MUSCLE-INDUCED PATELLOFEMORAL JOINT LOADING RAPIDLY AFFECTS CARTILAGE mRNA LEVELS IN A SITE SPECIFIC MANNER

2004 ◽  
Vol 08 (01) ◽  
pp. 1-12 ◽  
Author(s):  
Andrea L. Clark ◽  
Linda Mills ◽  
David A Hart ◽  
Walter Herzog
Keyword(s):  
Articular Cartilage ◽  
Mechanical Loading ◽  
Patellofemoral Joint ◽  
Mrna Level ◽  
Recovery Period ◽  
Cartilage Explants ◽  
Mrna Levels ◽  
Biological Responses

Mechanical loading of articular cartilage affects the synthesis and degradation of matrix macromolecules. Much of the work in this area has involved mechanical loading of articular cartilage explants or cells in vitro and assessing biological responses at the mRNA and protein levels. In this study, we developed a new experimental technique to load an intact patellofemoral joint in vivo using muscle stimulation. The articular cartilages were cyclically loaded for one hour in a repeatable and measurable manner. Cartilage was harvested from central and peripheral regions of the femoral groove and patella, either immediately after loading or after a three hour recovery period. Total RNA was isolated from the articular cartilage and biological responses were assessed on the mRNA level using the reverse transcriptase-polymerase chain reaction. Articular cartilage from intact patellofemoral joints demonstrated heterogeneity at the mRNA level for six of the genes assessed independent of the loading protocol. Cyclical loading of cartilage in its native environment led to alterations in mRNA levels for a subset of molecules when assessed immediately after the loading period. However, the increases in TIMP-1 and decreases in bFGF mRNA levels were transient; being present immediately after load application but not after a three hour recovery period.

scholarly journals Wnt/β-catenin signaling contributes to articular cartilage homeostasis through lubricin induction in the superficial zone

2019 ◽  
Vol 21 (1) ◽  
Author(s):  
Fengjun Xuan ◽  
Fumiko Yano ◽  
Daisuke Mori ◽  
Ryota Chijimatsu ◽  
Yuji Maenohara ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Mechanical Loading ◽  
Cartilage Degeneration ◽  
Mrna Levels ◽  
Superficial Zone ◽  
Gain Of Function ◽  
Wnt Ligands ◽  
Signaling Activation ◽  
Signaling Activity

Abstract Background Both loss- and gain-of-function of Wnt/β-catenin signaling in chondrocytes result in exacerbation of osteoarthritis (OA). Here, we examined the activity and roles of Wnt/β-catenin signaling in the superficial zone (SFZ) of articular cartilage. Methods Wnt/β-catenin signaling activity was analyzed using TOPGAL mice. We generated Prg4-CreERT2;Ctnnb1fl/fl and Prg4-CreERT2;Ctnnb1-ex3fl/wt mice for loss- and gain-of-function, respectively, of Wnt/β-catenin signaling in the SFZ. Regulation of Prg4 expression by Wnt/β-catenin signaling was examined in vitro, as were upstream and downstream factors of Wnt/β-catenin signaling in SFZ cells. Results Wnt/β-catenin signaling activity, as determined by the TOPGAL reporter, was high specifically in the SFZ of mouse adult articular cartilage, where Prg4 is abundantly expressed. In SFZ-specific β-catenin-knockout mice, OA development was significantly accelerated, which was accompanied by decreased Prg4 expression and SFZ destruction. In contrast, Prg4 expression was enhanced and cartilage degeneration was suppressed in SFZ-specific β-catenin-stabilized mice. In primary SFZ cells, Prg4 expression was downregulated by β-catenin knockout, while it was upregulated by β-catenin stabilization by exon 3 deletion or treatment with CHIR99021. Among Wnt ligands, Wnt5a, Wnt5b, and Wnt9a were highly expressed in SFZ cells, and recombinant human WNT5A and WNT5B stimulated Prg4 expression. Mechanical loading upregulated expression of these ligands and further promoted Prg4 transcription. Moreover, mechanical loading and Wnt/β-catenin signaling activation increased mRNA levels of Creb1, a potent transcription factor for Prg4. Conclusions We demonstrated that Wnt/β-catenin signaling regulates Prg4 expression in the SFZ of mouse adult articular cartilage, which plays essential roles in the homeostasis of articular cartilage.

