Gene expression profiling and functional analysis of angiogenic markers in murine collagen-induced arthritis

Introduction

Dysregulated angiogenesis is implicated in the pathogenesis of rheumatoid arthritis (RA). To provide a more profound understanding of arthritis-associated angiogenesis, we evaluated the expression of angiogenesis-modulating genes at onset, peak and declining phases of collagen-induced arthritis (CIA), a well-established mouse model for RA.

Methods

CIA was induced in DBA/1 mice with type II collagen. Functional capillary density in synovial tissue of knee joints was determined by intravital fluorescence microscopy. To assess the ability of arthritic joint homogenates to induce angiogenesis, an endothelial chemotaxis assay and an in vivo matrigel plug assay were employed. The temporal expression profile of angiogenesis-related genes in arthritic paws was analysed by quantitative real-time RT-PCR using an angiogenesis focused array as well as gene specific PCR. Finally, we investigated the therapeutic effect of a monoclonal antibody specifically blocking the binding of VEGF to neuropilin (NRP)-1.

Results

Although arthritic paw homogenates displayed angiogenic activity in vitro and in vivo, and synovia of arthritic paws appeared highly vascularised on histological examination, the functional capillary density in arthritic knee synovia was significantly decreased, whereas capillary diameter was increased. Of the 84 genes analysed, 41 displayed a differential expression in arthritic paws as compared to control paws. Most significant alterations were seen at the peak of clinical arthritis. Increased mRNA expression could be observed for VEGF receptors (Flt-1, Flk-1, Nrp-1, Nrp-2), as well as for midkine, hepatocyte growth factor, insulin-like growth factor-1 and angiopoietin-1. Signalling through NRP-1 accounted in part for the chemotactic activity for endothelial cells observed in arthritic paw homogenates. Importantly, therapeutic administration of anti-NRP1^B antibody significantly reduced disease severity and progression in CIA mice.

Conclusions

Our findings confirm that the arthritic synovium in murine CIA is a site of active angiogenesis, but an altered balance in the expression of angiogenic factors seems to favour the formation of non-functional and dilated capillaries. Furthermore, our results validate NRP-1 as a key player in the pathogenesis of CIA, and support the VEGF/VEGF receptor pathway as a potential therapeutic target in RA.     

L1 is sequentially processed by two differently activated metalloproteases and presenilin/gamma-secretase and regulates neural cell adhesion, cell migration, and neurite outgrowth

The immunoglobulin superfamily recognition molecule L1 plays important functional roles in the developing and adult nervous system. Metalloprotease-mediated cleavage of this adhesion molecule has been shown to stimulate cellular migration and neurite outgrowth. We demonstrate here that L1 cleavage is mediated by two distinct members of the disintegrin and metalloprotease family, ADAM10 and ADAM17. This cleavage is differently regulated and leads to the generation of a membrane bound C-terminal fragment, which is further processed through gamma-secretase activity. Pharmacological approaches with two hydroxamate-based inhibitors with different preferences in blocking ADAM10 and ADAM17, as well as loss of function and gain of function studies in murine embryonic fibroblasts, showed that constitutive shedding of L1 is mediated by ADAM10 while phorbol ester stimulation or cholesterol depletion led to ADAM17-mediated L1 cleavage. In contrast, N-methyl-d-aspartate treatment of primary neurons stimulated ADAM10-mediated L1 shedding. Both proteases were able to affect L1-mediated adhesion and haptotactic migration of neuronal cells. In particular, both proteases were involved in L1-dependent neurite outgrowth of cerebellar neurons. Thus, our data identify ADAM10 and ADAM17 as differentially regulated L1 membrane sheddases, both critically affecting the physiological functions of this adhesion protein.     

