Biomedical subjects

Mehdi Shakibaei

Publications and source records attributed to Mehdi Shakibaei.

At least 19 recordsLinked to original sources

PubMed2007

Accelerated mitochondrial adenosine diphosphate/adenosine triphosphate transport improves hypertension-induced heart disease.

BACKGROUND: Strong evidence suggests that mitochondrial malfunction, which leads to disturbed energy metabolism and stimulated apoptosis, is a linchpin in the induction and manifestation of cardiac failure. An adequate exchange of ATP and ADP over the inner mitochondrial membrane by the adenine nucleotide translocase (ANT) is thereby essential to guarantee the cellular energy supply. METHODS AND RESULTS: To explore the effect of an ameliorated mitochondrial ATP/ADP transportation on cardiac dysfunction, we generated transgenic rats overexpressing ANT1 in the heart (ANT rats) and crossed them with renin-overexpressing rats (REN rats) suffering from hypertension-induced cardiac insufficiency. Cardiac-specific ANT1 overexpression resulted in a higher ATP/ADP transportation and elevated activities of respiratory chain complexes. Increased ANT activity in double-transgenic (ANT/REN) animals did not influence excessive hypertension seen in REN rats. Hypertension-induced cardiac hypertrophy in the REN rats was prevented by parallel ANT1 overexpression, however, and left ventricular function remarkably improved. The ANT1 overexpression led to a reduction in fibrosis and an improvement in cardiac tissue architecture. Consequently, the survival rate of ANT/REN rats was enhanced. Further investigations into the cardioprotective mechanism of ANT1 overexpression revealed improved mitochondrial structure and function and significantly reduced apoptosis in ANT/REN rats, shown by lowered cytosolic/mitochondrial cytochrome c ratio, reduced caspase 3 level, and prevented DNA degradation. CONCLUSIONS: Myocardial ANT1 overexpression protects against hypertension-induced cardiac pathology. Thus, the improvement in mitochondrial function may be a basic principle for new strategies in treating heart disease.

Adenine Nucleotide Translocator 1↗

PubMed2006

Igf-I extends the chondrogenic potential of human articular chondrocytes in vitro: molecular association between Sox9 and Erk1/2.

Expansion of articular chondrocytes in monolayer culture leads to loss of the unique chondrocyte phenotype and the cells' redifferentiation capacity. Dedifferentiation of chondrocytes in monolayer culture is a challenging problem for autologous chondrocyte transplantation (ACT). It is well established that Igf-I exerts positive anabolic effects on chondrocytes in vivo and in vitro. Accordingly, in this study, we examined whether the anabolic insulin-like growth factor-I (Igf-I) is capable of extending the chondrogenic potential of dedifferentiated chondrocytes in vitro. Chondrocyte monolayers were cultured up to 10 passages. At each passage chondrocytes were stimulated with Igf-I (10ng/ml) and introduced to high-density cultures for up to 7 days. Expression of collagen type II, cartilage-specific proteoglycans, activated caspase-3, integrin beta1, extracellular signal-regulated kinase (Erk) and Sox9 was examined by Western blotting, immunoprecipitation and immunomorphological techniques. Monolayer chondrocytes rapidly lost their differentiated phenotype. When introduced to high-density cultures, only chondrocytes from P1-P4 redifferentiated. In contrast, Igf-I treated cells from P1 up to P7 redifferentiated and formed cartilage-like tissue in high-density culture. P8-P10 cells exhibited apoptotic alterations and produced significantly less matrix. Igf-I markedly increased expression of integrin beta1, Erk and Sox9. Immunoprecipitation revealed that phosphorylated Erk1/2 physically interacts with Sox9 in chondrocyte nuclei, suggesting a previously unreported functional association which was markedly enhanced by Igf-I. Treatment of chondrocyte cultures with Igf-I stabilizes chondrogenic potential, stimulates Sox9 and promotes molecular interactions between Erk and Sox9. These effects appear to be regulated by the integrin/MAPK signaling pathways.