scholarly journals Effect of GCSB-5, a Herbal Formulation, on Monosodium Iodoacetate-Induced Osteoarthritis in Rats

2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Joon-Ki Kim ◽  
Sang-Won Park ◽  
Jung-Woo Kang ◽  
Yu-Jin Kim ◽  
Sung Youl Lee ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Protein Expression ◽  
Nuclear Factor Κb ◽  
Interleukin 10 ◽  
Cartilage Explants ◽  
Therapeutic Effects ◽  
Monosodium Iodoacetate ◽  
Factor Α

Therapeutic effects of GCSB-5 on osteoarthritis were measured by the amount of glycosaminoglycan in rabbit articular cartilage explantsin vitro, in experimental osteoarthritis induced by intra-articular injection of monoiodoacetate in ratsin vivo. GCSB-5 was orally administered for 28 days.In vitro, GCSB-5 inhibited proteoglycan degradation. GCSB-5 significantly suppressed the histological changes in monoiodoacetate-induced osteoarthritis. Matrix metalloproteinase (MMP) activity, as well as, the levels of serum tumor necrosis factor-α, cyclooxygenase-2, inducible nitric oxide synthase protein, and mRNA expressions were attenuated by GCSB-5, whereas the level of interleukin-10 was potentiated. By GCSB-5, the level of nuclear factor-κB p65 protein expression was significantly attenuated but, on the other hand, the level of inhibitor of κB-α protein expression was increased. These results indicate that GCSB-5 is a potential therapeutic agent for the protection of articular cartilage against progression of osteoarthritis through inhibition of MMPs activity, inflammatory mediators, and NF-κB activation.

scholarly journals Altered aggrecan synthesis correlates with cell and nucleus structure in statically compressed cartilage

1996 ◽  
Vol 109 (2) ◽  
pp. 499-508 ◽  
Author(s):  
M.D. Buschmann ◽  
E.B. Hunziker ◽  
Y.J. Kim ◽  
A.J. Grodzinsky
Keyword(s):  
Mechanical Loading ◽  
Cellular Response ◽  
Functional Adaptation ◽  
Cartilage Explants ◽  
Spatial Inhomogeneity ◽  
Cartilage Tissue ◽  
Nucleus Structure ◽  
Nucleus Volume

Previous studies have shown that static equilibrium compression of cartilage tissue in vivo and in vitro decreases chondrocyte synthesis of aggrecan molecules. In order to identify mechanisms of cellular response to loading, we have investigated alterations in cell and nucleus structure and the accompanying changes in the synthesis of aggrecan in statically compressed cartilage explants. Using glutaraldehyde fixation and quantitative autoradiography of compressed and radiolabeled cartilage disks we spatially localized newly synthesized aggrecan. Using stereological tools to analyze these same specimens we estimated the cell and nucleus volume, surface area and directional radii. We found that aggrecan synthesis was reduced overall in compressed tissue disks. However, the compression induced a spatial (radial) inhomogeneity in aggrecan synthesis which was not present in uncompressed disks. This spatial inhomogeneity appeared to be directly related to mechanical boundary conditions and the manner in which the load was applied and, therefore, may represent a spatially specific functional adaptation to mechanical loading. Coincident with reduced aggrecan synthesis, we observed reductions in cell and nucleus volume and radii in the direction of compression which were in approximate proportion to the reduction in tissue thickness. Cell and nucleus dimensions perpendicular to the direction of compression did not change significantly. Therefore the observed deformation of the cell and nucleus in statically compressed cartilage approximately followed the dimensional changes imposed on external specimen surfaces. The strong correlation observed between local changes in aggrecan synthesis and alterations in cell and nucleus structure also lend support to certain hypotheses regarding the intracellular signal transduction pathways that may be important in the biosynthetic response of chondrocytes to mechanical loading.

