Hideaki Nagase, Ph.D.
Kennedy Institute of Rheumatology-Centre for Degenerative Diseases, University of Oxford, UK
Osteoarthritis: diagnosis, treatment and challenges
Hideaki Nagase1, Ngee Han Lim1, George Bou-Gharios1, Ernst Meinjohanns2 and Morten Meldal3
- Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, London, W6 8LH UK
- Carlsberg Laboratory, Copenhagen, Denmark,
- Nano-Science Center, Department of Chemistry, University of Copenhagen, Denmark
Osteoarthritis (OA) is the most prevalent age-related degenerative joint disease. With the expanding ageing population, it imposes a major socio-economic burden on society. A key feature of OA is a gradual loss of articular cartilage and deformation of bone, resulting in the impairment of joint function. Currently, there is no effective disease-modifying treatment except joint replacement surgery. There are many possible causes of cartilage loss (e.g. mechanical load, injury, reactive oxygen species, aging, etc.) and etiological factors (obesity, genetics), but the degradation of cartilage is primarily caused by elevated levels of active metalloproteinases. It is therefore attractive to consider proteinase inhibitors as potential therapeutics. However, there are several hurdles to overcome, namely early diagnosis and continuous monitoring of the efficacy of inhibitor therapeutics. We are therefore aiming at developing non-invasive probes to detect cartilage degrading metalloproteinase activities.
We have designed in vivo imaging probes to detect MMP-13 (collagenase 3) activity that participates in OA by degrade cartilage collagen II and MMP-12 (macrophage elastase) activity involved in inflammatory arthritis. These activity-based probes consist of a peptide that is selectively cleaved by the target proteinase, a near-infrared fluorophore and a quencher. The probe’s signal multiplies upon proteolysis. They were first used to follow the respective enzyme activity in vivo in the mouse model of collagen-induced arthritis and we found MMP-12 activity probe (MMP12AP) activation peaked at 5 days after onset of the disease, whereas MMP13AP activation was observed at 10-15 days. The in vivo activation of these probes was inhibited by specific low molecule inhibitors. We proceeded to test both probes in the mouse model of OA induced by the surgical destabilization of medial meniscus of the knee joints. In this model, degradation of knee cartilage is first detected histologically 6 weeks after surgery with significant erosion detectable at 8 weeks. Little activation of MMP12AP was detected, which was expected, as macrophage migration is not obvious in OA. MMP13AP, on the other hand, was significantly activated in the operated knee at 6 weeks compared with the non-operated contralateral knee, but there were no significant differences between the operated and sham-operated knees. At 8 weeks, however, the signals in the operated knees were significantly higher than both the contralateral and sham-operated controls. Activation of aggrecanases and MMP-13 are observed before structural changes of cartilage. We are therefore currently improving the MMP-13 probe for earlier detection by attaching it to polymers that are retained in cartilage.
K. Satyamoorthy, Ph.D.
Director, Life Sciences Centre, Manipal University, India
Epigenetic Changes due to DNA Methylation in Human Epithelial Tumors
Extensive global hypomethylation in the genome and hypermthylation of selective tumor specific suppressor genes appears to be a hallmark of human cancers. Data suggests that hypermethylation of promoter region in genes is more closely related to subsequent gene expression; contrary to gene-body DNA methylation. The intricate balance between these two may contribute to the progressive process of development, differentiation and carcinogenesis. Epigenetic changes encompass, apart from DNA methylation, chromatin modifications through post-translational changes in histones and control by miRNAs. At the genome level, effects from these are compounded by copy number variations (CNVs) which may ultimately influence protein functions. From clinical perspective, changes in DNA methylation occur very early which are reversible and are influenced by environmental factors. Therefore, these can be potential resource for identifying therapeutic targets as well as biomarkers for early screening of cancer. Our current efforts in profiling genome wide DNA methylation changes in oral, cervical and breast cancers through DNA methylation microarray analysis has revealed number of alterations critical for survival, progression and metastatic behavior of tumors. Bioinformatics and functional analysis revealed several key regulatory molecules controlled by DNA methylation and suggests that DNA methylation changes in several CpG islands appear to co-segregate in the regions of miRNAs as well as in the CNVs. We have validated the signatures for methylation of CpG islands through bisufite sequencing for essential genes in clinical samples and have undertaken transcriptional and functional analysis in tumor cell lines. These results will be presented.
Aswath Balakrishnan, Kapaettu Satyamoorthy and Manjunath B Joshi
Introduction
Insulin resistance is a hall mark of metabolic disorders such as diabetes. Reduced insulin response in vasculature leads to disruption of IR/Akt/eNOS signaling pathway resulting in vasoconstriction and subsequently to cardiovascular diseases. Recent studies have demonstrated that inflammatory regulator interleukin-6 (IL-6), as one of the potential mediators that can link chronic inflammation with insulin resistance. Accumulating evidences suggest a significant role of epigenetic mechanisms such as DNA methylation in progression of metabolic disorders. Hence the present study aimed to understand the role of epigenetic mechanisms involved during IL-6 induced vascular insulin resistance and its consequences in cardiovascular diseases.
Materials and Methods
Human umbilical vein endothelial cells (HUVEC) and Human dermal microvascular endothelial cells (HDMEC) were used for this study. Endothelial cells were treated in presence or absence of IL-6 (20ng/ml) for 36 hours and followed by insulin (100nM) stimulation for 15 minutes. Using immunoblotting, cell lysates were stained for phosphor- and total Akt levels to measure insulin resistance. To investigate changes in DNA methylation, cells were treated with or without neutrophil conditioned medium (NCM) as a physiological source of inflammation or IL-6 (at various concentrations) for 36 hours. Genomic DNA was processed for HPLC analysis for methyl cytosine content and cell lysates were analyzed for DNMT1 (DNA (cytosine-5)-methyltransferase 1) and DNMT3A (DNA (cytosine-5)-methyltransferase 3A) levels using immunoblotting.
Results
Endothelial cells stimulated with insulin exhibited an increase in phosphorylation of Aktser 473 in serum free conditions but such insulin response was not observed in cells treated with IL-6, suggesting chronic exposure of endothelial cells to IL-6 leads to insulin resistance. HPLC analysis for global DNA methylation resulted in decreased levels of 5-methyl cytosine in cells treated with pro-inflammatory molecules (both by NCM and IL-6) as compared to untreated controls. Subsequently, analysis in cells treated with IL-6 showed a significant decrease in DNMT1 levels but not in DNMT3A. Other pro-inflammatory marker such as TNF-α did not exhibit such changes.
Conclusion
Our study suggests: a) Chronic treatment of endothelial cells with IL-6 results in insulin resistance b) Neutrophil conditioned medium and IL-6 decreases methyl cytosine levels c) DNMT1 but not DNMT3a levels are reduced in cells treated with IL-6.