Sildenafil Reduces Expression and Release of IL-6 and IL-8 Induced by Reactive Oxygen Species in Systemic Sclerosis Fibroblasts

Oxidative stress linked to vascular damage plays an important role in the pathogenesis of systemic sclerosis (SSc). Indeed, vascular damage at nailfold capillaroscopy in patients with Raynaud’s Phenomenon (RP) is a major risk factor for the development of SSc together with the presence of specific autoantiobodies. Here, we investigated the effects of the phosphodiesterase type 5 inhibitor (PDE5i) sildenafil, currently used in the management of RP, in modulating the proinflammatory response of dermal fibroblasts to oxidative stress in vitro. Human fibroblasts isolated from SSc patients and healthy controls were exposed to exogenous reactive oxygen species (ROS) (100 µM H2O2), in the presence or absence of sildenafil (1 µM). Treatment with sildenafil significantly reduced dermal fibroblast gene expression and cellular release of IL-6, known to play a central role in the pathogenesis of tissue damage in SSc and IL-8, directly induced by ROS. This reduction was associated with suppression of STAT3-, ERK-, NF-κB-, and PKB/AKT-dependent pathways. Our findings support the notion that the employment of PDE5i in the management of RP may be explored for its efficacy in modulating the oxidative stress-induced proinflammatory activation of dermal fibroblasts in vivo and may ultimately aid in the prevention of tissue damage caused by SSc

Activation of the Gi protein-RHOA axis by non-canonical Hedgehog signaling is independent of primary cilia.

Primary cilia are solitary organelles that emanate from the plasma membrane during growth arrest in almost all mammalian cells. The canonical Hedgehog (HH) pathway requires trafficking of the G protein-coupled receptor SMOOTHENED (SMO) and the GLI transcription factors to the primary cilium upon binding of a HH ligand to PATCHED1. However, it is unknown if activation of the small GTPase RHOA by SMO coupling to heterotrimeric Gi proteins, a form of non-canonical HH signaling, requires localization of SMO in the primary cilium. In this study, we compared RHOA and Gi protein stimulation by activation of SMO or sphingosine 1-phosphate receptor (S1P) receptors in WT and KIF3A-deficient mouse embryonic fibroblasts that lack primary cilia. We found that activation of SMO in response to Sonic HH (SHH) or purmorphamine (PUR), a small molecule agonist of SMO, stimulates Gi proteins and RHOA independently of the presence of primary cilia, similar to the effects of S1P. However, while S1P induced a fast activation of AKT that is sensitive to the Gi inhibitor pertussis toxin, HH pathway activators did not significantly activate AKT, suggesting that RHOA activation is not downstream of AKT. Our findings demonstrate that early events in some forms of non-canonical HH signaling occur in extraciliary membranes, which might be particularly relevant for actively-cycling cells, for some cancers characterized by loss of primary cilia, and in ciliopathies

Sildenafil Counteracts the In Vitro Activation of CXCL-9, CXCL-10 and CXCL-11/CXCR3 Axis Induced by Reactive Oxygen Species in Scleroderma Fibroblasts

Oxidative stress plays a key role in systemic sclerosis (SSc) pathogenesis, and an altered redox homeostasis might be responsible for abnormal inflammatory status, fibrosis and tissue damage extension. In this study, we explored the effect of the phosphodiesterase type 5 inhibitor sildenafil in modulating the activation of the CXCL-9, -10, -11/CXCR3 axis, which is fundamental in the perpetuation of inflammation in different autoimmune diseases, in the cell culture of SSc human dermal fibroblasts exposed to a pro-oxidant environment. We observed that sildenafil significantly reduced gene expression and release of CXCL-9, -10 and -11, inhibited the CXCR3 action and suppressed the activation of STAT1-, JNK- and p38MAPK pathways. This in vitro study on dermal fibroblasts supports clinical studies to consider the efficacy of sildenafil in preventing tissue damage and fibrosis in SSc by targeting central biomarkers of disease progression, vascular injuries and fibrosis and reducing the pro-inflammatory activation induced by oxidative stress

SFRP4 Expression Is Linked to Immune-Driven Fibrotic Conditions, Correlates with Skin and Lung Fibrosis in SSc and a Potential EMT Biomarker

