Meniscal tissue explants response depends on level of dynamic compressive strain

SummaryObjectiveFollowing partial meniscectomy, the remaining meniscus is exposed to an altered loading environment. In vitro 20% dynamic compressive strains on meniscal tissue explants has been shown to lead to an increase in release of glycosaminoglycans from the tissue and increased expression of interleukin-1α (IL-1α). The goal of this study was to determine if compressive loading which induces endogenously expressed IL-1 results in downstream changes in gene expression of anabolic and catabolic molecules in meniscal tissue, such as MMP expression.MethodRelative changes in gene expression of MMP-1, MMP-3, MMP-9, MMP-13, A Disintegrin and Metalloproteinase with ThromboSpondin 4 (ADAMTS4), ADAMTS5, TNFα, TGFβ, COX-2, Type I collagen (COL-1) and aggrecan and subsequent changes in the concentration of prostaglandin E2 released by meniscal tissue in response to varying levels of dynamic compression (0%, 10%, and 20%) were measured. Porcine meniscal explants were dynamically compressed for 2h at 1Hz.Results20% dynamic compressive strains upregulated MMP-1, MMP-3, MMP-13 and ADAMTS4 compared to no dynamic loading. Aggrecan, COX-2, and ADAMTS5 gene expression were upregulated under 10% strain compared to no dynamic loading while COL-1, TIMP-1, and TGFβ gene expression were not dependent on the magnitude of loading.ConclusionThis data suggests that changes in mechanical loading of the knee joint meniscus from 10% to 20% dynamic strain can increase the catabolic activity of the meniscus

Detection of the glucocorticoid receptors in brain protein extracts by SDS-PAGE

Uncorrected proofGlucocorticoids are steroid hormones vital for organ system homeostasis and for the maintenance of essential biological processes. A significant part of these actions are mediated through glucocorticoid receptor (GR) that belongs to the nuclear receptor superfamily. To cover such variety of processes the different glucocorticoids act through different GR isoforms that are originated due to posttranscriptional and posttranslational mechanisms. For this reason when evaluating the levels of GRs we should preferentially determine protein levels instead of gene expression. Here, we describe the detection by Western blotting of the GR (a and ß isoforms) protein, using macrodissected brain tissue

Loss of receptor activity-modifying protein 2 in mice causes placental dysfunction and alters PTH1R regulation

Receptor activity-modifying protein 2 (Ramp2) is a single-pass transmembrane protein that heterodimerizes with several family B G-protein coupled receptors to alter their function. Ramp2 has been primarily characterized in association with calcitonin receptor-like receptor (Calcrl, CLR), forming the canonical receptor complex for the endocrine peptide adrenomedullin (Adm, AM). However, we previously demonstrated that Ramp2+/- female mice display a constellation of endocrine-related phenotypes that are distinct from those of Adm+/- and Calcrl+/- mice, implying that RAMP2 has physiological functions beyond its canonical complex. Here, we localize Ramp2 expression in the mouse placenta, finding that Ramp2 is robustly expressed in the fetal labyrinth layer, and then characterize the effects of loss of Ramp2 on placental development. Consistent with the expression pattern of Ramp2 in the placenta, Ramp2-/- placentas have a thinner labyrinth layer with significantly fewer trophoblast cells secondary to a reduction in trophoblast proliferation. We also find that absence of Ramp2 leads to failed spiral artery remodeling unaccompanied by changes in the uterine natural killer cell population. Furthermore, we assess changes in gene expression of other RAMP2-associated G-protein coupled receptors (GPCRs), concluding that Ramp2 loss decreases parathyroid hormone 1 receptor (Pthr1) expression and causes a blunted response to systemic parathyroid hormone (PTH) administration in mice. Ultimately, these studies provide in vivo evidence of a role for RAMP2 in placental development distinct from the RAMP2-CLR/AM signaling paradigm and identify additional pathways underlying the endocrine and fertility defects of the previously characterized Ramp2 heterozygous adult females

