Filomena Esteves

Nitric oxide producing neurones in the rat medulla oblongata that project to nucleus tractus solitarii

The production of nitric oxide in neurones of the rat medulla oblongata that project to the nucleus tractus solitarii (NTS) was examined by simultaneous immunohistochemical detection of nitric oxide synthase (NOS) and of cholera toxin B-subunit (CTb), which was injected into the caudal zone of the NTS. Neurones immunoreactive for CTb and neurones immunoreactive for NOS were widely co-distributed and found in almost all the anatomical divisions of the medulla. Dual-labelled cells, containing both CTb and NOS immunoreactivities were more numerous ipsilaterally to the injection sites. They were concentrated principally in the more rostral zone of the NTS, raphé nuclei, dorsal, intermediate and lateral reticular areas, spinal trigeminal and paratrigeminal nuclei and the external cuneate and medial vestibular nuclei. Isolated dual-labelled neurones were also scattered throughout most of the divisions of the reticular formation. These observations indicate that many areas of the medulla that are known to relay somatosensory and viscerosensory inputs contain NOS immunoreactive neurones that project to the NTS, and may, therefore, contribute to the dense NOS-immunoreactive innervation of the NTS. The release of nitric oxide from the axon terminals of these neurones may modulate autonomic responses generated by NTS neurones in relation to peripheral sensory stimuli, and thus ultimately regulate sympathetic and/or parasympathetic outflow.

A Rac/Cdc42 exchange factor complex promotes formation of lateral filopodia and blood vessel lumen morphogenesis

During angiogenesis, Rho-GTPases influence endothelial cell migration and cell–cell adhesion; however it is not known whether they control formation of vessel lumens, which are essential for blood flow. Here, using an organotypic system that recapitulates distinct stages of VEGF-dependent angiogenesis, we show that lumen formation requires early cytoskeletal remodelling and lateral cell–cell contacts, mediated through the RAC1 guanine nucleotide exchange factor (GEF) DOCK4 (dedicator of cytokinesis 4). DOCK4 signalling is necessary for lateral filopodial protrusions and tubule remodelling prior to lumen formation, whereas proximal, tip filopodia persist in the absence of DOCK4. VEGF-dependent Rac activation via DOCK4 is necessary for CDC42 activation to signal filopodia formation and depends on the activation of RHOG through the RHOG GEF, SGEF. VEGF promotes interaction of DOCK4 with the CDC42 GEF DOCK9. These studies identify a novel Rho-family GTPase activation cascade for the formation of endothelial cell filopodial protrusions necessary for tubule remodelling, thereby influencing subsequent stages of lumen morphogenesis.

Affimer proteins are versatile and renewable affinity reagents

Molecular recognition reagents are key tools for understanding biological processes and are used universally by scientists to study protein expression, localisation and interactions. Antibodies remain the most widely used of such reagents and many show excellent performance, although some are poorly characterised or have stability or batch variability issues, supporting the use of alternative binding proteins as complementary reagents for many applications. Here we report on the use of Affimer proteins as research reagents. We selected 12 diverse molecular targets for Affimer selection to exemplify their use in common molecular and cellular applications including the (a) selection against various target molecules; (b) modulation of protein function in vitro and in vivo; (c) labelling of tumour antigens in mouse models; and (d) use in affinity fluorescence and super-resolution microscopy. This work shows that Affimer proteins, as is the case for other alternative binding scaffolds, represent complementary affinity reagents to antibodies for various molecular and cell biology applications. DOI: http://dx.doi.org/10.7554/eLife.24903.001

Scleroderma fibroblasts suppress angiogenesis via TGF-β/caveolin-1 dependent secretion of pigment epithelium-derived factor