Secreted Frizzled Receptor Protein 4 (SFRP4) has been shown to be increased in Scleroderma (SSc). To determine its role in immune-driven fibrosis, we analysed SSc and sclerotic Chronic Graft Versus Host Disease (sclGVHD) biosamples; skin biopsies (n = 24) from chronic GVHD patients (8 with and 5 without sclGVHD), 8 from SSc and 3 healthy controls (HC) were analysed by immunofluorescence (IF) and SSc patient sera (n = 77) assessed by ELISA. Epithelial cell lines used for in vitro Epithelial-Mesenchymal-Transition (EMT) assays and analysed by Western Blot, RT-PCR and immunofluorescence. SclGVHD skin biopsies resembled pathologic features of SSc. IF of fibrotic skin biopsies indicated the major source of SFRP4 expression were dermal fibroblasts, melanocytes and vimentin positive/caveolin-1 negative cells in the basal layer of the epidermis. In vitro studies showed increased vimentin and SFRP4 expression accompanied with decreased caveolin-1 expression during TGFβ-induced EMT. Additionally, SFRP4 serum concentration correlated with severity of lung and skin fibrosis in SSc. In conclusion, SFRP4 expression is increased during skin fibrosis in two different immune-driven conditions, and during an in vitro EMT model. Its serum levels correlate with skin and lung fibrosis in SSc and may function as biomarker of EMT. Further studies are warranted to elucidate the role of SFRP4 in EMT within the pathogenesis of tissue fibrosi

Silencing of Histone Deacetylase 6 Decreases Cellular Malignancy and Contributes to Primary Cilium Restoration, Epithelial-to-Mesenchymal Transition Reversion, and Autophagy Inhibition in Glioblastoma Cell Lines

Glioblastoma multiforme, the most common type of malignant brain tumor as well as the most aggressive one, lacks an effective therapy. Glioblastoma presents overexpression of mesenchymal markers Snail, Slug, and N-Cadherin and of the autophagic marker p62. Glioblastoma cell lines also present increased autophagy, overexpression of mesenchymal markers, Shh pathway activation, and lack of primary cilia. In this study, we aimed to evaluate the role of HDAC6 in the pathogenesis of glioblastoma, as HDAC6 is the most overexpressed of all HDACs isoforms in this tumor. We treated glioblastoma cell lines with siHDAC6. HDAC6 silencing inhibited proliferation, migration, and clonogenicity of glioblastoma cell lines. They also reversed the mesenchymal phenotype, decreased autophagy, inhibited Shh pathway, and recovered the expression of primary cilia in glioblastoma cell lines. These results demonstrate that HDAC6 might be a good target for glioblastoma treatment

Database Species-Area Relationships in Palaearctic Grasslands.

The database collects the data resulting from the sampling of species-area relationships (SARs) in grassland communities in the Palaearctic. The core features of the database are the complete data from the EDGG Research Expeditions, but similar data from other studies are also included. Main features of the majority of relevés are: (i) they are part of nested-plot series (typically 0.0001-100 m²), (ii) bryophytes and lichens are treated comprehensively; (iii) detailed soil and other environmental data

Scale-dependent plant diversity in Palaearctic grasslands: a comparative overview

Here we present an extensive overview of plant diversity values in Palaearctic grasslands for seven standard grain sizes from 0.0001 to 100 m². The data originate from 20 studies, including the Field Workshops of the Eurasian Dry Grassland Group (EDGG), ranging geographically from Spain in the west to Siberia in the east, from Sicily in the south to Estonia in the north and from the sea coast up to 3100 m a.s.l. The majority of data is from dry grasslands (Festuco-Brometea, Koelerio-Corynephoretea, Cleistogenetea squarrosae), but there are also some mesic, wet, saline, acidic, alpine and Mediterranean grasslands included. Among others, we compiled data from 1795 1-m², 1109 10-m² and 338 100-m² plots. In all cases we present mean, minimum and maximum richness for the seven grain sizes, plus, in cases where also terricolous bryophytes and lichens had been recorded, the same values for total “plant” species richness, non-vascular plant species richness and fraction of non-vascular plants. The maximum richness values were 82, 101 and 134 for all “plants”, and 79, 98 and 127 vascular plants at grain sizes of 1 m², 10 m² and 100 m², respectively (all in Transylvania, Romania). Our overview comprises new, hitherto unpublished world records of vascular plant species richness at the scales of 0.0001 m² (9) and 0.001 m² (19, both shoot presence), from meso-xeric, basiphilous grasslands in Navarre, Spain, which is much higher than the previously known maxima. The highest values of non-vascular plant richness at 1 m², 10 m² and 100 m², respectively, were 49, 64 and 64, respectively (all in Sedo-Scleranthenea communities of Öland, Sweden, and Saaremaa, Estonia). In general, the dry, alpine and Mediterranean grasslands were much richer than the studied mesic, wet or saline grasslands at any spatial scale. The presented set of mean, minimum and maximum values and their metadata is publically available and will be continuously updated. These data can serve as a reference of “normal” richness, both in fundamental and applied research. To facilitate the application, we provide an easy formula based on the power-law species-area relationship that allows the estimation of richness values at intermediate grain sizes not included in our dataset. In conclusion, our data emphasise the role of Palaearctic grasslands as global hotspot of small-scale vascular plant diversity, while at the same time highlighting that in some grassland types also the bryophyte and lichen diversity can be extraordinarily high

