Mehdi Shakibaei

INTEGRINS AND STRETCH ACTIVATED ION CHANNELS; PUTATIVE COMPONENTS OF FUNCTIONAL CELL SURFACE MECHANORECEPTORS IN ARTICULAR CHONDROCYTES

Perception of mechanical signals and the biological responses to such stimuli are fundamental properties of load bearing articular cartilage in diarthrodial joints. Chondrocytes utilize mechanical signals to synthesize an extracellular matrix capable of withstanding high loads and shear stresses. Recent studies have shown that chondrocytes undergo changes in shape and volume in a coordinated manner with load induced deformation of the matrix. These matrix changes, together with alterations in hydrostatic pressure, ionic and osmotic composition, interstitial fluid and streaming potentials are, in turn, perceived by chondrocytes. Chondrocyte responses to these stimuli are specific and well coordinated to bring about changes in gene expression, protein synthesis, matrix composition and ultimately biomechanical competence. In this hypothesis paper we propose a chondrocyte mechanoreceptor model incorporating key extracellular matrix macromolecules, integrins, mechanosensitive ion channels, the cytoskeleton and subcellular signal transduction pathways that maintain the chondrocyte phenotype, prevent chondrocyte apoptosis and regulate chondrocyte‐specific gene expression.

Apoptosis and the loss of chondrocyte survival signals contribute to articular cartilage degradation in osteoarthritis.

Apoptotic death of articular chondrocytes has been implicated in the pathogenesis of osteoarthritis (OA). Apoptotic pathways in chondrocytes are multi-faceted, although some cascades appear to play a greater in vivo role than others. Various catabolic processes are linked to apoptosis in OA cartilage, contributing to the reduction in cartilage integrity. Recent studies suggest that beta1-integrin mediated cell-matrix interactions provide survival signals for chondrocytes. The loss of such interactions and the inability to respond to IGF-1 stimulation may be partly responsible for the hypocellularity and matrix degradation that characterises OA. Here we have reviewed the literature in this area of cartilage cell biology in an effort to consolidate the existing information into a plausible hypothesis regarding the involvement of apoptosis in the pathogenesis of OA. Understanding of the interactions that promote chondrocyte apoptosis and cartilage hypocellularity is essential for developing appropriately targeted therapies for inhibition of chondrocyte apoptosis and the treatment of OA.

INTEGRIN EXPRESSION AND COLLAGEN TYPE II IMPLICATED IN MAINTENANCE OF CHONDROCYTE SHAPE IN MONOLAYER CULTURE: AN IMMUNOMORPHOLOGICAL STUDY

Chondrocytes grown in monolayer culture at low density, with serum added, either dedifferentiate after several days whereby their cell shape changes or they are overgrown by fibroblast‐like cells. The aim of this study was to optimize the cultivation of chondrocytes in monolayer culture and to slow down their transformation or their overgrowth by fibroblast‐like cells. For this purpose freshly isolated chondrocytes of cartilage anlagen from 17‐day‐old mouse embryos were grown on plastic or collagen type II‐coated substrates. With this model: (a) chondrocytes grown on plastic substrates had almost completely changed to fibroblast‐like cells after 5 days in culture. (b) When grown on collagen type II, the chondrocytes maintained their round phenotype for more than 2 weeks in culture. (c) Immunomorphological investigations showed that chondrocytes produce collagen type II and fibronectin and express specific surface receptors (integrins of the β1‐group) on the membrane from day 1 until the end of the culture period when grown on collagen type II. (d) Treatment with β1‐integrin antibodies clearly reduces chondrocyte adhesion on collagen type II by about 70%. Hence, these data indicate that the most probable influence of collagen type II on cellular behaviour depends on the integrins participating in a chondrocyte—collagen type II interaction, and this model represents a pure chondrocyte culture which allows cell growth for an extended period.

DIFFERENTIATION OF MESENCHYMAL LIMB BUD CELLS TO CHONDROCYTES IN ALGINATE BEADS

Manyin vitromodels of embryonic material used for the cultivation of chondrocytes yield mixed cultures consisting of chondrocytes and fibroblast‐like cells. For the optimization of cartilage cell cultures, alginate, a semisolid medium, was employed to obtain pure chondrocyte cultures.

