Step based physical activity guidelines for preschool-aged children

OBJECTIVE Public health organizations recommend that preschool-aged children accumulate at least 3h of physical activity (PA) daily. Objective monitoring using pedometers offers an opportunity to measure preschooler's PA and assess compliance with this recommendation. The purpose of this study was to derive step-based recommendations consistent with the 3h PA recommendation for preschool-aged children. METHOD The study sample comprised 916 preschool-aged children, aged 3 to 6years (mean age=5.0+/-0.8years). Children were recruited from kindergartens located in Portugal, between 2009 and 2013. Children wore an ActiGraph GT1M accelerometer that measured PA intensity and steps per day simultaneously over a 7-day monitoring period. Receiver operating characteristic (ROC) curve analysis was used to identify the daily step count threshold associated with meeting the daily 3hour PA recommendation. RESULTS A significant correlation was observed between minutes of total PA and steps per day (r=0.76, p/=3h of total PA was 9099 steps per day (sensitivity (90%) and specificity (66%)) with area under the ROC curve=0.86 (95% CI: 0.84 to 0.88). CONCLUSION Preschool-aged children who accumulate less than 9000 steps per day may be considered Insufficiently Active

Urocortin protects chondrocytes from acute trauma in articular cartilage

Purpose: Single injurious mechanical impact to joints, as seen in sports injury, is known to be associated with a high risk of later development of osteoarthritis (OA). Irreversible chondrocyte death is initiated following impact and results in progressive breakdown of articular cartilage. Promotion of chondrocyte survival is therefore a necessary target in the prevention of development of further OA. Urocortin1 (Ucn) has recently been shown to have a pro-survival effect on chondrocytes in both the presence and absence of pro-apoptotic stimuli. The aim of this study was to investigate the role of Ucn in response to mechanical impact. Methods: Full thickness porcine cartilage explants from lateral and medial femoral condyles were impacted with a 500g weight using a custom-made drop tower device. Impacted explants were treated with human Ucn (10-7M, 30 minutes prior to or following impact), non-selective cation channel blocker Gadolinium (500μM, 30 minutes following impact), or mechanosensitive ion channel blocker GsMTx4 (40μM, 2 hours prior to impact) and cultured for 72hrs. Cell viability was assessed with a CMFDA:PI live dead assay, using both macro confocal microscopy, as a means of avoiding bias, and high magnification confocal microscopy techniques. Lactate dehydrogenase (LDH) assays were conducted at 72hrs to provide a quantitative read out of cell death for each explant. Calcium influx was assessed 1hr post impact by Fluo-4 AM staining and visualised by multiphoton microscopy. Results: Porcine explants were impacted from a range of heights (10-70mm) in order to establish the correct energy of impact for inducing cell death without excess damage and fracture of the cartilage explant. Cell death increased in a dose dependent manner with height. 50mm was chosen as optimal height for all further experiments. Pre-treatment of explants with Ucn had a significant increase on cell viability when compared to impacted alone, as seen by quantification of the CMFDA:PI ratio on macro confocal (p=0.0042) and by high magnification confocal microscopy (p=0.0015). This increase in cell viability was confirmed by LDH assay (p=0.0063). Post-impact treatment of the explants with Ucn also resulted in a significant increase in cell survival, seen in both CMFDA:PI ratio (p=0.0009) and LDH assay (p=0.0053). We have previously found in primary human chondrocytes that Ucn exerts its protective effect on chondrocytes by the inhibition of excessive calcium influx. Calcium influx was therefore visualised in impacted explants treated pre- and post-impact with Ucn. A significant decrease in the levels of calcium staining was seen in both of these conditions when compared to explants impacted in the absence of Ucn (p=0.0073, p=0.05). In vitro work also highlighted a role for the mechanosensitive ion channel Piezo1 in cell death associated with the absence of Ucn. Explants were therefore treated with both Gadolinium and GsMTx4 post-impact and cell viability assessed by multiphoton microscopy. An increase in cell viability was observed in both conditions, comparable to those seen with addition of Ucn. Conclusions: A novel role for Ucn in the protection of articular chondrocytes maintained in cartilage explants has been shown and validated by two microscopy techniques and LDH assay. Ucn has also been shown to reduce calcium influx in impacted explants at 60 minutes post impact, and increased cell viability was observed by blocking mechanosensitive ion channels. These data suggest the importance of regulation of calcium influx in Ucn-mediated cell protection. The pro-survival effect of Ucn was seen both pre- and post-impact, highlighting the potential prophylactic and therapeutic use of Ucn for treating sudden injury to cartilage, and subsequent progression to post-traumatic OA