Biomarkers↗

PubMed2006

Effects of 2,3,7,8-tetrachloro-dibenzo-p-dioxin on the extracellular matrix of the thymus in juvenile marmosets (Callithrix jacchus).

2,3,7,8-Tetrachloro-dibenzo-p-dioxin (TCDD) is an ubiquitously distributed xenobiotic. It has been postulated that the effects of TCDD on T-lymphocytes are mediated by modulation of the thymic microenvironment. There is growing evidence of a modified interplay between thymocytes and thymic epithelium related to changes in extracellular matrix (ECM) proteins. Eighteen male marmosets (Callithrix jacchus) were treated with single subcutaneous TCDD doses (1, 10, 100 ng/kg body weight) or vehicle and sacrificed 2 or 4 weeks thereafter. Thymus samples were stained with fluorescein isothiocyanate-conjugated antibodies for ECM proteins and examined immunohistochemically by semi-quantitative image analysis. Thymus samples of animals treated with 1 and 10 ng/kg were additionally analysed by Western blotting for ECM proteins, transforming growth factor-beta(1) (TGF-beta(1)) and integrin chain content (CD49a, CD49e, CD49f and CD29). Monkeys showed no overt signs of toxicity after TCDD treatment. Immunohistochemistry revealed an increase of ECM proteins at 100 ng/kg after 2 and 4 weeks. Western blotting confirmed immunohistochemistry showing a dose-dependent increase of several ECM proteins in animals treated with the lower TCDD doses of 1 and 10 ng/kg. Additionally, dose-dependent increases were observed in integrin chain and TGF-beta(1) contents. We demonstrated changes of thymic ECM in marmosets following low single TCDD doses. Combined with altered integrin expression and enhanced TGF-beta(1) stimulation our findings suggest a modified interplay between thymic epithelium and thymocytes. The extracellular matrix may play a more central role in mediating adverse effects of TCDD in organs than yet acknowledged.

Animals↗

PubMed2006

Simulated weightlessness changes the cytoskeleton and extracellular matrix proteins in papillary thyroid carcinoma cells.

Studies of astronauts, experimental animals, and cells have shown that, after spaceflights, the function of the thyroid is altered by low-gravity conditions. The objective of this study was to investigate the cytoskeleton and extracellular matrix (ECM) protein synthesis of papillary thyroid cancer cells grown under zero g. We investigated alterations of ONCO-DG 1 cells exposed to simulated microgravity on a three-dimensional random-positioning machine (clinostat) for 30 min, 24 h, 48 h, 72 h, and 120 h (n=6, each group). ONCO-DG 1 cells grown under microgravity exhibited early alterations of the cytoskeleton and formed multicellular spheroids. The cytoskeleton was disintegrated, and nuclei showed morphological signs of apoptosis after 30 min. At this time, vimentin was increased. Vimentin and cytokeratin were highly disorganized, and microtubules (alpha-tubulin) did not display their typical radial array. After 48 h, the cytoskeletal changes were nearly reversed. The formation of multicellular spheroids continued. In parallel, the accumulation of ECM components, such as collagen types I and III, fibronectin, chondroitin sulfate, osteopontin, and CD44, increased. The levels of both transforming growth factor beta-1 (TGF-beta(1)) and TGF-beta receptor type II proteins were elevated from 24 h until 120 h clinorotation. Gene expression of TGF-beta(1) was clearly enhanced during culture under zero g. The amount of E-cadherin was enhanced time-dependently. We suggest that simulated weightlessness rapidly affects the cytoskeleton of papillary thyroid carcinoma cells and increases the amount of ECM proteins in a time-dependent manner.

Carcinoma, Papillary↗

PubMed2006

Tissue engineering of autologous human heart valves using cryopreserved vascular umbilical cord cells.