Targeting human plasmacytoid dendritic cells through BDCA2 prevents skin inflammation and fibrosis in a novel xenotransplant mouse model of scleroderma

Objectives: Plasmacytoid dendritic cells (pDCs) have been implicated in the pathogenesis of autoimmune diseases, such as scleroderma (SSc). However, this has been derived by indirect evidence using ex vivo human samples or mouse pDC in vivo. We have developed human-specific pDC models to directly identify their role in inflammation and fibrosis, as well as attenuation of pDC function with BDCA2-targeting to determine its therapeutic application. Methods: RNA-seq of human pDC with TLR9 agonist ODN2216 and humanised monoclonal BDCA2 antibody, CBS004. Organotypic skin rafts consisting of fibroblasts and keratinocytes were stimulated with supernatant from TLR9-stimulated pDC and with CBS004. Human pDCswere xenotransplanted into SCID mice treated with Aldara (inflammatory model), or bleomycin (fibrotic model) with CBS004 or human IgG control. Punch biopsy of skin was used to assess gene and protein expression. Results: RNA-seq shows TLR9-induced activation of human pDC goes beyond type I interferon (IFN) secretion, which is functionally inactivated by BDCA2 targeting. Consistent with these findings, we show that BDCA2 targeting of pDC can completely suppress in vitro skin IFN-induced response. Most importantly, xenotransplantation of human pDC significantly increased in vivo skin IFN-induced response to TLR agonist and strongly enhanced fibrotic and immune response to bleomycin compared with controls. In these contexts, BDCA2 targeting suppressed human pDC- specific pathological responses. Conclusions: Our data indicate that human pDC plays a key role in inflammation and immune-driven skin fibrosis, which can be effectively blocked by BDCA2 targeting, providing direct evidence supporting the development of attenuation of pDC function as a therapeutic application for SSc

Role of cellular senescence and NOX4-mediated oxidative stress in systemic sclerosis pathogenesis.

Systemic sclerosis (SSc) is a systemic autoimmune disease characterized by progressive fibrosis of skin and numerous internal organs and a severe fibroproliferative vasculopathy resulting frequently in severe disability and high mortality. Although the etiology of SSc is unknown and the detailed mechanisms responsible for the fibrotic process have not been fully elucidated, one important observation from a large US population study was the demonstration of a late onset of SSc with a peak incidence between 45 and 54 years of age in African-American females and between 65 and 74 years of age in white females. Although it is not appropriate to consider SSc as a disease of aging, the possibility that senescence changes in the cellular elements involved in its pathogenesis may play a role has not been thoroughly examined. The process of cellular senescence is extremely complex, and the mechanisms, molecular events, and signaling pathways involved have not been fully elucidated; however, there is strong evidence to support the concept that oxidative stress caused by the excessive generation of reactive oxygen species may be one important mechanism involved. On the other hand, numerous studies have implicated oxidative stress in SSc pathogenesis, thus, suggesting a plausible mechanism in which excessive oxidative stress induces cellular senescence and that the molecular events associated with this complex process play an important role in the fibrotic and fibroproliferative vasculopathy characteristic of SSc. Here, recent studies examining the role of cellular senescence and of oxidative stress in SSc pathogenesis will be reviewed

Disturbance indicator values for European plants

Motivation Indicator values are numerical values used to characterize the ecological niches of species and to estimate their occurrence along gradients. Indicator values on climatic and edaphic niches of plant species have received considerable attention in ecological research, whereas data on the optimal positioning of species along disturbance gradients are less developed. Here, we present a new data set of disturbance indicator values identifying optima along gradients of natural and anthropogenic disturbance for 6382 vascular plant species based on the analysis of 736,366 European vegetation plots and using expert-based characterization of disturbance regimes in 236 habitat types. The indicator values presented here are crucial for integrating disturbance niche optima into large-scale vegetation analyses and macroecological studies. Main types of variables contained We set up five main continuous indicator values for European vascular plants: disturbance severity, disturbance frequency, mowing frequency, grazing pressure and soil disturbance. The first two indicators are provided separately for the whole community and for the herb layer. We calculated the values as the average of expert-based estimates of disturbance values in all habitat types where a species occurs, weighted by the number of plots in which the species occurs within a given habitat type. Spatial location and grain Europe. Vegetation plots ranging in size from 1 to 1000 m(2). Time period and grain Vegetation plots mostly sampled between 1956 and 2013 (= 5th and 95th quantiles of the sampling year, respectively). Major taxa and level of measurement Species-level indicator values for vascular plants. Software format csv file