Silvia Cetrullo

Hydroxytyrosol prevents chondrocyte death under oxidative stress by inducing autophagy through sirtuin 1-dependent and -independent mechanisms

BACKGROUNDnHydroxytyrosol (HT), a major phenolic antioxidant found in olive oil, can afford protection from oxidative stress in several types of non-tumoral cells, including chondrocytes. Autophagy was recently identified as a protective process during osteoarthritis (OA) development and critical for survival of chondrocytes. Therefore we have investigated the possibility to modulate chondrocyte autophagy by HT treatment.nnnMETHODSnDNA damage and cell death were estimated in human C-28/I2 and primary OA chondrocytes exposed to hydrogen peroxide. Autophagic flux and mitophagy were monitored by measuring levels and location of autophagy markers through western blot, immunostaining and confocal laser microscopy. Late autophagic vacuoles were stained with monodansylcadaverine. The involvement of sirtuin 1 (SIRT-1) was evaluated by immunohistochemistry, western blot and gene silencing with specific siRNA.nnnRESULTSnHT increases markers of autophagy and protects chondrocytes from DNA damage and cell death induced by oxidative stress. The protective effect requires the deacetylase SIRT-1, which accumulated in the nucleus following HT treatment. In fact silencing of this enzyme prevented HT from promoting the autophagic process and cell survival. Furthermore HT supports autophagy even in a SIRT-1-independent manner, by increasing p62 transcription, required for autophagic degradation of polyubiquitin-containing bodies.nnnCONCLUSIONSnThese results support the potential of HT as a chondroprotective nutraceutical compound against OA, not merely for its antioxidant ability, but as an autophagy and SIRT-1 inducer as well.nnnGENERAL SIGNIFICANCEnHT may exert a cytoprotective action by promoting autophagy in cell types that may be damaged in degenerative diseases by oxidative and other stress stimuli.

Hydroxytyrosol modulates the levels of microRNA-9 and its target sirtuin-1 thereby counteracting oxidative stress-induced chondrocyte death

OBJECTIVEnNutraceutical compounds, such as hydroxytyrosol (HT), have been found to exert protective effects in osteoarthritis (OA) by affecting a variety of key molecular and cellular processes in chondrocytes. However, to our knowledge, no relationship has been reported between nutraceuticals and microRNA (miR) network in OA models. Here, we identified a miR that is implicated in HT-mediated chondroprotection following oxidative stress condition by targeting sirtuin-1 (SIRT-1).nnnMETHODSnHuman primary and C-28/I2 chondrocytes were pre-treated with 100xa0μM HT 30xa0min before 100xa0μM H2O2 addition. In silico analyses were exploited to select putative candidate miRs able to target SIRT-1 mRNA. Luciferase-based gene reporter assay was employed to demonstrate the direct link between miR-9 and its putative mRNA target. Transient transfection approach was performed to examine the effects of miR-9 levels on caspase activity, cell viability and expression of OA-related genes.nnnRESULTSnMiR-9 was identified and confirmed as a post-transcriptional regulator of SIRT-1. MiR-9 and SIRT-1 levels showed opposite changes in chondrocytes following H2O2 and HT treatment. Moreover mir-9 silencing inhibited cell death induced by H2O2 partly through down-regulation of SIRT-1, whereas miR-9 overexpression markedly reduced the protective effect of HT. The manipulation of miR-9 levels also resulted in the modulation of OA-related gene expression, including MMP-13, VEGF and RUNX-2.nnnCONCLUSIONSnThese results show that miR-9 is a critical mediator of the deleterious and OA-related effects of oxidative stress in chondrocytes and that modulation of miR expression may be a crucial mechanism underlying the protective action of HT.