BACKGROUND: Tissue engineering of autologous heart valves with the potential to grow and to remodel represents a promising concept in pediatric cardiovascular surgery. Currently we are exploring the impact of cryopreserved human umbilical cord cells (CHUCCs) for the fabrication of tissue-engineered heart valves for patients diagnosed prenatally with congenital heart lesions, potentially enabling heart valve replacement in the early years of life. METHODS: Human umbilical cord cells were isolated from vascular segments of umbilical cords and cryopreserved in a cell bank. After 12 weeks the cryopreserved cells were again expanded in culture and characterized by histology, immunohistochemistry, and proliferation assays. Trileaflet heart valve scaffolds were fabricated from a porous polymer (P4HB, Tepha Inc, Cambridge, MA) and sequentially seeded with CHUCCs (n = 10). Five of the heart valve constructs were grown for 7 days in a pulse duplicator and, as a control, five constructs were grown under static cell culture conditions for 7 days. Analysis of all tissue-engineered heart valves included histology, immunohistochemistry, electron microscopy, functional analysis, and biomechanical and biochemical examination. RESULTS: We found that CHUCCs remained viable after 12 weeks of cryopreservation and showed a myofibroblast-like morphology that stained positive for alpha-actin and fibroblast specific marker. Histology of the tissue-engineered heart valves showed layered tissue formation, including connective tissue between the inside and the outside of the porous scaffold. Immunohistochemistry was positive for collagen (types I, III, and IV), desmin, laminin, and alpha-actin. Electron microscopy showed that the cells had grown into the pores and formed a confluent tissue layer during maturation in the pulsatile flow system. Biochemical examination showed an increase of extracellular matrix formation in constructs after pulsatile flow exposure compared with the static control group. Functional analysis demonstrated a physiological increase of the intracellular Ca2+ concentration of the recultivated cells and the conditioned constructs after stimulation with histamine. CONCLUSIONS: This study demonstrates in vitro generation of viable and functional human heart valves based on CHUCCs and biomimetic flow culture systems. The CHUCCs demonstrated excellent growth potential and abilities of in vitro tissue formation. These findings suggest the potential benefit of establishing autologous human cell banks for pediatric patients diagnosed intrauterinely with congenital defects that will potentially require heart valve replacement in the early years of life.

Actins↗

PubMed2005

A hypothesis for the origin and pathogenesis of rheumatoid diseases.

It is well established that a correlation exists between rheumatoid arthritis (RA) and microbial damage. Material analyses have suggested that bacteria may be causative agents. This study was undertaken to further characterize the microbial agent responsible for pathogenesis of RA. In order to investigate whether substances in moist building materials can affect human cartilage, extracts from moist building materials were analysed for microbial components. Exposure of chondrocyte cultures to extracts in vitro showed that they were damaging the cultures. A direct correlation between strength of damage and concentration of MMP3 demonstrated that the effect was dose-dependent. High quantities of LPS were detected in the extracts. Experiments after deactivation with Polymyxin B showed that LPS are the causative agents. The present study leads to the hypothesis that LPS may bind to procollagen, as they bind to scavenger receptors. This procollagen endotoxin complex may block tropocollagen synthesis.

Air Pollution, Indoor↗

PubMed2005

Fluoroquinolones cause changes in extracellular matrix, signalling proteins, metalloproteinases and caspase-3 in cultured human tendon cells.

Antimicrobial therapy with fluoroquinolones can be associated with tendinitis and other tendon disorders as an adverse reaction associated with this class of antimicrobials. Here we investigated aspects of the mechanism of quinolone-induced tendotoxicity in human tenocytes focussing mainly on the question whether fluoroquinolones may induce apoptosis. Monolayers of human tenocytes were incubated with ciprofloxacin or levofloxacin at different concentrations (0, 3, 10, 30 and 100mg/L medium) for up to 4 days. Ultrastructural changes were studied by electron microscopy, and alterations in synthesis of specific proteins were determined using immunoblotting. At concentrations, which are achievable during quinolone therapy, 3mg ciprofloxacin/L medium significantly decreased type I collagen; similar changes were observed with 3mg ciprofloxacin or 10mg levofloxacin/L medium for the beta(1)- integrin receptors. Effects were intensified at higher concentrations and longer incubation periods. Cytoskeletal and signalling proteins, such as activated shc or erk 1/2, were significantly reduced by both fluoroquinolones already at 3mg/L. Furthermore, time- and concentration-dependent increases of matrix metalloproteinases as well as of the apoptosis marker activated caspase-3 were found. Apoptotic changes were confirmed by electron microscopy: both fluoroquinolones caused typical alterations like condensed material in the nucleus, swollen cell organelles, apoptotic bodies and bleb formation at the cell membrane. Our results provide evidence that besides changes in receptor and signalling proteins apoptosis has to be considered as a final event in the pathogenesis of fluoroquinolone-induced tendopathies.