Effects of Curcumin (Diferuloylmethane) on Nuclear Factor κB Signaling in Interleukin‐1β‐Stimulated Chondrocytes

Abstract: Curcumin (diferuloylmethane) is a nontoxic dietary pigment in tumeric and curry and a potent inhibitor of the common transcription factor Nuclear Factor κB (NF‐κB) in several cell types. It is well established that some of the catabolic effects of the proinflammatory cytokines interleukin‐1β (IL‐1β) and tumor necrosis factor‐α in osteoarthritis are regulated by the activation of NF‐κB. Therefore, the aim of this study was to determine whether curcumin modifies the catabolic response of chondrocytes to IL‐1β. Human articular chondrocytes were prestimulated with 10 ng/mL IL‐1β for 0, 4, 8, 12, or 24 h and then cotreated with 50 μM curcumin for 0, 12, 24, 36, or 48 h. Synthesis of the cartilage‐specific collagen type II and matrix‐degrading enzyme matrix metalloproteinase‐3 (MMP‐3) was investigated in chondrocytes by Western blot analysis. Activation and nuclear translocation of NF‐κB were observed by immunofluorescence microscopy. IL‐1β induced a decrease in collagen type II and upregulation of MMP‐3 in a time‐dependent manner. Upregulation of MMP‐3 was inhibited by curcumin in a time‐dependent manner. In addition, IL‐1β‐induced a decrease in type II collagen, which was relieved by curcumin treatment. In response to IL‐1β, NF‐κB translocated to the nucleus, but translocation was inhibited by curcumin, as revealed by immunofluorescence microscopy. Taken together, these results confirmed an IL‐1β‐mediated upregulation of proinflammatory MMP‐3 in chondrocytes via an NF‐κB activation mechanism. Curcumin protected chondrocytes from the catabolic effects of IL‐1β, such as MMP‐3 upregulation, and interestingly also relieved cytokine‐induced suppression of matrix protein synthesis. Therefore, curcumin antagonizes crucial catabolic effects of IL‐1β signaling that are known to contribute to the pathogenesis of osteoarthritis.

Integrins on joint cartilage chondrocytes and alterations by ofloxacin or magnesium deficiency in immature rats

Abstract Recently, we showed that magnesium deficiency induces lesions in knee joint cartilage from 5-week-old rats that are very similar to ofloxacin-induced cartilage defects. We concluded that quinolone-induced arthropathy is probably due to chelation of magnesium and thus a deficit in functionally available magnesium in joint cartilage (Stahlmann et al. 1995). As magnesium deficiency in joint cartilage could impair chondrocyte-matrix interaction which is mediated by cation-dependent integrin receptors of the β1-subfamily, we investigated integrin expression in joint cartilage from untreated, ofloxacin-treated and magnesium-deficient Wistar rats. With immunohistochemical methods using monoclonal and polyclonal antibodies, we showed that the integrin pattern in joint cartilage from rats corresponded largely to integrin expression described for human cartilage tissue: β1, α1, α3 and αν subunits and the α5β1 and ανβ3 heterodimers were consistently expressed. Joint cartilage lesions were detected in ofloxacin-treated and magnesium-deficient rats. Lesions were more pronounced in the quinolone-treated group. Expression of several integrins was reduced in the vicinity of lesions after oral treatment with 2 × 600 mg ofloxacin/kg for 1 day. Gross-structural lesions (e. g., cleft formation, unmasked collagen fibres) in magnesium-deficient rats were very similar but changes in integrin expression were less pronounced. On the other hand, changes in cartilage matrix composition showed similar alterations in ofloxacin-treated and magnesium-deficient rats: fibronectin deposition in the cartilage matrix increased in both groups while glycosaminoglycan content decreased. In summary, similar defects occur in ofloxacin-treated and magnesium-deficient rats and with immunohistochemical methods subtle differences are demonstrable.

Early onset of chondroitin sulfate and osteopontin expression in angiotensin ii-dependent left ventricular hypertrophy

BACKGROUND Chondroitin sulfate proteoglycan (CSPG) is expressed during embryonic heart development and osteopontin (OPN) is an important mediator of the profibrotic effects of angiotensin II (Ang II). The objective of this study was to analyze extracellular matrix protein (ECMP) expression in Ang II-dependent left ventricular (LV) hypertrophy (LVH), LV dysfunction, and to investigate right ventricular changes. METHODS We used the hypertensive transgenic rat line TGR(mRen2)27 (Ren2), which provides a well-established model of Ang II-driven cardiac remodeling and progressive LV dysfunction and compared young Ren2 rats at the age of 10 weeks with normotensive Sprague-Dawley (SD) rats (n = 15, each group). RESULTS Systolic blood pressure and LV weight were elevated in Ren2 compared to SD rats (P < .001). Left ventricular end-diastolic pressure was not altered in Ren2, but +dP/dt(max) and -dP/dt(max) were decreased in Ren2 compared to SD rats (P < .01). Cardiomyocyte widths, interstitial and perivascular fibrosis were increased in left and right ventricles of Ren2 in comparison to SD rats (P < .05). The LV mRNA expression of atrial natriuretic factor, OPN, and collagen I were increased in Ren2 as compared to SD rats (P < .05, respectively). The LV CSPG, collagen I, collagen III, fibronectin, laminin, and OPN contents were elevated in Ren2 compared to SD rats as measured by image analysis and Western blotting (P < .01). CONCLUSIONS Reactivated expression of CSPG in the adult heart may be an important component of LV ECMP remodeling in LVH. Elevated cardiac OPN expression could mediate the alterations in LV ECMP pattern in Ang II-dependent LVH, thus contributing to the development of contractile dysfunction in young Ren2 rats.