Abstract 14720: Pre-clinical Safety Assessment of SLN360, a Novel Short Interfering Ribonucleic Acid Targeting LPA

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
David Rider ◽  
Daniel Swerdlow ◽  
Mona Eisermann ◽  
Kathrin Loeffler ◽  
Judith Hauptmann ◽  
...  
Keyword(s):  
Target Genes ◽  
Clinical Chemistry ◽  
Recovery Period ◽  
Human Hepatocytes ◽  
Saline Control ◽  
Mrna Levels ◽  
Clinical Safety ◽  
Primary Human Hepatocytes

Introduction: SLN360 is a liver-targeted N-acetyl galactosamine (GalNAc)-conjugated siRNA with a promising profile for addressing Lp(a)-related cardiovascular risk. We previously reported the pharmacodynamic effects of SLN360 in female (F) cynomolgus monkeys (cynos) 1 . Here we describe the findings from key pre-clinical safety studies. Methods: SLN360 was tested in vitro in primary human hepatocytes for LPA knockdown and effects on hypothetical off targets identified through in silico screening. An in vivo biodistribution study was performed in rats over 21 days (4 males [M]/4 F per timepoint; single 10 mg/kg s.c. injection). In a GLP safety study, healthy cynos received 5 once weekly s.c. injections of saline control or SLN360 (3 M / 3 F per group, up to 200 mg/kg) followed by an 8-week recovery period (2 M / 2 F in saline and top dose). A standard battery of safety assessments was performed. Results: In vitro, SLN360 reduced LPA expression in primary human hepatocytes with no effects on any hypothetical liver-expressed human off-target genes at concentrations >300-fold the human LPA IC 50 . SLN360 showed a typical rodent GalNAc distribution pattern, with significant levels in liver and kidney (peak 126 or 246 mg/g tissue at 6h respectively; estimated liver half-life of 56 hours). Levels in other organs (including reproductive organs) were <1% of peak liver levels. In cynos, no clinical observations were observed following 5 s.c. doses of SLN360. Liver LPA mRNA levels were significantly reduced by 98% at day 30 and 95% after the 8-week recovery period, while serum Lp(a) was undetectable at both timepoints. Findings were restricted to the liver (increased weight and diffuse hepatocyte hypertrophy) and lymph nodes (vacuolated macrophages) at day 30. These were considered non-adverse due to reversibility after recovery. No dose-related changes in clinical chemistry, hematology, circulatory and ECG parameters, respiratory rate, neurobehavior, plasma cytokines, complement activation or CRP were noted. The NOAEL was defined as 200 mg/kg, >60-fold the active dose in cynos. Conclusions: Overall, the off-target profile, biodistribution and NOAEL of SLN360 indicate suitability for entry into clinical studies. 1 Aleku et al., 2019. Circulation. 2019, 140:A9538.

Cisplatin Nano-Liposomes Promoting Apoptosis of Retinoblastoma Cells Both In Vivo and In Vitro

2020 ◽  
Vol 12 (4) ◽  
pp. 536-542
Author(s):  
Lijuan Zhao ◽  
Fei Wang ◽  
Wei Fan
Keyword(s):  
Protein Expression ◽  
Nude Mice ◽  
Caspase 3 ◽  
Mrna Level ◽  
Control Group ◽  
Mrna Levels ◽  
Bax Protein ◽  
Experimental Group