Apoptosis↗

PubMed2005

Intraluminal application of vascular endothelial growth factor enhances healing of microvascular anastomosis in a rat model.

BACKGROUND: Early reconstitution after injury to the endothelium is an important feature for reducing a number of vessel wall pathologies. We investigated the effect of vascular endothelial growth factor (VEGF) and its impact on the vascular remodeling process and reendothelialization after microsurgery. METHODS AND RESULTS: Microvascular anastomosis was performed in the rat femoral artery. One group was treated with intraluminal administration of VEGF and the other with vehicle. We investigated morphological, ultrastructural and immunohistochemical changes of the vascular wall and the reendothelialization process. After 10 days, reendothelialization was significantly faster in VEGF-treated rats. Transmission electron microscopy revealed a complete healing in contrast to vehicle-treated vessels. Moreover, extracellular matrix proteins, such as fibronectin, collagen types I, III and IV, were significantly increased. Furthermore, VEGF treatment significantly induced VEGF receptor 2, flk-1, osteopontin and TGF-beta(1) proteins. CONCLUSIONS: Our data clearly document for the first time that intraluminal treatment with VEGF is beneficial to the healing process in vascular microsurgery. Osteopontin and TGF-beta(1), both induced by VEGF, may play an important role in the vascular remodeling process. Our results provide clear evidence that VEGF application may represent a useful strategy in accelerating reendothelialization and improving vascular healing after microsurgery.

Administration, Topical↗

PubMed2005

Ultrastructural changes observed in rat ovaries following in utero and lactational exposure to low doses of a polybrominated flame retardant.

Polybrominated diphenyl ethers (PBDEs), used as flame retardants in textiles, plastics and electrical appliances, have been shown to interfere with thyroid homeostasis. We evaluated the effects of environmentally relevant concentrations (low doses) of 2,2',4, 4',5-pentabromodiphenyl ether (PBDE-99) on the female reproductive system. A single dose of either 60 microg or 300 microg PBDE-99/kg body weight (BW) was administered on gestation day 6 to gravid Wistar rats. A reference control was treated with 6-n-propyl-2-thiouracil (PTU) on gestation days 7-21. Ultrastructural changes compatible with altered mitochondrial morphology were observed in the ovaries of the F1 offspring. No statistically significant changes in ovarian follicle counts were observed. Mating of the F1 females with untreated males revealed resorption rates in the PBDE groups greater than the limits considered normal for our controls. External and skeletal anomalies were detected in offspring (F2) from two different dams (F1) with early developmental exposure to 300 microg PBDE-99/kg BW. Exposure to PBDE-99 resulted in female reproductive tract changes in the F1 generation which were apparent at adulthood.

Animals↗

PubMed2005

Increase of fibronectin and osteopontin in porcine hearts following ischemia and reperfusion.