Accelerated development with increased bone mass and skeletal response to loading suggest receptor activity modifying protein-3 as a bone anabolic target

Knockout technologies provide insights into physiological roles of genes. Studies initiated into endocrinology of heteromeric G protein-coupled receptors included deletion of receptor activity modifying protein-3, an accessory protein that alters ligand selectivity of calcitonin and calcitonin-like receptors. Initially, deletion of Ramp3-/- appeared phenotypically silent, but it has emerged that mice have a high bone mass phenotype, and more subtle alterations to angiogenesis, amylin homeostasis, and a small proportion of the effects of adrenomedullin on cardiovascular and lymphatic systems. Here we explore in detail, effects of Ramp3-/- deletion on skeletal growth/development, bone mass and response of bone to mechanical loading mimicking exercise. Mouse pups lacking RAMP3 are healthy and viable, having accelerated development of the skeleton as assessed by degree of mineralisation of specific bones, and by microCT measurements. Specifically, we observed that neonates and young mice have increased bone volume and mineralisation in hindlimbs and vertebrae and increased thickness of bone trabeculae. These changes are associated with increased osteoblast numbers and bone apposition rate in Ramp3-/- mice, and increased cell proliferation in epiphyseal growth plates. Effects persist for some weeks after birth, but differences in gross bone mass between RAMP3 and WT mice lose significance in older animals although architectural differences persist. Responses of bones of 17-week old mice to mechanical loading that mimics effects of vigorous exercise is increased significantly in Ramp3-/- mice by 30% compared with WT control mice. Studies on cultured osteoblasts from Ramp3-/- mice indicate interactions between mRNA expression of RAMPs1 and 3, but not RAMP2 and 3. Our preliminary data shows that Ramp3-/- osteoblasts had increased expression β-catenin, a component of the canonical Wnt signalling pathway known to regulate skeletal homeostasis and mechanosensitivity. Given interactions of RAMPs with both calcitonin and calcitonin-like receptors to alter ligand selectivity, and with other GPCRs to change trafficking or ligand bias, it is not clear whether the bone phenotype of Ramp3-/- mice is due to alterations in signalling mediated by one or more GPCRS. However, as antagonists of RAMP-interacting receptors are growing in availability, there appears the likelihood that manipulation of the RAMP3 signalling system could provide anabolic effects therapeutically

Accelerated Development With Increased Bone Mass and Skeletal Response to Loading Suggest Receptor Activity Modifying Protein-3 as a Bone Anabolic Target

Knockout technologies provide insights into physiological roles of genes. Studies initiated into endocrinology of heteromeric G protein-coupled receptors included deletion of receptor activity modifying protein-3, an accessory protein that alters ligand selectivity of calcitonin and calcitonin-like receptors. Initially, deletion of Ramp3-/- appeared phenotypically silent, but it has emerged that mice have a high bone mass phenotype, and more subtle alterations to angiogenesis, amylin homeostasis, and a small proportion of the effects of adrenomedullin on cardiovascular and lymphatic systems. Here we explore in detail, effects of Ramp3-/- deletion on skeletal growth/development, bone mass and response of bone to mechanical loading mimicking exercise. Mouse pups lacking RAMP3 are healthy and viable, having accelerated development of the skeleton as assessed by degree of mineralisation of specific bones, and by microCT measurements. Specifically, we observed that neonates and young mice have increased bone volume and mineralisation in hindlimbs and vertebrae and increased thickness of bone trabeculae. These changes are associated with increased osteoblast numbers and bone apposition rate in Ramp3-/- mice, and increased cell proliferation in epiphyseal growth plates. Effects persist for some weeks after birth, but differences in gross bone mass between RAMP3 and WT mice lose significance in older animals although architectural differences persist. Responses of bones of 17-week old mice to mechanical loading that mimics effects of vigorous exercise is increased significantly in Ramp3-/- mice by 30% compared with WT control mice. Studies on cultured osteoblasts from Ramp3-/- mice indicate interactions between mRNA expression of RAMPs1 and 3, but not RAMP2 and 3. Our preliminary data shows that Ramp3-/- osteoblasts had increased expression β-catenin, a component of the canonical Wnt signalling pathway known to regulate skeletal homeostasis and mechanosensitivity. Given interactions of RAMPs with both calcitonin and calcitonin-like receptors to alter ligand selectivity, and with other GPCRs to change trafficking or ligand bias, it is not clear whether the bone phenotype of Ramp3-/- mice is due to alterations in signalling mediated by one or more GPCRS. However, as antagonists of RAMP-interacting receptors are growing in availability, there appears the likelihood that manipulation of the RAMP3 signalling system could provide anabolic effects therapeutically

