a direct effect of this transmitter on the colon.4. Acetylcholine causes an increase in potential difference, a small decrease in resistance and hence a rise in the current generated by both normal and stripped everted sacs of rat colon.5. In the absence of sodium, the calculated current change produced by acetylcholine is reduced, and the removal of chloride has a similar inhibitory effect. The absence of bicarbonate does not significantly affect the response.6. Acetylcholine virtually abolished net sodium movement and induced net chloride secretion and these changes accounted for the increased short-circuit current.7. Acetylcholine had no effect on oxygen consumption by rings of colon.8. Tracts staining for acetylcholinesterase were observed running from the submucous plexus towards the mucosal epithelium.9. This study shows that acetylcholine can influence ion movement by rat colonic mucosa and suggests that the autonomic nervous system might be involved in the regulation of transport mechanisms in this tissue.     

多重耐药基因在睫状体上皮细胞容积调节中的作用

目的：研究多重耐药（ＭＤＲ１）基因与细胞容积调节的关系。方法：用反义寡核苷酸阻抑牛眼睫状体非色素上皮细胞ＭＤＲ１基因表达,在激光共聚焦显微镜下检测ＭＤＲ１基因反义寡核苷酸（ＭＤＲ－ａｎｔｉ）的导入,用反射／散射技术测量由低渗液引起的细胞容积变化。结果：阳离子脂质体促进ＭＤＲ－ａｎｔｉ导入细胞,ＭＤＲ－ａｎｔｉ导入量与剂量呈依赖性增强关系（ｒ＝０９７,Ｐ＜００１）。人ＭＤＲ－ａｎｔｉ使低渗液引起的细胞调节性容积减小反应延迟、缓慢,容积恢复不完全（６０％）,而鼠ＤＭＲ－ａｎｔｉ没有此作用。结论：人ＭＤＲ－ａｎｔｉ抑制细胞调节性容积减小,提示ＭＤＲ１基因参与细胞容积调节。     

Osmotic regulation of neuronal activity: a new role for taurine and glial cells in a hypothalamic neuroendocrine structure

Maintenance of osmotic pressure is a primary regulatory process essential for normal cell function. The osmolarity of extracellular fluids is regulated by modifying the intake and excretion of salts and water. A major component of this regulatory process is the neuroendocrine hypothalamo-neurohypophysial system, which consists of neurons located in the paraventricular and supraoptic nuclei. These neurons synthesize the neurohormones vasopressin and oxytocin and release them in the blood circulation. We here review the mechanisms responsible for the osmoregulation of the activity of these neurons. Notably, the osmosensitivity of the supraoptic nucleus is described including the recent data that suggests an important participation of taurine in the transmission of the osmotic information. Taurine is an amino acid mainly known for its involvement in cell volume regulation, as it is one of the major inorganic osmolytes used by cells to compensate for changes in extracellular osmolarity. In the supraoptic nucleus, taurine is highly concentrated in astrocytes, and released in an osmodependent manner through volume-sensitive anion channels. Via its agonist action on neuronal glycine receptors, taurine is likely to contribute to the inhibition of neuronal activity induced by hypotonic stimuli. This inhibitory influence would complement the intrinsic osmosensitivity of supraoptic neurons, mediated by excitatory mechanoreceptors activated under hypertonic conditions. These observations extend the role of taurine from the regulation of cell volume to that of the whole body fluid balance. They also point to a new role of supraoptic glial cells as active components in a neuroendocrine regulatory loop.     

Healing of experimentally produced lesions in articular cartilage following chondrocyte transplantation

Articular cartilage is known to have limited ability to heal once injured, and attempts to heal lesions in cartilage have yielded equivocal results. The following experiments were performed to investigate healing in cartilage transplantation of chondrocytes grown in vitro. The knee joint of the New Zealand White rabbit was used as the experimental model. An initial baseline study was made to determine the intrinsic capability of cartilage for healing defects that do not fracture the subchondral plate. A second experiment examined the effects of autologous in vitro grown chondrocytes on the healing rates of these defects. The results were evaluated by qualitative and quantitative light microscopy. In control defects not grafted with chondrocytes, 6 weeks after the initial defect was created, there was little repair. Macroscopic and histological findings were consistent with an osteoarthritic pathology such as synovitis and "cell nests." Macroscopic results from grafted specimens displayed a marked decrease in synovitis and other degenerative changes. Defects which had received transplants had a significant amount of cartilage reconstituted (82%) compared to ungrafted controls (18%). Controls showed a healing rate comparable to that obtained in the initial baseline study.     