Following a severe ischemic injury or myocardial infarction, the extracellular matrix (ECM) of the heart is involved in pathophysiological conditions such as dilatation and cardiac dysfunction. Osteopontin (OPN) has been shown to interact with fibronectin suggesting its possible role in matrix organization, stability and wound healing. There is increased expression of OPN in several tissues in response to injury. Therefore, we tested the hypothesis that acute ischemia (2 h), followed by reperfusion (4 h) may induce early OPN and fibronectin in an isolated hemoperfused working porcine heart model. Twenty hearts were prepared and connected to a perfusion system. After 1 h of perfusion, these hearts were randomized to two groups: ten infarcted (MI, ramus circumflexus) and ten non-infarcted hearts (C). In addition, cardiac fibroblasts derived from infarcted, remote and control myocardium were investigated. In both groups, the heart rate, electrolytes, pH, blood gases, and lactate remained similar. The LVEDP and perfusion pressure of MI hearts increased significantly (P<0.05). The total fibronectin and OPN volume contents were clearly elevated in the infarct area. The matrix metalloproteinases (MMP-1 and MMP-8), fibronectin, OPN, TGF-beta1 proteins and the mRNAs for fibronectin, TGF-beta1, and OPN were significantly elevated in the infarct area as compared to the remote area and the non-infarcted hearts. Simultaneously, circulating carboxyterminal propeptide of type I procollagen (PICP) was released in the perfusion medium (threefold versus C). Fibroblast-like cells originating from the infarct area exhibited an enhanced OPN and fibronectin gene and protein expression compared to fibroblasts derived from control myocardium. Our data demonstrate the early appearance of the MMPs (increased collagen degrading enzymes) and PICP (a collagen synthesis marker) following ischemia and reperfusion. Moreover, OPN, fibronectin and TGF-beta1 protein and gene expression are elevated after ischemia and reperfusion in the ex vivo working hemoperfused porcine heart model.

Animals↗

PubMed2005

Curcumin protects human chondrocytes from IL-l1beta-induced inhibition of collagen type II and beta1-integrin expression and activation of caspase-3: an immunomorphological study.

Interleukin 1beta (IL-1beta) is a pleiotropic pro-inflammatory cytokine that plays a key role in mediating cartilage degradation in osteoarticular disorders such as osteoarthritis (OA) and rheumatoid arthritis (RA). At the cellular level, IL-1beta activates matrix degrading enzymes, down-regulates expression of matrix components and induces chondrocyte apoptosis. Curcumin (diferuloylmethane) is an anti-inflammatory phytochemical agent that has recently been shown to antagonize the pro-inflammatory effects of cytokines in chondrocytes and other cells. To test the hypothesis that curcumin also protects chondrocytes from morphological alterations induced by IL-1beta, we investigated its in vitro effects on apoptotic signalling proteins and key cartilage-specific matrix components in IL-1beta-stimulated chondrocytes. Human articular chondrocytes were pre-treated with 10 ng/mI IL-1beta alone for 30 min before being co-treated with IL-1beta and 50 microM curcumin for 5, 15 or 30 min, respectively. The ultrastructural morphology of chondrocytes was investigated by transmission electron microscopy. The production of collagen type II, the adhesion and signal transduction receptor beta1-integrin, the apoptosis marker activated caspase-3 was analysed by immunohistochemistry, immunoelectron microscopy and Western blotting. Transmission electron microscopy of chondrocytes stimulated with IL-1beta revealed early degenerative changes which were relieved by curcumin co-treatment. The suppression of collagen type II and beta1-integrin synthesis by IL-1beta was inhibited by curcumin. Additionally, curcumin antagonized IL-1beta-induced caspase-3 activation in a time-dependent manner. This study clearly demonstrates that curcumin exerts anti-apoptotic and anti-catabolic effects on IL-1beta-stimulated articular chondrocytes. Therefore curcumin may have novel therapeutic potential as an adjunct nutraceutical chondroprotective agent for treating OA and related osteoarticular disorders.

Caspase 3↗

PubMed2005

Nutraceutical therapies for degenerative joint diseases: a critical review.

