Crucial role of miR-9 and miR-155 in cartilage homeostasis and osteoarthritis pathology

Several studies have reported beneficial effects of autophagy in preventing chondrocyte death, OA-like changes in gene expression and cartilage degeneration. Many microRNAs have been identified as key modulators of autophagy pathway. So far, to our knowledge no relationship has been revealed between nutraceuticals and microRNA network in OA. First aim of this thesis is to evaluate molecular mechanisms of action of hydroxytyrosol (HT) a promising compound already tested for protective efficacy in OA chondrocytes. HT increases markers of autophagy and protects chondrocytes from DNA damage and cell death induced by oxidative stress. The protective effect requires SIRT-1, which accumulated in the nucleus following HT treatment. Second aim consists in identifying a microRNA implicated in HT-mediated protective response to oxidative stress and examining the effects after modulation of miR levels by approach of transient transfection. After in silico analysis we identify miR-9 as a speculative candidate able to target SIRT-1 and confirm this hypothesis by means of luciferase gene-reporter assay. Moreover miR-9 mediates cell death induced by H2O2 and the protective effect of HT, as observed in human primary chondrocytes and C/28-I2 cell line. Third aim is to investigate the potential role of miR-155, found to be one of the most highly upregulated microRNAs in human OA knee cartilage, in autophagic pathway. Autophagy flux induced by rapamycin and 2-DG was significantly increased by miR-155 LNA, and significantly decreased after miR-155 mimic transfection in T/C28a2 cells and in human primary chondrocytes. These effects of miR-155 on autophagy were related to suppression of gene and protein expression of key autophagy. The identification of a single microRNA, involved simultaneously in several disease-related pathways, discloses a potent therapeutic target. Indeed the unveiling of bioactive compounds, exerting a beneficial effect through induction of epigenetic changes, may open a new topic of research not yet well explored

Effect of oxidative stress and 3-hydroxytyrosol on DNA methylation levels of miR-9 promoters

No abstract availabl

MicroRNA-155 suppresses autophagy in chondrocytes by modulating expression of autophagy proteins

Objective: Autophagy dysfunction has been reported in osteoarthritis (OA) cartilage. The objective of this study was to investigate the role of microRNA-155 (miR-155), which is overexpressed in OA, in the regulation of autophagy in human chondrocytes. Design: Rapamycin (50 nM) and 2-deoxyglucose (2-DG) (5 mM) were used to stimulate autophagy in primary human articular chondrocytes and in the T/C28a2 human chondrocyte cell line. Cells were transfected with LNA GapmeR or mimic specific for miR-155 and autophagy flux was assessed by LC3 western blotting and by Cyto-ID® dye quantification in autophagic vacuoles. Expression of predicted miR-155 targets in the autophagy pathway were analyzed by real-time PCR and western blotting. Results: Autophagy flux induced by rapamycin and 2-DG was significantly increased by miR-155 LNA, and significantly decreased after miR-155 mimic transfection in T/C28a2 cells and in human primary chondrocytes. These effects of miR-155 on autophagy were related to suppression of gene and protein expression of key autophagy regulators including Ulk1, FoxO3, Atg14, Atg5, Atg3, Gabarapl1, and Map1lc3. Conclusion: MiR-155 is an inhibitor of autophagy in chondrocytes and contributes to the autophagy defects in OA

NOTCH1: A Novel Player in the Molecular Crosstalk Underlying Articular Chondrocyte Protection by Oleuropein and Hydroxytyrosol

Osteoarthritis (OA) is the most common joint disease, but no effective and safe disease-modifying treatment is available. Risk factors such as age, sex, genetics, injuries and obesity can concur to the onset of the disease, variably triggering the loss of maturational arrest of chondrocytes further sustained by oxidative stress, inflammation and catabolism. Different types of nutraceuticals have been studied for their anti-oxidative and anti-inflammatory properties. Olive-derived polyphenols draw particular interest due to their ability to dampen the activation of pivotal signaling pathways in OA. Our study aims to investigate the effects of oleuropein (OE) and hydroxytyrosol (HT) in in vitro OA models and elucidate their possible effects on NOTCH1, a novel therapeutic target for OA. Chondrocytes were cultured and exposed to lipopolysaccharide (LPS). Detailed analysis was carried out about the OE/HT mitigating effects on the release of ROS (DCHF-DA), the increased gene expression of catabolic and inflammatory markers (real time RT-PCR), the release of MMP-13 (ELISA and Western blot) and the activation of underlying signaling pathways (Western blot). Our findings show that HT/OE efficiently attenuates LPS-induced effects by firstly reducing the activation of JNK and of the NOTCH1 pathway downstream. In conclusion, our study provides molecular bases supporting the dietary supplementation of olive-derived polyphenols to revert/delay the progression of OA