Objectives Systemic sclerosis (SSc) is characterised by tissue fibrosis and vasculopathy with defective angiogenesis. Transforming growth factor beta (TGF-β) plays a major role in tissue fibrosis, including downregulation of caveolin-1 (Cav-1); however, its role in defective angiogenesis is less clear. Pigment epithelium-derived factor (PEDF), a major antiangiogenic factor, is abundantly secreted by SSc fibroblasts. Here, we investigated the effect of TGF-β and Cav-1 on PEDF expression and the role of PEDF in the ability of SSc fibroblasts to modulate angiogenesis. Methods PEDF and Cav-1 expression in fibroblasts and endothelial cells were evaluated by means of immunohistochemistry on human and mouse skin biopsies. PEDF and Cav-1 were silenced in cultured SSc and control fibroblasts using lentiviral short-hairpin RNAs. Organotypic fibroblast–endothelial cell co-cultures and matrigel assays were employed to assess angiogenesis. Results PEDF is highly expressed in myofibroblasts and reticular fibroblasts with low Cav-1 expression in SSc skin biopsies, and it is induced by TGF-β in vitro. SSc fibroblasts suppress angiogenesis in an organotypic model. This model is reproduced by silencing Cav-1 in normal dermal fibroblasts. Conversely, silencing PEDF in SSc fibroblasts rescues their antiangiogenic phenotype. Consistently, transgenic mice with TGF-β receptor hyperactivation show lower Cav-1 and higher PEDF expression levels in skin biopsies accompanied by reduced blood vessel density. Conclusions Our data reveal a new pathway by which TGF-β suppresses angiogenesis in SSc, through decreased fibroblast Cav-1 expression and subsequent PEDF secretion. This pathway may present a promising target for new therapeutic interventions in SSc.

Transforming Growth Factor β Activation Primes Canonical Wnt Signaling Through Down-Regulation of Axin-2

Aberrant activation of Wnt signaling has been observed in tissues from patients with systemic sclerosis (SSc). This study aimed to determine the role of transforming growth factor β (TGFβ) in driving the increased Wnt signaling, through modulation of axis inhibition protein 2 (Axin‐2), a critical regulator of the Wnt canonical pathway.

SAT0022 Palmoplantar specific long NCRNA hotair drives myofibroblasts specific signature in systemic sclerosis

Background The key cellular elements in the pathogenesis of tissue fibrosis are myofibroblasts. It is widely accepted that the number of myofibroblasts is increased in Scleroderma (SSc) and it correlates with the severity of tissue fibrosis. The mechanisms underlying their increased number in SSc are unknown. The heterogeneity in their frequency may explain the inconsistency of some in vitro studies published on SSc fibroblast biology and may mirror the clinical heterogeneity of SSc patients both in natural history and severity of skin fibrosis. Objectives The purpose of this study was to unravel the specific transcriptome of myofibroblasts derived from SSc skin biopsies. Methods Four patients with diffuse rapidly progressive SSc, within 18 months from skin involvement and before any immunosuppression, were enrolled in the study. Skin biopsy on forearm was performed and the fibroblasts subcultured for three passages. 250 acetone fixed alpha-SMA positive cells were isolated by laser capture microdissection (LCM) for mRNA analysis by Affymetrix Gene array. qRT-PCR and in situ hybridization were employed for validation of the results. Pathway analysis was conducted according to David-NIH software. Immunofluorescence (IF) followed by confocal laser scanning microscopy (CLSM) was conducted as well. Normal dermal fibroblasts were utilized to evaluate the effects of TGF-beta stimulation both at mRNA and protein level. Results qRT-PCR for alpha-SMA showed, in average, 3.7 fold increased expression in alpha-SMA in the LCM captured cells. Microarray analisis identified 269 genes upregulated more than 2 fold in the myofibroblasts. Of these, 24 were clearly reconducible to profibrotic activation, including alpha-SMA, Collagens I, VI and XI, Fibronectin, several Integrin genes, FGF7, CD36, IGF and Rho; 16 were ribosomial genes; 14 were mitochondrial genes involved in oxidative phosphorylation, including COX1,2, 3 and 6 ND1 to 6, CYT-b and F-type ATP-ase; 28 genes were involved in cell to cell adhesion including, JAM2, ERM, and MLC and 7 in antigen processing and presentation including RAB13, B2-microgrobiulin, cathepsin, HSPs, calnexin, and calreticulin. The remaining genes were not classifiable in any specific functional pathway. IF studies followed by CLSM confirmed the specific pattern of protein expression in myofibroblasts vs alpha-SMA negative fibroblasts from the same donor. Long non coding RNA tiling array of the same cells indicated that alpha-SMA positive fibroblasts had a specific up-regulation of HOX-A9 also known as HOTAIR, which is usually expressed mainly in the dermis of the palmo-plantar region. qRT-PCR and in situ hybridization confirmed the increased expression of HOTAIR in SSc skin. Consistently with these results, skin biopsies from the forearms of SSc patients showed an increased level of the palmo-plantar specific Keratin-9. Conclusions Myofibroblast secretome displayed, besides predictable genes involved in the increased ECM production and TGF-beta pathway activation, a specific gene expression non TGF-inducible and suggestive of a distinct and stable differentiation lineage usually present only in the palmo-plantar region. This distinct differentiation status may account for the dermal and epidermal changes in SSc. Disclosure of Interest None Declared