There is growing recognition of the importance of nutritional factors in the maintenance of bone and joint health, and that nutritional imbalance combined with endocrine abnormalities may be involved in the pathogenesis of osteoarthritis (OA) and osteochondritis dissecans (OCD). Despite this, dietary programs have played a secondary role in the management of these connective tissue disorders. Articular cartilage is critically dependent upon the regular provision of nutrients (glucose and amino acids), vitamins (particularly vitamin C), and essential trace elements (zinc, magnesium, and copper). Therefore, dietary supplementation programs and nutraceuticals used in conjunction with non-steroidal, anti-inflammatory drugs (NSAIDs) may offer significant benefits to patients with joint disorders, such as OA and OCD. This article examines the available clinical evidence for the efficacy of nutraceuticals, antioxidant vitamin C, polyphenols, essential fatty acids, and mineral cofactors in the treatment of OA and related joint disorders in humans and veterinary species. This article also attempts to clarify the current state of knowledge. It also highlights the need for additional targeted research to elucidate the changes in nutritional status and potential alterations to the expression of plasma membrane transport systems in synovial structures in pathophysiological states, so that current therapy and future treatments may be better focused.

Amino Acids↗

PubMed2004

Glycosaminoglycan polymerization may enable osmotically inactive Na+ storage in the skin.

Osmotically inactive skin Na(+) storage is characterized by Na(+) accumulation without water accumulation in the skin. Negatively charged glycosaminoglycans (GAGs) may be important in skin Na(+) storage. We investigated changes in skin GAG content and key enzymes of GAG chain polymerization during osmotically inactive skin Na(+) storage. Female Sprague-Dawley rats were fed a 0.1% or 8% NaCl diet for 8 wk. Skin GAG content was measured by Western blot analysis. mRNA content of key dermatan sulfate polymerization enzymes was measured by real-time PCR. The Na(+) concentration in skin was determined by dry ashing. Skin Na(+) concentration during osmotically inactive Na(+) storage was 180-190 mmol/l. Increasing skin Na(+) coincided with increasing GAG content in cartilage and skin. Dietary NaCl loading coincided with increased chondroitin synthase mRNA content in the skin, whereas xylosyl transferase, biglycan, and decorin content were unchanged. We conclude that osmotically inactive skin Na(+) storage is an active process characterized by an increased GAG content in the reservoir tissue. Inhibition or disinhibition of GAG chain polymerization may regulate osmotically inactive Na(+) storage.

Animals↗

PubMed2004

Loss of chondrogenic potential in dedifferentiated chondrocytes correlates with deficient Shc-Erk interaction and apoptosis.

OBJECTIVE: If dedifferentiated chondrocytes could be induced to redifferentiate in vitro, then we might thereby be furnished with a population of phenotypically stable cells for autologous implantation in reconstructive surgery. We therefore investigated the redifferentiation capabilities of chondrocytes which, having migrated from alginate beads to form a monolayer, were subsequently passaged. We also characterized the molecular traits of irreversibly dedifferentiated cells. METHODS: Human chondrocytes that had migrated from alginate beads to form a monolayer (passage 1) were passaged seven times (passages 2-8). Cells from each passage were then recultivated in alginate beads. We assessed the synthesis of type-II collagen, cartilage-specific proteoglycans, adhesion molecules (integrins), signaling proteins (Src-homology collagen [Shc] and extracellular-signal-regulated kinase [Erk]) and the apoptosis marker 'activated' caspase-3 in monolayer or secondary alginate cultures. RESULTS: The synthesis of cartilage-specific type-II collagen, alpha 3-integrin, Shc and activated Erk1/2 decreased rapidly after four passages in monolayer culture. Up to passage 4, cells redifferentiated in alginate culture. However, between passages 5 and 8, cells began to produce activated caspase-3; these cells not only failed to redifferentiate when recultivated in alginate, but underwent apoptosis. CONCLUSION: We conclude that the loss of chondrogenic potential by chondrocytes maintained in monolayer culture is associated with a decrease in the synthesis of cartilage markers and with a suppressed activation of key signaling proteins in the Ras-mitogen-activated protein kinase pathway (Shc and Erk1/2). These events lead to apoptosis. A decrease in Shc/Erk expression/interaction could serve as a recognition marker for irreversibly dedifferentiated chondrocytes in tissue engineering.

Adaptor Proteins, Signal Transducing↗

PubMed2004

[Effect of a Harpagophytum procumbens DC extract on matrix metalloproteinases in human chondrocytes in vitro].