This study was established to investigate the effects of cisplatin nano-liposomes on the apoptosis of the human retinoblastoma (RB) cell line Y79 in vitro and in vivo. Y79 cells were cultured and then exposed to Annexin V/PI to test their apoptosis, tested with the Caspase-3 activity detection kit to examine the change in activity of Caspase-3, and subjected to western blotting to test Bcl-2 and Bax protein expression. Y79-cell-transplanted tumor model in nude mice was also established and divided into three groups, with five nude mice in each. Cisplatin nano-liposomes were applied to the experimental group, cisplatin was injected into the control group, while saline was administered to the blank group, after which the nude mice were killed and the tumor was removed. Tumor volumes and weights in the three groups were compared. Nucleic acid extraction from magnetic beads was adopted to extract DNA, RT-PCR was employed to test Bcl-2 and Bax mRNA levels in tumor tissues, and in situ cell death assay kit was applied to test apoptotic cells. In comparison to the cisplatin solution and DMSO groups, the cisplatin liposome group showed higher Y79 apoptotic rate, Caspase-3 activity, and Bax protein expression, and lower Bcl-2 protein expression (all P < 0 05). In comparison with the control and blank groups, the experimental group showed lower tumor volume, weight, and Bcl-2 mRNA level of nude mice. In addition, in comparison with the control group, the experimental group showed higher cellular apoptotic rate and Bax mRNA level. In terms of the clinical effects of cisplatin nano-liposomes on a tumor transplant in nude mice with cervical cancer, they were shown to promote tumor apoptosis.

Hyaline Characteristics of Mechanically Induced Cartilaginous Tissues

2007 ◽  
Author(s):  
Kristy T. S. Palomares ◽  
Thomas A. Einhorn ◽  
Louis C. Gerstenfeld ◽  
Elise F. Morgan
Keyword(s):  
Articular Cartilage ◽  
Mrna Expression ◽  
Mechanical Loading ◽  
Biochemical Composition ◽  
Hyaline Cartilage ◽  
Cartilage Explants ◽  
Tissue Structure ◽  
In Vivo Model ◽  
Extracellular Matrix Components

The mechanical properties of hyaline cartilage depend heavily on tissue structure and biochemical composition. Glycosaminoglycans (GAGs) and collagen fibrils are the key extracellular matrix components of hyaline cartilage that bestow compressive and tensile stiffness, respectively.[1–2] In articular cartilage, a decline in GAG content and collagen organization with injury or with diseases such as osteoarthritis is intimately linked with a decline in mechanical function.[3] In tissue-engineered cartilage and articular cartilage explants, mechanical loading in vitro results in increased aggrecan mRNA expression, GAG content, and increased stiffness.[4–6] These findings suggest that mechanical loading could be applied in vivo to promote cartilage repair via modulation of gene expression, tissue structure, and tissue composition. We have previously developed an in vivo model of skeletal repair in which application of a controlled bending motion to a healing osteotomy gap results in formation of cartilage within the gap.[6] Using this model, we sought to characterize the biochemical composition and collagen structure of the mechanically induced cartilaginous tissue. The objectives of this study were: 1) to quantify the total GAG content and aggrecan mRNA expression; and 2) to characterize the collagen fiber orientation.

scholarly journals Nutritional and hormonal factors control the gene expression of FoxOs, the mammalian homologues of DAF-16

2003 ◽  
Vol 30 (2) ◽  
pp. 253-262 ◽  
Author(s):  
M Imae ◽  
Z Fu ◽  
A Yoshida ◽  
T Noguchi ◽  
H Kato
Keyword(s):  
Gene Expression ◽  
Mrna Level ◽  
Fold Increase ◽  
Mrna Levels ◽  
Hormonal Factors ◽  
Protein Levels ◽  
Nuclear Exclusion ◽  
Protein Free Diet