Lack of oxygen in articular cartilage: consequences for chondrocyte biology

Controlling the chondrocytes phenotype remains a major issue for cartilage repair strategies. These cells are crucial for the biomechanical properties and cartilage integrity because they are responsible of the secretion of a specific matrix. But chondrocyte dedifferentiation is frequently observed in cartilage pathology as well as in tissue culture, making their study more difficult. Given that normal articular cartilage is hypoxic, chondrocytes have a specific and adapted response to low oxygen environment. While huge progress has been performed on deciphering intracellular hypoxia signalling the last few years, nothing was known about the particular case of the chondrocyte biology in response to hypoxia. Recent findings in this growing field showed crucial influence of the hypoxia signalling on chondrocytes physiology and raised new potential targets to repair cartilage and maintain tissue integrity. This review will thus focus on describing hypoxia‐mediated chondrocyte function in the native articular cartilage.     

The role of metabolism in chondrocyte dysfunction and the progression of osteoarthritis

Osteoarthritis (OA) is a degenerative joint disease characterized by low-grade inflammation and high levels of clinical heterogeneity. Aberrant chondrocyte metabolism is a response to changes in the inflammatory microenvironment and may play a key role in cartilage degeneration and OA progression. Under conditions of environmental stress, chondrocytes tend to adapt their metabolism to microenvironmental changes by shifting from one metabolic pathway to another, for example from oxidative phosphorylation to glycolysis. Similar changes occur in other joint cells, including synoviocytes. Switching between these pathways is implicated in metabolic alterations that involve mitochondrial dysfunction, enhanced anaerobic glycolysis, and altered lipid and amino acid metabolism. The shift between oxidative phosphorylation and glycolysis is mainly regulated by the AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) pathways. Chondrocyte metabolic changes are likely to be a feature of different OA phenotypes. Determining the role of chondrocyte metabolism in OA has revealed key features of disease pathogenesis. Future research should place greater emphasis on immunometabolism and altered metabolic pathways as a means to understand the pathophysiology of age-related OA. This knowledge will advance the development of new drugs against therapeutic targets of metabolic significance.     

The role of the splanchnic circulation in the regulation of total intravascular volume during alpha adrenergic receptor stimulation

Previous studies have not defined the contribution of the splanchnic circulation to the total intravascular volume change associated with selective alpha adrenergic receptor stimulation. Since the splanchnic circulation is responsible for the total volume changes associated with other types of selective autonomic receptor stimulation, the present study was undertaken to examine the influence of alpha adrenergic receptor stimulation on splanchnic intravascular volume, the hemodynamic mechanism responsible for the splanchnic volume change, and the contribution of the splanchnic volume change to the change in total volume. In 35 anesthetized dogs, blood from the vena cavae was drained into an extracorporeal reservoir and returned to the right atrium at a constant rate so that changes in total intravascular volume could be measured as reciprocal changes in reservoir volume. Phenylephrine infusion (100 g/min) for 20 min in 28 dogs was associated with a decrease in total volume of 64±17 (SEM) ml (P<0.0001). The response was abolished by either alpha adrenergic blockade or evisceration but was not attenuated by beta adrenergic blockade, sinoaortic baroreceptor denervation, ganglionic blockade, or splenectomy. In 5 animals with separate splanchnic perfusion and drainage, total and splanchnic volumes decreased 59±8 ml (P<0.0001) and 317±20 ml (P<0.0001), respectively, while transhepatic vascular resistance increased 17±4 cm H₂O·min/l (P<0.0001). These responses were abolished after alpha adrenergic blockade. Thus, splanchnic volume decreases with alpha adrenergic receptor stimulation, despite an increase in hepatic resistance to splanchnic, venous outflow. The splanchnic volume decrement is entirely responsible for the total volume decrement.The study was supported by NHLBI Grant 1 R23-HL27185, Grant 11-203-812 from the American Heart Association of Greater Hartford, Inc., and the Duberg Cardiovascular Research Fund. Dr. Rutlen was the Duberg Scholar in Cardiovascular Disease when the study was performed.This work was presented in part at the 1982 Scientific Sessions of the American Heart Association (Circ. 66:II-311)