Receptor activity modifying proteins (RAMPs) interact with the VPAC 2 receptor and CRF 1 receptors and modulate their function: RAMP interactions with VPAC2and CRF1receptors

Although it is established that the receptor activity modifying proteins (RAMPs) can interact with a number of GPCRs, little is known about the consequences of these interactions. Here the interaction of RAMPs with the glucagon-like peptide 1 receptor (GLP-1 receptor), the human vasoactive intestinal polypeptide/pituitary AC-activating peptide 2 receptor (VPAC2) and the type 1 corticotrophin releasing factor receptor (CRF1) has been examined

Influences of light and humidity on carbonyl sulfide-based estimates of photosynthesis

Understanding climate controls on gross primary productivity (GPP) is crucial for accurate projections of the future land carbon cycle. Major uncertainties exist due to the challenge in separating GPP and respiration from observations of the carbon dioxide (CO2) flux. Carbonyl sulfide (COS) has a dominant vegetative sink, and plant COS uptake is used to infer GPP through the leaf relative uptake (LRU) ratio of COS to CO2 fluxes. However, little is known about variations of LRU under changing environmental conditions and in different phenological stages. We present COS and CO2 fluxes and LRU of Scots pine branches measured in a boreal forest in Finland during the spring recovery and summer. We find that the diurnal dynamics of COS uptake is mainly controlled by stomatal conductance, but the leaf internal conductance could significantly limit the COS uptake during the daytime and early in the season. LRU varies with light due to the differential light responses of COS and CO2 uptake, and with vapor pressure deficit (VPD) in the peak growing season, indicating a humidity-induced stomatal control. Our COS-based GPP estimates show that it is essential to incorporate the variability of LRU with environmental variables for accurate estimation of GPP on ecosystem, regional, and global scales.Peer reviewe

Fetal-derived adrenomedullin mediates the innate immune milieu of the placenta

The remodeling of maternal uterine spiral arteries (SAs) is an essential process for ensuring low-resistance, high-capacitance blood flow to the growing fetus. Failure of SAs to remodel is causally associated with preeclampsia, a common and life-threatening complication of pregnancy that is harmful to both mother and fetus. Here, using both loss-of-function and gain-of-function genetic mouse models, we show that expression of the pregnancy-related peptide adrenomedullin (AM) by fetal trophoblast cells is necessary and sufficient to promote appropriate recruitment and activation of maternal uterine NK (uNK) cells to the placenta and ultimately facilitate remodeling of maternal SAs. Placentas that lacked either AM or its receptor exhibited reduced fetal vessel branching in the labyrinth, failed SA remodeling and reendothelialization, and markedly reduced numbers of maternal uNK cells. In contrast, overexpression of AM caused a reversal of these phenotypes with a concomitant increase in uNK cell content in vivo. Moreover, AM dose-dependently stimulated the secretion of numerous chemokines, cytokines, and MMPs from uNK cells, which in turn induced VSMC apoptosis. These data identify an essential function for fetal-derived factors in the maternal vascular adaptation to pregnancy and underscore the importance of exploring AM as a biomarker and therapeutic agent for preeclampsia

Evaluation of carbonyl sulfide biosphere exchange in the Simple Biosphere Model (SiB4)