Transcription factors of the FoxO family in mammals are orthologues of the Caenorhabditis elegans forkhead factor DAF-16, which has been characterized as a target of insulin-like signalling. Three members of this family have been identified in rodents: FoxO1, FoxO3 and FoxO4, originally termed FKHR, FKHRL1 and AFX respectively. A number of in vitro studies have revealed that FoxOs are regulated through phosphorylation in response to insulin and related growth factors, resulting in their nuclear exclusion and inactivation. To clarify the mechanisms involved in the regulation of these factors in vivo, we investigated in the present study whether or not, and if so how, their mRNA levels in rat liver respond to the stimuli of several nutritional and hormonal factors. Imposed fasting for 48 h significantly elevated mRNA levels of FoxO1 (1.5-fold), FoxO3 (1.4-fold), and FoxO4 (1.6-fold). Refeeding for 3 h recovered the induced mRNA levels of FoxO1 and FoxO3 to the control levels, but did not affect that of FoxO4. FoxO1 and FoxO4 mRNA levels were proved to be highly reflective of their protein levels measured by Western immunoblotting. Of the three FoxO genes, FoxO4 only showed altered levels of mRNA (a 1.5-fold increase) in response to a protein-free diet. Streptozotocin-induced diabetes for 28 days decreased hepatic mRNA levels of FoxO1 and FoxO3 and increased the level of FoxO4 mRNA, but short-term (7 days) diabetes had fewer effects on the expression of these genes. Insulin replacement partially restored the FoxO1 and FoxO4 mRNA levels, but had no effect on the FoxO3 mRNA level. Daily administration for 1 week of dexamethasone, a synthetic glucocorticoid, increased the mRNA levels of FoxO1 (1.8-fold) and FoxO3 (2.4-fold). These results show that the FoxO genes respond differently to nutritional and hormonal factors, suggesting a new mechanism for the regulation of FoxO-dependent gene expression by these factors. Moreover, changes of FoxO1 and FoxO4 in the nucleus in response to fasting also suggest that the regulation of nucleus/cytoplasm translocation actually functions in vivo.

scholarly journals The Releasate of Avascular Cartilage Demonstrates Inherent Pro-Angiogenic Properties In Vitro and In Vivo

Cartilage ◽  
2021 ◽  
pp. 194760352110476
Author(s):  
Yannick Nossin ◽  
Eric Farrell ◽  
Wendy J.L.M. Koevoet ◽  
Frank Datema ◽  
Rodrigo A. Somoza ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Nasal Septum ◽  
Healthy Adult ◽  
Cartilage Explants ◽  
Cartilage Tissue ◽  
Human Articular Cartilage ◽  
Cam Assay ◽  
Related Factors

Objective Cartilage is avascular and numerous studies have identified the presence of single anti- and pro-angiogenic factors in cartilage. To better understand the maintenance hyaline cartilage, we assessed the angiogenic potential of complete cartilage releasate with functional assays in vitro and in vivo. Design We evaluated the gene expression profile of angiogenesis-related factors in healthy adult human articular cartilage with a transcriptome-wide analysis generated by next-generation RNAseq. The effect on angiogenesis of the releasate of cartilage tissue was assessed with a chick chorioallantoic membrane (CAM) assay as well as human umbilical vein endothelial cell (HUVEC) migration and proliferation assays using conditioned media generated from tissue-engineered cartilage derived from human articular and nasal septum chondrocytes as well as explants from bovine articular cartilage and human nasal septum. Experiments were done with triplicate samples of cartilage from 3 different donors. Results RNAseq data of 3 healthy human articular cartilage donors revealed that the majority of known angiogenesis-related factors expressed in healthy adult articular cartilage are pro-angiogenic. The releasate from generated cartilage as well as from tissue explants, demonstrated at least a 3.1-fold increase in HUVEC proliferation and migration indicating a pro-angiogenic effect of cartilage. Finally, the CAM assay demonstrated that cartilage explants can indeed attract vessels; however, their ingrowth was not observed. Conclusion Using multiple approaches, we show that cartilage releasate has an inherent pro-angiogenic capacity. It remains vessel free due to anti-invasive properties associated with the tissue itself.

scholarly journals Labisia pumilaUpregulates Peroxisome Proliferator-Activated Receptor Gamma Expression in Rat Adipose Tissues and 3T3-L1 Adipocytes

2013 ◽  
Vol 2013 ◽  
pp. 1-7
Author(s):  
Fazliana Mansor ◽  
Harvest F. Gu ◽  
Claes-Göran Östenson ◽  
Louise Mannerås-Holm ◽  
Elisabet Stener-Victorin ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Polycystic Ovary ◽  
Mrna Level ◽  
Water Extract ◽  
Peroxisome Proliferator ◽  
Mrna Levels ◽  
Peroxisome Proliferator Activated Receptor ◽  
Protein Levels

