chondrocytes (ACs) and its regulatory mechanisms remain unclear. This study aimed to explore epigenetic regulatory mechanisms of age-related SOX9 expression in ACs of mice, spanning from the embryonic stage to 18 months of age. Methods: The hip and shoulder joints of wild type BALB/c mice were harvested at embryonic day 16.5 and 1, 2, 6, 12 and 18 months for histopathological and immunohistochemical analyses. Femoral and humeral head cartilage from the same age groups was used for chondrocyte isolation, gene expression, methylated DNA immunoprecipitation or chromatin immunoprecipitation assays to examine epigenetic changes in the promoter region of the Sox9 gene. siRNA-mediated knockdown of the histone lysine-specific demethylases-1 gene (Lsd1) and 5-azacytidine treatment were performed in cultured ACs. Results: Sox9 mRNA and protein were highly expressed in ACs during joint development but significantly decreased at 2-18 months of age. No histopathological features of osteoarthritis were observed in examined joints by 18 months. Epigenetic DNA methylation and histone methylation are both associated with the age-dependent SOX9 expression. Knockdown of Lsd1 is sufficient to up-regulate SOX9 expression in ACs of adult mice through increased recruitment of H3K4me2 (a histone modification for transcriptional activation) in the promoter region of the Sox9 gene. However, the reduction of DNA methylation in the Sox9 promoter region induced by 5-azacytidine treatment in cultured ACs did not increase Sox9 expression. The data suggest that reduction of SOX9 expression in ACs of adult mice is primarily regulated by H3K4me2. Conclusions: These results suggest that SOX9 expression in mouse ACs is significantly decreased after the completion of joint development due to reduced demands for SOX9. This developmental switch in SOX9 expression in mouse articular cartilage is primarily regulated by epigenetic histone methylation.

BackgroundCartilage is an avascular and aneural tissue. Chondrocytes thrive in this restricted environment of low oxygen tension and poor nutrient availability which has led to suggestions that hypoxia may be a protective mechanism against the development of osteoarthritis (OA). There is also a growing body of evidence to support the role of epigenetic factors in the pathogenesis of OA. However, few studies have investigated the epigenetic-OA process within a hypoxic environment. The current study has investigated the effects of hypoxia on gene expression and DNA methylation of anabolic and catabolic genes involved in the pathogenesis of OA.MethodsChondrocytes extracted from OA femoral heads were incubated in normoxia and hypoxia (20% and 2% oxygen concentrations respectively). Interleukin 1-beta (IL-1β) plus oncostatin M (OSM), 5-azadeoxycytidine (5-aza-dC) or media alone (control) were added twice weekly to the incubated samples. After 5 weeks, levels of Collagen type IX (COL9A1), IL1B, and matrix metalloproteinase-13 (MMP13) gene expression were measured using SYBR Green-based qRT-PCR and were correlated with methylation status analysed by pyrosequencing methodology.ResultsHypoxia resulted in a >50-fold and >10-fold increase in relative expression of COL9A1 and IL1B respectively. This was inversely correlated to the DNA methylation status of these genes. Expression of MMP13 was reduced at 2% oxygen tension in control cells. Relative expression of MMP13 increased in cells stimulated with IL-1β and 5-aza-dC in normoxic conditions, and this effect was eliminated at low oxygen tension although no correlation with methylation status was observed.ConclusionsThese findings demonstrate a role for hypoxia in the regulation of anabolic and catabolic gene expression and the influence of changes in DNA methylation. These results further support the role of epigenetics in OA and, critically, highlight the complex relationship between the physiological environment of cartilaginous cells and the osteoarthritic process with implications for therapeutic intervention and our understanding of OA pathophysiology.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2474-15-431) contains supplementary material, which is available to authorized users.

, "Heart-rate sensitive optical coherence angiography for measuring vascular changes due to posttraumatic brain injury in mice," J. Biomed. Opt. Abstract. Traumatic brain injury (TBI) results in direct vascular disruption, triggering edema, and reduction in cerebral blood flow. Therefore, understanding the pathophysiology of brain microcirculation following TBI is important for the development of effective therapies. Optical coherence angiography (OCA) is a promising tool for evaluating TBI in rodent models. We develop an approach to OCA that uses the heart-rate frequency to discriminate between static tissue and vasculature. This method operates on intensity data and is therefore not phase sensitive. Furthermore, it does not require spatial overlap of voxels and thus can be applied to preexisting datasets for which oversampling may not have been explicitly considered. Heart-rate sensitive OCA was developed for dynamic assessment of mouse microvasculature post-TBI. Results show changes occurring at 5-min intervals within the first 50 min of injury. © The Authors. Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full attribution of the original publication, including its DOI.

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