Chondrotoxicity of ciprofloxacin in immature beagle dogs: immunohistochemistry, electron microscopy and drug plasma concentrations.

Abstract The systemic effects of ciprofloxacin in immature Beagles were studied. Dogs of 10–11 weeks were dosed orally for 5 days with 0 (n=3), 30 (n=5) and 200 (n=5) mg ciprofloxacin/kg body wt. Plasma concentrations were measured by high-performance liquid chromatography (HPLC) 1 h after dosing (assuming to be peak concentrations). In view of the high doses used, the plasma concentrations were rather low and declined during the study period. For example, plasma concentrations in the high dose group were 6.6 ± 0.9 mg/l (day 1), 3.9 ± 1.4 mg/l (day 3), and 2.6 ± 1.6 mg/l (day 5). In control dogs and in dogs treated with the low dose of ciprofloxacin no pathological changes were seen by light microscopy. However, cleft formation and erosions were observed in joint cartilage from two of five dogs treated with 200 mg/kg. It is noteworthy that despite the high dose used cartilage lesions were not detectable in all five dogs of this group by light microscopy. Using antibodies against cell membrane receptors (e.g. the α5β1-integrin) or matrix components (fibronectin, collagen II) the articular cartilage effects were studied in detail by immunohistochemistry. The most sensitive alteration was an increase in fibronectin which was detectable in the vicinity of the lesions in cartilage samples from the group of dogs administered the high dose. No clear-cut changes were seen with the use of antibodies against other matrix components. Electron microscopy revealed typical alterations in chondrocytes from dogs treated with ciprofloxacin: e.g., swollen mitochondria and enlarged rough endoplasmic reticulum. These changes were much more pronounced in dogs from the high dose group than in dogs from the low dose group. Our main conclusion is that after oral administration ciprofloxacin exhibits rather low chondrotoxicity, even in the most sensitive species known to date. This correlates with the findings in humans that ciprofloxacin seems to be less chondrotoxic than pefloxacin or other quinolones.

Quinolone-induced cartilage lesions are not reversible in rats

Abstract The reversibility of quinolone-induced cartilage lesions has not been studied in detail. We treated five groups of five to seven juvenile Wistar rats (male and female; age: 5 weeks) with 2×600 mg ofloxacin/kg by gastric intubation on 1 day only (9:00 a.m. and 5:00 p.m.) and studied the knee joints histologically 3 days, 1, 3, 8 and 17 weeks later. In addition, joint cartilage specimens from vehicle-treated control rats (n=21) at corresponding age were examined. Cartilage lesions such as matrix swelling, loss of proteoglycans and horizontal clefts were found in nearly all knee joints (26 of 27 joints; incidence: 96%) of the ofloxacin-treated rats. Within the observation period of 4 months the size of these lesions in knee joint cartilage did not decrease significantly. The diameter of the lesions at the time points of evaluation was 1146±535, 1713±309, 1250 ±585, 1406±356, and 1542±467 μm, respectively (mean values±sd). Chondrocyte clusters producing glycosaminoglycans were observed 3 weeks after dosing and at later time points. They are considered to reflect the onset of repair but chondrocyte organization did not normalize during the study period, thus indicating the irreversibility of the effect under the experimental conditions. In principle, long-term joint cartilage damage has to be taken into account when the use of quinolones in children is considered. More detailed pharmacokinetic data are necessary for a reasonable risk assessment approach.

Molecular anatomy of the perivascular sheath in human placental stem villi: the contractile apparatus and its association to the extracellular matrix

Abstract. In previous studies, we have shown that smooth muscle cells and myofibroblast subpopulations of the perivascular stem villous sheath of the human placenta contain focal adhesion plaques and talin immunoreactivity. The close association of these cells to elastic and collagen fibres have led to the assumption of a functional myofibroelastic unit within the perivascular stem villous sheath. Interactions between the extracellular matrix and smooth muscle cells depend on a variety of structural protein assemblies. In the present study, we examined, by immunocytochemistry, whether the molecular assembly of extracellular matrix proteins and molecules of focal adhesions, known to be essential for signal transduction in smooth muscle cells, are also found in smooth muscle cells of the perivascular stem villous sheath of the human placenta. Vascular and extravascular smooth muscle cells were immunoreactive for α-actinin, vinculin, paxillin and tensin, the integrin chains α1 and β1, and the basement membrane components laminin and heparan/-chondroitin sulfate proteoglycan perlecan. pp125FAK did not react. In the extracellular matrix of blood vessel walls and the perivascular stem villous sheath, we found immunoreactivity of fibronectin and collagen types I, VI and undulin (collagen type XIV). From our data we conclude that within the perivascular stem villous sheath, there exists a system of signal transduction molecules, indicating a cross talk between the smooth muscle cells of this sheath and their surrounding extracellular matrix.