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated transcription factor that regulates lipid and glucose metabolism. We investigated the effects ofLabisia pumila(LP) standardized water extract on PPARgamma transcriptional activity in adipocytesin vitroandin vivo. We used a rat model of dihydrotestosterone- (DHT-) induced polycystic ovary syndrome (PCOS), a condition characterized by insulin resistance. At 9 weeks of age, the PCOS rats were randomly subdivided into two groups: PCOS-LP (50 mg/kg/day of LP) and PCOS-control (1 mL of deionised water) for 4-5 weeks on the same schedule. Real-time RT-PCR was performed to determine the PPARgamma mRNA levels. LP upregulated PPARgamma mRNA level by 40% in the PCOS rats. Western blot analysis further demonstrated the increased PPARgamma protein levels in parallel with upregulation in mRNA. These observations were further proven by adipocytes culture. Differentiated 3T3-L1 adipocytes were treated with final concentration of 100 μg/mL LP and compared to untreated control and 10 μM of rosiglitazone (in type of thiazolidinediones). LP increased PPARgamma expressions at both mRNA and protein levels and enhanced the effect of glucose uptake in the insulin-resistant cells. The data suggest that LP may ameliorate insulin resistance in adipocytes via the upregulation of PPARgamma pathway.

The Influence of a Single Intra-Articular Lidocaine Injection on the Viability of Articular Cartilage in the Knee

Cartilage ◽  
2020 ◽  
pp. 194760352090475
Author(s):  
Klemen Ravnihar ◽  
Tomaž Marš ◽  
Sergej Pirkmajer ◽  
Armin Alibegović ◽  
Gordana Koželj ◽  
...  
Keyword(s):  
Articular Cartilage ◽  
Cartilage Explants ◽  
Protective Mechanism ◽  
Chondrocyte Viability ◽  
Intact Cartilage ◽  
Lidocaine Injection ◽  
Liquid Chromatography Tandem Mass ◽  
And Control

Objective To evaluate the in vivo effect of a single intra-articular injection of local anesthetic (LA) lidocaine on the viability of articular cartilage in the intact or osteoarthritic (OA) human knees, and to measure the synovial postinjection concentration of lidocaine in the knee Design This study includes 3 interconnected experiments: (A) Synovial LA concentration measurement after a 2% lidocaine injection before knee arthroscopy in 10 patients by liquid chromatography–tandem mass spectrometry (LC-MS/MS). (B) Human osteochondral explants ( N = 27) from intact knees procured at autopsies were incubated for different time intervals (30 minutes, 2 hours, 24 hours) with 2% lidocaine, 0.04% lidocaine (measured), or culture medium (control), and later evaluated for cell viability by LIVE/DEAD staining. (C) Ten out of 19 matched patients scheduled for knee replacement received a single intra-articular injection of 2% lidocaine approximately 30 minutes prior to the procedure; 9 patients served as control. Osteochondral samples with OA changes were harvested during surgery and analyzed for chondrocyte viability by LIVE/DEAD staining. Results (A) The synovial LA concentration was significantly lower than the primary concentration injected: average 0.23 mg/mL (0.02%), highest measured 0.37 mg/mL (0.04%). (B) In vitro exposure to a reduced LA concentration had no significant influence on chondrocyte viability in intact cartilage explants (24-hour averages: control, 93%; 0.04% lidocaine, 92%; 2% lidocaine, 79%). (C) Viability of chondrocytes in OA knees was similar between 2% lidocaine injection (85%) and control (80%). Conclusions A single intra-articular knee injection of 2% lidocaine did not influence the chondrocyte viability neither in healthy nor in OA cartilage. A fast postinjection reduction of synovial LA concentration (more than 40 times) is the most likely protective mechanism.

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