Positive Reinforcement Training in Squirrel Monkeys Using Clicker Training

Nonhuman primates in research environments experience regular stressors that have the potential to alter physiology and brain function, which in turn can confound some types of research studies. Operant conditioning techniques such as positive reinforcement training (PRT), which teaches animals to voluntarily perform desired behaviors, can be applied to improve behavior and reactivity. PRT has been used to train rhesus macaques, marmosets, and several other nonhuman primate species. To our knowledge, the method has yet to be used to train squirrel monkeys to perform complex tasks. Accordingly, we sought to establish whether PRT, utilizing a hand‐box clicker (which emits a click sound that acts as the conditioned reinforcer), could be used to train adult male squirrel monkeys (Saimiri boliviensis, N = 14). We developed and implemented a training regimen to elicit voluntary participation in routine husbandry, animal transport, and injection procedures. Our secondary goal was to quantify the training time needed to achieve positive results. Squirrel monkeys readily learned the connection between the conditioned reinforcer (the clicker) and the positive reinforcer (food). They rapidly developed proficiency on four tasks of increasing difficulty: target touching, hand sitting, restraint training, and injection training. All subjects mastered target touching behavior within 2 weeks. Ten of 14 subjects (71%) mastered all tasks in 59.2 ± 2.6 days (range: 50–70 days). In trained subjects, it now takes about 1.25 min per monkey to weigh and administer an intramuscular injection, one‐third of the time it took before training. From these data, we conclude that clicker box PRT can be successfully learned by a majority of squirrel monkeys within 2 months and that trained subjects can be managed more efficiently. These findings warrant future studies to determine whether PRT may be useful in reducing stress‐induced experimental confounds in studies involving squirrel monkeys. Am. J. Primatol. 74:712–720, 2012. © 2012 Wiley Periodicals, Inc.     

Analysis of the copy number profiles of several tumor samples from the same patient reveals the successive steps in tumorigenesis

We present a computational method, TuMult, for reconstructing the sequence of copy number changes driving carcinogenesis, based on the analysis of several tumor samples from the same patient. We demonstrate the reliability of the method with simulated data, and describe applications to three different cancers, showing that TuMult is a valuable tool for the establishment of clonal relationships between tumor samples and the identification of chromosome aberrations occurring at crucial steps in cancer progression.     

Ecological indicators based on trophic spectrum as a tool to assess ecosystems fishing impacts

Trophic indicators were used to compare two Malian freshwater reservoirs whose main differences are based on their different fishing pressures. Data were collected from a scientific survey of small-scale fishery landings conducted in 2002/2003. The trophic levels of fish species caught by artisanal fisheries are estimated from observations of scientific fishing or from the metabase Fishbase. Important differences exist in the trophic structure of both reservoirs. In Selingue (with high fishing pressure), very few top predators are found in the catches while the low trophic level fishes increase in total catches. In Manantali (with low fishing pressure), the top predators contribute twice as much to catches compared to Selingue. Hence, the mean trophic level of catches in Selingue (2.80) is lower than in Manantali (2.97). When comparing these results with those of study made in 1994/1995, it clearly appears that the effects of the fishing pressure in Selingue are obvious through a decrease of 0.12 in the mean trophic level while in Manantali this mean level has increased by 0.33 due to a recent strategic targeting of top predators. Trophic spectra seem to be relevant tools to characterize exploited fish communities from multi-specific and multi-gear small-scale fisheries catch data.     

Precipitation-dependent flowering of Globularia alypum and Erica multiflora in Mediterranean shrubland under experimental drought and warming, and its inter-annual variability

Background and Aims

Relationships between autumn flowering, precipitation and temperature of plant species of Mediterranean coastal shrublands have been described, but not analysed experimentally. These relationships were analysed for two species of co-occurring, dominant, autumn-flowering shrubs, Globularia alypum and Erica multiflora, over 4 years and in experimentally generated drought and warming conditions. The aim was to improve predictions about the responses and adaptations of flowering of Mediterranean vegetation to climate change.

Methods

Beginning of anthesis and date of maximum flowering intensity (‘peak date’) were monitored over 4 years (2001–2004) on a garrigue land type in the noth-east of the Iberian Peninsula. Two experimental treatments were applied, increased temperature (+0·73°C) and reduced soil moisture (–17%) relative to untreated plots.

Key Results

Flowering of Globularia alypum and Erica multiflora differed greatly between years depending on the precipitation of the previous months and the date of the last substantial rainfall (>10 mm). Globularia alypum flowered once or twice (unimodal or bimodal) as the result of differences in the distribution and magnitude of precipitation in late-spring and summer (when floral buds develop). The drought treatment delayed and decreased flowering of Globularia alypum in 2001 and delayed flowering in 2002. Warming extended the period between the beginning of flowering and the end of the second peak for autumn flowering in 2001 and also increased peak intensity in 2002. Flowering of Erica multiflora was unaffected by either treatment.