The uptake of carbonyl sulfide (COS) by terrestrial plants is linked to photosynthetic uptake of CO2 as these gases partly share the same uptake pathway. Applying COS as a photosynthesis tracer in models requires an accurate representation of biosphere COS fluxes, but these models have not been extensively evaluated against field observations of COS fluxes. In this paper, the COS flux as simulated by the Simple Biosphere Model, version 4 (SiB4), is updated with the latest mechanistic insights and evaluated with site obser- vations from different biomes: one evergreen needleleaf forest, two deciduous broadleaf forests, three grasslands, and two crop fields spread over Europe and North America. We improved SiB4 in several ways to improve its representation of COS. To account for the effect of atmospheric COS mole fractions on COS biosphere uptake, we replaced the fixed atmospheric COS mole fraction boundary condition originally used in SiB4 with spatially and temporally varying COS mole fraction fields. Seasonal amplitudes of COS mole fractions are similar to 50-200 ppt at the investigated sites with a minimum mole fraction in the late growing season. Incorporating seasonal variability into the model reduces COS uptake rates in the late growing season, allowing better agreement with observations. We also replaced the empirical soil COS uptake model in SiB4 with a mechanistic model that represents both uptake and production of COS in soils, which improves the match with observations over agricultural fields and fertilized grassland soils. The improved version of SiB4 was capable of simulating the diurnal and seasonal variation in COS fluxes in the boreal, temperate, and Mediterranean region. Nonetheless, the daytime vegetation COS flux is underestimated on average by 8 +/- 27 %, albeit with large variability across sites. On a global scale, our model modifications decreased the modeled COS terrestrial biosphere sink from 922 Gg S yr(-1) in the original SiB4 to 753 Gg S yr(-1) in the updated version. The largest decrease in fluxes was driven by lower atmospheric COS mole fractions over regions with high productivity, which highlights the importance of accounting for variations in atmospheric COS mole fractions. The change to a different soil model, on the other hand, had a relatively small effect on the global biosphere COS sink. The secondary role of the modeled soil component in the global COS budget supports the use of COS as a global photosynthesis tracer. A more accurate representation of COS uptake in SiB4 should allow for improved application of atmospheric COS as a tracer of local- to global-scale terrestrial photosynthesis.Peer reviewe

Insight into glucocorticoid receptor signalling through interactome model analysis

Glucocorticoid hormones (GCs) are used to treat a variety of diseases because of their potent anti-inflammatory effect and their ability to induce apoptosis in lymphoid malignancies through the glucocorticoid receptor (GR). Despite ongoing research, high glucocorticoid efficacy and widespread usage in medicine, resistance, disease relapse and toxicity remain factors that need addressing. Understanding the mechanisms of glucocorticoid signalling and how resistance may arise is highly important towards improving therapy. To gain insight into this we undertook a systems biology approach, aiming to generate a Boolean model of the glucocorticoid receptor protein interaction network that encapsulates functional relationships between the GR, its target genes or genes that target GR, and the interactions between the genes that interact with the GR. This model named GEB052 consists of 52 nodes representing genes or proteins, the model input (GC) and model outputs (cell death and inflammation), connected by 241 logical interactions of activation or inhibition. 323 changes in the relationships between model constituents following in silico knockouts were uncovered, and steady-state analysis followed by cell-based microarray genome-wide model validation led to an average of 57% correct predictions, which was taken further by assessment of model predictions against patient microarray data. Lastly, semi-quantitative model analysis via microarray data superimposed onto the model with a score flow algorithm has also been performed, which demonstrated significantly higher correct prediction ratios (average of 80%), and the model has been assessed as a predictive clinical tool using published patient microarray data. In summary we present an in silico simulation of the glucocorticoid receptor interaction network, linked to downstream biological processes that can be analysed to uncover relationships between GR and its interactants. Ultimately the model provides a platform for future development both by directing laboratory research and allowing for incorporation of further components, encapsulating more interactions/genes involved in glucocorticoid receptor signalling