MicroRNAs and Autophagy: Fine Players in the Control of Chondrocyte Homeostatic Activities in Osteoarthritis

Osteoarthritis (OA) is a debilitating degenerative disease of the articular cartilage with a multifactorial etiology. Aging, the main risk factor for OA development, is associated with a systemic oxidative and inflammatory phenotype. Autophagy is a central housekeeping system that plays an antiaging role by supporting the clearance of senescence-associated alterations of macromolecules and organelles. Autophagy deficiency has been related to OA pathogenesis because of the accumulation of cellular defects in chondrocytes. Microribonucleic acids (microRNAs or miRs) are a well-established class of posttranscriptional modulators belonging to the family of noncoding RNAs that have been identified as key players in the regulation of cellular processes, such as autophagy, by targeting their own cognate mRNAs. Here, we present a state-of-the-art literature review on the role of miRs and autophagy in the scenario of OA pathogenesis. In addition, a comprehensive survey has been performed on the functional connections of the miR network and the autophagy pathway in OA by using “microRNA,” “autophagy,” and “osteoarthritis” as key words. Discussion of available evidence sheds light on some aspects that need further investigation in order to reach a more comprehensive view of the potential of this topic in OA.

Soft TCPTP Agonism-Novel Target to Rescue Airway Epithelial Integrity by Exogenous Spermidine

A reparative approach of disrupted epithelium in obstructive airway diseases, namely asthma and chronic obstructive pulmonary disease (COPD), may afford protection and long-lasting results compared to conventional therapies, e.g., corticosteroids or immunosuppressant drugs. Here, we propose the polyamine spermidine as a novel therapeutic agent in airways diseases, based on a recently identified mode of action: T-cell protein tyrosine phosphatase (TCPTP) agonism. It may include and surpass single-inhibitors of stress and secondary growth factor pathway signaling, i.e., the new medicinal chemistry in lung diseases. Enhanced polyamine biosynthesis has been charged with aggravating prognosis by competing for L-arginine at detriment of nitric oxide (NO) synthesis with bronchoconstrictive effects. Although excess spermine, a higher polyamine, is harmful to airways physiology, spermidine can pivot the cell homeostasis during stress conditions by the activation of TCPTP. In fact, the dephosphorylating activity of TCPTP inhibits the signaling cascade that leads to the expression of genes involved in detachment and epithelial-to-mesenchymal transition (EMT), and increases the expression of adhesion and tight junction proteins, thereby enhancing the barrier functionality in inflammation-prone tissues. Moreover, a further beneficial effect of spermidine may derive from its ability to promote autophagy, possibly in a TCPTP-dependent way. Since doses of spermidine in the micromolar range are sufficient to activate TCPTP, low amounts of spermidine administered in sustained release modality may provide an optimal pharmacologic profile for the treatment of obstructive airway diseases.

Emerging Players at the Intersection of Chondrocyte Loss of Maturational Arrest, Oxidative Stress, Senescence and Low-Grade Inflammation in Osteoarthritis

The prevalence of Osteoarthritis (OA) is increasing because of the progressive aging and unhealthy lifestyle. These risk factors trigger OA by removing constraints that keep the tightly regulated low turnover of the extracellular matrix (ECM) of articular cartilage, the correct chondrocyte phenotype, and the functionality of major homeostatic mechanisms, such as mitophagy, that allows for the clearance of dysfunctional mitochondria, preventing increased production of reactive oxygen species, oxidative stress, and senescence. After OA onset, the presence of ECM degradation products is perceived as a “danger” signal by the chondrocytes and the synovial macrophages that release alarmins with autocrine/paracrine effects on the same cells. Alarmins trigger innate immunity in the joint, with important systemic crosstalks that explain the beneficial effects of dietary interventions and improved lifestyle. Alarmins also boost low-grade inflammation: the release of inflammatory molecules and chemokines sustained by continuous triggering of NF-κB within an altered cellular setting that allows its higher transcriptional activity. Chemokines exert pleiotropic functions in OA, including the recruitment of inflammatory cells and the induction of ECM remodeling. Some chemokines have been successfully targeted to attenuate structural damage or pain in OA animal models. This represents a promising strategy for the future management of human OA.