Conclusions

Autumn flowering of Globularia alypum and Erica multiflora is more dependent on water availability than on temperature. Considerable inter-annual plasticity in the beginning of anthesis and peak date and on unimodal or bimodal flowering constitutes a ‘safe strategy’ for both species in relation to varying precipitation and temperature. However, severe changes in precipitation in spring and summer may severely affect flowering of Globularia alypum but not Erica multiflora, thus affecting development/structure of the ecosystem if such conditions persist.Key words: Globularia alypum, Erica multiflora, autumn flowering, drought, global warming, Mediterranean     

Receptor association and tyrosine phosphorylation of S6 kinases

Ribosomal protein S6 kinase (S6K) is activated by an array of mitogenic stimuli and is a key player in the regulation of cell growth. The activation process of S6 kinase involves a complex and sequential series of multiple Ser/Thr phosphorylations and is mainly mediated via phosphatidylinositol 3-kinase (PI3K)-3-phosphoinositide-dependent protein kinase-1 (PDK1) and mTor-dependent pathways. Upstream regulators of S6K, such as PDK1 and protein kinase B (PKB/Akt), are recruited to the membrane via their pleckstrin homology (PH) or protein-protein interaction domains. However, the mechanism of integration of S6K into a multi-enzyme complex around activated receptor tyrosine kinases is not clear. In the present study, we describe a specific interaction between S6K with receptor tyrosine kinases, such as platelet-derived growth factor receptor (PDGFR). The interaction with PDGFR is mediated via the kinase or the kinase extension domain of S6K. Complex formation is inducible by growth factors and leads to S6K tyrosine phosphorylation. Using PDGFR mutants, we have shown that the phosphorylation is exerted via a PDGFR-src pathway. Furthermore, src kinase phosphorylates and coimmunoprecipitates with S6K in vivo. Inhibitors towards tyrosine kinases, such as genistein and PP1, or src-specific SU6656, but not PI3K and mTor inhibitors, lead to a reduction in tyrosine phosphorylation of S6K. In addition, we mapped the sites of tyrosine phosphorylation in S6K1 and S6K2 to Y39 and Y45, respectively. Mutational and immunofluorescent analysis indicated that phosphorylation of S6Ks at these sites does not affect their activity or subcellular localization. Our data indicate that S6 kinase is recruited into a complex with RTKs and src and becomes phosphorylated on tyrosine/s in response to PDGF or serum.     

Proteomic Profiling of the TRAF3 Interactome Network Reveals a New Role for the ER-to-Golgi Transport Compartments in Innate Immunity

Tumor Necrosis Factor receptor-associated factor-3 (TRAF3) is a central mediator important for inducing type I interferon (IFN) production in response to intracellular double-stranded RNA (dsRNA). Here, we report the identification of Sec16A and p115, two proteins of the ER-to-Golgi vesicular transport system, as novel components of the TRAF3 interactome network. Notably, in non-infected cells, TRAF3 was found associated with markers of the ER-Exit-Sites (ERES), ER-to-Golgi intermediate compartment (ERGIC) and the cis-Golgi apparatus. Upon dsRNA and dsDNA sensing however, the Golgi apparatus fragmented into cytoplasmic punctated structures containing TRAF3 allowing its colocalization and interaction with Mitochondrial AntiViral Signaling (MAVS), the essential mitochondria-bound RIG-I-like Helicase (RLH) adaptor. In contrast, retention of TRAF3 at the ER-to-Golgi vesicular transport system blunted the ability of TRAF3 to interact with MAVS upon viral infection and consequently decreased type I IFN response. Moreover, depletion of Sec16A and p115 led to a drastic disorganization of the Golgi paralleled by the relocalization of TRAF3, which under these conditions was unable to associate with MAVS. Consequently, upon dsRNA and dsDNA sensing, ablation of Sec16A and p115 was found to inhibit IRF3 activation and anti-viral gene expression. Reciprocally, mild overexpression of Sec16A or p115 in Hec1B cells increased the activation of IFNβ, ISG56 and NF-κB -dependent promoters following viral infection and ectopic expression of MAVS and Tank-binding kinase-1 (TBK1). In line with these results, TRAF3 was found enriched in immunocomplexes composed of p115, Sec16A and TBK1 upon infection. Hence, we propose a model where dsDNA and dsRNA sensing induces the formation of membrane-bound compartments originating from the Golgi, which mediate the dynamic association of TRAF3 with MAVS leading to an optimal induction of innate immune responses.