Urocortin – From Parkinson’s disease to the skeleton

Urocortin (Ucn 1), a 40 amino acid long peptide related to corticotropin releasing factor (CRF) was discovered 19 years ago, based on its sequence homology to the parent molecule. Its existence was inferred in the CNS because of anatomical and pharmacological discrepancies between CRF and its two receptor subtypes. Although originally found in the brain, where it has opposing actions to CRF and therefore confers stress-coping mechanisms, Ucn 1 has subsequently been found throughout the periphery including heart, lung, skin, and immune cells. It is now well established that this small peptide is involved in a multitude of physiological and pathophysiological processes, due to its receptor subtype distribution and promiscuity in second messenger signalling pathways. As a result of extensive studies in this field, there are now well over one thousand peer reviewed publications involving Ucn 1. In this review, we intend to highlight some of the less well known actions of Ucn 1 and in particular its role in neuronal cell protection and maintenance of the skeletal system, both by conventional methods of reviewing the literature and using bioinformatics, to highlight further associations between Ucn 1 and disease conditions. Understanding how Ucn 1 works in these tissues, will help to unravel its role in normal and pathophysiological processes. This would ultimately allow the generation of putative medical interventions for the alleviation of important diseases such as Parkinson's disease, arthritis, and osteoporosis

Chondroprotection by urocortin involves blockade of a mechanosensitive piezo1 ion channel: novel, exploitable pathways for the treatment and prevention of osteoarthritis?

Purpose: Currently, the only treatments for Osteoarthritis (OA) are steroidal and non-steroidal anti-inflammatory drugs, and in severe cases total joint replacement surgery. However, these strategies only ameliorate symptoms but do not address the fundamental cause of the disease. Clearly therefore, there is a need for better therapies which address the underlying cause of OA. Importantly, this includes a reduction in the number of viable chondrocytes in articular cartilage, as the severity of cartilage damage has been shown to correlate negatively with the number of remaining chondrocytes. To develop a treatment strategy at a more fundamental level therefore, it is crucial to understand the nature of this chondrocyte cell death. We have previously found that the small endogenous peptide Urocortin (Ucn) and its two cognate G protein coupled receptors (CRF-R1 and CRF-R2) are expressed by human chondrocytes and crucially, its removal from the surrounding milieu using a Ucn depleting antibody or a pan receptor antagonist (as Ucn can bind to both receptor subtypes) causes profound chondrocyte cell death. Therefore, we believe that Ucn is an essential autocrine/paracrine pro-survival factor for human chondrocytes. Here we examine the pathways involved in this chondroprotective effect of Ucn. Methods: Selective pharmacological antagonists to CRF-R1 (CP-154526, 1–50μM; Tocris) and CRF-R2 (astressin2B 1–50μM; Tocris) were used to determine the receptor subtype responsible for the pro-survival effect of Ucn in human primary articular chondrocytes. Cell death was detected using the Annexin V-FITC Apoptosis Detection Kit I (BD Bioscience) and Western immunoblotting was used to determine the status of pro-apoptotic markers. Downstream signalling pathways involved in antagonist induced cell death were investigated using the adenylate cyclase activator forskolin (0.1μM; Tocris), the phospholipase C (PLC) activator m3M3FBS (0.1μM; Tocris), and the phospholipase A2 (PLA2) inhibitor OBAA (0.1μM; Tocris). Changes in intracellular Ca2+ were detected using Fluo-4 AM permeant dye (Thermo Fisher Scientific) and this effect was studied using the non-selective cation channel blocker Gadolinium (Gd3+ 100μM; Tocris). Human chondrocytes were transfected with siRNAs derived from a selected panel (Dharmacon), to knock down and identify specific ion channel species involved in chondrocyte survival/death. Results: We found that only inhibition of CRF-R1 using CP 154526 caused antagonist induced cell death, suggesting that the chondrocyte pro-survival effect is caused by Ucn binding to CRF-R1 alone (p < 0.05). This cell death was associated with an increased expression of p53 and cleavage of both caspases 9 and 3 but not caspase 8, implicating the intrinsic apoptotic pathway in this process. Furthermore, we were able to rescue chondrocyte cell death in the presence of antagonist, with the adenylate cyclase activator forskolin and the phospholipase A2 inhibitor OBAA but not with the phospholipase C activator m3M3FBS, suggesting a role for cAMP and PKA activation and a decrease in PLA2 activity in this process. Antagonist induced cell death initially involved a large inward flux of Ca2+ resulting in Ca2+ overload. This inward movement of Ca2+ and subsequent cell death could be prevented by Gd3+ p < 0.05, implying that this process involves a non-selective cation channel and that when Ucn is present, this channel is in a closed conformation. Using an siRNA array panel, we identified Piezo1 as the target ion channel responsible for chondrocyte cell death in the absence of Ucn. Conclusions: These findings are of great interest because Piezo1 is a novel type of mechanosensor and has recently been found to be highly expressed in chondrocytes and responsible for chondrocyte cell death in a porcine acute mechanical injury model of OA. This is the first study to bring together all of these critical mediators of chondrocyte survival/death and elucidating fully the relationship between Ucn receptor activation and gating of Piezo1, may highlight novel targets as potential therapy nodes for the treatment/prevention of OA. Additionally, because of the crucial role of Ucn in chondrocyte survival, a study of the molecular status of this peptide and/or its receptor could represent novel biomarkers of OA severity and progression