Modulation of Fatty Acid-Related Genes in the Response of H9c2 Cardiac Cells to Palmitate and n-3 Polyunsaturated Fatty Acids

While high levels of saturated fatty acids are associated with impairment of cardiovascular functions, n-3 polyunsaturated fatty acids (PUFAs) have been shown to exert protective effects. However the molecular mechanisms underlying this evidence are not completely understood. In the present study we have used rat H9c2 ventricular cardiomyoblasts as a cellular model of lipotoxicity to highlight the effects of palmitate, a saturated fatty acid, on genetic and epigenetic modulation of fatty acid metabolism and fate, and the ability of PUFAs, eicosapentaenoic acid, and docosahexaenoic acid, to contrast the actions that may contribute to cardiac dysfunction and remodeling. Treatment with a high dose of palmitate provoked mitochondrial depolarization, apoptosis, and hypertrophy of cardiomyoblasts. Palmitate also enhanced the mRNA levels of sterol regulatory element-binding proteins (SREBPs), a family of master transcription factors for lipogenesis, and it favored the expression of genes encoding key enzymes that metabolically activate palmitate and commit it to biosynthetic pathways. Moreover, miR-33a, a highly conserved microRNA embedded in an intronic sequence of the SREBP2 gene, was co-expressed with the SREBP2 messenger, while its target carnitine palmitoyltransferase-1b was down-regulated. Manipulation of the levels of miR-33a and SREBPs allowed us to understand their involvement in cell death and hypertrophy. The simultaneous addition of PUFAs prevented the effects of palmitate and protected H9c2 cells. These results may have implications for the control of cardiac metabolism and dysfunction, particularly in relation to dietary habits and the quality of fatty acid intake

Hydroxytyrosol Prevents Increase of Osteoarthritis Markers in Human Chondrocytes Treated with Hydrogen Peroxide or Growth-Related Oncogene \u3b1

Hydroxytyrosol (HT), a phenolic compound mainly derived from olives, has been proposed as a nutraceutical useful in prevention or treatment of degenerative diseases. In the present study we have evaluated the ability of HT to counteract the appearance of osteoarthritis (OA) features in human chondrocytes. Pre-treatment of monolayer cultures of chondrocytes with HT was effective in preventing accumulation of reactive oxidant species (ROS), DNA damage and cell death induced by H2O2 exposure, as well as the increase in the mRNA level of pro-inflammatory, matrix-degrading and hypertrophy marker genes, such as iNOS, COX-2, MMP-13, RUNX-2 and VEGF. HT alone slightly enhanced ROS production, but did not enhance cell damage and death or the expression of OA-related genes. Moreover HT was tested in an in vitro model of OA, i.e. three-dimensional micromass cultures of chondrocytes stimulated with growth-related oncogene \u3b1 (GRO\u3b1), a chemokine involved in OA pathogenesis and known to promote hypertrophy and terminal differentiation of chondrocytes. In micromass constructs, HT pre-treatment inhibited the increases in caspase activity and the level of the messengers for iNOS, COX-2, MMP-13, RUNX-2 and VEGF elicited by GRO\u3b1. In addition, HT significantly increased the level of SIRT-1 mRNA in the presence of GRO\u3b1. In conclusion, the present study shows that HT reduces oxidative stress and damage, exerts pro-survival and anti-apoptotic actions and favourably influences the expression of critical OA-related genes in human chondrocytes treated with stressors promoting OA-like features

Small Extracellular Vesicles from Inflamed Adipose Derived Stromal Cells Enhance the NF-κB-Dependent Inflammatory/Catabolic Environment of Osteoarthritis

The last decade has seen exponentially growing efforts to exploit the effects of adipose derived stromal cells (ADSC) in the treatment of a wide range of chronic degenerative diseases, including osteoarthritis (OA), the most prevalent joint disorder. In the perspective of developing a cell-free advanced therapy medicinal product, a focus has been recently addressed to the ADSC secretome that lends itself to an allogeneic use and can be further dissected for the selective purification of small extracellular vesicles (sEVs). sEVs can act as "biological drug carriers" to transfer information that mirror the pathophysiology of the providing cells. This is important in the clinical perspective where many OA patients are also affected by the metabolic syndrome (MetS). ADSC from MetS OA patients are dysfunctional and "inflammatory" primed within the adipose tissue. To mimic this condition, we exposed ADSC to IL-1 beta, and then we investigated the effects of the isolated sEVs on chondrocytes and synoviocytes, either cultured separately or in co-culture, to tease out the effects of these "IL-1 beta primed sEVs" on gene and protein expression of major inflammatory and catabolic OA markers. In comparison with sEVs isolated from unstimulated ADSC, the IL-1 beta primed sEVs were able to propagate NF-kappa B activation in bystander joint cells. The effects were more prominent on synoviocytes, possibly because of a higher expression of binding molecules such as CD44. These findings call upon a careful characterization of the "inflammatory fingerprint" of ADSC to avoid the transfer of an unwanted message as well as the development of in vitro "preconditioning" strategies able to rescue the antiinflammatory/anticatabolic potential of ADSC-derived sEVs