Inflammatory articular cartilage diseases such as arthritis and osteoarthritis are characterized by a loss of articular cartilage due to an imbalance between synthesis and degradation of the extracellular cartilage matrix. These diseases are accompanied by an increased induction of cytokines such as interleukin 1beta (IL-1beta) and tumor necrosis factor alpha (TNF-alpha). The increased release of cytokines leads to an enhanced production of matrix-degrading enzymes e.g. the matrix metalloproteinases (MMPs). In this study the direct antirheumatic effects of an extract of the secondary root of the African devil's claw (Harpagophytum procumbens DC) on the production of MMPs in IL-1beta-stimulated human chondrocytes were examined. A detailed evaluation by immunomorphological methods and Western blot analysis showed that the extracts of Harpagophytum decreased significantly the production of MMPs (MMP-1, MMP-3, MMP-9) in chondrocytes. The IL-1beta-induced production of MMPs was also significantly reduced by both a JM-extract (Jucurba) containing 210 mg dry extract and JF-extract (Jucurba forte) containing 480 mg dry extract. After all it could be shown that the effect of JF-extract on the MMP-synthesis was more pronounced in untreated and cytokine-stimulated chondrocytes when compared with the effect of the JM-extract. The capability of the JM-extract to suppress the MMP-production via the inhibition of the synthesis of inflammatory cytokines could explain its therapeutic effect in arthritic inflammations. In these in vitro experiments the JF-extract showed a higher efficacy than the JM-extract.

Antirheumatic Agents↗

PubMed2004

Effects of curcumin (diferuloylmethane) on nuclear factor kappaB signaling in interleukin-1beta-stimulated chondrocytes.

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

Blotting, Western↗

PubMed2004

Longterm conditions of mimicked weightlessness influences the cytoskeleton in thyroid cells.

Weightlessness influences the human immune and hormone system, reduces bone mass, leads to muscle atrophy and cardiac atrophy. Effects on control mechanisms for proliferation, programmed cell death and differentiation are well documented. The principal aim of this study was to investigate changes of the cytoskeleton in thyroid cells cultured in vector-averaged gravity under clinostat rotation. After 12 hours the formation of multicellular spheroids started. An increase of extracellular matrix proteins and beta 1-integrin was observed. Laser scanning confocal microscopy of ML-1 follicular thyroid carcinoma cells and normal thyroid HTU-5 cells immunostained with anti-cytokeratin to demonstrate these intermediate filaments revealed that cytokeratin filaments extended from centers, were thickened, coalesced and shortened as compared to control cells. Moreover, vimentin was highly disorganized. The vimentin network formed a coiled aggregate closely associated with the nucleus. Western blot analyses of talin, alpha- and beta-tubulin showed a clear increase of these proteins in cells cultured under simulated 0 g. Our data suggest that the effects of microgravity on cultured human thyroid cells are accompanied by noticeable functional cellular changes. Future studies to clarify the pathway that regulate the observed integrin activation and the mechanisms by which they function have to be performed.

Cell Differentiation↗

PubMed2004

Vascular endothelial growth factor inhibits programmed cell death of endothelial cells induced by clinorotation.

The principal aim of this study was to investigate short- and long-term effects of clinorotation on human endothelial cells (EA hy 926 cell line) using a three-dimensional random positioning machine. Moreover, the impact of vascular endothelial growth factor (VEGF) was addressed. Immediately, within one hour and after four and twenty-four hours an increase of apoptotic cells was detected. VEGF significantly inhibited the amount of apoptotic endothelial cells (EC). VEGF reduced the amount of fas-positive EC. Moreover, after 24 hours, proliferating EC grew in form of three-dimensional multicellular spheroids and also as monolayers. The initially formed spheroids (maximum diameter 3 mm) remained stable up to the 15th day of clinorotation. Some spheroids revealed tubular structures. In addition, a clear increase of extracellular matrix proteins such as osteopontin and fibronectin was measured. The three-dimensional clinostat represents an important tool for cell biological experiments. VEGF significantly attenuated the changes of endothelial cells induced by simulated weightlessness in a cell protective manner.

Apoptosis↗