Phenotypic and genetic characterization of a family carrying two Xq21.1-21.3 interstitial deletions associated with syndromic hearing loss

Sensorineural hearing impairment is a common pathological manifestation in patients affected by X-linked intellectual disability. A few cases of interstitial deletions at Xq21 with several different phenotypic characteristics have been described, but to date, a complete molecular characterization of the deletions harboring disease-causing genes is still missing. Thus, the aim of this study is to realize a detailed clinical and molecular analysis of a family affected by syndromic X-linked hearing loss with intellectual disability

Gain-of-function defects of astrocytic Kir4.1 channels in children with autism spectrum disorders and epilepsy

Dysfunction of the inwardly-rectifying potassium channels Kir4.1 (KCNJ10) represents a pathogenic mechanism contributing to Autism-Epilepsy comorbidity. To define the role of Kir4.1 variants in the disorder, we sequenced KCNJ10 in a sample of affected individuals, and performed genotype-phenotype correlations. The effects of mutations on channel activity, protein trafficking, and astrocyte function were investigated in Xenopus laevis oocytes, and in human astrocytoma cell lines. An in vivo model of the disorder was also explored through generation of kcnj10a morphant zebrafish overexpressing the mutated human KCNJ10. We detected germline heterozygous KCNJ10 variants in 19/175 affected children. Epileptic spasms with dysregulated sensory processing represented the main disease phenotype. When investigated on astrocyte-like cells, the p.R18Q mutation exerted a gain-of-function effect by enhancing Kir4.1 membrane expression and current density. Similarly, the p.R348H variant led to gain of channel function through hindrance of pH-dependent current inhibition. The frequent polymorphism p.R271C seemed, instead, to have no obvious functional effects. Our results confirm that variants in KCNJ10 deserve attention in autism-epilepsy, and provide insight into the molecular mechanisms of autism and seizures. Similar to neurons, astrocyte dysfunction may result in abnormal synaptic transmission and electrical discharge, and should be regarded as a possible pharmacological target in autism-epilepsy. Supplementary information accompanies this paper in the files section.peer-reviewe

Ontogenetic Profile of the Expression of Thyroid Hormone Receptors in Rat and Human Corpora Cavernosa of the Penis

Introduction. In the last few years, various studies have underlined a correlation between thyroid function and male sexual function, hypothesizing a direct action of thyroid hormones on the penis. Aim. To study the spatiotemporal distribution of mRNA for the thyroid hormone nuclear receptors (TR) alpha 1, alpha 2 and beta in the penis and smooth muscle cells (SMCs) of the corpora cavernosa of rats and humans during development. Methods. We used several molecular biology techniques to study the TR expression in whole tissues or primary cultures from human and rodent penile tissues of different ages. Main Outcome Measure. We measured our data by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) amplification, Northern blot and immunohistochemistry. Results. We found that TR alpha 1 and TR alpha 2 are both expressed in the penis and in SMCs during ontogenesis without development-dependent changes. However, in the rodent model, TR beta shows an increase from 3 to 6 days post natum (dpn) to 20 dpn, remaining high in adulthood. The same expression profile was observed in humans. While the expression of TR beta is strictly regulated by development, TR alpha 1 is the principal isoform present in corpora cavernosa, suggesting its importance in SMC function. These results have been confirmed by immunohistochemistry localization in SMCs and endothelial cells of the corpora cavernosa. Conclusions. The presence of TRs in the penis provides the biological basis for the direct action of thyroid hormones on this organ. Given this evidence, physicians would be advised to investigate sexual function in men with thyroid disorders. Carosa E, Di Sante S, Rossi S, Castri A, D'Adamo F, Gravina GL, Ronchi P, Kostrouch Z, Dolci S, Lenzi A, and Jannini EA. Ontogenetic profile of the expression of thyroid hormone receptors in rat and human corpora cavernosa of the penis. J Sex Med 2010;7:1381-1390